1. No category

Title
Author(s)
The functional roles of the polymorphisms of a secretary
candiate tumor suppressor, serum amyloid A1 (SAA1), in
nasopharyngeal carcinoma(NPC)
Yeung, Man-chung; 楊敏聰
Citation
Issue Date
URL
Rights
2011
http://hdl.handle.net/10722/144785
The author retains all proprietary rights, (such as patent
rights) and the right to use in future works.
The functional roles of the polymorphisms of a
secretary candidate tumor suppressor, serum amyloid
A1 (SAA1), in nasopharyngeal carcinoma (NPC)
by
YEUNG Man Chung
B.Sc., HKUST
A thesis submitted in partial fulfillment for the requirements for
the degree of Master of Philosophy
at The University of Hong Kong
October 2011
i
Abstract of thesis entitled
The functional roles of the polymorphisms of a secretary
candidate tumor suppressor, serum amyloid A1 (SAA1), in
nasopharyngeal carcinoma (NPC)
Submitted by
Yeung Man Chung
For the degree of Master of Philosophy
The University of Hong Kong
In October 2011
Nasopharyngeal carcinoma (NPC) is a cancer which occurs in high frequency in
Southern China. The identification of diagnostic and prognostic markers will be
highly beneficial for early stage NPC detection. Previous microcell-mediated
chromosome transfer (MMCT) studies showed that the transfer of an intact human
chromosome 11 suppresses the in vivo tumor growth of a NPC cell line (HONE1) in
nude mouse tumorigenicity assays. By using a 19K oligo-microarray analysis, one
candidate tumor suppressor gene (TSG), Serum Amyloid A 1 (SAA1), which showed
differential expression in the tumor-suppressive hybrid cells versus their matched
tumorigenic revertants, was chosen for further study in this project. Frequent
down-regulation of SAA1 expression was observed in both NPC cell lines and clinical
ii
tumor biopsies, and promoter hypermethylation is one major gene silencing
mechanism. It is of interest to investigate whether physiological levels of SAA1
expressed locally in the normal nasopharyngeal tissue play a novel protective role
against tumor development.
SAA encodes an acute phase HDL-associated apolipoprotein, whose levels are
greatly elevated following injury, inflammation, and cancer. This plasma protein is
mainly synthesized in the liver. SAA is a generic term for a family of acute-phase
proteins encoded by different genes with a high allelic variation. SAA1 maps in a 150
kb region of chromosome 11p15.1. It has five coding alleles, SAA1.1, SAA1.2, SAA1.3,
SAA1.4, and SAA1.5. A number of studies have demonstrated the association between
the increase in plasma SAA level and tumor development. However, the functional
role of SAA1 in tumor progression has not been well-characterized. This study aims
to determine the relationship between SAA1 polymorphisms and the risk of NPC and
to determine the usefulness of SAA1 variants as a biomarker for NPC diagnosis. This
study also aims to investigate the anti-tumor activities of SAA1 variant proteins and
to study the functional roles of ectopically-expressed SAA1 proteins in NPC cell
lines.
My results of SAA1 genotyping showed that only three SAA1 isoforms (SAA1.1,
1.3, and 1.5) were observed in both Hong Kong NPC patients and healthy people. The
iii
frequency of SAA1.5/1.5 genotype in NPC patients was higher compared to that in
healthy individuals, which indicates that the SAA1.5/1.5 genotype may associate with
a higher risk of NPC development. Reintroduction of the three SAA1 isoforms in
tetracycline-regulated HONE1-2 cell lines and lentivirus-controlled expression
systems inhibited angiogenesis in vitro. Treatment of SAA1 recombinant proteins also
inhibited endothelial cell viability, adhesion, and angiogenesis. Differential inhibitory
effects were observed among the three SAA1 isoforms. Results of the functional
studies showed that the SAA1.5 recombinant proteins consistently elicit the lowest
inhibitory effects on endothelial cell viability and tube formation ability among the
three isoforms.
In conclusion, the current functional evidence indicates that a single variation of
an amino acid residue can significantly reduce the activities of SAA1. The secreted
SAA1.5 protein might contribute to the weakest protective effects against NPC
formation, which may be associated with a higher risk of NPC development.
Word count: 483
iv
DECLARATION
I declare that the thesis and the research work thereof represent my own work,
except where due acknowledgement is made, and that it has not been previously
included in a thesis, dissertation or report submitted to this University or to any other
institution for a degree, diploma or other qualifications.
Signed: ____________________________
Man Chung YEUNG
v
Acknowledgements
It is one of the greatest achievements in my life to complete the MPhil study
under the supervision of Dr. Hong Lok Lung and Prof. Maria Li Lung in these two
years. Many people provided help to me throughout my study; without their help I
would not be able to accomplish as much.
First, I would like to thank my supervisors, Dr. Hong Lok Lung and Prof. Maria
Lung, for providing me the opportunity to be a MPhil student. From them, I learned
the scientific thinking and writing skills, and the serious and persistent attitudes
towards science.
My gratitude also goes to all my labmates in Prof. Lung’s Laboratory, Dr.
Josephine Ko, Villus Kwong, Dr. Paulisally Lo, Dr. Victor Wong, Dr. Candy Chan,
Valentine Yu, Evan Law, and Gerry Huang. They all provided me reliable support and
assistance in my research. I shall particularly like to thank Dr. Victor Wong and Dr.
Paulisally Lo. They did not only teach me lots of technical skills and knowledge, but
also gave me encouragement and valuable advice for my research throughout these
two years. My thanks also go to Prof. Didier Trono, for the supply of the lentiviral
vectors, pWPI, pMD2.G, and psPAX2 for our research project; Prof. Eugene
Zabarovsky for providing the inducible transfection vector system; Prof. George Tsao
for providing the immortalized nasopharyngeal epithelial cell lines, NP69 and NP460,
vi
and the immortalized esophageal cell lines, NE1 and NE3 cell lines; Dr. Carol Szeto
and Dr. Ka Ming Lee for analyzing the 3D structure of the SAA1 protein peptides.
Last but not least, I thank my family for their support and encouragement during my
MPhil study.
vii
Table of Contents
Abstract
Declarations
Acknowledgements
Table of Contents
List of Figures
Page
i
v
vi
viii
xiii
List of Tables
List of Abbreviations
xvi
xvii
1
Chapter 1: Introduction
1.1
Nasopharyngeal carcinoma (NPC)
1.1.1 Histology of NPC
1
1.1.2 Epidemiology
3
1.1.3 Risk factors for NPC
3
1.1.3.1 Diet
3
1.1.3.2 Epstein-Barr Virus
6
1.1.3.3 Smoking
7
1.1.4 Gene predisposition of NPC
1.2
1.3
1
9
1.1.4.1 Gene polymorphisms
9
1.1.4.2 Familial aggregation
10
1.1.4.3 Cytogenetic alterations
11
Molecular genetics of cancer
14
1.2.1 Multi-step progression model
14
17
Angiogenesis
1.3.1 Pro-angiogenic factors
19
1.3.2 Anti-angiogenic factors
20
viii
Page
1.4
Oncogenes
20
1.5
Tumor suppressor genes (TSGs)
23
1.6
1.7
1.8
1.9
1.10
1.5.1 p53 pathway
24
1.5.2 p16/pRB pathway
25
Mechanisms of TSG inactivation
27
1.6.1 Loss of heterozygosity (LOH)
28
1.6.2 Mutations
28
1.6.3 Epigenetic silencing
29
29
Methods to identify TSGs
1.7.1 Positional cloning
30
1.7.2 Functional cloning
32
Various chromosome transfer techniques
33
1.8.1 Whole and truncated chromosome transfer
33
1.8.2 YAC transfer (YACT)
34
1.8.3 BAC and P1-derived artificial chromosome (PAC) transfer
35
The current MMCT-based approach in identification TSGs in NPC
35
1.9.1 Role of chromosome 11 in NPC
37
1.9.2 Chromosome 11 NPC genes identified by MMCT
39
Serum amyloid A (SAA) family
41
1.10.1
SAA1 isoforms in amyloidosis
41
1.10.2
SAA1 and cancers
44
1.10.3
Functions of SAA1
45
48
1.11 Aims of the project
ix
Page
Chapter 2: Materials and Methods
53
2.1 Tissue culture
53
2.2 Tissue specimens and blood samples
54
2.3 RNA extraction
57
2.4 DNA extraction from mammalian cell lines
58
2.5 DNA extraction from patient tissue samples
58
2.6 DNA extraction from blood of healthy individuals
59
2.7 Reverse transcription polymerase chain reaction (RT-PCR)
60
2.8 DNA sequencing
60
2.9 Real-time quantitative reverse transcription PCR (Q-PCR)
64
2.10 Bisulfite treatment
65
2.11 Methylation-specific PCR (MSP)
66
2.12 Bisulfite genomic sequencing (BGS)
69
2.13 5-aza-2’ deoxycytidine treatment
70
2.14 Plasmid construction
70
2.15 Stable transfection
75
2.16 Recombinant SAA1 protein expression and purification
77
2.17 Preparation of total protein lysates and conditioned media from mammalian
79
cells
2.18 Western blot analysis
79
2.19 HUVEC and HMEC tube formation assays
81
2.20 Real-time cell viability assay
83
2.21 Cell adhesion assay
84
2.22 Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)
85
x
Page
assay
2.23 Statistical analysis
86
Chapter 3: Results
87
3.1 Genotyping of SAA1 in NPC patients versus healthy individuals
87
3.2 Gene expression analysis of SAA1 in chromosome 11 MCHs and their matched
89
TSs
3.3 Gene expression analysis of SAA1 in other NPC and esophageal squamous cell
91
carcinoma (ESCC) cell lines and clinical biopsies
3.4 Mechanism of gene silencing of SAA1
101
3.5 The anti-angiogenic effects of the endogenous SAA1 isoforms
106
3.5.1 Establishment of SAA1 stable transfectants
106
3.5.2 The secretary SAA1 proteins from the stable transfectants inhibit endothelial
108
cell tube formation in vitro
3.6 The biological activities of the SAA1 recombinant variant proteins
112
3.6.1 Production and purification of SAA1 recombinant proteins
112
3.6.2 The inhibitory effects of the recombinant SAA1 isoforms on endothelial cell
114
tube formation
3.6.3 The endothelial cell viability was differentially inhibited by the exogenous
116
SAA1 variants
3.6.4 The effects of SAA1 isoforms on endothelial cell-NPC cell adhesion
120
3.6.5 SAA1 induced-apoptosis in endothelial cells
124
3.7 The direct anti-tumor effects of the recombinant SAA1 variant proteins in
xi
126
Page
NPC cells
3.7.1 The effects of the recombinant SAA1 on NPC cell viability
126
3.7.2 SAA1 induced-apoptosis in NPC cells
129
Chapter 4: Discussion
132
4.1 Expression of SAA1 in the chromosome 11 MCHs and their matched TSs and
134
NPC and ESCC cell lines
4.2 Mechanism to silence SAA1 expression
135
4.3 Genotyping of SAA1 in NPC patients versus healthy people
136
4.4 The anti-angiogenic properties of SAA1 proteins
138
4.5 The direct anti-tumor effects of the recombinant SAA1 variant proteins on
140
NPC cells
4.6 General discussion
141
Chapter 5: Conclusions
147
Chapter 6: References
149
Appendix:
171
Table A1) Genotyping of SAA1 isoforms in 80 NPC patients.
171
Buffer list
174
xii
List of Figures
Figure 1
Figure 2
Figure 3
Figure 4
Page
Anatomy of the human nasopharynx.
2
Age-specific incidence rate and mortality rate of NPC in 2007.
5
Proposed mechanisms of Epstein-Barr virus (EBV) latent proteins
8
in NPC development.
Figure 5
Figure 6
Figure 7
LOH frequencies of 36 microdissected primary NPC tumors
detected by microsatellite analysis.
CGH analysis of 20 primary NPC biopsies.
Hallmarks of cancer and their potential therapeutic targets.
A genetic model of multi-step pathway in colorectal cancer from
Figure 8
Figure 9
Figure 10
normal epithelium to metastatic tumor.
Proposed pathogenesis model for NPC.
Well-studied gatekeeper TSG pathways.
Summary of approaches used for TSG identification.
Figure 11
12
13
15
16
18
26
31
Outline of our strategy to identify TSG by using
microcell-mediated chromosome transfer (MMCT).
The nucleotide and amino acid differences among the five SAA1
isoforms and the amino acid sequence.
Region of the SAA1 CpG sites containing sequence in the
promoter.
The vector map of pETE-Bsd.
The vector map of pWPI.
The vector map of pET-28a-(+).
36
90
Figure 22
The chromosome 11 19K oligo-microarray results showing the
SAA1 expression in MCHs and their matched TSs.
The SAA1 gene expression in HONE1, 556.15, the four MCH cell
lines and their TSs.
The SAA1 gene expression in six NPC cell lines.
Real-time PCR performed with 42 pairs of NPC tissues.
The SAA1 gene expression in 15 ESCC cell lines and 3 pairs of
ESCC tissue samples.
MSP analysis of SAA1 promoter methylation in seven NPC cell
Figure 23
lines and two immortalized NP cell lines.
BGS analysis of 5 CpG sites in SAA1 promoter region of seven
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17
Figure 18
Figure 19
Figure 20
Figure 21
xiii
43
67
71
73
74
93
95
96
100
102
104
Figure 24
Figure 25
Figure 26
Figure 27
Figure 28
Figure 29
Figure 30
Figure 31
Figure 32
Figure 33
Figure 34
NPC cell lines and the immortalized NP460 cell line.
Page
The SAA1 gene expression in four NPC and five ESCC cell lines 105
before and after treatment with 5-aza-2’-deoxycytidine.
The SAA1 RNA expression in the HONE1-pETE-Bsd-SAA1.1, 107
1.3, and 1.5 (H1-pB-SAA1.1, -1.3, and 1.5) transfectants and the
Western blot analysis of SAA1 protein in conditioned media of
these transfectants.
The GFP expression of C666-pWPI-SAA1.1, 1.3, and 1.5, and the 109
vector-alone transfectants and the Western blot analysis of SAA1
protein in conditioned media of SAA1.1, 1.3, and 1.5 expressed
cells.
HUVEC tube formation assay of the HONE1-2 SAA1 variant
transfectants and vector-alone control.
HUVEC tube formation assay of C666 SAA1 transfectants and
vector-alone control.
The SDS PAGE analysis of His-tag SAA1.1 recombinant proteins
and the Western blot analysis of the purified His-tag SAA1.1, 1.3,
and 1.5 variant proteins.
The effects of the SAA1.1, 1.3, and 1.5 recombinant proteins on
HUVEC tube formation.
The effects of the SAA1.1, 1.3, and 1.5 recombinant proteins on
HMEC tube formation.
The real-time HMEC cell viability assay studying the effects of
0.5 mg/ml SAA1.1, 1.3, and 1.5 proteins and various
concentrations of SAA1.3 protein.
The real-time cell viability analysis for studying the effects of 0.5
mg/ml SAA1.1, 1.3, and 1.5 proteins on NP460.
Figure 38
The cell adhesion assays for studying the effects of the exogenous
SAA1.1, 1.3, and 1.5 proteins on HUVEC-HONE1 cell adhesion.
The cell adhesion assays for studying the effects of the exogenous
SAA1.1, 1.3, and 1.5 proteins on HMEC-HONE1 cell adhesion.
The TUNEL assay for the apoptosis detection of HUVECs treated
with the exogenous SAA1.3 protein.
Western blot analysis of caspase 3 expression in the SAA1.3
protein-treated HUVEC cells.
Real-time cell viability analysis for studying the effects of 0.5
Figure 39
mg/ml SAA1.1, 1.3, and 1.5 proteins on HONE1 cells.
The TUNEL assays for studying apoptosis induced in HONE1
Figure 35
Figure 36
Figure 37
xiv
111
113
115
117
118
119
121
122
123
125
127
128
130
Page
Figure 40
Figure 41
cells by the recombinant SAA1.1, 1.3, and 1.5 proteins.
Western blot analysis of caspase 3 expression in the SAA1.3
protein treated-HONE1 cells and buffer control.
The 3D model of the secondary structure of the human SAA1.1,
1.3, and 1.5 peptides predicted by PyMol.
xv
131
143
List of Tables
Table 2
Table 3
Page
Sex ratio, median age, and cumulative risk for 20 leading cancer
4
sites for incidence and mortality rates in 2007.
Oncogenic proteins over-expressed in NPC.
22
Background information and culture conditions for cell lines used 55
Table 4
Table 5
Table 6
Table 7
in this study.
RT-PCR primer sequences, conditions, and product sizes.
Primers for DNA cloning.
Primers used for the methylation study of SAA1.
Antibodies used in Western blot analysis.
Table 1
Table 8
Table 9
Table 10
Table 11
61
63
68
82
A comparison of SAA1 genotypes between NPC patients and
healthy groups.
Microarray detection of SAA1 in HONE1 versus MCHs and MCHs
versus TSs.
88
Correlation between SAA1 expression assessed by Q-PCR in Hong
Kong NPC biopsies and clinicopathological parameters.
Comparison of SAA1 isoforms between tumor tissues with
increased and decreased SAA1 expression by Q-PCR.
97
xvi
92
99
List of Abbreviations
AA-amyloidosis
ATM
BAC
Bcl2
BGS
BL
Amyloid A protein-amyloidosis
Ataxia telangiectasia mutated (ATM) checkpoint kinase
Bacterial artificial chromosome
B-cell lymphoma protein 2
Bisulfite genomic sequencing
Burkitt’s lymphoma
BLU/ZMYND10
bp
BRCA1
BRCA2
BSA
Zinc finger, MYND-type containing 10
Base pair
Breast cancer 1, early onset
Breast cancer 2, early onset
Bovine serum albumin
BZLF1
CADM1
CCL2
CD55
BamHI W leftward reading frame 1
Cell ADhesion Molecule 1
Chemokine (C-C motif) ligand 2
Complement decay-accelerating factor
CDK
CFDA
CGH
CRC
CRYAB
CYP2E1
Da
DMEM
DNA
Cyclin dependent kinase
Methacrylated carboxyfluorescein diacetate
Comparative genomic hybridization
Colorectal cancer
Alpha-crystallin B chain
Cytochrome P450 2E1
Dalton
Dulbecco’s modified eagle medium
Deoxyribonucleic acid
dNTP
dox
E. coli
EA
EBERs
EBNA
EBV
EC
ECL
Deoxyribonucleotide peptide
Doxcycline
Escherichia coli
Early antigen
Epstein-Barr encoded RNAs
Epstein-Barr nuclear antigen
Epstein-Barr virus
Esophageal cancer
Enhanced chemiluminescence
ECM
EDTA
Extracellular matrix
Ethylenediaminetetraacetic acid
xvii
EGF
EGFR
Erk
FGFRs
FGFs
FPRL-1
GAPDH
GlPS
GSTM1
Epidermal growth factor
EGF receptor
Extracellular-signal-regulated kinases
FGF receptors
Fibroblast Growth Factors
Formyl peptide receptor like-1
Glyceraldehyde 3-phosphate dehydrogenase
Ganoderma ludicum polysaccharides
Glutathione S-transferase M1
HD
HEPES
HL
HLA
HMEC
Homozygous deletion
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Hodgkin’s lymphoma
Human leukocyte antigen
Human microvascular endothelial cell
HPV16E6/E7
hTERT
HUVEC
IgA
Human papillomavirus type 16 E6/E7
Telomerase reverse transcriptase
Human umbilical vein endothelial cell
Immunoglobulin A
IgG
IPTG
LB
LMP1
LOH
MCH
MIPOL1
MMCT
MSP
Immunoglobulin G
Isopropyl-B-D-thiogalactopyranoside
Luria Broth
Latent membrane protein 1
Loss of heterozygosity
Microcell hybrid
Mirror image polydactyly 1
Microcell-mediated chromosome transfer
Methylation-specific PCR
NF-κB
NMR
NPC
NSCLC
NTRK
PAC
PBS
PCR
PDGF
Nuclear factor kappa-light-chain-enhancer of activated B cells
Nuclear magnetic resonance
Nasopharyngeal carcinoma
Non-small cell lung cancer
Neurotrophic tyrosine kinase
P1-derived artificial chromosome
Phosphate-buffered saline
Polymerase chain reaction
Platelet-Derived Growth Factor
PEG
PLGF
Polyethylene glycol
PlacentaL Growth Factor
xviii
PTPRG
PVDF
Q-PCR
RAGE
RASSF1A
Rb
RNA
RPMI
RT-PCR
Receptor-type tyrosine-protein phosphatase gamma
Polyvinylidene fluoride
Quantitative reverse transcription PCR
Receptor for advanced glycation end products
Ras association domain family 1, isoform A
Retinoblastoma
Ribonucleic acid
Roswell Park Memorial Institute medium
Reverse transcription PCR
SAA
SAGE
SDS
SNP
SR-B1
Serum amyloid A
Serial analysis of gene expression
Sodium dodecyl sulfate
Single nucleotide polymorphism
Scavenger receptor class B member 1
TBS
TGF-β
THY1
TLR
Tris-Buffered Saline
Transforming Growth Factor-β
Thy-1 cell surface antigen
Toll-like receptor
TNF
TSG
TSLC1
TSs
TUNEL
VCA
VEGF
VEGFR
Wnt
Tumor necrosis factor
Tumor suppressor genes
Tumor Suppressor in Lung Cancer 1
Tumor segregants
Terminal deoxynucleotidyl transferase dUTP nick end labeling
viral capsid antigen
Vascular Endothelial Growth Factor
VEGF receptor
Wingless-type MMTV integration site family
YAC
YACT
Yeast artificial chromosome
YAC transfer
xix
Chapter 1: Introduction
1.1 Nasopharyngeal carcinoma (NPC)
1.1.1 Histology of NPC
The nasopharynx is a fibromuscular tube located in the uppermost part of the
pharynx behind the nasal cavity (Figure 1). NPC is a squamous cell carcinoma
originating from the epithelial cells around the nasopharynx. There are mainly three
histological subtypes of NPC, which all show distinct clinical behaviors and
characteristics (Type I, keratinizing squamous carcinoma with varying degree of
differentiation; Type II, differentiated non-keratinizing carcinoma; Type III,
undifferentiated non-keratinizing carcinoma). Among the three types of NPC, type III
is most common and is strongly associated with Epstein-Barr virus (EBV) infection.
Types II and III have a better prognosis and are more sensitive to radiotherapy
treatment (Goh and Lim 2009). Radiotherapy is the primary treatment choice for NPC
(Spano et al 2003) and together with adjuvant chemotherapy improves the overall
survival rates (Teo et al 1999, Wei and Sham 2005). Surgery is usually the last choice
for NPC as the nasopharynx is located deep inside the skull. Stages I and II are
sensitive to a combination of radiotherapy and chemotherapy. However, as early
diagnosis of NPC is difficult, most NPC patients are diagnosed at late stages. The
1
Adapted from: American Society of Clinical Oncology
(http://www.cancer.net/patient/Cancer+Types/Nasopharyngeal+Cancer/ci.Nasopharyn
geal+Cancer.printer)
Figure 1: Anatomy of the human nasopharynx.
2
identification of diagnostic and prognostic markers will be highly beneficial for early
stage detection.
1.1.2 Epidemiology
NPC is a unique disease which shows a distinct racial and geographic
distribution. It is more frequently found in Southeast China, Southeast Asia, North
Africa, Middle East, and Arctic regions, but is rare in most parts of the world
(Jeyakumar et al 2006, Wei and Sham 2005). NPC is also named as the ‘Canton
tumor’, as it is more common among the Cantonese-speaking group. In Hong Kong,
NPC ranks as the seventh leading cancer in terms of incidence rates (Table 1, Figure
2), with a male to female ratio of 2.6:1. For the mortality rate, NPC ranks eighth in
terms of mortality (Table 1), with a male to female ratio of 3.2:1. NPC is a common
cancer among those aged between 20-44 years and the median age for diagnosis for
NPC is 50, which is earlier compared with other cancers. NPC is one of the serious
health problems in Hong Kong due to its high incidence rate, mortality rate, and early
age of occurrence.
1.1.3 Risk factors for NPC
1.1.3.1 Diet
3
A.
B.
(Adapted from: Hong Kong Cancer registry, Hospital Authority, HKSAR, 2007)
Table 1: Sex ratio, median age, and cumulative risk for 20 leading cancer sites for A)
incidence and B) mortality rates in 2007.
4
A.
B.
(Adapted from: Hong Kong Cancer registry, Hospital Authority, HKSAR, 2007)
Figure 2: Age-specific A) incidence rate and B) mortality rate of NPC in 2007.
5
In previous epidemiological studies in NPC, consumption of preserved food such
as salted fish, salted shrimps, and salted vegetables in endemic areas is associated
with development of NPC (Armstrong et al 1983, Ho et al 1978, Yuan et al 2000).
Early exposure and high frequency of consumption promote the risk for NPC. The
nitrosamines and nitrites inside the preserved food are effective carcinogens and are
capable of inducing tumor formation in animal experiments (Huang et al 1978, Yu et
al 1989).
1.1.3.2 Epstein-Barr Virus
Epstein-Barr Virus is a DNA-containing herpesvirus. Some of its genes impact
cell cycle control and induce tumor formation. It is associated with many human
cancers such as Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), gastric
carcinoma, and NPC (Pagano 1999, Shah and Young 2009, Young and Rickinson
2004). Previous studies showed that NPC patient sera contain high levels of IgA and
IgG antibodies against EBV viral capsid antigen (VCA), early antigen (EA) and
Epstein-Barr nuclear antigen (EBNA) (Lung et al 1993, Tsai et al 1998, Vokes et al
1997). Besides, EBV-DNA and Epstein-Barr encoded RNAs (EBERs) are frequently
detected in NPC tissues. These studies suggest that EBV plays an important role in
NPC tumorigenesis (Tsai et al 1998). The EBV latent membrane protein (LMP1) and
6
EBNA-1 are frequently over-expressed in EBV-positive NPC (Vokes et al 1997).
LMP-1 is oncogenic in rodent fibroblasts and can inhibit the differentiation of human
epithelial cells. On the other hand, EBNA-1 induces genomic instability and promotes
oncogenesis in B cells of Burkitt’s lymphoma. These EBV latent proteins interact
with each other and contribute to the pathogenesis of NPC by promoting cell
proliferation, survival, invasion, and inhibiting apoptosis (Figure 3) (Chou et al 2008,
Everly et al 2009, Mainou et al 2005, Valentine et al 2010). Though there is no direct
evidence to prove EBV is the cause of NPC development, it is believed that EBV
infection plays an important role in NPC tumorigenesis (Lo and Huang 2002).
Previous studies indicated that EBV-A genome is present in NPC tissues, which may
contribute to the early development of NPC.
1.1.3.3 Smoking
Cigarette smoking is found to contribute to various cancers including lung,
stomach, oral, and pancreatic cancers. Cigarettes contain carcinogens such as
nitrosamine and benzopyrene. Besides, cigarettes contain nicotine which may
suppress individual immunity and contribute to cancer development (Hecht et al
2003). However, cigarette smoking is only weakly associated with NPC. It is found to
be a weak or moderate risk factor in Taiwan (Cheng et al 1999), Philippines
7
anddddddd
Adapted from Chou et al. 2008
Figure 3: Proposed mechanisms of Epstein-Barr virus (EBV) latent proteins in NPC
development.
8
(West et al 1993) and Southern China (Yu et al 1981). No direct correlation between
cigarette smoking and NPC is found in Singapore (Lee et al 1994).
1.1.4 Genetic predisposition of NPC
1.1.4.1 Gene polymorphisms
The high incidence rate of NPC observed in Southern Chinese suggests that
genetic and environmental factors may play a role in NPC development. Previous
studies of genetic markers and familial aggregation showed that genetic
polymorphisms of some genes are associated with cancer development. Cytochrome
P450 2E1 (CYP2E1) and glutathione S-transferase M1 (GSTM1) are good examples
to demonstrate the association between genetic polymorphisms and NPC development.
CYP2E1 is a cytochrome enzyme responsible for metabolic activation of nitrosamine
and is one of the variants associated with high risk for NPC development (Hildesheim
et al 1995). GSTM1 is a phase II enzyme, which conjugates with the activated
xenobiotic metabolites with charged species and detoxifies the carcinogen (eg.
1-chloro-2,4-dinitrobenzene) in tobacco. Previous studies show the null genotype of
GSTM1 is associated with high risk of NPC development (Nazar-Stewart et al 1999).
Besides, some human leukocyte antigen (HLA) haplotypes such as A2, B46, and B17,
were reported to have reduced efficiency in activating cytotoxic T-cell recognition and
9
host immune responses to EBV infection (Lu et al 1990) and may play important
roles in development of NPC. Previous studies indicated that the Chinese population
with these HLA alleles are associated with high risk of NPC development.
1.1.4.2 Familial aggregation
Familial aggregation is frequently observed in different cancers such as breast,
retinoblastoma, and colon cancers. The genetic defects and alterations can pass to the
offspring and increase their risk of cancer development. The studies of familial
aggregation provide hints for identification of oncogenes and TSGs involved in the
development of cancer. NPC familial aggregation was observed in endemic areas such
as Hong Kong, Guangdong, Singapore, and Taiwan (Chen et al 1990, Lee et al 1994,
Ung et al 1999, Yu et al 1986, Yu et al 1990). Previous studies show that chromosome
regions 3p21.3, 4p15.1-q12, and 5p13 are associated with familial aggregation of
NPC development (Feng et al 2002, Hu et al 2008, Hui et al 1999a, Zeng et al 2006).
Recent studies also show that chromosome 6p is associated with NPC development in
Southern Chinese (Li et al 2011). Genome-wide association studies have identified
the NPC-associated region at 6p21.3 within the HLA region in Taiwanese (Tse et al
2009).
The loci identified in the NPC familial aggregation studies may contain
genes associated with NPC pathogenesis. Gamma-amino butyric acid B receptor 1,
10
HLA-A , and HLA complex group 9, located on chromosome 6p, were found to be
highly associated with NPC in Southern Chinese (Li et al 2011). On the other hand,
another chromosome 6p HLA haplotype A1-B37-DR6 was reported to be associated
with predisposition for NPC (Coffin et al 1991). HLA-DPB1 was also reported to be
associated with NPC in Guangdong Province Hans (Wang et al 2010).
1.1.4.3 Cytogenetic alterations
Comparative genomic hybridization (CGH) and loss of heterozygous (LOH)
studies on NPC specimens have identified genetic alterations and chromosome
regions which are important in NPC development. In LOH studies, allelic losses were
detected on chromosomes 9q, 11q, 13q, and 14q (Figure 4). Highest frequencies of
allelic loss were detected on chromosomes 3p (75%) and 9p (87%) in primary NPC
tumors (Mutirangura et al 1997). In a GCH study, high frequencies of loss were
observed on chromosomes 3p, 9p, 9q, 11q, 13q, 14q, and 16q (Figure 5) (Hui et al
1999a). This study also identifies multiple minimally deleted regions 3p14-24.2,
11q21-23, 13q12-14, 13q31-32, 14q24-32, and 16q22-23 (Lo et al 2000a). These
regions may contain tumor suppressor genes that are inactivated during the
development of NPC. All these findings suggest genetic alterations play an important
role in NPC development.
11
Adapted from: Lo et al. 2000
Figure 4: LOH frequencies of 36 microdissected primary NPC tumors detected by
microsatellite analysis. High frequencies of LOH are observed for chromosomes 3p,
9p, 9q, 11q, 12q, 13q, 14q, and 16q.
12
Adapted from: Hui et al. 1999
Figure 5: CGH analysis of 20 primary NPC biopsies. The vertical lines to the left of
the chromosome represent loss of that region, while the lines on the right represent
the gain of that region. High frequencies of loss are observed on chromosomes 3p,
9p, 9q, 11q, 13q, and 14q.
13
1.2 Molecular genetics of cancer
1.2.1 Multi-step progression model
Cancer is a complex genetic disease which involves sequential acquisition of
genetic alterations in oncogenes and tumor suppressor genes (TSGs) so as to
transform a normal cell to a malignant one (Vogelstein and Kinzler 2004). The
progression of cancer can be classified into different steps. Hanahan and Weinberg
define ten hallmarks of cancer that govern the process of carcinogenesis. The process
includes sustaining proliferative signaling, evading growth suppressors, avoiding
immune destruction, enabling replicative immortality, tumor-promoting inflammation,
activating invasion and metastasis, inducing angiogenesis, genome instability and
mutation, resisting cell death, and deregulating cellular energetics (Figure 6)
(Hanahan and Weinberg 2011). In colorectal cancer, a complete genetic model for
normal colon epithelial cells to progress to adenomas and the subsequent carcinoma
was reported (Fearon and Vogelstein 1990, Vogelstein 1990). This genetic model with
multi-step genetic alterations for colorectal cancer (CRC) tumorigenesis is shown in
Figure 7. The alterations of three types of genes are involved in tumorigenesis: the
oncogenes, TSGs, and the DNA stability genes. In brief, the alteration of oncogenes
and TSGs provide the growth advantages to a subpopulation of cells with altered cell
cycle and death control. The mutated cells with growth advantages overgrow the
14
ttttttwas
Adapted from: Hanahan and Weinberg 2011
Figure 6: Hallmarks of cancer and their potential therapeutic targets.
15
Modified from: Fearon et al. 1994
Figure 7: A genetic model of multi-step pathway in colorectal cancer from normal
epithelium to metastatic tumor.
16
normal cells and finally resulted in cancer formation. A pathogenesis model of NPC
was proposed by Lo and Huang (2002) (Figure 8). Genome-wide studies show loss of
TSGs located on chromosomes 3p, 9p, and 14q and inactivation of TSGs Ras
association domain family 1, isoform A (RASSF1A), p16, and p14 are the early events
of NPC development (Chan et al 2000, Chen et al 2002). Inactivation of these TSGs
may contribute to dysregulation of the DNA repair system and the release of growth
control. Subsequent, overexpression of the oncogene B-cell lymphoma protein 2
(Bcl2), dysregulation of telomerase, and EBV infection may further transform the
neoplastic tissues. Followed by the loss of chromosomes 11q, 13q, 16q, the neoplastic
tissues may develop into invasive NPC.
1. 3 Angiogenesis
Angiogenesis is an important process for cancer development, since tumors
cannot grow beyond 2 mm diameter without blood vessels (Folkman 1971).
Angiogenesis involves the formation of primary vascular plexus, resulting in the
formation of new blood capillaries. The blood vessels are necessary for cancer cell
survival, as they provide nutrients and oxygen to the cells and remove their metabolic
wastes. Angiogenesis is also important for tumors to invade other tissues and
metastasize to other organs. The ‘angiogenic switch’ is an important process in cancer
17
Adapted from: Lo and Huang 2002
Figure 8: Proposed pathogenesis model for NPC.
18
progression; it refers to the ability of a tumor to proceed from the non-angiogenic to
angiogenic phenotype. If the tumor cannot go through the angiogenic switch, blood
vessels will not be able to form and the size of tumor will remain constrained to a
non-lethal state (Baeriswyl and Christofori 2009). The overall process of angiogenesis
depends on the net balance between pro-and anti-angiogenic factors.
1.3.1 Pro-angiogenic factors
The
Vascular
Endothelial
Growth
Factor
(VEGF)
is
a
well-studied
pro-angiogenic factor. The VEGF family consists of five members: VEGF-A,
VEGF-B, VEGF-C, VEGF-D, and PlacentaL Growth Factor (PLGF) (Ferrara 2002).
Multiple isoforms of VEGF are generated by alternative splicing of VEGF-A
pre-mRNAs. VEGF has positive effects on angiogenesis; it increases vascular
permeability, promotes endothelial cell growth, migration, and survival (Roskoski
2007). Other pro-angiogenic factors such as Fibroblast Growth Factors (FGFs),
Transforming Growth Factor-β (TGF-β), and Platelet-Derived Growth Factor (PDGF)
also have positive effects on angiogenesis (Makrilia et al 2009). FGFs are
heparin-binding proteins, which interact with FGF receptors (FGFRs) on the cell
surface and promote angiogenesis, wound healing, and embryonic development.
TGF-β is a secretary protein, which causes immune-suppression and angiogenesis in
19
cancer, making the cancer more invasive. The PDGF stimulates endothelial cell
proliferation and migration and inhibits apoptosis (Makrilia et al 2009).
1.3.2 Anti-angiogenic factors
In contrast to pro-angiogenic factors, anti-angiogenic factors act as a negative
regulator of angiogenesis. Angiostatin and endostatin are well-studied anti-angiogenic
factors produced from proteolysis of larger molecules. Angiostatin induces apoptosis
of endothelial cells, while endostatin inhibits endothelial cell migration, induces cell
cycle arrest, and disrupts cell-to-cell adhesion (Ribatti 2009). In addition, other
anti-angiogenic proteins such as thrombospondin (Haviv et al 2005), vasostatin, and
calreticulin can also suppress angiogenesis by inhibition of cell proliferation and cell
migration and induction of apoptosis (Pike et al 1998). Other inhibitors such as
platelet factor-4 inhibit angiogenesis by inhibiting the binding of VEGF to endothelial
cells (Yamaguchi et al 2005). RGD or YIGSR motifs are always found in proteins
involved in anti-angiogenesis. For example, the anastellin is an anti-angiogenic
protein containing the RGD motif (Carmeliet and Jain 2000) and some laminin-1
peptides contain the YIGSR motif and also inhibit angiogenesis (Malinda et al 1999).
1.4 Oncogenes
20
The activation of oncogenes from mutation or amplification of proto-oncogenes
is a gain of function and is dominant. Proto-oncogenes are usually positive regulators
and are tightly controlled. However, the activated oncogenes promote cell cycle,
proliferation, invasion, and inhibit apoptosis. Oncogene activation is usually due to
chromosomal translocations, gene amplifications, or mutations (Vogelstein and
Kinzler 2004). Six classes of oncogenes are classified based on their functions, which
include growth factors, growth factor receptors, plasma membrane G proteins,
intracellular protein kinases, transcription factors, and cell cycle regulators (Bertram
2000). The oncogenic proteins over-expressed in NPC are listed in Table 2. Normal
cells respond to external growth factors and proliferate by tightly controlled
processes.
Common external growth factors which induce cell proliferation includes
epidermal growth factor (EGF), FGF, VEGF, PGDF, and Wingless-type MMTV
integration site family (Wnt) (Bertram 2000). Normal cells can be transformed with
inappropriate synthesis of growth factors. For example, Wnt and VEGF are the
growth factors which are commonly found to be up-regulated in NPC tumor tissues
(Shi et al 2006). VEGF is a growth factor found to be over-expressed in 40-70% of
NPC tumor tissues. VEGF over-expression is associated with the high rate of
metastatic relapse and reduced overall survival rate
21
Table 2: Oncogenic proteins over-expressed in NPC.
Abnormal protein
NPC with overexpression (%)
NF-κB
100
Survivin
100
Intranulear β-catenin
92
Wnt protein
93
hTERT
91
Telomerase
85
Bcl-2
75-86
C-myc
90
Cyclin D1
66
Erk
53
EGFR
49
Data from Chou et al. 2008
22
(Krishna et al 2006, Li et al 2008b, Segawa et al 2009). External growth factors exert
their proliferative action and induce a cascade of responses within a cell by binding to
appropriate receptors on the cell membrane. These receptors include EGF receptor
(EGFR), VEGF receptor (VEGFR), and neurotrophic tyrosine kinase (NTRK).
Inappropriate expression of growth receptors can enhance the sensitivity of cells to
the growth factors. The growth factor receptors are usually over-expressed in cancer
cells and contribute to uncontrolled cell proliferation. For example, EGFR is
over-expressed in more that 80% of NPC tissues (Fujii et al 2002, Leong et al 2004).
It was found that over-expressed EGFR level is associated with poorer overall
survival and tumor progression (Ma et al 2003). Signal transducers are located in the
plasma membrane. They respond to the growth signal from the cell surface receptors
and trigger the multiple phosphorylation events. The overall result will stimulate cell
proliferation. The final class of oncogenes are nuclear transcription factors, which
mediate the transcription of genes that contribute to tumorigenicity, including c-myc,
cyclins, and β-catenin.
1. 5 Tumor suppressor genes (TSGs)
TSGs are negative regulators of cell growth and suppress malignant
transformation. The mutations of TSGs are loss-of-function events and can contribute
23
to cancer development. The mutation of TSGs can occur as a result of deletions,
insertions, missense or nonsense mutations, or epigenetic silencing (Vogelstein and
Kinzler 2004). According to Knudson two-hit hypothesis, mutation or loss on both
copies (two-hits) of the TSG are necessary to contribute to tumor formation. Usually a
TSG is inactivated by deletion of one allele coupled with mutation of another allele to
loss its effect (Knudson 2001a, Knudson 2001b), although exceptions occur in
haploinsufficiency and with dominant-negative effects (Payne and Kemp 2005). TSGs
can be classified into gatekeeper and caretaker TSGs. Gatekeepers are TSGs, which
regulate cell proliferation and death; inactivation of gatekeepers will lead to
uncontrolled cell growth and tumor development. Well-studied gatekeepers include
TP53 and pRb. Caretakers are DNA repair genes, which regulate genome stability;
loss of caretakers will lead to more mutations in the genome. Examples of caretakers
include ATM, breast cancer 1 (BRCA1), and breast cancer 2 (BRCA2). Re-expression
of gatekeepers can suppress tumor formations, while re-expression of caretakers
cannot.
1.5.1 p53 pathway
p53 is a transcription factor, which is involved in inducing cell cycle arrest, cell
senescence, apoptosis, inhibits cell proliferation, cell differentiation, and DNA repair
24
(Aurelio et al 1998, Chipuk et al 2005, Jin and Levine 2001, Levine et al 1991, Lowe
et al 2004, Maddika et al 2007, Pietsch et al 2006). p53 suppresses cell growth and
initiates cell death, preventing cells with DNA damage to overgrow into cancer. p53
mutations are found in more than 50% of cancers (Pietsch et al 2006), mostly as point
mutations, which disrupt its binding ability to its cognate recognition sequence. The
p53-related growth arrest pathway is mediated through p21 (WAF1/CIP1), which is a
cyclin-dependent kinase inhibitor. p21 inhibits the phosphorylation of pRB by
inhibiting the binding of cyclins A or E to CDK2 and cyclins D1 or D2 to CDK4.
However, the mutational inactivation of p53 in NPC is rare, and overexpression of
p53 was observed in NPC patient tumors. On the other hand, EBV infection also plays
an important role in p53 activity and NPC development. The EBV protein, BZLF1
can directly interact with the p53 protein and inhibit its cellular function. Besides,
EBV nuclear antigen 3C can initiate p53 degradation through the MDM2-mediated
p53 ubiquitination and degradation mechanisms. A summary of the p53 pathway is
shown in Figure 9.
1.5.2 p16/pRB pathway
The p16/pRB pathway is an important pathway controlling the G1 to S phase
transition during the cell cycle. The mutation of the retinoblastoma (Rb) gene is
25
Adapted from : Vogelstein and Kinzler 2004
Figure 9: Well-studied gatekeeper TSG pathways. A) pRB/p16 and B) p53 signaling
pathways.
26
associated with retinal tumors. Rb mutations are found in other human cancers such as
small cell lung cancer and osteosarcoma (Classon and Harlow 2002). However, the
mutational inactivation of pRB in NPC is rare. pRb is a key negative regulator for cell
cycle. In brief, the unphosphorylated pRb tightly binds to the E2F family members,
which are transcription factors for genes involved in G1-S phase transitions.
Cyclin-dependent kinase can phosphorylate pRb and lead to the release of E2F to
allow the cell to pass through the cell cycle checkpoint (Ohtani et al 2004, Vogelstein
and Kinzler 2004). However, overexpression of cyclin D1 was observed in many NPC
patients. The overexpression of cyclin D1 increased the phosphorylation of pRb, when
it binds to CDK4, resulting in uncontrolled cell growth.
On the other hand, p16 also helps to control the p53 activity. The p16 is a
member of cyclin-dependent kinase inhibitor family, which can inhibit the
phosphorylation of pRB by cyclin D/CDK4 complex during G1-S phase transition
(Wang et al 1999). The expression of p16 can induce cell cycle arrest and inhibit cell
proliferation. High frequency of LOH and down-regulation of p16 are observed in
NPC patients, suggesting that loss of p16 is important for NPC development. A
summary of the pRB/p16 pathway is shown in Figure 9.
1.6 Mechanisms of TSG inactivation
27
1.6.1 Loss of heterozygosity (LOH)
Loss of heterozygosity is the loss of one allele of a gene in a heterozygous state
to leave it in a homozygous state, which is commonly observed in tumorigenesis and
TSG inactivation (Knudson 2001b). LOH can be detected by using polymorphic
microsatellite markers and comparing their heterozygosity in the genomic DNA of
normal and matched tumor samples. The studies of LOH are useful for identification
of critical regions responsible for tumor progression and candidate TSGs.
1.6.2 Mutations
Mutation is an important event in tumorigenesis. Truncating point mutations are
frequently observed in tumor development, caused by deletions and nonsense
mutations. Missense and nonsense mutations frequently occur in oncogenes and TSGs
in various types of cancers. Missense and nonsense mutations are also observed in
TSG inactivation (eg. ATM and TP53). Missense mutation altering the DNA sequence
results in a different amino acid which is essential for its activity. A nonsense mutation
converts a coding codon into a stop codon, resulting in a truncated protein product
(Vogelstein and Kinzler 2004). Silent mutation alters DNA sequences without
affecting the protein sequence. Missense and nonsense mutations affect the function
of TSGs and are often involved in tumorigenesis.
28
1.6.3 Epigenetic silencing
Epigenetic alterations are an important mechanism for TSG inactivation and are
commonly observed in cancers. Epigenetic alterations result in a change of expression
level without changing the genetic sequence. Promoter hypermethylation and histone
deacetylation are mechanisms for epigenetic inactivation of TSGs (Egger et al 2004,
Gronbaek et al 2007). Promoter hypermethylation occurs at cytosines located on the 5’
end of guanine of the promoter regions in CpG islands. The cytosine is then converted
to 5-methylcytosine by DNA methyltransferases. The 5-methylcytosine inhibits the
gene transcription by blocking the binding of promoter elements essential for
initiation of transcription (Luczak and Jagodzinski 2006). On the other hand, histone
deacetylation is also a common mechanism in TSG silencing. Histone deacetylases
can remove the acetyl group from the N-terminal histone wrapped around the DNA in
chromatin. The change of chromatin configuration will make the promoter region
inaccessible to transcription machinery, and, thus, the gene transcription is repressed
(Gronbaek et al 2007).
1.7 Methods to identify TSGs
There are several approaches which can be used to identify candidate TSGs.
Positional cloning of the regions showing loss of heterozygosity (LOH) and
29
homozygous deletion (HD) is one of the methods to identify candidate regions
contributing to tumor suppression. On the other hand, chromosome transfer
techniques such as yeast artificial chromosome (YAC) and bacterial artificial
chromosome (BAC) transfer allow us to analyze the tumor suppressing activity of
particular chromosome regions. Furthermore, after the identification of critical
regions for tumor suppression, transfection of candidate genes from these regions into
cancer cells allows us to obtain functional evidence for the tumor suppressing ability
of these genes (Murakami 2002). Figure 10 summarizes the common approaches used
in recent studies of TSGs.
1.7.1 Positional cloning
Positional cloning of chromosome regions identified in linkage analysis of
hereditary cancer is a traditional method to identify TSGs for further studies. For
example, BRCA1 and BRCA2 are high-penetrance breast cancer predisposition genes
identified by genome-wide linkage analysis and positional cloning (Turnbull and
Rahman 2008). Another example, RB1, is a well-studied TSG identified by linkage
analysis and its mutation contributes to retinoblastoma and osteosarcoma (Friend et al
1986). In tumor development, inactivation of TSGs associated with LOH on specific
chromosome regions is responsible for multi-step carcinogenesis. One of the
30
Adapted from: Murakami 2002
Figure 10: Summary of approaches used for TSG identification.
31
approaches to identify a TSG is positional cloning of a common region of LOH
observed in primary tumors, but it is difficult to identify a single responsible gene
from the candidate genes identified by this approach unless the target gene is
associated with a homozygous deletion.
1.7.2 Functional cloning
Another approach to identify a TSG is by functional cloning, which is a
technique based on genetic complementation of defective cancer cells with DNA
fragments of normal cells. The first step of functional complementation of a TSG is to
introduce a normal DNA fragment into a cancer cells lacking the function of the
relevant gene, which can be performed by different chromosome transfer methods,
such as whole cell fusion, microcell fusion for intact or fragmented individual
chromosomes, spheroplast fusion of yeast artificial chromosome (YAC), lipofection
of bacteriophage P1, P1-derived artificial chromosome (PAC), bacterial artificial
chromosome (BAC) or plasmids carrying the target genes, etc. Then the next step is to
examine the tumorigenicity of the malignant recipient cell line after the DNA transfer
process. Functional cloning is useful for identification of regions contributing to
tumor suppression, which may harbor potential TSGs (Murakami 2002). However, if
the region identified contains multiple genes, further verification is required to
32
demonstrate the role of those genes identified in those critical regions.
CADM1/TSLC1 in chromosome 11q22-23 is the TSG, which was originally identified
by functional cloning in non-small cell lung cancer.
1.8 Various chromosome transfer techniques
1.8.1 Whole and truncated chromosome transfer
Whole and truncated chromosome transfer can be performed by whole cell
fusion and microcell-mediated chromosome transfer (MMCT) (Murakami 2002).
Whole cell fusion in early cancer studies are performed by using the Sendai virus to
enable the production of hybrid cells. Sendai virus and polyethylene glycol (PEG) can
be used as fusogens for the construction of hybrid cells. However, multiple
chromosomes are often transferred to the host cell, and the resultant hybrid cells are
polyploid and unstable and some specific chromosomes will be lost. To transfer a
particular chromosome into a host cell, the MMCT technique can be used to
investigate the functional role of a specific chromosome in tumor suppression. By
using this approach, a single human chromosome can be transferred into cancer cell
lines. In this approach, a microcell donor, which contains a human chromosome of
interest, is used as a donor to transfer selected chromosomes into tumor cells.
Microcells presumably with one chromosome within a nuclear envelope and plasma
33
membrane are generated after disruption of the cytoskeleton of the donor cells
(Prescott et al 1972). By fusing the microcell with the host cell, hybrid cells
containing an extra chromosome can be produced and can be selected by endogenous
markers (Fournier and Ruddle 1977). To transfer a fragment of a chromosome into a
host cell, the donor chromosomes in the microcells are irradiated into smaller
fragments before cell fusion. These experiments enable the transfer of a single copy of
a chromosome into recipient cells, and the genes are expressed under the control of
their endogenous promoters, enhancers, and are regulated in their native environment.
Thus, MMCT provides an ideal method for studying gene expression closely
mimicking its physiological level and regulation control under its native environment.
1.8.2 YAC transfer (YACT)
YACT enables the cloning of a long genomic DNA (several megabases) to study
the genes in a particular region (Murakami 2002). YAC transfer in mammalian cells
can be performed by using PEG, micro-injection, and spheroblast fusion (Pachnis et
al 1990, Pavan et al 1990, Schedl et al 1993). The advantage of using YACT to study
gene functions is that only a single copy of DNA is transferred into the host cells, and
the genes in the central portion of the YAC are expressed by the endogenous
promoters. However, the YAC is less stable compared with chromosome fragments.
34
Gel purification of YACs often cause breakage or rearrangement (Reeves et al 1990).
1.8.3 BAC and P1-derived artificial chromosome (PAC) transfer
By using the bacteriophage P1 cloning system, BACs and PACs may be used to
clone smaller sizes of DNA fragments (80-200 kb) (Murakami 2002). Those
fragments are more stable and easier to be manipulated, when compared with the
whole chromosome and YAC. The BAC and PAC containing the target genes and
their promoters can be transferred into the host cells by lipofection, but the
disadvantage is that the copy number of the integrated DNA cannot be controlled.
1.9 The current MMCT-based approach in identification TSGs in NPC
MMCT is a useful approach which has been used in our laboratory for years to
study the tumor suppressing functions of various chromosomes in NPC (Figure 11).
MMCT is the technique which allows the transfer of an intact or a truncated human
chromosome into a cancer cell line. It aims to determine whether the exogenous
chromosome is able to complement the defects in cancer cells. After the transfer, the
resultant cell line contains an extra exogenous chromosome, which are called
microcell hybrids (MCHs). The loss of the tumorigenic phenotype of MCHs provides
functional evidence that the exogenously transferred chromosome contains tumor
35
Figure 11: Outline of our strategy to identify TSG by using microcell-mediated
chromosome transfer (MMCT).
36
suppressing activities. The in vivo nude mouse assay is used to assess the
tumorigenicity of the MCHs. Most of the tumor-suppressing hybrids only form
tumors after a delayed lag phase. As those cells are under high selective pressure in
the mice, some genes, presumably candidate TSGs, may be inactivated or eliminated
in the emerging mouse tumors. Tumor tissues obtained from these nude mice are
established in cell culture; these cell lines are called tumor segregants (TSs). Detailed
microsatellite typing comparison between MCH and TS cell lines was performed to
search for commonly deleted critical regions contributing to tumor suppression. After
critical regions are identified, genes in these regions which are up-regulated in the
MCHs but down-regulated in their matched TSs, are selected as candidate TSGs for
further analysis such as global gene expression analysis, as described in the later
section. In our previous studies, several NPC critical regions including 3p21.3, 11q13,
11q22-23, 13q12, 14q11.2-13.1, and 14q32.1 were identified by this MMCT-based
approach (Cheng et al 1998, Cheng et al 2000, Cheng et al 2002, Cheng et al 2003,
Cheng et al 2004, Lung et al 2004).
1.9.1 Role of chromosome 11 in NPC
LOH in chromosome 11q was reported in several genome-wide studies,
indicating that loss of chromosome 11 may be one of the major genetic changes
37
during NPC development (Chien et al 2001, Fan et al 2000, Fang et al 2001, Hui et al
1999a, Lo et al 2000a, Mutirangura et al 1997, Shao et al 2000, Shao et al 2001). A
CGH study with 20 primary NPC tumors identified that chromosomes 1p, 3p, 9p, 9q,
11q, 13q, 14q, and 16q are commonly lost in NPC. A high incidence of loss (70%)
was observed on chromosome 11q (Hui et al 1999b). Previous LOH study using
high-resolution allelotyping with 382 microsatellite markers and microdissected
primary NPC tumors indicated that high frequencies (74.1%) of allelic imbalance
were observed in 11q (Lo et al 2000b). These studies indicated that chromosome 11
may play an important role in early stage NPC and may harbor candidate TSGs.
Using MMCT, the MCHs containing intact chromosome 11 in the NPC HONE1 cell
lines were shown to suppress tumor growth in nude mice, indicating that chromosome
11 contains gene(s) that suppress(s) tumor formation. It was found that 11q13 and
11q22-23 are critical regions for tumor suppression in NPC (Cheng et al 2002, Lung
et al 2004). These critical regions were identified by using MMCT approaches with
the NPC cell line HONE1, followed by detailed investigation of MCHs and their
matched TSs using microsatellite analysis to narrow down the region of
tumor-suppressive activity. Fluorescence in situ hybridization was also performed
with BAC and cosmid probes to confirm the microsatellite data. TSGs located within
the critical regions are harbored and their functions are often related to NPC
38
development such as angiogenesis, invasion, and metastasis (Lung et al 2004, Lung et
al 2005, Lung et al 2006, Lung et al 2010).
1.9.2 Chromosome 11 NPC genes identified by MMCT
By using the technique of MMCT and the genotyping analysis of the MCHs/TSs,
we have successfully refined three critical regions of 0.36 Mb, 0.44 Mb, and 0.3 Mb
for tumor suppression at 11q22-23. In the third region with high allelic loss, a TSG,
Tumor Suppressor in Lung Cancer 1 (TSLC1 or is now called Cell ADhesion
Molecule 1 (CADM1)), is located (Lung et al 2004). The CADM1/TSLC1 gene was
originally identified as a tumor suppressor in non-small cell lung cancer (NSCLC) by
combinatorial analyses of YACT into human NSCLC cells with a tumorigenicity
assay in nude mice (Kuramochi et al 2001). Down-regulation of CADM1/TSLC1 was
found in 83% of the lymph node metastatic NPC samples (Lung et al 2006) and the
down-regulation is caused by promoter hypermethylation (Hui et al 2003). Further
functional studies indicated that over-expression of CADM1/TSLC1 in NPC can
suppress tumorigenicity by cell growth inhibition and apoptosis induction (Lung et al
2006). These findings suggest that CADM1/TSLC1 is a tumor suppressor gene in NPC,
which is significantly associated with lymph node metastases.
As described in the above section, the known TSG CADM1/TSLC1 was
39
identified as a candidate gene in NPC from the genotyping analyses of the
non-tumorigenic chromosome 11 MCHs and their matched TSs (Lung et al 2004).
Differential expression analysis of 19K genes was performed in oligonucleotide
microarray hybridization for these chromosome 11 MCHs/TSs pairs to hunt for some
other unknown NPC genes. Another interesting gene was identified from this
approach, Thy-1 cell surface antigen (THY1), which is located close to the previously
defined 11q22-23 NPC CR (Lung et al 2005). By using a tissue microarray and
immunohistochemical staining, it was found that down-regulated expression of THY1
was observed in NPC cases and its down-regulation is significantly associated with
the lymph node metastasis. After transfection of THY1 gene into HONE1 cells, a
dramatic reduction of colony formation ability was observed. Further studies
indicated that the restoration of THY1 in HONE1 cells also suppresses tumorigenicity
by arresting cell in G0-G1 phase and reduces cell anchorage-independent growth and
invasiveness (Lung et al 2005, Lung et al 2010). These findings suggest that THY1 is
a tumor suppressor gene in NPC, which is involved in invasion and shows an
association with tumor metastasis. By using the same 19K oligonucleotide
microarrays which identified THY1 as a candidate gene, SAA1 was also shown to be
differentially expressed in the MCHs and TSs. This is the reason why SAA1 is chosen
as a candidate gene for further analyses in this project.
40
1.10 Serum amyloid A (SAA) family
The SAA gene family is located at chromosome 11p15.1 and encodes a number of
differentially-expressed apolipoproteins, which are synthesized by the liver (Yamada
1999). There are mainly two classes of SAA, the acute phase SAA (A-SAA) and
constitutive SAA (C-SAA), which are based on their responsiveness to inflammation.
There are four members in the SAA family, SAA1, SAA2, SAA3, and SAA4, but only
SAA1, SAA2, and SAA4 encode SAA proteins (Uhlar and Whitehead 1999). SAA1 and
SAA2 are coordinately induced during the acute phase response. They share more
than 90% sequence identity. SAA1 has five isoforms, SAA1.1, SAA1.2, SAA1.3,
SAA1.4, and SAA1.5. SAA2 has two alleles, SAA2.1 and SAA2.2, which encode distinct
proteins with a few amino acid changes. SAA3 is a pseudogene in the human genome.
Finally, SAA4 gene is a constitutively expressed gene, sharing only around 53-55%
identity with SAA1 and SAA2. All the SAA isoforms map in a 150 kb region of
chromosome 11p15.1.
1.10.1
SAA1 isoforms in amyloidosis
As mentioned earlier, SAA1 has at least five alleles (SAA1.1, SAA1.2, SAA1.3,
SAA1.4, and SAA1.5) that encode distinct proteins (Uhlar and Whitehead 1999). The
amino acids, which distinguish these five SAA1 isoforms, are summarized in Figure
41
12A. Previous studies indicate that SAA1 allelic polymorphisms may be associated
with risk of diseases such as amyloid A protein-amyloidosis (AA-amyloidosis).
AA-amyloidosis is a systemic complication of chronic inflammatory diseases caused
by the aggregation of amyloid A protein into insoluble anti-parallel beta-pleated sheet
fibrils. There are many symptoms of amyloidosis, such as heart failure, arrhythmia,
vomiting, diarrhea, skin lesion, and petechiae (Reed and Morris 1992). SAA1.3
protein has been reported to be associated with higher risk, while SAA1.1 protein is
associated with lower risk of developing AA-amyloidosis in Japanese rheumatoid
patients (Baba et al 1995, Moriguchi et al 1999). Moreover, it has been reported that
the SAA1.3 protein is associated with higher levels of SAA, which in turn are
associated with the progression of AA-amyloidosis in Japanese rheumatoid patients.
Indeed, later studies indicated that SAA1.3 is associated with increased transcriptional
activity of the gene in Japanese rheumatoid patients (Ajiro et al 2006, Moriguchi et al
2005). In contrast, SAA1.1 is associated with higher risk of developing
AA-amyloidosis in Caucasians (Booth et al 1998). As the function of SAA is closely
associated with inflammation, the amino acid sequence differences in the SAA
isoforms may contribute to the altered severity of inflammation, and may cause
diverse risk levels of the disease.
42
A.
B.
SAA1.1 protein amino acid sequence
MKLLTGLVFCSLVLGVSSRSFFSFLGEAFDGARDMWRAYSDMREANYIGSDK
YFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANE
WGRSGKDPNHFRPAGLPEKY
Figure 12: A) The nucleotide and amino acid differences among the five SAA1
isoforms. The five human SAA1 proteins are distinguished by several amino acid
changes at positions 70, 75, 78, and 90. B) The amino acid sequence of SAA1.1
protein, the YIGSR- and RGD-like like motifs are highlighted in red and the SNP
sites are labeled with grey color.
43
1.10.2 SAA1 and cancers
Serum SAA protein levels are elevated up to 1000-fold in patient sera in
response to a number of different stimuli such as injury, inflammation, and cancers
(Rosenthal and Franklin 1975). The increased serum SAA protein levels in cancer
patients suggest that SAA1 is associated with tumor development, but the functional
role of SAA1 protein in cancer progression is still unclear. In addition, how each
SAA1 variant is associated with the elevation of serum SAA level and each of their
roles in tumor development remain unknown. SAA levels are higher in cancer
patients with distant metastases and SAA levels are inversely correlated with patient
survival (Biran et al 1986, Weinstein et al 1984) and response to chemotherapy
(Kaneti et al 1984, Rosenthal and Sullivan 1979). These studies indicated that SAA1
may play a role in the metastasis process in cancers. Previous studies indicated that
SAA level is best correlated with colorectal carcinoma occurrence among all the acute
phase proteins (Glojnaric et al 2001). SAA was also found to be the most significant
protein differentiating sera of lung cancer patients compared with sera of healthy
individuals (Howard et al 2003). Serum SAA elevation was also reported in other
cancer types such as, renal, pancreatic, prostate, breast, and gastric cancers (Chan et al
2007, Kaneti et al 1984, Kimura et al 2001, Le et al 2005, O'Hanlon et al 2002). As
can be seen, the serum SAA elevation may be an important biomarker in cancer
44
development. In NPC studies, protein profiling studies of Hong Kong NPC patients
sera showed that two SAA isoforms levels were significantly elevated at relapse
compared with patients in remission, but the elevation was not observed in early NPC
patients (Cho et al 2004). However, in early NPC SAA levels were not substantially
elevated. Serum SAA is elevated in NPC patients, particularly in association with
lymph node metastasis (Liao et al 2008).
Besides studying the serum SAA levels in cancer patients, the expression of SAA
directly in tumors was also studied in colon and gastric cancers. The SAA mRNA
level was barely detectable in normal-looking colonic epithelium, but increased when
epithelial cells progressed through dysplasia to neoplasia (Gutfeld et al 2006). In
contrast, other studies showed that SAA protein was undetectable in the gastric cancer
tissues, although they also observed the elevation of SAA in patient sera and its
association with tumor stage, recurrence, and survival (Chan et al 2007). Previous
serial analysis of gene expression (SAGE) studies showed that SAA expression was
reduced in metastatic colorectal cancer cell lines compared with primary colorectal
cancer cell lines (Parle-McDermott et al 2000).
1.10.3 Functions of SAA1
In vitro analyses of the biological activities of recombinant SAA proteins
45
synthesized by bacteria are widely used to assess the physiological functions of serum
SAA in vivo (Badolato et al 1994, Liang and Sipe 1995, Liang et al 1996,
Preciado-Patt et al 1994, Preciado-Patt et al 1996, Xu et al 1995). It has been reported
that SAA1 is involved in several functions, which include induction of extracellular
matrix (ECM) degrading enzymes for tissue repair (Mitchell et al 1991, Strissel et al
1997), recruitment of immune cells to inflammation sites (Badolato et al 1994,
Preciado-Patt et al 1996, Xu et al 1995), and lipid transport and metabolism (Cabana
et al 1989, Hoffman and Benditt 1982, Liang and Sipe 1995, Liang et al 1996,
Lindhorst et al 1997).
Both functional YIGSR-like and RGD-like motifs are present in the SAA protein
within close proximity (YIGSDKYFHARGNY; residues 29-42, Figure 12B)
(Preciado-Patt et al 1994). Previous studies indicated that proteins with YIGSR and/or
RGD motifs can inhibit angiogenesis, tumor cell adhesion to ECM, tumor growth, and
metastasis (Chiang et al 1995, Iwamoto et al 1996, Nicosia and Bonanno 1991). It
was found that the RGD motif is involved in integrin-dependent TGF-β activation. A
latency associated peptide (LAP, a protein derived from the N-terminal region of the
TGF-β gene product) containing an RGD motif can be recognized by αV containing
integrins, such as the αVβ6 integrin. The αVβ6 integrin was the first integrin to be
identified as TGF-β1 activator; it binds to the RGD motif in the LAP of the TGF-β
46
latent complex and induces a adhesion-mediated cell force, which then translates into
biochemical signals and will cause the activation of TGF-β. As TGF-β plays an
important role in anti-proliferation and apoptosis in normal epithelial cells and at the
early stages of oncogenesis (Blobe et al 2000), the activation of TGF-β by the RGD
motif may help to suppress tumorigenesis. A previous study by Nicosia and Bonanno
(1991) showed that the RGD containing the synthetic peptides can suppress
microvessel formation in the mouse aortic explant, which indicates the
anti-angiogenic function of the RGD-motifs. On the other hand, YIGSR-like motif
was also found to be involved in anti-angiogenesis, -tumor growth, and -metastasis. A
previous study by Iwamoto et al. (1996) showed that co-injection of human
fibrosarcoma cells with the synthetic peptides containing YIGSR-like sequences from
laminin can inhibit angiogenesis, tumor growth, and experimental metastasis. As
SAA1 proteins contain both the RGD and YIGSR-like motifs in close proximity, it
may also play a role in inhibiting angiogenesis, tumorigenesis, and metastasis. The
above evidence suggests augmentation of SAA protein during metastasis is functional
and relates to the body’s homeostatic mechanisms. The acute-phase protein SAA
might be involved, either directly or via its peptide fragments, in inhibition of
inflammatory reactions or metastatic processes. Thus, the elevation of serum SAA
may play an important role in combating malignancies.
47
On the other hand, SAA was found to play a role in mediating the anti-tumor
activities of Ganoderma ludicum polysaccharides (GlPS) (Li et al 2008a). Their
results showed that GlPS up-regulated the SAA protein expression and inhibited the
tumor growth and tumor cell adhesion to vascular endothelial cells. In contrast, some
studies suggest that low concentration of SAA protein can increase angiogenesis
processes such as endothelial cell tube formation and human umbilical vein
endothelial cell (HUVEC) migration in vitro (Mullan et al 2006).
1.11 Aims of the project
NPC occurs with the highest frequencies among the Southern Chinese,
suggesting there may be a genetic predisposition to NPC. Multiple genetic alterations
in this cancer have been widely documented. Loss or inactivation of TSGs is
necessary for neoplastic initiation and tumor progression. Compared with other
common human cancers, not much is known about the molecular basis for NPC
development. NPC remains a top priority for cancer studies in China and Hong Kong.
We have previously used a functional complementation approach to generate
chromosome 11 MCHs. Oligonucleotide microarray hybridization profiling of
tumor-suppressive hybrids versus the tumorigenic recipient NPC cell line and
tumorigenic revertants identified an interesting differentially-expressed gene, SAA1,
48
as a candidate gene in tumorigenesis. Its expression was elevated in all
tumor-suppressive hybrid cells versus the recipient cell line and down-regulated in all
tumorigenic revertants versus their corresponding hybrids. The SAA1 gene expression
in the chromosome 11 MCHs and their TSs was verified in this study. I also examined
the expression of SAA1 in other NPC cell lines and clinical tumor biopsies and its
gene silencing mechanism(s). I found that SAA1 was indeed frequently
down-regulated in the tumorigenic NPC cell lines and tumor tissues. It is of interest to
investigate whether physiological levels of SAA1 expressed locally in the normal
nasopharyngeal tissue play a novel protective role against tumor development.
Three SAA1 allelic variants, SAA1.1, 1.3, and 1.5, encoding distinct proteins with
a few amino acid differences, were identified in NPC/normal tissues/cell lines and
healthy Chinese individuals. SAA may be an important biomarker for human cancers.
It is elevated up to 1000-fold in patient sera in response to a number of different
cancers. However, how each SAA1 variant is associated with NPC development
remains unknown. I attempted to compare the genotypes and allelic frequencies
between the Hong Kong NPC patients and healthy individuals by direct DNA
sequencing. How the SAA1 polymorphisms are associated with NPC risk were
assessed.
In NPC patients, significant elevations of SAA levels are demonstrated in
49
patients with lymph node metastases. What is less clear, however, is what possible
role systemic serum SAA1 protein may actually play in tumor development. It is
suggested that the elevation of serum SAA may play a major role in combating
malignancies. In order to investigate whether the elevated serum SAA proteins in
NPC patients is beneficial, the anti-tumor activities, including inhibition of tumor cell
growth, adhesion, and anti-angiogenic properties of exogenous recombinant SAA1.1,
1.3, and 1.5 proteins were used to study their physiological relevance during
tumorigenesis. Previous studies indicate the benefit of using recombinant SAA1
proteins which include: 1) the concentrations of protein and duration of treatment can
be precisely controlled, 2) most recombinant proteins can retain their biological
activities, 3) high yields of purified proteins can be obtained. The concentrations I
applied in various cell growth and angiogenesis assays were based on the reported
concentrations of elevated SAA levels in serum (200–2000 µg/ml) of NPC patients.
The synthesis of the SAA1.1, 1.3, and 1.5 recombinant proteins was performed in a
bacterial-inducible expression with a His-tag protein purification system. These
recombinant SAA1 variant proteins were produced by me because they are not
commercially available. Since SAA1 contains the functional YIGSR- and RGD-like
angiogenesis-inhibiting motifs within close proximity, I aimed to determine the role
of SAA1 and its variant forms in inhibition of angiogenesis and tumor development.
50
This study on the tumorigenesis-associated activities of SAA1 will give us
insight as whether its dramatic elevation plays any functional role in cancers. There is,
as yet, no report describing the preferential association of any SAA1 variants with
cancers. I aimed to provide genomic evidence for an allelic variant of human SAA1 that
is strongly associated with NPC. Previous studies focused only on the quantitative
changes of SAA levels in different cancers. This study aimed to investigate both
quantitative changes and qualitative differences of SAA1 in NPC. Taken together,
understanding the genetic and functional roles and activities of each SAA1 variant in
tumorigenesis will provide new insight as to how SAA1 may serve as an useful
prognostic biomarker for tumor progression and be a potential therapeutic target to
improve control of this cancer.
In brief, the aims of this project were to:
1. Determine whether there was any relationship between SAA1 polymorphisms and
the risk of NPC and determine if SAA1.1, 1.3, or 1.5 variants could be used as a
biomarker for NPC diagnosis
2. Investigate the anti-angiogenic and anti-tumor activities of SAA1.1, 1.3, and 1.5
variants in assays using recombinant SAA1 variant proteins and determine if they
could be used as potential therapeutic targets in cancer
51
3. Investigate the functional roles of ectopically-expressed SAA1 variant proteins in
two NPC cell lines
4. Validate the clinical relevance of SAA1 gene expression and protein levels in
tumor versus normal tissues and the mechanism of SAA1 gene silencing in NPC.
52
Chapter 2: Materials and methods
2.1 Tissue culture
The tumorigenic NPC cell lines, HONE1 (Glaser et al 1989, Yao et al 1990),
HK1 (Huang et al 1980), HNE1 (Glaser et al 1989), CNE1, CNE2 (Teng et al 1996),
C666 (Cheung et al 1999), and SUNE1 were used as the NPC cell lines for the gene
expression and functional studies of SAA1. The immortalized nasopharyngeal
epithelial cell lines, NP69 (Zhang et al 2004) and NP460 (Li et al 2006), were used as
positive controls of SAA1 gene expression in experiments. The NP69 cell line was
immortalized by transfection of a viral oncogene, SV40T antigen, while NP460 was
immortalized by over-expressing telomerase.
The recipient NPC HONE1 cell line, donor chromosome 11 mouse hybrid cell
line (MCH556.15), four HONE1/chromosome 11 MCH cell lines (HK11.8, HK11.12,
HK11.13, and HK11.19), and their TSs (HK11.8-3TS, HK11.12-2TS, HK11.13-1TS,
and HK11.19-4TS) were used (Cheng et al 2000) for the verification of the 19K
oligomicrarray analysis.
The esophageal cancer (EC) cell lines, SLMT-1 S1 (Tang et al 2001), 81T (Hu et
al 1984), TTn, HKESC2 (Hu et al 2002), EC1, EC18 (Pan 1989), KYSE30, KYSE70,
KYSE140, KYSE150, KYSE180, KYSE410, KYSE450, KYSE510, and KYSE520
53
(Shimada et al 1992) were used for the SAA1 expression analysis. The immortalized
esophageal epithelial cell lines, NE1, NE3, and NE083, were used as positive controls
and a template for the cloning of SAA1.5. The NE1 and NE3 cell lines were kindly
given by Prof. George. Tsao (HKU). The NE1 and NE3 were immortalized by
overexpression of human papillomavirus type 16 E6/E7 (HPV16E6/E7) (Deng et al
2004), while the NE083 was immortalized by over-expressing telomerase (Zhang et al
2006).
Human umbilical vein endothelial cell (HUVEC) and human microvascular
endothelial cell (HMEC) were used for the in vitro angiogenesis assays. The HUVECs
are isolated from normal human umbilical vein and are responsive to cytokine
stimulation for the expression of cell adhesion molecules and the formation of blood
vessels. HUVECs (C-003-5C, Cascade Biologics) were cultured on 30 µg/ml collagen
type 1 coated culture plates. The HMECs are isolated from human macrovascular
tissues and are also responsible for blood vessel formation. HMEC (Titz et al 2004)
were cultured on 30 µg/ml fibrolectin-coated culture plates. The culture conditions of
all the cell lines used in this study are listed in Table 3.
2.2 Tissue specimens and blood samples
Eighty matched normal nasopharyngeal and NPC tumor tissue biopsies from
54
Table 3: Background information and culture conditions for cell lines used in this
study.
Cell lines
Ethnic origin
Differentiation status
Culture conditions
Origin
Selection drugs
HONE1
Chinese
Poor
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
HONE1-2
Chinese
Poor
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
500 ug/ml G418
HK1
Hong Kong
Well
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
NA
HNE1
Chinese
Poor
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
NA
CNE1
Chinese
Well
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
NA
CNE2
Chinese
Poor
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
NA
C666
Hong Kong
Poor
RPMI1640 + 10% FBS + 1% P/S
NPC
NA
SUNE1
NA
NA
DMEM + 5% FBS + 5%NCS + 1% P/S
NPC
NA
NP69
Hong Kong
Immortalized normal
Keratinocyte-Serum Free medium
Immortalized NP
NA
NP460
Hong Kong
Immortalized normal
50% Defined Keratinocyte-Serum Free
Immortalized NP
NA
medium + 50% Epilife medium
SLMT-1 S1
Hong Kong
Well
DMEM + 5% FBS + 5%NCS + 1% P/S
EC
NA
TTn
Japanese
Moderate
DMEM + 5% FBS + 5%NCS + 1% P/S
EC
NA
81T
Taiwanese
Well
DMEM + 5% FBS + 5%NCS + 1% P/S
EC
NA
HKESC2
Hong Kong
Moderate
DMEM + 5% FBS + 5%NCS + 1% P/S
EC
NA
EC1
Hong Kong
NA
RPMI1640 + 10% FBS + 1% P/S
EC
NA
EC18
Hong Kong
NA
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE70
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE140
Japanese
Moderate
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE180
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE410
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE510
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE520
Japanese
Moderate
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE30
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE450
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
KYSE150
Japanese
Well
RPMI1640 + 10% FBS + 1% P/S
EC
NA
NE1
Hong Kong
Immortalized normal
Keratinocyte-Serum Free medium
Immortalized EC
NA
NE083
Hong Kong
Immortalized normal
Keratinocyte-Serum Free medium
Immortalized EC
NA
55
HUVEC
NA
NA
M199 + ECGF + Heparin sulfate + 20%
Umbilical vein
FBS
endothelial cell
NA
line
HMEC
NA
NA
Endothelial growth medium MV
Human
NA
macrovascular
endothelial cell
line
293T
NA
NA
DMEM + 5% FBS + 5%NCS + 1% P/S
Human embryonic
NA
kidney cell
Chr. 11
NA
NA
DMEM + 5% FBS + 5%NCS + 1% P/S
MCHs
Cell lines
500 μg/ml G418
established from
MMCT
Chr. 11TSs
NA
NA
DMEM + 5% FBS + 5%NCS + 1% P/S
Tumor revetant of
the chr. 11 MCHs
56
NA
NPC patients were collected from Queen Mary Hospital in Hong Kong from 2006 to
2010. All the biopsies were stored in RNAlater solution (Ambion, Austin, TX, USA)
before RNA/DNA extraction. This study protocol was approved by the Hospital
Institutional Review Board and written consents were obtained from all the patients.
Fiberoptic nasopharyngoscopy and biopsy of tumor and normal epithelium were
performed. All the biopsies were verified histologically to identify the presence of
tumor cells. Three hundred blood samples from healthy individuals without infectious
diseases and cancers were obtained from the Hong Kong Red Cross, and human
research ethics approval from the institute have been obtained.
2.3 RNA extraction
Total RNA extraction of patient biopsies was performed by using the Trizol
reagent (Invitrogen, CA, USA). The procedures were as described in the
manufacturer’s manual. In brief, 1 ml Trizol reagent was added to each sample, then
0.2 ml chloroform was added to each sample to separate RNA from DNA and cell
protein. The sample was then centrifuged at 12000 g for 15 mins; the mixture was
separated into different layers. The upper aqueous phase was removed for RNA
extraction. After that, 0.5 ml of isopropyl alcohol was added to each sample to
precipitate RNA, then the sample was centrifuged at 12000 g for 10 mins. The RNA
57
pellet was washed with 1 ml 75% ethanol and then redissolved in RNase-free water.
2.4 DNA extraction from mammalian cell lines
Cells cultured in 25 cm2 tissue culture flasks at 90% confluence were lysed in 3
ml homogenizing buffer (50 mM Tris-Cl, 100 mM NaCl, and 10 mM sodium EDTA,
0.75% SDS, and 0.22 mg/ml proteinase K (Roche Diagnostics, Basel, Switzerland)) at
55℃ overnight. Three ml of phenol /chloroform/ isopropanol (25:24:1) was added to
extract the DNA. Then the mixture was centrifuged at 4000 rpm for 15 mins to
separate the aqueous layer from organic layer. The aqueous layer was obtained and
the DNA inside was precipitated by adding 2 volumes of ice-cold 100% ethanol and
0.1 volume of 2 M sodium acetate. The precipitated DNA was washed with 75%
ethanol followed by centrifugation at 14000 rpm for 10 mins. The ethanol was
removed and the DNA pellet was air-dried. Then the air-dried DNA pellet was
dissolved in 100 μl TE buffer (10mM Tris pH 8 and 1 mM EDTA pH8).
2.5 DNA extraction from patient tissue samples
The same tissue biopsies were for both RNA and DNA extraction by using the
Trizol reagent (Invitrogen, CA, USA). In brief, after the phenol/chloroform phase
separation, the upper aqueous phase was removed for RNA extraction. The
58
phenol/chloroform phase and the interphase were used for DNA extraction; 0.3 ml of
100% ethanol was added to this remaining mixture for DNA precipitation, followed
by centrifugation at 2000 g for 5 mins. The DNA pellet was then washed with 1 ml
0.1 M sodium citrate in 10% ethanol. After the wash, DNA was redissloved in 8 mM
NaOH with 0.1 M HEPES.
2.6 DNA extraction from blood of healthy individuals
The blood samples of healthy individuals obtained from the Red Cross were used
for DNA extraction and genotyping of SAA1 in this study. The DNA extraction was
performed by using the blood genomic Prep Mini Spin (GE Healthcare), following the
manufacturer’s protocol. In brief, 2 ml of blood from each sample were separated into
four 500 μl samples. For each 500 μl blood sample, 20 μl of Proteinase K and 400 μl
lysis buffer were added to lyse the blood cells. The mixture was vortexed for 15 mins.
Then, the lysate was loaded to the mini-column and followed by centrifugation at
11000 g for 1 min. The collecting tube containing the flow-through was removed and
a new collecting column was replaced. Then, 500 μl of lysis buffer and 500 μl wash
buffer were sequentially added to the column and removed after centrifugation at
11000 g for 1 min. The purification column was then transferred to a fresh DNase-free
microcentrifuge tube and 200 μl of 70 pre-heated elution buffer was then added to the
59
column. The buffer was allowed to incubate with the column for 1 min in room
temperature. Then the column was centrifuged for 1 min at 11000 g and the purified
genomic DNA from the blood sample was obtained.
2.7 Reverse transcription polymerase chain reaction (RT-PCR)
The RNAs extracted from tissue samples were reverse transcribed into
complementary DNAs (cDNAs). The cDNAs were reverse transcribed by using 1μg
of total RNA, 40 units MMLV (USB, OH, USA), 100 units of RNase OUT inhibitor
(Invitrogen, CA, USA), 10 mM Oligo dT (USB, OH, USA), 500 μM dNTP (USB, OH,
USA), and RNase-free water. The reaction mixture was kept at 37℃ for 1 hour
followed by 99℃ for five minutes.
To detect the expression level of the target gene, cDNAs of samples were
amplified by PCR reactions. PCR reactions were carried out with 1X PCR buffer, 0.2
mM dNTP, 1.5 mM MgCl2, 0.2 μM primers, one unit of Platinum Taq DNA
polymerase (Invitrogen, CA, USA), water, and 1 μl of cDNA. The SAA1 primers,
SAA1-RT-F1 and -R2, and the GADPH primers, GADPH-RT-F1 and -R1, were used
(Table 4). The amount of PCR products was analyzed by agarose gel electrophoresis.
2.8 DNA sequencing
60
Table 4: RT-PCR primer sequences, conditions, and product sizes.
Primer
Product size (bp)
Annealing temperature (°C)
Primer sequence (5’-3’)
SAA1-RT-F1
294
60
AGCCAATTACATCGGCTCAG
SAA1-RT-R2
GAPDH-RT-F1
TACCCATTGTGTACCCTCTCC
548
60
GAGTCAACGGATTTGGTCGT
GAPDH-RT-R1
GAPDH-RT-F2
ATCCACAGTCTTCTGGGTGG
220
55-60
GAAGGTGAAGGTCGGAGTC
GAPDH-RT-R2
GAAGATGGTGATGGGATTTC
61
DNA sequencing was performed to study the SAA1 allelic frequencies in Hong
Kong NPC patients versus the healthy Chinese. Both NPC tumor tissues collected
from NPC patients and blood samples collected from healthy individuals were used in
this study. For genotyping the tumor tissue samples, the total RNA was obtained as
mentioned in Section 2.3. After the total RNA of the tissues was extracted, cDNAs of
the samples were produced by RT-PCR as mentioned in Section 2.7. The cDNAs were
used as templates for PCR reactions. Amplification was carried out with 100 ng of
cDNA, 1X PCR buffer, 2.5 mM MgCl2, 200 μM dNTP, 0.1 unit of AmpliTaq Gold
DNA polymerase (Applied Biosystems, CA), 0.5 μM of forward and reverse primers
(NheI-SAA1-F (Table 5) and SAA1-RT-R2 (Table 4)). The amplified SAA1 cDNA
fragments (which contain the polymorphic region) from clinical tissues were
sequenced by using the reverse primer SAA1-RT-R2. One minute of heat denaturation
at 95°C was performed, followed by 40 amplification cycles of denatuation at 96°C
for 10 sec, annealing at 50°C for 5 sec, and extension at 60°C for 4 min. G50 columns
were used to remove excess dye and the samples were denatured before loading into
the genetic analyzer. The DNA sequencing signals were detected by using the ABI
PRISMTM 3100 Genetic Analyzer (Applied Biosystems, CA, USA).
For the SAA1 genotyping of healthy individuals, the genomic DNA of the blood
samples from healthy individuals was extracted by using the Blood genomicPrep Mini
62
Table 5: Primers for DNA cloning.
Primer
Annealing temperature (°C)
Primer sequence (5’-3’)
NheI-SAA-F
55
TACGGCTAGCATGAAGCTTCTCAC
SAA1-BamHI-rev
55
CGACGGATCCTCAGTATTTCT
PacI-SAA1-F
60
TACGTTAATTAAGCCACCATGAAGCTTCTCAC
Pet-NheI-SAA-F
60
TACGGCTAGCATGCGAAGCTTCTTTTCGTT
63
Spin (GE Healthcare), as mentioned in Section 2.6. The genomic DNA obtained was
used as templates for PCR reactions; amplifications were carried out with 100 ng of
genomic DNA, 1X PCR buffer, 2.5 mM MgCl2, 200 μM dNTP, 0.1 unit of AmpliTaq
Gold DNA polymerase (Applied Biosystems, CA), 0.5 μM of forward and reverse
primers (NheI-SAA1-F (Table 5) and SAA1-RT-R2 (Table 4)). The PCR conditions
were described in the previous paragraph. The PCR products obtained were purified
by using the ExoSAP-IT (USB, OH, USA) following the manufacturer’s protocol.
The purified DNAs together were sequenced by using the reverse primer
SAA1-RT-R2. The sequencing process was performed in the Genome Research
Center in The University of Hong Kong.
2.9 Real-time quantitative reverse transcription PCR (Q-PCR)
The expression levels of SAA1 in normal and tumor nasopharyngeal biopsies
were analyzed by Q-PCR. GAPDH was used as an internal control for equal loading.
The SAA1 primers, SAA1-RT-F1 and -R2, and the GADPH primers, GADPH-RT-F2
and -R2 (Table 4), were used for this Q-PCR analysis. The SYBR Green PCR master
mix (Applied Biosystems, CA, USA) and the Step-One Plus real-time PCR machine
(Applied Biosystems, CA, USA) were used for the real-time PCR. In brief, 1X SYBR
Green PCR master mix, 0.2 μM primers, and 1 μl cDNAs of samples in 20 μl reaction
64
volume were amplified in the following conditions: 95°C for 10 mins, followed by 40
cycles of 95°C for 30s, and 60℃ for 1 min. The 2-ΔΔCT method was used for the
relative quantification of gene expression levels (Livak and Schmittgen 2001). The
expression of SAA1 in NPC tumor samples was compared with the matched normal
biopsies.
2.10 Bisulfite treatment
Genomic DNAs of samples used in this study were treated with sodium
metabisulfite to convert the unmethylated cytosine residues into uracil and leaving the
methylated cytosine residue unaffected. The methylation of DNAs was studied by
methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). In brief, 2
μg of genomic DNAs diluted in 10 μl of water were denatured in 1.1 μl of 3N NaOH
at 37℃ for 15 mins. After that, 104 μl of urea/metabisulfite solution (6.24 M urea and
2 M sodium metabisulfite (Sigma, MO, USA) and 6 μl of 10 mM hydroquinone
(Sigma, MO, USA) were added to the DNAs and incubated at 55°C for no more than
15 hours. Then, the treated DNAs were purified by QIAquick PCR purification Kit
(Qiagen, CA, USA), following the manufacturer’s protocol and eluted with 200 μl
elution buffer. The purified DNAs were then denatured in 23 μl of 3N NaOH at 37°C
for 15 mins. The DNAs were then precipitated in 20 μg of tRNA (USB, OH, USA),
65
50 μl of 10M ammonium acetate, and 500 μl of 100% ethanol at -20°C for 30 mins.
Then the DNAs were centrifuged at 12000 rpm at 4°C for 20 mins. The DNA pellets
were then washed with 500 μl of 70% ethanol and centrifuged at full speed at 4°C for
20 mins. Finally, the DNA pellets were dissolved in 50 μl of 10 mM Tris-Cl (pH8.5)
solution.
2.11 Methylation-specific PCR (MSP)
The SAA1 promoter is identified based on previous studies (Kumon et al 2002,
Thorn et al 2003) and from sequence information from the UCSC Genome Browser
(http://genome.ucsc.edu). The GC content, the observed/expected CpG ratio, and the
number of CpG sites in the SAA1 promoter were determined by computer analysis
(http://www.genomatix.de). The CpG sites of SAA1 in the promoter region were
identified at the region from -1 bp to -390 bp across the transcription start sites
(Figure 13). The MSP primers overlapping the CpG island of SAA1 were designed by
MethPrimer (www.urgene.org/methprimer). The MSP primer sequences are listed in
Table 6. MSP PCR reactions were performed with 1X PCR buffer, 2.5 mM MgCl 2,
200 μM dNTP, 0.2 units of AmpliTaq Gold polymerase (Applied Biosystems, CA,
USA), 0.3 μM of primers mix, and 1 μl of bisulfate-treated DNA in 15 μl reaction
volume. For the PCR reaction performed with the SAA1-MSP-M-F and
66
Figure 13: Region of the SAA1 CpG sites containing sequence in the promoter.
Positions of the BGS PCR primers are indicated by thin arrows. Positions of the MSP
forward and reverse primers are indicated by thick arrows. The CpG sites within the
promoter region are labeled in red.
67
Table 6: Primers used for the methylation study of SAA1.
Primer
Product size (bp)
Annealing temperature (℃)
Primer sequence (5’-3’)
SAA1-MSP-M-F
153
60
TTACGGGGTTTTTATTTTTAATTTC
SAA1-MSP-M-R
SAA1-MSP-U-F
AATACCAATAATTTCTTCATCCCG
152
62
ATGGGGTTTTTATTTTTAATTTTGT
SAA1-MSP-U-R
SAA1-BGS-F
AAATACCAATAATTTCTTCATCCCA
285
55-60
TGATTGGTAGAGTTAGGAGTTGGTT
SAA1-BGS-R
TCCCTAAAAAAAATCCCTACAAATC
68
SAA1-MSP-M-R primers, the annealing temperature of the PCR reaction was 60°C
and the PCR reaction was performed for 42 cycles. For the PCR reaction performed
with the SAA1-MSP-U-F and SAA1-MSP-U-R primers, the annealing temperature of
the PCR reaction was 62°C and the PCR reaction was performed for 40 cycles. The
PCR products were analyzed by agarose gel electrophoresis.
2.12 Bisulfite genomic sequencing (BGS)
The BGS primers of the SAA1 promoter were designed by MethPrimer
(www.urgene.org/methprimer) (Figure 13). In brief, a promoter region of 285 bp of
the bisulfite-treated DNAs was amplified by SAA1-BGS-F and SAA1-BGS-R (Table
6) for 30 cycles. The PCR products were then purified by QIAquick PCR purification
Kit (Qiagen, CA, USA) and eluted in 30 μl of water. Electrophoresis of PCR products
was performed on a 2% agarose gel. The purified PCR products were then cloned into
the TA cloning pMD18T Simple Vector (TakaRa Biotechnology, Dalian, China) and
transformed into competent cell DH5α by heat-shock transformation. The plasmid
DNAs in the competent cell were extracted by using QIAprep spin miniprep kit
(Qiagen, CA, USA). The SAA1 promoter fragment in the plasmid was then amplified
by BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA)
with the pMD18/T7 forward sequencing primer. The sequencing signals were
69
detected using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, CA,
USA). The sequencing results of at least 15 clones from each cell line was analyzed
by the QUMA program (http://quma.cdb.riken.jp/top/index.html).
2.13 5-aza-2’ deoxycytidine treatment
To investigate the silencing mechanism of candidate tumor genes, the effects of
dermethylation on candidate gene expression were studied by 5-aza-2’ deoxycytidine
treatment. Cells were treated with 5 μM 5-aza-2’ deoxycytidine (Sigma, MO, USA)
for five days. Freshly diluted drugs in medium were changed daily. After the
treatment, the cells were harvested for RNA extraction and the expression levels of
candidate genes were analyzed by RT-PCR.
2.14 Plasmid construction
The pETE-Bsd vector (Figure 14) constructed by Protopopov and colleagues
(Protopopov et al 2002) was used in this study for the establishment of
tetracycline-regulated SAA1 stable transfectants. Full open reading frames of SAA1.1,
1.3, and 1.5 were amplified from cDNAs of cell lines NP460, MCH556.15, and
NE083, respectively, by Platinum Pfx DNA polymerase (Invitrogen, CA, USA) with
NheI-SAA1-F and SAA1-BamHI-rev primers. The cloning primers’ sequences are
70
Figure 14: The vector map of pETE-Bsd.
Adapted from: Protopopov et al. 2002
71
listed in Table 5. For construction of pETE-Bsd-SAA1.1, -SAA1.3, and -SAA1.5, the
SAA1 PCR products s were excised by restriction enzymes Nhe1 and BamH1 (New
England Biolabs, MA), and was ligated into the pETE-Bsd vector. The ligation
products were transformed into DH5 α competent cells, the plasmids in the
competent cells were then extracted by Plasmid Mini Kit (Qiagen, CA, USA). Direct
DNA sequencing was used to verify the full length of the SAA1 insert sequences in the
pETE-Bsd vector.
The pWP1 vector (Addgene plasmid 12254, Figure 15) was a lentivirus
expression vector we used to establish SAA1 stable transfectants in C666 cells. Full
open reading frames of SAA1.1, 1.3, and 1.5 were amplified from plasmids of
pETE-Bsd-SAA1.1, -SAA1.3, and -SAA1.5 by Platinum Pfx DNA polymerase
(Invitrogen, CA, USA) with PacI-SAA1-F and SAA1-BamH1-rev primers (Table 5).
A Kozak sequence, GCCACC, was added to the 5’ end of the start condon of the
forward cloning primer in order to increase the efficiency of gene expression. For
construction of pWP1-SAA1.1, -SAA1.3, and -SAA1.5, the 5’ end of the PCR amplified
fragments were excised by Pac1 (New England Biolabs, MA) and the 3’ end was left
as a blunt end, the cleaved DNA fragments were ligated into the pWP1 vector. Direct
DNA sequencing was used to ensure successful cloning of the full length of the SAA1
inserts in the transformants.
72
Figure 15: The vector map of pWPI.
Adapted from: Addgene
(http://www.addgene.org/pgvec1?f=c&plasmidid=12254&cmd=showmap)
73
Figure 16: The vector map of pET-28a-(+).
Adapted from: EcoliWiki (http://ecoliwiki.net/colipedia/index.php/pET-28a(%2B)
74
The pET-28a(+) (Figure 16) was used in this study for recombinant His-tag
SAA1 protein production. The three SAA1 isoforms, SAA1.1, 1.3 and 1.5 were cloned
into the pET-28a(+) vector. The three SAA1 isoforms with their signaling peptide
equence removed were amplified from plasmids of pETE-BSD-SAA1.1, -1.3, and -1.5
by PCR reactions with Pet-Nhe1-SAA1F and SAA1-BamH1-rev primers (Table 5).
For construction of pET-28a(+)-SAA1.1, -SAA1.3, and -SAA1.5, the PCR product was
excised by restriction enzymes Nhe1 and BamH1 (New England BioLabs, MA) and
was ligated to the pET-28a(+) vector.
2.15 Stable transfection
The tTA-expressing HONE1-2 cell line (Protopopov et al 2002) was transfected
with pETE-Bsd-SAA1.1, -SAA1.3, and -SAA1.5, and pETE-Bsd vector-alone for
establishing of the tetracycline-regulated SAA1 stable transfectants and vector-alone,
respectively. One μg of DNA was added into the cells on a 6-well plate with 5 l
Lipofectamine 2000 reagent and 1 ml DMEM. The cells were incubated at 37°C for 6
hours. Then the mixed DNA and DMEM were removed and replaced by DMEM
supplemented with serum. After overnight incubation, the cells were transferred onto
100 mm diameter dishes for selection. The cells were treated with selection medium
with 5 µg/ml blasticidin after 48 hours of transfection. Independent clones were
75
picked after 3-4 weeks of selection. The total RNA was extracted from the clones,
since SAA1 is a secretary protein, conditioned media was also obtained. RT-PCR,
Q-PCR, and Western blot analyses, were used to screen for positive clones. The
vector-alone was used as a control.
For the establishment of SAA1 stable transfectants in the EBV-positive C666
cells, a lentivirus expression system was used. In brief, a 293T cell line was
transfected with the envelope and packaging vectors, pMD2.G and psPAX2 (Addgene
plasmids 12259 and 12260), and expression vectors pWP1-SAA1.1, -SAA1.3, -SAA1.5,
or the vector-alone, to generate viral particles. The 293T cells were seeded in a T75
flask with 50% confluency the day before transfection. The transfection mixture was
prepared (4 μg of pMD2.G and psPAX2 and 30 l Lipofectamine 2000 reagent in
DMEM of 800 l total volume) and incubated at room temperature for at least 20
mins. After 20 mins, the medium of the 293T cells was removed and 9 ml of fresh
complete medium and 800 µl of the transfection mixture were added to the cells. The
cells were incubated at 37°C for 48 hours. After 48 hours incubation, the medium
containing the viral particles was transferred into a fresh 15 ml Falcon tube and
centrifuged at 3000 rpm for 5 mins. To concentrate the medium containing the
lentivirus, 2.5 ml cold (4°C), PEG-it virus precipitation solution (LV810-A1, SBI)
was added to 10 ml of lentivirus-containing medium, followed by refrigeration
76
overnight. Then the mixture was centrifuged at 1500 g for 30 mins at 4°C; the
supernatant was removed and the residual mixture was spun down by centrifugation at
1500 g for 5 mins. The pellet was then resuspended in 1/10 of original volume with
cold and sterile PBS at 4°C.
The medium with concentrated lentivirual particules was used for the
transduction of C666 cells. The C666 cell line was seeded in a 6-well plate with 50%
confluence. The medium was then removed, and 2 ml of flash medium containing 8
µg/ml protamine sulfate and the virus-containing medium were added to the cells
followed by a 48 hours incubation at 37°C. The pWPI plasmid is a bicistronic vector,
which allows for simultaneous expression of a transgene and green fluorescent protein
(GFP) marker to facilitate tracking of transduced cells. The C666 cells were observed
by fluorescence microscopy to observe the transduction efficiency. The medium was
removed from the transduced cells and fresh medium was added to the cells followed
by 24 hours incubation at 37°C. The conditioned media of the SAA1 isoforms
expressing and the vector-alone cells were obtained, and Western blot analysis was
used to screen for positive cells.
2.16 Recombinant SAA1 protein expression and purification
The recombinant SAA1 protein was produced by a bacterial inducible system
77
and purified by a His-tag protein purification system. The pET-28a(+)-SAA1.1, 1.3,
and 1.5 plasmids were transformed into E coli (BL21) and cultured at 37°C overnight
on Luria broth (LB) plates with antibiotic selection. The colonies on the LB plates
were picked and cultured in 20 ml LB at 37℃ in a shaker incubator overnight. Then
the starter culture was poured into 1 l of LB containing antibiotic and incubated at
37°C in a shaker incubator for 3 hours until the OD600 reached 0.6-0.8. To induce
protein expression, 1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) (USB,
Cleveland, OH) was then added into the culture. After an induction of 3 hours, the
bacterial culture was centrifuged at 14000 g for 15 min at 4°C. The supernatant was
discarded; 30 ml of lysis buffer (50 mM NaH2PO4 (pH 8.0), 150 mM NaCl, 20 mM
imidazole) was added to resuspend the pellet. The lysate was sonicated and
centrifuged at 18000 rpm at 4°C for 20 mins. Two ml of Ni-NTA beads (Qiagen,
Hilden, Germany) was washed by 20 ml PBS to remove the ethanol. The supernatant
was collected and added to the beads; the mixture was then transferred into a new
tube and mixed in 4°C overnight. The mixture was then centrifuged at 2000 rpm for 1
min at room temperature; the supernatant was removed while the beads were washed
by 60 ml wash buffer (50 mM NaH2PO4 (pH 8.0), 150 mM NaCl, 40 mM imidazole) .
Two ml of elution buffer (50 mM NaH2PO4 (pH 8.0), 150 mM NaCl, 500 mM
imidazole) was added to the beads and vortexed gently. The mixture was centrifuged
78
at 2000 rpm for 2 min to collect the eluted proteins. The purified His-tagged SAA1
protein collected was then used to perform SDS-PAGE analysis in a 15% acrylamide
gel to check the purity of the protein. Western blot analysis was also used to detect the
purified SAA1 proteins.
2.17 Preparation of total protein lysates and conditioned media from mammalian
cells
To collect the protein in the mammalian cell lysates, cells in 60 mm2 tissue
culture dishes were lysed in 200 μl lysis buffer (50 mM Tris-Cl pH7.5, 100 mM NaCl,
1% Trition X-100, 0.5% sodium deoxycholate, 0.1% SDS and proteinase inhibitor
(Roche Diagnostics, Basel, Switerland)) at 4°C for 1 hour. Then the cell lysates were
centrifuged at 4°C for 5 min at maximum speed. The supernatant was collected.
To collect the total protein in conditioned medium of a cell line, cells were
grown in a T75 culture flask until 80% confluence. The culture medium was removed
and replaced by 10 ml serum-free DMEM. Conditioned medium was collected after
16 hours incubation. The conditioned medium collected was centrifuged at 1000 rpm
for 1 min to remove the cell debris. The supernatant was collected.
2.18 Western blot analysis
79
Western blot analysis hybridization was used to determine the protein expression
of the target proteins in the total lysates, conditioned media, or the purified protein
preparations. An equal volume of 2X protein loading buffer (100 mM pH 6.8 Tris-Cl,
200 mM β-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was
added to the protein samples. Then the protein mixtures were denatured at 100°C for
10 mins and then cool on ice for 10 mins. The protein samples were separated in
10-15% SDS polyacrylamide gels by electrophoresis with 1X protein running buffer
(20 mM Tris, 250 mM pH 8.3 glycine, 0.1% SDS). The SDS gel with separated
proteins was transferred onto a 0.45 μM polyvinylidene fluoride (PVDF) membrane
(Millipore, MA, USA) by Mini Trans-Blot cell system (Biorad, Hercules, CA, USA)
The PVDF membrane was activated by immersing in methanol. The activated
membrane and the SDS gel were immersed in 1X transfer buffer for 10 mins. Then
the filter papers, the gels, and the membrane were used to set up the transferring
sandwich. The transferring sandwich was loaded on the transfer cell with 1X transfer
buffer, and the transfer process was performed under 100 V for one hour. After the
transfer process was completed, the membranes were taken out and immersed in
blocking solution (5% non-fat skimmed milk in PBS-T buffer (PBS+0.1% Tween 20)),
for one hour. Membranes were washed with PBS-T for three times and incubated with
diluted primary antibodies at 4℃ overnight. The primary antibodies used in this study
80
are listed in Table 7. Then the membrane was washed in PBS-T three times and
probed by Amersham ECL-HRP conjugated secondary antibody (1:3000) (Amersham
Biosciences, Uppsala, Sweden) at room temperature for 30 mins. Then the membrane
was washed in PBS-T for three times. The targeted proteins on the membranes were
detected by using Amersham ECL reagent (Amersham Bioscieces, Uppsala, Sweden).
The signals on the membrane were detected on a X-ray film. When the target was a
phosphorylated protein, TBS-T (TBS+0.1% Tween 20) buffer was used instead of
PBS-T for the incubation and washing steps after the transfer.
2.19 HUVEC and HMEC tube formation assays
Endothelial cell tube formation assay was used to investigate the effects of SAA1
recombinant proteins or conditioned media of the SAA1 transfectants on in vitro
angiogensis. In brief, HUVECs and HMECs were incubated in serum-free DMEM for
one hour. Then 50 μl of growth factor-reduced Matrigel Matrix (BD Biosciences, MA,
USA) was coated on a 96-well plate and solidified at 37°C for 30 min. The
HUVECs/HMECs were harvested and resuspended in conditioned medium from the
SAA1 transfectants and the vector-alone controls. Then 100 μl cells were added to the
wells coated with Matrigel. For the tube formation assays performed with the
recombinant SAA1 proteins, conditioned medium from HONE1 were used to culture
81
Table 7: Antibodies used in Western blot analysis
Antibody
Host
Dilution
Protein size (kDa)
Catalog no.
Source
SAA1
Mouse
1:2500
~14
Ab81483
Abcam
α-tubulin
Mouse
1:5000
60
#CP06
Calbiochem
Caspase 3
Mouse
1:1000
37, 17,19
#9668
Cell signalling
Caspase 9
Mouse
1:1000
48, 37, 10
#9508
Cell signalling
Phospho-Akt
Rabbit
1:1000
60
#4058
Cell signalling
Phospho-Erk1/2
Rabbit
1:1000
42, 44
#4370
Cell signalling
82
HUVECs/HMECs. Ten μl of SAA1 proteins were added together with the cells
followed by 4 hours of incubation for HUVECs and 12 hours incubation for HMECs
at 37°C. The cells were observed by stereomicroscopy (Nikon TMS) at 100X
magnification. The tube-forming ability was determined by measuring the total
branch number.
2.20 Real-time cell viability assay
The effects of SAA1 recombinant proteins on cell viability were analyzed using
the xCELLigence System (Roche, Springer Medizin, Germany) for real-time cell
analysis. The xCELLigence System is composed of four main components: the
16-well electronic microtiter plate (E-plate); the Real-time Cell Analysis (RTCA) DP
station, which accommodates the E-Plate and is placed in a tissue culture incubator;
the RTCA Control unit, which operates the software and continuously acquires and
displays the data; the RTCA analyzer, which sends and receives signals between
RTCA control unit and RTCA DP station. In brief, the cells are seeded in E-plates that
are integrated with gold micro-electrode arrays. Application of low voltage (less than
20 mV) alternating current signal leads to the generation of an electric field between
the electrodes, which interacts with the environment of growth medium inside the
wells and is differentially modulated by the number of cells covering the electrodes,
83
the morphology, and the strength of cell attachment. The impedance readout allows
the detection and quantification of the cells inside the wells, and, therefore, allows the
study of the effect of drugs in cell viability assay. The number of cells in the well is
measured as a cell index and is shown on the RTCA control unit.
The experiment was carried out following the manufacturer’s protocol. In brief,
50 l of medium was added to each well of the E-plate and incubated at 37°C for 15
min. Then the plate was loaded on the machine and the first measurement was taken.
Then, 2 x 104 cells in 50 l medium were seeded to each well. The plate was loaded
on the machine and the second measurement was taken, a continuous measurement of
cell viability was performed every 15 min for 24 hours. Then different concentrations
of SAA1 protein, control proteins, and solvent control in 100 μl medium were added
to the wells and incubated in 37°C for another 150 hours, and the cell viability in the
form of cell index was measured every 5 min.
2.21 Cell adhesion assay
In order to investigate the effects of the recombinant SAA1 proteins on the cell
adhesion ability between HONE1 and HUVEC/HMEC cells, the cell adhesion assay
was performed. HONE1 cells were seeded on 24-well plates until confluent. Then
HUVEC/HMEC
cells
were
labeled
with
84
fluorescent
tracer
methacrylated
carboxyfluorescein diacetate (CFDA, Sigma) (100 mg/L) at 37°C for 30 mins. Then
the cells were harvested and washed by 10 ml 1X PBS three times. Then 5 X 104
fluorescence-labeled HUVECs/HMECs were resuspended in 500 μl DMEM with
10% FBS and 10% SAA1 proteins, and added to the HONE1 cells followed by 1.5
hour incubation at 37℃. Then the unbounded HUVECs/HMECs were washed away
by 1X PBS three times. The cell adhesion ability was determined by the number of
fluorescence-labeled cells observed under the microscope at 492 nm.
2.22 Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)
assay
To investigate the relationship between SAA1 recombinant protein and
programmed cell death, TUNEL assay was performed by using the In situ Cell Death
Detection kit (Roche, Mannheim, Germany). Cells were seeded on a 96-well plate
until 80% confluence. Then the cells were air-dried and fixed with freshly prepared
fixation solution (4% paraformaldehyde in PBS, pH7.4) at 25℃ for 1 hour. The cells
were washed by 1X PBS three times and incubated in freshly prepared
permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min on ice.
After that the cells were rinsed with PBS and allowed to air-dry. Fifty μl TUNEL
reaction mixture were added to each sample. The 96-well plate was then incubated in
85
the dark for 60 min at 37℃. After that the cells were washed by PBS three times. The
cells were observed by fluorescence microscopy at 450-500 nm.
2.23 Statistical analysis
The statistical analyses of the functional assays were made by Student’s t test. All
p values <0.05 were considered statistically significant.
86
Chapter 3: Results
3.1 Genotyping of SAA1 in NPC patients versus healthy individuals
To study the relationship between the SAA1 allelic variants and the risk of NPC
development, the allelic frequencies of SAA1 isoforms in NPC patients and healthy
Hong Kong Chinese people were investigated by direct sequencing of cDNA from
NPC tissues and blood DNA from healthy people. A total of 80 Hong Kong NPC
patients and 308 healthy individuals were included for this SAA1 genotyping study.
The results of sequencing are shown in Table 8. It can be seen that only SAA1.1, 1.3,
and 1.5 were observed in both Hong Kong NPC patients and healthy people, whereas
SAA1.2 and 1.4 were not detected in any of those samples. The allele distribution
analysis of SAA1 isoforms show that the frequency of SAA1.5 was the highest (60%)
among the NPC patients, while the frequencies of the three isoforms were about the
same among the healthy individuals. When compared the genotype of the NPC group
versus the healthy group, the current results showed that the frequency of SAA1.5/1.5
genotype in NPC patients was higher (p-value = 0.0507), whereas SAA1.1/1.5
genotype was lower (p-value = 0.0833) than in healthy individuals. The p-value of the
disproportionate frequency of SAA1.5/1.5 genotype between the disease and healthy
groups is very close to the significant difference level (when the significance level is
87
Table 8: A comparison of SAA1 genotypes between NPC patients and healthy groups.
SAA1 allele
NPC patients
Healthy group
SAA1.1
36/80 (45%)
164/308 (53.2%)
0.1885
SAA1.3
41/80 (51.3%)
178/308 (57.8%)
0.2930
SAA1.5
48/80 (60%)
167/308 (54.2%)
0.3542
SAA1 genotype
NPC patients
Healthy group
p-value (healthy verse NPC)
SAA1.1/1.1
9/80 (11.3%)
33/308 (10.7%)
0.8907
SAA1.3/1.3
8/80 (10%)
32/308 (10.4%)
0.9187
SAA1.5/1.5
18/80 (22.5%)
42/308 (13.6%)
0.0507
SAA1.1/1.3
15/80 (18.8%)
57/308 (18.5%)
0.9602
SAA1.3/1.5
18/80 (22.5%)
70/308 (22.7%)
0.9655
SAA1.1/1.5
12/80 (15%)
74/308 (24%)
0.0833
88
p-value (healthy verse NPC)
set to 0.05).
3.2 Gene expression analysis of SAA1 in chromosome 11 MCHs and their
matched TSs
By using the same 19K oligonucleotide microarrays, which identified THY1 as
a NPC candidate TSG (Lung et al 2005), SAA1 was also shown to be differentially
expressed in the chromosome 11 MCHs and TSs (Figure 17). In total, sixteen 19K
microarrays were used: the recipient HONE1 cells versus four HONE1/intact
chromosome 11 MCHs (HK11.8, HK11.12, HK11.13, and HK11.19), and the four
hybrids versus their TSs (HK11.8-3TS, HK11.12-2TS, HK11.13-1TS, and
HK11.19-4TS). Genes up-regulated in MCHs after chromosome transfer and
down-regulated in the TSs are presumably putative TSGs (Robertson et al 1999).
Stringency was set so that only data sets with expression ratios higher than 1.2 in
hybrids/HONE1 cells and ratios less than 0.8 in hybrids/TSs for both duplicates were
selected for further studies. Under the above criteria, SAA1 located at 11p15.1, was
found to be one of the most differentially expressed genes among the 19K transcripts.
The expression ratios of each duplicate from the hybrid pairs are summarized in Table
9. A comparison of the expression profile of HONE cells with the four hybrids,
89
Figure 17: The chromosome 11 19K oligo-microarray results show that the SAA1
expression is differentially expressed in MCHs and their matched TSs.
90
showed an increased expression of SAA1 in all the four hybrid clones. On the other
hand, the mRNA levels of SAA1 were down-regulated in all four TSs, when compared
with the corresponding MCHs.
In order to verify the SAA1 gene expression, semi-quantitative RT-PCR was
performed for MCHs and TSs. The same batch of RNAs used in microarray analysis
was used in this confirmation analysis and quantification revealed a good correlation
between the fold-changes obtained in the microarray and RT-PCR for all four genes
(Table 9 and Figure 18). Undetectable levels of SAA1 expression were observed in
HONE1 cells and after transfer of an exogenous chromosome 11, the gene expression
of SAA1 was induced in all four MCHs (Figure 18). When comparing the gene
expression between these MCHs and their TSs, down-regulation of SAA1 expression
was observed in all TSs, and complete loss of mRNA expression of SAA1 was
observed in their HK11.8-3TS, HK11.13-1TS, and HK11.19-4TS (Figure 18). The
human SAA1 genes was expressed in the mouse chromosome 11 donor cells, MCH
556.15, and this cell line was used as a positive control for this gene. Different
batches of RNAs from the same set of cell lines at different passages also reveal
similar results (data not shown).
3.3 Gene expression analysis of SAA1 in other NPC and esophageal squamous cell
carcinoma (ESCC) cell lines and clinical biopsies
91
Table 9: Microarray detection of SAA1 in HONE1 versus MCHs and MCHs versus
TSs.
Expression ratio*
Cell lines
Average
Cy5/Cy3 Cy3/Cy5
11.8/HONE1
2.65
2.20
2.425
11.8-3TS/11.8
0.21
0.14
0.175
11.12/HONE1
2.72
5.95
4.335
11.12-2TS/11.12
0.44
0.45
0.445
11.13/HONE1
1.14
1.00
1.07
11.13-1TS/11.13
0.46
0.74
0.6
11.19/HONE1
1.72
1.70
1.71
11.19-4TS/11.19
0.26
0.36
0.31
*expression ratio is Cy5/Cy3, for dye-swap Cy3/Cy5 the expression ratio is the
reciprocal
92
Figure 18: The SAA1 gene expression in HONE1, 556.15, the four MCH cell lines and
their TSs. GAPDH was used as an internal control.
93
Besides HONE1 cells, I also studied the SAA1 expression in other NPC cell lines,
which include HK1, HNE1, CNE1, C666, SUNE1, and CNE2 cells. No expression of
SAA1 was detected in three NPC cell lines (HK1, C666, and CNE2), and barely
detectable levels were observed in HNE1 and SUNE1 (Figure 19). The SAA1 gene
expression was clearly detected in CNE1 and the level was slightly less than the
NP460 and 556.15. To study the clinical relevance of SAA1 in NPC, Q-PCR was
performed to determine the SAA1 expression level in NPC patient biopsies. In a total
of 42 patients in different cancer stages, 23 of 42 (54.8%) tumor tissue samples
showed down-regulated SAA1 expression of 1.7-fold or above, compared with their
matched normal tissues (Figure 20). Among 42 pairs of NPC tissue samples, nine
decreased by more that 10-fold in SAA1 expression. We also studied the association of
SAA1 expression in the tumor tissues with their clinical information (Table A1 in
Appendix). Only one pair of samples shows an increase by more than 10-fold of SAA1
expression. There was no significant association of SAA1 down-regulation with age
and stage of tumor (Table 10). However, there are more SAA1 down-regulated cases
found in male NPC patients than the female and the result is statistically significant
(p-value = 0.0113).
In order to determine the usefulness of SAA1.1, 1.3, or 1.5 variants as a
biomarker for NPC diagnosis, we analyzed the 42 samples from the above Q-PCR
94
Figure 19: The SAA1 gene expression in six NPC cell lines. GAPDH is used as an
internal control. MCH556.15 and NP460 cells were used as normal controls.
95
Figure 20: Real-time PCR was performed with 42 pairs of NPC tissues to study the
fold-change of SAA1 expression in normal versus tumor tissues. A total of 42 pairs of
NPC tissue samples was examined by real-time PCR, 23 (54.8 %) show a
down-regulation of SAA1. Among 42 pairs of NPC tissue samples, nine show
decreases of more that 10-fold in SAA1 expression.
96
Table 10: Correlation between SAA1 expression assessed by Q-PCR in Hong Kong
NPC biopsies and clinicopathological parameters.
Parameter
Sex
Age
Stage
Number of cases
Ratio of down-regulated expression
p-value
Male
25/40*
17/25 (68%)
0.0113
Female
15/40*
4/15 (26.7)
60≥
18/42
12/18 (66.7%)
>60
24/42
9/24 (37.5%)
I-II
15/42
8/15 (53.3)
III-IV
27/42
15/27 (55.6
*the information of gender of two NPC patients were not available
97
0.0614
0.9401
assay with the SAA1 genotyping data (Table A1 in Appendix). The SAA1 gene
expression for 42 genotyped normal and tumor tissue pairs; 54.8% of NPC tissues
showed down-regulation of SAA1. Among those samples with decreased
SAA1expression, SAA1.1 (61.1%) and SAA1.5 (65.4%) isoforms are frequently found
(Table 11). In addition, increased SAA1 gene expression in tumor tissues is frequently
observed in NPC patients with SAA1.1/1.1 (75%) and SAA1.3/1.3 (80%) genotypes,
suggesting that SAA1.1/1.1 and SAA1.3/1.3 genotypes may be associated with an
increase of SAA1 gene expression. On the other hand, a high percentage of SAA1.1/1.5
genotype (85.7%) shows decreased gene expression in tumor tissues, which suggests
that the decrease in SAA1.1/1.5 expression may be correlated with the NPC tumor
development. However, the difference is not statistically significant because of the
small sample size.
The SAA1 gene expression was also studied in another aerodigestive tract cancer,
esophageal squamous cell carcinoma (ESCC). The global gene expression analysis of
the tumor suppressive ESCC chromosome 14 MCHs (Ko et al 2005) versus their
matched TSs was performed, SAA1 was found to be differentially expressed (Data not
shown). These results suggest that SAA1 may also play an important role in ESCC
tumor development.The expression of SAA1 in 15 ESCC cell lines was studied by
semi-quantitative RT-PCR. The expression of SAA1 was down-regulated in 14 out of
98
Table-11: Comparison of SAA1 isoforms between tumor tissues with increased and
decreased SAA1 expression by Q-PCR.
SAA allele
Tissues with increased expression
Tissues with decreased expression
SAA1.1
7/18(38.8%)
11/18(61.1%)
SAA1.3
10/20(50%)
10/20(50%)
SAA1.5
9/26(34.6%)
17/26(65.4%)
SAA1
Tissues with increased expression
Tissues with decreased expression
SAA1.1/1.1
3/4(75%)
1/4(25%)
SAA1.3/1.3
4/5(80%)
1/5(20%)
SAA1.5/1.5
5/11(45.5%)
6/11(54.5%)
SAA1.1/1.3
3/7(42.9%)
4/7(57.1%)
SAA1.3/1.5
3/8(37.5%)
5/8(62.5%)
SAA1.1/1.5
1/7(14.3%)
6/7(85.7%)
genotype
99
A.
B.
Figure 21: The SAA1 gene expression in (A) 15 ESCC cell lines and (B) 3 pairs of
ESCC tissue samples. GAPDH is used as an internal control. NE1 was used as a
normal control. SAA1 expression is down-regulated in 14/15 (93%) of the NPC cell
lines. Down-regulated SAA1 expression was demonstrated in three selected ESCC
paired tumor (T)/normal tissues (N).
100
15 ESCC cell lines compared with immortalized normal esophageal epithelial cell line,
NE1 (Figure 21). The SAA1 expression was only detected in two ESCC cell lines,
SLMT-1S1 and EC1, and the expression level of the SLMT-1S1 was down-regulated,
whereas there was no obvious change in SAA1 expression for EC1. The SAA1
expression in three chosen ESCC tumor/normal tissue pairs was also studied by
semi-quantitative RT-PCR. The SAA1 gene expression was down-regulated in all three
ESCC tumor tissues, when compared with their matched normal controls.
3.4 Mechanism of gene silencing of SAA1
The SAA1 promoter hypermetylation status was studied by methylation-specific
PCR (MSP) and bisulfite genomic sequencing (BGS) assays in seven NPC cell lines
(HONE1, HK1, HNE1, CNE1, CNE2, C666, and SUNE1). Methylated alleles were
observed in five out of seven NPC cell lines (HONE1, HK1, HNE1, CNE2, and
C666); barely detectable levels were observed in CNE1 and SUNE1 (Figure 22).
Unmethylated alleles were observed in all seven NPC cell lines. Hence, heterozygous
methylation was observed in these five NPC cell lines, but no homozygous
methylation was observed in any of them. The results suggest that a heterozygous
pattern of promoter methylation was observed in NPC cell lines. The immortalized
epithelial cell lines NP69 and NP460 were use as normal controls in this assay; only
101
ee
Figure 22: MSP analysis of SAA1 promoter methylation in seven NPC cell lines
(HONE1, HK1, HNE1, CNE1, CNE2, C666, and SUNE1) and two immortalized NP
cell lines (NP69 and NP460). Size of PCR amplicons is shown on the right.
102
unmethylated alleles were observed.
Five CpG sites -297, -289, -268, -163, and -121 in the promoter region of SAA1in
the seven NPC cell lines and NP460 were studied by clonal BGS. The results of the
BGS study are shown in Figure 23. High frequencies of methylation were observed in
the CpG position -297 and -289 in six NPC cell lines (HONE1, HK1, HNE1, CNE1,
CNE2, C666, and SUNE1), moderate frequencies were observed in SUNE1, and low
frequencies were observed in CNE1 and NP460. The percentages of methylation in
other three CpG sites -268, -163, and -121 are generally much lower in all seven NPC
cell lines; no methylation was observed in NP460 for these three CpG sites.
A total of four NPC cell lines (HONE1, HK1, HNE1, and CNE1) were chosen
for the demethylation treatment analysis. Semi-quantitative RT-PCR was used to
examine the SAA1 gene expression after the treatment. Treatment with the
demethylation agent, 5-Aza-2’-deoxycytidine, resulted in the restoration of SAA1
expression in three NPC cell lines (HONE1, HK1, and HNE1), compared with the
untreated controls (Figure 24). There was no change in SAA1 expression for CNE1.
Taken together, it is likely that promoter hypermethylation is an important silencing
mechanism to inactivate SAA1 in NPC cells.
When five selected ECSS cell lines (TTn, 81T, HKESC, KYSE140, and
KYSE180)
with
down-regulated
SAA1
103
expression
were
treated
with
rrrrrrrrrrrrrrrrrrrrrr
Figure 23: BGS analysis of 5 CpG sites in SAA1 promoter region of seven NPC cell
lines and the immortalized NP460 cell line. Each row represents an individual allele
and the circles represent a single CpG dinucleotide. The CpG sites -297, -289, -268,
-163, and -121 are shown from left to right, unmethylation (○) and methylation (●)
status of CpG sites are as indicated.
104
A)
B)
Figure 24: The SAA1 gene expression in (A) four NPC and (B) five ESCC cell lines
before and after treatment with 5-aza-2’-deoxycytidine. GAPDH serves as an internal
control. NP460 and NE3 were used as normal controls for comparison.
105
5-Aza-2’-deoxycytidine, increased expression was observed in all of them. Promoter
methylation also seems to be involved in gene silencing in ESCC (Figure 24).
3.5 The anti-angiogenic effects of the endogenous SAA1 isoforms
3.5.1 Establishment of SAA1 stable transfectants
As mentioned in the previous section, three SAA1 isoforms (SAA1.1, SAA1.3, and
SAA1.5) were identified in NPC/normal tissues/cell lines and healthy Hong Kong
Chinese individuals in the present study. To study the functional roles of the three
SAA1 isoforms in NPC development, SAA1.1, 1.3, and 1.5 were ectopically expressed
in HONE1 cells with down-regulated SAA1 expression. The pETE-Bsd-SAA1.1,
-SAA1.3, -SAA1.5, and pETE-Bsd vector-alone clones were transfected into the
tetracycline-regulated HONE1-2 NPC cell lines. Stable SAA1 transfected clones
which express the SAA1 proteins were chosen for further studies. The SAA1
expression of the stable transfectants was confirmed by both Q-PCR and Western
blotting. Two stable clones were obtained for each SAA1 isoform in HONE1-2 cells,
namely,
H1-pB-SAA1.1-C1
and
-C2,
H1-pB-SAA1.3-C1
and
-C2,
and
H1-pB-SAA1.5-C1 and -C2. In Q-PCR analysis, all SAA1.1, 1.3, and 1.5 transfected
clones showed over-expression of SAA1 compared to the vector-alone, the levels of
over-expression was comparable with the NP460 (Figure 25A). In the presence of dox,
106
A.
10000.00
Fold-change
1000.00
100.00
10.00
1.00
B.
Figure 25: A) The SAA1 RNA expression in the HONE1-pETE-Bsd-SAA1.1, 1.3, and
1.5 (H1-pB-SAA1.1, -1.3, and 1.5) transfectants. In the absence of dox, the expression
of SAA1 in all of the clones were higher compared the plus dox conditions. B)
Western blot analysis of SAA1 protein in conditioned media of the same SAA1.1, 1.3,
and 1.5 transfectants. Secretary SAA1 proteins were detected in all the transfected
clones. Coommassie blue staining of total protein in the conditioned media served as
internal control.
107
the SAA1 gene expression was significantly reduced in all SAA1 stable clones when
the transgenes were switched off. The Western blot analysis for the conditioned media
of the stable clones was performed, as SAA1 is presumably a secretary protein in the
NPC cell lines. The protein expression of SAA1 in conditioned media was observed
in all of the SAA1 transfectants (Figure 25B). The levels of the secreted SAA1
protein in the two SAA1.5 clones were the highest, whereas the SAA1.3 clones were
the lowest. These results were consistently observed in at least three sets of Western
blot.
In addition, SAA1 stable transfectants were also established by a lentivirus
expression system. The lentiviral plasmids pWP1-SAA1.1, -SAA1.3, -SAA1.5, and the
vector-alone were transduced into the EBV-positive C666 cell line. SAA1 is not
expressed in C666 cells as described in previous section (Figure 19). Results of
transduction showed that more than 90% of C666 cells expressed the GFP reporter
gene, observed by fluorescence microscopy (Figure 26A). The SAA1 protein
expression of the C666-pWPI-SAA1.1, 1.3, and 1.5 stable transfectants was confirmed
by Western blotting. The expression of SAA1 proteins in conditioned media was
observed in all of the transfectants of C666 cells (Figure 26B).
3.5.2 The secretary SAA1 proteins from the stable transfectants inhibit
108
A.………..
B.
Figure 26: A) The GFP expression of C666-pWPI-SAA1.1, 1.3, and 1.5, and the
vector-alone transfectants. The C666 cells without any pWPI vector were used as a
negative control. B) Western blot analysis of SAA1 protein in conditioned media of
SAA1.1, 1.3, and 1.5 expressed cells. Secretary SAA1 proteins were detected in the
C666-pWPI- SAA1.1, 1.3, and 1.5 cells. Commassie blue staining of total protein in
the conditioned media served an internal control.
109
endothelial cell tube formation in vitro
Successful blood vessel formation is important for tumor growth, since cancer
cannot grow beyond 2 mm diameter without blood vessels (Folkman 1971). The
‘angiogenic switch’ is an important process in cancer progression; it refers to the
ability of a tumor to proceed from the non-angiogenic to angiogenic phenotype. To
study the effects of secretary SAA1 protein on tube-forming ability of human
endothelial cells, the HUVEC tube formation assay was performed. The HUVECs are
endothelial cells which will undergo tube formation in response to angiogenic
stimulation. The conditioned media from the H1-pB-SAA1.1-C1, 1.1-C2, 1.3-C1,
1.3-C2, 1.5-C1, 1.5-C2, and H1-pB-C1 vector-alone transfectants were incubated with
HUVECs for 4 hours. The expression of the secreted SAA1 proteins in the
conditioned media of SAA1 stable transfectants was verified by Western blot analysis
(Figure 25). HUVECs are able to differentiate into tube-like structure mimicking
blood vessel formation in vitro. The total branch numbers of each sample were
measured and compared with the vector control. The two SAA1.1 transfectants
showed the highest inhibitory effect on tube formation ability, while the two SAA1.5
transfectants showed the lowest inhibitory effect (Figure 27). Only one clone of each
SAA1 isoform statistically significantly reduced the tube formation ability of
HUVECs (H1-pB-SAA1.1-C1, H1-pB-SAA1.3-C2 and H1-pB-SAA1.5-C2) (Figure
110
Figure 27: HUVEC tube formation assay of the HONE1-2 SAA1 variant transfectants
and vector-alone control. A) Representative results of the HUVEC tube formation
assay of the SAA1.1, 1.3, and 1.5, and vector-alone transfectants (100 X
magnification). B) The percentage of tube formation ability of the SAA1.1, 1.3, and
1.5 clones, as compared to their vector-alone control. The clones which showed a
statistically significant inhibition on tube formation ability compared with the vector
control are marked with a (*) on top of the column.
111
27B).
Besides using HONE1 as a recipient cell line for transfection studies, we also
successfully obtained stable transfectants of the EBV-positive C666 NPC cells with
the pWPI lentiviral vector system (Figure 26). In vitro tube formation of HUVEC was
performed for these C666-pWPI-SAA1.1, 1.3, and 1.5 transfectants. After incubation
of the conditioned media with HUVEC cells, their tube formation ability was
differentially inhibited; C666-pWPI-SAA1.5 could not significantly reduce the tube
formation, whereas C666-pWPI-SAA1.1 and -SAA1.5 dramatically reduced by about
70% to 80% of the HUVEC tube formation and the differences were statistically
significant (Figure 28).
3.6 The biological activities of the SAA1 recombinant variant proteins
3.6.1 Production and purification of SAA1 recombinant proteins
To study the biological activities and to fine-tune the differential effects of the
three SAA1 isoforms in NPC development, the recombinant His-tagged proteins of
the three SAA1 isoforms, SAA1.1, SAA1.3, and SAA1.5, were produced. The use of
these recombinant proteins allows accurate quantification of each SAA1 isoform used
in various assays and offers an advantage over the use of conditioned medium
obtained from the SAA1 stable transfectants.
112
A.---
B.
Figure 28: HUVEC tube formation assay of C666 SAA1 transfectants and
vector-alone control. A) Representative results of the HUVEC tube formation assay of
the SAA1.1, 1.3, and 1.5, and vector-alone transfectants (100 X magnification). B) The
percentage of tube formation ability of the SAA1.1, 1.3, and 1.5 clones, as compared
to their vector-alone control. The clones, which showed a statistically significant
inhibition on tube formation ability compared with the vector control, are marked
with a (*) on top of the column.
113
To further study the effects of SAA1 at the protein level, SAA1.1, 1.3, and 1.5
were subcloned into the pET-28a(+) vector and were expressed in the BL21 bacterial
cells to produce the three His-tagged SAA1 variant proteins. The purified proteins
were eluted in three times from the affinity column. A clear single band with the size
of 14kDa was obtained after the purification. One liter of bacterial culture can
approximately generate 8 mg of purified protein and the average overall purity was
around 98% (Figure 29A). Western blot analysis was performed to confirm the
presence of the SAA1 proteins (Figure 29B).
3.6.2 The inhibitory effects of the recombinant SAA1 isoforms on endothelial cell
tube formation
To compare the inhibitory activities of the three SAA1 isoforms on angiogenesis,
the effects on HUVEC and HMEC tube formation assays after treatment with the
recobminant SAA1.1, 1.3, or 1.5 proteins was examined. Identical quantity of the
three SAA variant proteins was used to treat the HUVECs and HMECs and was used
to compare the relative inhibitory effects of the three SAA1 variant proteins on the
tube formation ability of these two endothelial cell lines. The conditioned medium of
HONE1 cells was used to stimulate the tube formation of both HUVEC and HMEC.
When 4 mg/ml and 1 mg/ml of SAA1 proteins were used for the HUVEC and HMEC
114
A.
B.
Figure 29: A) The SDS PAGE analysis of His-tag SAA1.1 recombinant proteins
produced by the His-tagged purification system. The His-tag SAA1.1 proteins from
the three final elutions are circled in red and the overall purity of the protein is 98%. B)
Western blot analysis of the purified His-tag SAA1.1, 1.3, and 1.5 variant proteins.
115
tube formation assays, respectively, the SAA1.3 protein treated samples showed the
greatest suppression in both HUVEC and HMEC tube formation abilities (43%
inhibition for HUVECs and 47% inhibition for HMECs), while the SAA1.5
recombinant protein treatment showed the least reduction in both cell lines (19%
inhibition for HUVECs and 5% inhibition for HMECs) (Figures 30 and 31). The
recombinant BSA protein was used as a negative control protein for both the HUVEC
and HMEC tube formation assays. Cells treated with BSA of same concentrations (4
mg/ml for HUVEC and 1 mg/ml for HMEC) did not show any significant reduction
(>10%) of tube formation ability of HUVEC and HMEC. Thus, the recombinant
SAA1 variants differentially suppressed the endothelial cell tube formation in vitro.
3.6.3 The endothelial cell viability was differentially inhibited by the exogenous
SAA1 variants
Since the suppressive activities of the three exogenous SAA1 isoforms on the
endothelial cell tube formation was clearly demonstrated, we planned to study the
effects of those recombinant SAA1 proteins on cell viability of HMEC by using the
Roche´s xCELLigence System for real-time cell viability analysis. The results show
that reduction of cell viability was observed in HMECs treated with 0.5 mg/ml
recombinant SAA1.1, 1.3, and 1.5 proteins (Figure 32A). Among the three SAA1
isoforms, SAA1.3 was the most suppressive (~70% in average) in cell viability, while
116
A.
B.
Figure 30: The effects of the SAA1.1, 1.3, and 1.5 recombinant proteins on HUVEC
tube formation. A) Microscopic views at 40X magnification showing the tube
formation ability of HUVECs with or without the treatment of 4 mg/ml SAA1 variant
proteins. B) The percentage of tube formation of the SAA1.1, 1.3, and 1.5 proteins
treated HUVEC, as compared to the solvent control. BSA was used as a negative
control protein.
117
A.
B.
Figure 31: The effects of the SAA1.1, 1.3, and 1.5 recombinant proteins on HMEC
tube formation. A) Microscopic views at 40X magnification showing the tube
formation ability of HMECs with or without the treatment of 1 mg/ml SAA1 variant
proteins. B) The percentage of tube formation of the SAA1.1, 1.3, and 1.5
protein-treated HMEC, as compared to the solvent control.
118
A.
B.
Figure 32: The real-time HMEC cell viability assay was used to study the effects A)
of 0.5 mg/ml SAA1.1, 1.3, and 1.5 proteins and B) of various concentrations (0.2 to 1
mg/ml) of SAA1.3 protein. The solvent and same concentration of BSA were used as
controls. The arrow indicates the addition of SAA1 proteins.
119
SAA1.5 was the least (~50% in average). In addition, the inhibitory effects of the
recombinant SAA1.3 protein were dose-dependent; 0.2 mg/ml of SAA1.3 protein was
still effective in reducing the HMEC cell viability (Figure 32B). The inhibitory effects
of SAA1 proteins is likely to be cell type-specific, as 0.5 mg/ml of the three SAA1
protein slightly increased the cell viability of NP460 for the first 72 hours. The cell
viability was slightly decreased thereafter, but still remained at high levels, when
compared with the solvent control (Figure 33).
3.6.4 The effects of SAA1 isoforms on endothelial cell-NPC cell adhesion
The effects of the three SAA1 isoform proteins on endothelial cell-NPC cells
were studied. HONE1 cells were seeded on a 96-well plate until the cell density
reached confluence, then fluorescence-labeled HMECs/HUVECs and SAA1 proteins
were incubated with the HONE1 cells for 3 hours. The number of HMECs/HUVECs
attached to the HONE1 cells after the incubation was compared with samples without
the SAA1 treatment. The recombinant SAA1.1- and SAA1.3-treated samples showed
the much higher reduction on the number of attached endothelial cells than SAA1.5
for both HUVECs and HMECs (Figures 34 and 35). Both SAA1.1 and 1.3 proteins
could reduce the adhesion of endothelial cell/HONE1 cell by at least 60% (Figures
34B and 35B). The sample treated with BSA did not show significant effects on the
120
Figure 33: The real-time cell viability analysis was used to study the effects of 0.5
mg/ml SAA1.1, 1.3, and 1.5 proteins on NP460. The solvent control was used for
comparison. The arrow indicates the addition of SAA1 proteins.
121
A.
B.
Cell adhesion ability
(%)
Cell adhesion assay
120
100
80
60
40
20
0
Buffer
control
BSA
1.1
1.3
1.5
Figure 34: The cell adhesion assays study the effects of the exogenous SAA1.1, 1.3,
and 1.5 proteins on HUVEC-HONE1 cell adhesion. A) Microscopic views at 40X
magnification showing the CFDA-labeled HUVECs with or without the recombinant
SAA1 protein treatment (1 mg/ml). B) The percentage of attached HUVEC after the
treatment with the SAA1.1, 1.3, and 1.5 proteins, compared with the solvent control.
Same concentration of BSA was used as a negative control protein.
122
A.
Cell adhesion ability (%)
B.
120
100
80
60
40
20
0
Cell adhesion assay
Buffer
control
BSA
1.1
1.3
1.5
Figure 35: The cell adhesion assays study the effects of the exogenous SAA1.1, 1.3,
and 1.5 proteins on HMEC-HONE1 cell adhesion. A) Microscopic views at 40-X
magnification showing the CFDA-labeled HMECs with or without the recombinant
SAA1 protein treatment (1 mg/ml). B) The percentage of attached HMEC after the
treatment with the SAA1.1, 1.3, and 1.5 proteins, compared with the solvent control.
Same concentration of BSA was used as a negative control protein.
123
cell adhesion, showing that the inhibitory effects of the SAA1 proteins are specific.
3.6.5 SAA1 induced-apoptosis in endothelial cells
The treatment of SAA1 proteins suppressed cell growth as shown by the
real-time cell viability assay. To investigate whether this is due to the induction of
programmed cell death, the possibility of SAA1-induced apoptosis in endothelial cells
was studied by the TUNEL assay. The TUNEL assay detects apoptotic events by
detecting DNA fragmentation and labeling the terminal ends of the nucleic acid.
When the cells are undergoing apoptosis, the DNA inside the nucleus will be
fragmented. The fragmented DNA can be labeled by terminal deoxynucleotidyl
transferase, which catalyzes polymerization of fluorescence-labeled nucleotides to the
free 3’-DNA ends. The fluorescein labels incorporated in nucleotide polymers can be
detected and quantified by fluorescence microscopy.
The HUVECs were grown on 96-well plates and treated with 1 mg/ml of
SAA1.3 protein for 20 hours before staining for TUNEL assay. Untreated cells are
treated with DNAase digestion to generate DNA fragmentation in order to obtain a
positive control (data not shown). Stained cells were observed by fluorescence
microscopy. The observed fluorescence signals represent the DNA fragmentation
inside the nucleus, indicating the cells have undergone apoptosis. The results show
124
A.
B.
Figure 36: The TUNEL assay for the apoptosis detection of HUVECs treated with the
exogenous SAA1.3 protein. A) Microscopic views at 40X magnification showing the
fluorescence-labeled HUVECs with DNA breakage after treatement of 1 mg/ml of
SAA1 proteins. B) The number of apoptotic cells induced by the treatment with
SAA1 variant proteins. The same concentration of BSA was used as a negative
control protein.
125
that fluorescence signals were only detected in the recombinant SAA1.3 treated
sample, but not detected in the buffer control, indicating that the apoptosis was
induced by the SAA1.3 protein (Figure 36).
To further study the role of the recombinant SAA1 protein in the apoptotic
pathway, HUVECs were treated with the most prominent SAA1.3 protein (1 mg/ml)
and their cell lysates were obtained for the detection of the apoptosis effector
molecule caspase 3. Caspase 3 was detected by Western blot analysis. Results show
cleaved caspase 3 was induced after treatment with the SAA1.3 protein (Figure 37).
The results indicated that SAA1.3 protein induced the activation of caspase 3 in
HUVECs.
3.7 The direct anti-tumor effects of the recombinant SAA1 variant proteins in
NPC cells
3.7.1 The effects of the recombinant SAA1 on NPC cell viability
The direct anti-tumor effects of the recombinant SAA1.1, 1.3, and 1.5 proteins
on the cell viability of HONE1 cells were studied by using the real-time cell analysis.
The results show that reduction of cell viability was observed in HONE1 cells treated
with the three exogenous SAA1 proteins (0.5 mg/ml) (Figure 38). Among the three
SAA1 isoforms, highest reduction on cell viability (~60% in average) was observed in
126
Figure 37: Western blot analysis of caspase 3 expression in the SAA1.3
protein-treated HUVEC cells. The HUVECs were treated with 1 mg/ml SAA1.3
protein for 2 hours. α-tubulin was used as an internal control. Active caspase 3 was
detected after the treatment of SAA1.3 protein.
127
Figure 38: Real-time cell viability analysis was used to study the effects of 0.5 mg/ml
SAA1.1, 1.3, and 1.5 proteins on HONE1 cells. The solvent control was used for
comparison. The arrow indicates the addition of SAA1 proteins.
128
the SAA1.3-treated cells, while the lowest reduction (~30% in average) was observed
with the use of the SAA1.5 protein. The number of HONE1 cells dramatically
dropped from 5 to 10 hours after the addition of the three exogenous SAA1 proteins
and the cell number increased thereafter. The cytotoxic effects of the recombinant
SAA1 proteins appear to be transient.
3.7.2 SAA1 induced-apoptosis in NPC cells
The TUNEL assay was used to determine whether the recombinant SAA1.1, 1.3,
and 1.5 proteins would induce apoptosis in HONE1 cells. The results show that the
apoptotic fluorescence signals were only detected in HONE1 cells treated with the
three SAA1 proteins (1 mg/ml), but not detected in the BSA control. All the three
SAA1 proteins are able to induce apoptosis in HONE1 cells (Figure 39). The SAA1.3
protein was chosen for further apoptotic pathway analysis. The activated caspase 3
was detected after 2 hours treatment with the 1 mg/ml SAA1.3 protein (Figure 40).
The results indicated that SAA1.3 protein was able to induce the activation of caspase
3 in HONE1 cells.
129
A.
B.
Figure 39: The TUNEL assays for studying apoptosis induced in HONE1 cells by the
recombinant SAA1.1, 1.3, and 1.5 proteins. A) Microscopic views at 40 X
magnification showing the fluorescence-labeled HONE1 cells with DNA breakage
after treatment of all three SAA1 proteins (1 mg/ml). B) The number of apoptotic
HONE1 cells induced by the treatment with SAA1 variant proteins. Same
concentration of BSA was used as a negative control protein.
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Figure 40: Western blot analysis of caspase 3 expression in the SAA1.3 protein
treated-HONE1 cells and buffer control. α-tubulin was used as an internal control.
Active caspase 3 was detected after the treatment of 1mg/ml SAA1.3 protein for 2
hours.
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Chapter 4: Discussion
Our previous MMCT studies show that the transfer of an intact chromosome 11
suppressed the tumorigenicity of the NPC HONE1 cells (Cheng et al 2000).
Differentially expressed genes were identified by the 19K oligo-microarray analysis.
Genes which are down-regulated in tumorigenic cells (HONE1 and TSs) and
up-regulated in non-tumorigenic cells (MCHs) were selected as candidate tumor
suppressor genes for further analysis. SAA1 was one of the candidate TSGs identified
and was selected for further study in this project.
MMCT approaches were successfully used to identify CRs associated with tumor
suppression in the NPC HONE1 cell line system and to test candidate TSGs mapping to
these regions for their role in tumor suppression. We discovered that BLU/ZMYND10
(Yau et al 2006), PTPRG (Cheung et al 2008) at 3p14-21, MIPOL1 (Cheung et al
2009), and CRIP2 (Cheung et al 2011) at 14q11-13 and 14q32, are genes found to
map into CRs and are important for tumor suppression. The monochromosome
transfer approach identified a 3p14 gene, ADAMTS9, as a candidate TSG, which is
down-regulated in both NPC and EC cell lines and tumors (Lung et al 2008). It
contributes to cancer development via its anti-angiogenesis role. Using this approach
we also identified several important chromosome 11 genes in NPC including,
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CADM1/TSLC1, THY1, and CRYAB (Lung et al 2005, Lung et al 2006, Mineva et al
2005). Gene expression profiling using the 19K oligonucleotide microarray was
utilized to identify candidate genes within our critical tumor suppressive regions. THY1
was identified using this approach, which is established in our laboratory.
The present genotyping analysis of SAA1 indicated that out of the five SAA1
variants, it seems like only three of them, SAA1.1, 1.3, and 1.5, are expressed in the
Hong Kong Chinese. Thus, these three SAA1 isoforms were selected for further
functional analyses in the present study. By analyzing the amino acid sequences of the
three SAA1 variant proteins chosen in the present study, they all contain the YIGSRand RGD-like motifs with close proximity (Preciado-Patt et al 1994). Proteins with
YIGSR and/or RGD motifs can inhibit angiogenesis (Chiang et al 1995, Iwamoto et
al 1996, Nicosia and Bonanno 1991), thus, the anti-angiogenic potential of the three
Hong Kong Chinese expressed SAA1 isoforms was examined by the in vitro
endothelial cell tube formation assays. Although numerous studies have already
indicated that the serum SAA level is dramatically induced in patients with various
types of cancer, our gene expression analyses clearly show that SAA1 was consistently
down-regulated in all the tumorigenic NPC cell lines and the chromosomes 11 TSs.
These results suggest that the physiological levels of SAA1 expressed locally in the
normal nasopharyngeal tissue play a novel protective role against tumor development.
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The functional relevance of the locally expressed SAA1.1, 1.3, and 1.5 was
examined by ectopically expressing these three SAA1 isoforms in HONE1 and the
EBV-positive C666 cells. Whether the effects of exogenous serum SAA1 (presumably
to be expressed in the liver) in cancer patients’ sera are similar to the locally expressed
SAA1, the anti-angiogenic properties of exogenous recombinant SAA1.1, 1.3, and 1.5
proteins with physiological levels were examined. The concentrations of the
recombinant SAA1 proteins applied in various cell growth and angiogenesis assays
are based on the reported concentrations of elevated SAA levels in serum (200–2000
μg/ml) of NPC patients.
In addition, the genotypes and allelic frequencies of the three SAA1 isoforms
between the Hong Kong NPC patients and healthy individuals were compared. The
association of the SAA1 polymorphisms with NPC risk were assessed to determine
whether this association would play any functional role.
4.1 Expression of SAA1 in the chromosome 11 MCHs and their matched TSs and
NPC and ESCC cell lines
Based on the stringency setting of the present microarray analysis of the
differentially expressed genes in the chromosome 11 MCHs and their matched TSs,
the role of SAA1 in NPC was investigated. SAA1 might be a possible candidate TSG
134
in NPC. The differential gene expression levels identified by microarray analysis of
SAA1 were validated by semi-quantitative RT-PCR. SAA1 was found to be
down-regulated in all four TSs compared with their corresponding MCHs, suggesting
that the inactivation of SAA1 may be associated with the alteration of tumorigenicity
in MCHs. On the other hand, semi-quantitative PCR studies showed that SAA1 was
reduced in most NPC cell lines compared with the normal immortalized epithelial cell
line and the chromosome 11 donor cell line. The current Q-PCR studies of the
normal/NPC tissue pairs also showed that SAA1 was frequently down-regulated in the
NPC biopsy specimens. Down-regulation of SAA1 was also detected in another
aerodigestive tract cancer, ESCC. Barely detectable and low SAA1 expression levels
were detected in the ESCC cell lines, TTn and 81T, respectively (Figure 24B), these
low levels of gene expression sometimes are undetectable under the same PCR
conditions (Figure 21A). It is likely that the aberrant SAA1 expression might not be
specific to NPC. These results further indicate that the SAA1 down-regulation may
contribute to the tumorigenicity in NPC.
4.2 Mechanism to silence SAA1 expression
From the results of the MSP studies, SAA1 promoter methylation was observed
in most NPC cell lines. Methylated alleles at the nucleotide positions -163 and -61 of
135
the SAA1 promoter were frequently observed in NPC cells. Although an unmethylated
allele (CpG sites at positions -163 and -61) was present in HK1, C666, and CNE2
cells by MSP, undetectable expression of SAA1 was observed in these cell lines. Their
loss of SAA1 gene expression can be explained by the BGS results. The current BGS
results indicate that CpG sites at positions -297 and -289 were heavily methylated in
most of the NPC cell lines which include those three cell lines. Although very few
CpG sites are found in the SAA1 promoter, methylation of these two CpG sites may
represent important regulation of SAA1 transcription; the CpG sites at positions -297
and -289 actually overlap with the putative YY-1 and NFκB transcription factor
binding sequences (Kumon et al 2002, Thorn and Whitehead 2002). Taken together
with the demethylation analysis, it is likely that promoter hypermathylation is an
important gene silencing mechanism in the NPC cell lines.
4.3 Genotyping of SAA1 in NPC patients versus healthy people
The SAA1 polymorphisms have been reported as risk factors in certain
inflammatory diseases. SAA1.3 is associated with increased risk and SAA1.1 with
decreased risk of AA-amyloidosis in Japanese rheumatoid arthritis patients (Baba et al
1995, Moriguchi et al 1999). However, SAA1.1 polymorphism is a risk factor for
developing AA-amyloidosis in Caucasian populations (Booth et al 1998). We studied
136
whether the SAA1 polymorphism is associated with NPC development or not. Direct
sequencing of NPC tissue samples from NPC patients and blood samples from healthy
Chinese individuals in Hong Kong allows the study of isoform distribution. SAA1.1,
SAA1.3, and SAA1.5 were identified in all the NPC patients and healthy people with
Chinese ethnic background. In Caucasians the SAA1.3 allele occurs only at a
frequency of 5.3% (Booth et al 1998), whereas SAA1.1 and 1.3 alleles occur in similar
frequencies in Japanese (Yamada, Okuda et al. 2003). Thus, it is clear that ethnic
differences occur. The allelic frequencies of the three SAA1 isoforms do not show a
significant difference between NPC patients and healthy groups. Interestingly, the
frequency of SAA1.5/1.5 genotype in NPC patients was 65% higher than healthy
group; this suggests that SAA1.5/1.5 is associated with higher risk of NPC
development. However, this difference is not statistically significant (p-value =
0.0507). The p-value of this disproportionate frequency of SAA1.5/1.5 genotypes
between the disease and healthy groups is very close to the significant difference level
(when the significance level is set to 0.05). This indicates that Hong Kong people with
the SAA1.5/1.5 homozygous genotype may be associated with an increased risk for
NPC. To verify the frequency distribution of this SAA1 isoforms, the sample size of
NPC patients will be increased in future studies.
137
4.4 The anti-angiogenic properties of SAA1 proteins
Angiogenesis is essential for successful tumor development (Hanahan and
Weinberg 2011). The regulation of angiogenesis is a balance between the expression
of pro-angiogenic and anti-angiogenic factors (Baeriswyl and Christofori 2009).
Conditioned media of the SAA1 stable transfectants of HONE1 and C666 cells
reduced the HUVEC tube formation abilities in vitro, suggesting the restoration of the
secretary SAA1 in the NPC cell lines can inhibit angiogenesis. However, it is difficult
to precisely control the levels of SAA1 protein secreted by the stable transfectants in
the conditioned media, as reflected by the Western blot analysis of SAA1 in the
conditioned media. Apparently, the stable clones expressing SAA1.1 seem to be the
most anti-angiogenic, while the SAA1.5 expressing cells were the least. The use of
recombinant SAA1 proteins is able to accurately compare the inhibitory effects of
angiogenesis among the three SAA1 isoforms. All three SAA1 recombinant proteins
were able to inhibit the HUVEC and HMEC tube formation abilities, which is
consistent with the results of using the conditioned media of the stable transfectants.
Among the three SAA1 isoforms, SAA1.3 showed the greatest inhibitory effects,
while SAA1.5 showed the least inhibitory effects on the tube formation of HUVEC
and HMEC. The results of tube formation assays indicate that SAA1 variant proteins
could differentially inhibit the NPC cells-induced angiogenesis.
138
The real-time cell analysis results clearly show that the treatment with all three
recombinant SAA1 proteins significantly reduced the viability of both HUVEC and
HMEC. SAA1.3 showed the strongest inhibitory effects on the HMEC cell viability.
None of the three SAA1 variant proteins resulted in any significant effect on the cell
viability of the normal epithelial cell line, NP460. These results suggest that the
inhibitory effects of the SAA1 proteins on cell viability are cell type-specific. These
results suggest that the inhibition of the endothelial cell viability may contribute to the
reduction in their tube formation abilities after the SAA1 treatment.
Results of our cell adhesion assay show that treatment of SAA1 recombinant
proteins can reduce the HUVEC and HMEC adhesion ability in HONE1 cells.
SAA1.3 was also the most effective in reducing the adhesion ability of both cell lines,
whereas SAA1.5 was the least. These results are in agreement with the endothelial
cell tube formation and real-time cell viability assays. In addition, I demonstrated that
SAA1.3 could induce apoptosis in HUVECs. In addition, we also attempted to study
the cleavage of caspase 3 in response to the SAA1.3 treatment in HUVECs, as
caspase 3 is one of the major effector molecules in apoptosis (Pradelli et al 2010). The
Western blot results clearly show that active caspase 3 was detected in the
SAA1.3-treated HUVECs; it is likely that SAA1.3 induced HUVEC apoptosis via a
caspase 3-dependent pathway. Taken together, the angiogensis assays results clearly
139
show that high concentrations of SAA1 proteins (0.2-4 mg/ml) are inhibitory on the
vascular endothelial cells and this is probably due to the induction of apoptosis. In
contrast, it has been reported that low concentrations of SAA protein significantly
increased angiogenesis processes including endothelial cell tube formation and
HUVEC migration in vitro (Mullan et al 2006). Their conclusions were based on the
use of a commercially available recombinant SAA1 protein. However, this human
SAA1 protein differs to the actual SAA1 protein by substitution of two amino acid
residues. Whether this discrepancy is due to the change of amino acid sequence or is
due the low SAA1 concentrations they used, further investigation are required to
answer this question.
4.5 The direct anti-tumor effects of the recombinant SAA1 variant proteins on
NPC cells
Direct treatment with the three recombinant SAA1 proteins reduced the cell
viability of HONE1 cells for the first few hours; the HONE1 cells numbers increased
thereafter. This is in contrast to the inhibitory effects on the endothelial cells. It is
likely that these temporary inhibitory effects are cancer cell-specific, since NP460
with the same treatments did not show any significantly effects as mentioned in the
previous section. All three SAA1 variant proteins could induce DNA fragmentation
140
and caspase 3 activation after the addition of SAA1.3. The SAA1 protein induced
apoptosis in the NPC cell line. Furthermore, the anti-tumor effects of SAA1.1, 1.3,
and 1.5 on HONE cells are similar, which is in contrast to their differential
anti-angiogenic effects on the vascular endothelial cells.
4.6 General discussion
From the results of the SAA1 genotyping study, the SAA1.5/1.5 genotype is
associated with higher risk of NPC development. Although SAA1 has been reported
to interact with various cell surface receptors, which might contribute to enhancement
of cell proliferation and angiogenesis, those receptors include FPRL-1, TLR-4, TLR-2,
SR-B1, TAINS, CD55, and RAGE (Malle et al 2009). However, the present study
showed that the SAA1 proteins are inhibitory in tumor cell growth and angiogenesis.
Based on the results of the various functional assays of SAA1.1, 1.3, and 1.5, we
clearly show that SAA1.1 and SAA1.3 consistently elicit the high inhibitory effects,
while minimal effects are observed for SAA1.5; the difference in activities between
the SAA1.1/1.3 and SAA1.5 can be as high as 5-fold. It is likely that an individual,
who carries the homogenous SAA1.5/1.5 genotype, possesses the least protective
effects against tumor formation. That may explain the higher occurrence of
SAA1.5/1.5 in the NPC patients, when compared with the healthy people. People with
141
this SAA1 genotype are more susceptible to cancer development including NPC.
Among the three SAA1 isoforms, all of them contain the same numbers of amino
acids and complete RGD- and YIGSR- like motifs. The major differences among
these isoforms come from the amino acids located at the positions 70 and 75. It was
found that SAA1.3 was the most prominent SAA1 variant with respect to its in vitro
anti-angiogenic and anti-cancer activities. Its amino acids at positions 70 and 75 are
both alanine residues. Based on the 3D model of the secondary structure of the
SAA1.3 peptide predicted by PyMol, we found that the side chain of alanine 70 and
75 are facing opposite to each other (Figure 41). When alanine 75 is replaced with a
more hydrophobic valine residue in SAA1.5, it will contribute to increase of
hydrophobic force on this side-chain facing side and probably would result in the
reduced activities of this SAA1 variant. In the case of SAA1.1, the side chain of
valine 70 is facing the hydrophilic side of the helix, which will cause an increase in
hydrophobicity of this side. The increase in hydrophobic force on the hydrophobic
side of valine 75 in SAA1.5 is more prominent than the hydrophilic side of valine 70
in SAA1.1, with respect to their biological activities. The change of a single amino
acid residue may not contribute to a major conformational change of the protein
structure of SAA1 itself, but may affect to its binding partner(s) on the affected cells.
In addition, the two polymorphic amino acid residues are not located within the RGD142
Figure 41: The 3D model of the secondary structure of the human SAA1.1, 1.3, and
1.5 peptides predicted by PyMol (http://robetta.bakerlab.org/). The region with the
polymorphic amino acids at positions 70 and 75 is illustrated for each isoform.
143
and YIGSR-like motifs (Figure 12B), the change in amino acid residues 70 and/or 75
may affect the exposure of these implicit functional motifs, and contributes to the
discrepancies in the biological activities among the three SAA1 isoforms. We also
tried to predict the tertiary structures of the three SAA1 variant proteins, but the
results are not consistent as the c-scores of all the predicted models with I-TASSER
(http://zhanglab.ccmb.med.umich.edu/I-TASSER/)
or
Robetta
(http://robetta.bakerlab.org/) (data not shown) were too low. Nuclear magnetic
resonance (NMR) and X-ray crystallography are the methods which can be used to
accurately determine the 3D structures of the SAA1 variant proteins.
Early diagnosis and surgery of NPC are difficult, as the nasopharynx is located
deep inside the skull. Most NPC cases are diagnosed at the late stage due to the
non-specific and late symptoms including headache, nasal, weight loss and aural
dysfunction (Wei and Sham 2005). It is important to identify diagnostic and
prognostic markers for early detection and treatment of NPC. Previous studies
indicated that EBV infection is associated with the development of NPC. There is
convincing evidence suggesting that EBV infection is strongly associated with the
development of NPC (Chan and Lo 2002, Cho 2007, Feng et al 2011, Ng et al 2006).
Indeed the EBV genome is present in almost all NPC tissues, which makes it an ideal
tumor marker for NPC. Quantitative analyses of EBV antibodies and plasma EBV
144
DNA have been demonstrated to be clinically useful for early detection, monitoring,
and prognosis of NPC (Cho 2007). Other potential NPC biomarkers include matrix
metalloproteinase-9 (MMP-9) (He et al 2011), fibulin-3 (Hwang et al 2010), and
serum CCL2 (chemokine C-C motif ligand 2 or monocyte chemotactic protein-1
(MCP-1)) and serum tumor necrosis factor alpha (TNF-alpha) (Lu et al 2011) are new
potential biomarkers identified in recent studies for NPC prognosis. Previous studies
of protein profiling have identified the serum SAA level was significantly elevated at
relapse compared to patients in remission. However, in early NPC SAA levels were
not substantially elevated. Serum SAA is elevated in NPC patients, particularly in
association with lymph node metastases (Cho et al 2004, Liao et al 2008). The
elevated serum SAA level might not be directly associated with NPC tumor lesions,
but could be a secondary product produced by the hepatocytes. This was suggested by
the studies showing that the protein was not detected in a tumor biopsy section,
whereas substantial staining was observed in a control liver resection from a
non-cancer patient (Cho et al 2004). Besides NPC, SAA has been shown to be
elevated in various types of cancer, including breast cancer (Chan et al 2007), lung
cancer (Liu et al 2007, Liu et al 2011, Sreseli et al 2010, Sung et al 2011), gastric
cancer (Chan et al 2007), pancreatic cancer (Bunger et al 2011), ovarian cancer
(Urieli-Shoval et al 2010), and renal cancer (Ramankulov et al 2008, Vermaat et al
145
2010). As mentioned above, numerous studies focused only on the quantitative
changes of serum SAA levels in different cancers. To my knowledge, this is the first
report to describe the preferential association of SAA1 variants with cancers. For future
study, our research team wants to determine whether the elevated SAA levels in NPC
are associated with any of the SAA1 genotypes and allele distributions of those
patients, and clinicopathological parameters and the protein expression levels will be
analyzed to identify any statistically significant associations. We aim to investigate
both quantitative changes and qualitative differences of SAA1 in NPC, that will
probably provide new insight as to how SAA1 may serve as an useful prognostic
biomarker for tumor progression and metastasis.
146
Chapter 5: Conclusions
This project aims to study the functional role of the candidate TSG, SAA1, which
was identified by a monochromosome transfer approach in NPC. SAA1
down-regulation was observed in NPC cell lines and tumor tissues. Promoter
hypermethylation is one of the major mechanisms contributing to the SAA1 silencing.
Results of SAA1 genotyping in the Hong Kong NPC patients versus the normal
healthy individuals showed that the SAA1.5/1.5 genotype appears to be associated
with a higher risk of NPC development (p-value = 0.0507). The sample size of the
NPC patients in the genotyping study is small; more samples need to be collected and
genotyped in the future. Both endogenous and exogenous SAA1 were able to suppress
angiogenesis in vitro. The anti-angiogenic effects among the three SAA1 isoforms
identified in the current NPC and healthy individuals were fairly different. Among the
three isoforms, the recombinant SAA1.3 protein consistently showed the greatest
inhibitory effects, while these activities of SAA1.5 were the weakest. These findings
support the SAA1.5 genotype results, which suggest that Hong Kong Chinese with
this SAA1 isoform are relatively more susceptible to NPC development. In conclusion,
the current functional evidence indicates that a single variation of an amino acid
residue can significantly reduce the activities of SAA1. The secreted SAA1.5 protein
147
might contribute to weaker protective effects against NPC development. For future
studies, in vivo tumorigenicity assays of SAA1 transfected clones will be performed
to study the inhibition of tumor formation of endogenous SAA1 expression. The
matrigel plug assay will also be performed to study the in vivo inhibitory effect of
angiogenesis of endogenous and exogenous SAA1 proteins. Further investigation of
each SAA1 variant protein structure and the identification its binding partners will
definitely provide a new insight into the tumor suppression anti-angiogenesis
mechanism(s) of SAA1 in NPC.
148
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Appendix
Table A1) Genotyping of SAA1 isoforms in 80 NPC patients.
Sample
1
SAA1.1
SAA1.3
√
√
SAA1.5
Gender
Stage
Age
NA*
III(T1N2)
NA*
2
√
M
III(T1N1)
73
3
√
M
II(T2N0)
51
√
M
I(T1N0)
55
NA*
III(T2N2)
NA*
M
IVb(T1N3)
78
F
II(T2N0)
48
M
IVa(T4N2)
83
M
II(T2bN1)
54
F
III(T3N1)
30
5
√
6
√
√
7
8
√
10
√
20
√
√
14
√
√
√
√
22
√
√
M
II(T2bN1)
36
25
√
√
F
III(T3N1)
46
F
II(T2N2)
33
M
III(T2bN2)
51
M
T2bN2M0
64
M
T3N2M0
59
√
M
T1N0M0
47
√
M
T2bN0M0
56
M
T3N3M0
70
27
√
38
√
42
√
43
√
√
√
√
√
44
45
√
48
√
50
√
√
F
T2bN1M0
55
52
√
√
F
T2bN2M0
56
53
√
M
T2bN1M0
66
M
III (T1N2M0)
43
54
√
√
55
√
√
M
IV (T4N1M0)
83
56
√
√
M
III (T2bN2M0)
49
57
√
M
III (T3N2M0)
43
58
√
M
II (T1N1M0)
58
59
√
√
M
III (T3N2M0)
46
60
√
√
M
I (T1N0M0)
56
61
√
√
F
II (T2aN1M0)
45
M
IVa (T4N2M0)
NA*
M
III (T3N0M0)
50
62
√
63
√
√
171
√
64
F
III (T2bN2M0)
50
M
IVa (T4N2M0
68
√
M
III (T1N2M0)
73
√
M
III (T3N2M0)
50
√
F
III (T3N1M0)
49
√
NA*
IVa (T4N1M0)
NA*
√
NA*
IVa(T4N2M0)
NA*
71
√
M
I (T1N0M0)
60
72
√
F
II (T1N1M0)
52
73
√
F
III (T2bN2M0)
68
65
√
√
66
67
√
√
68
69
70
√
74
√
F
I (T1N0M0)
53
75
√
M
I (T1N0M0)
53
76
√
M
III(T1N2M0)
42
M
I (T1N0M0)
62
√
77
78
√
M
IVa (T4N0M0)
59
79
√
M
IVa (T4N1M0)
54
80
√
F
T3N0M0 (III)
76
√
F
T2aN2 (III)
53
F
T1N3a (Ivb)
74
M
T2N1 stage II
50
M
T3N1 stage III
63
√
M
T1N2 stage III
36
√
M
T3N2 stage III
61
F
T3N2 stage III
34
F
T3N2 stage III
52
M
T2N3bM1 stage Ivc
80
M
T2N2 stage III
64
81
√
82
√
83
√
√
84
85
86
√
87
√
√
88
√
89
√
90
√
91
√
√
M
T2N0 stage II
46
92
√
√
M
T1N0 stage I
75
√
93
√
√
F
T2N1 stage II
44
94
√
√
M
T1N0 stage I
76
F
T1N2 stage III
56
√
F
T1N1 stage II
72
√
95
96
97
√
√
M
T4N1 stage Iva
61
98
√
√
M
T1N0 stage I
49
99
√
M
T3N3 stage Ivb
55
F
T1N0 stage I
49
100
√
√
172
101
√
√
M
T3N1 stage III
71
102
√
√
F
T2N1 stage II
82
√
M
T4N1 stage Iva
80
√
M
T1N3M1 stage Ivc
60
√
M
T2N1 stage II
47
√
F
T1N3 stage Ivb
42
103
104
√
105
√
106
107
√
√
F
T2N1 stage II
39
108
√
√
M
T1N3 stage Ivb
49
√
F
T2bN0
53
√
M
T1N3b
35
√
M
T1N3b
35
110
111
112
√
√
*the information of the patient was not available
173
Appendix
Buffer List
Freezing buffer
- 10% DMSO
- 50% Fetal bovine serum
- 40% non-selective growth medium
Homogenizing buffer
- 50 mM Tris
- 100 mM NaCl
- 10 mM sodium EDTA
TE buffer
- 10 mM Tris
- 1 mM EDTA
6X DNA gel loading buffer
- 0.25% (w/v) bromophenol blue
- 0.25% (w/v) xylene cyanol FF
- 40% (v/v) glycerol
Denaturing solution
- 1.5 M NaCl
- 0.5 M NaOH
PBS, pH 7.4
- 2.67 mM potassium chloride (KCl)
- 1.47 mM potassium phosphate monobasic (KH2PO4)
- 137.93 mM sodium chloride (NaCl)
- 8.10 mM sodium phosphate dibasic (Na2HPO4) anhydrous
Protein lysis buffer
- 50 mM Tris-Cl (pH7.5)
174
- 100 mM NaCl
- 1% Triton X-100
- 0.5% sodium deoxycholate
- 0.1% SDS
2X Protein loading buffer
- 100 mM Tris-Cl (pH6.8)
- 200 mM β-mercaptoethanol
- 4% (w/v) SDS
- 0.2% (w/v) bromophenol blue
- 20% (v/v) glycerol
1X Protein running buffer
- 25 mM Tris
- 250 mM glycine (electrophoresis grade) (pH8.3)
- 0.1 % (w/v) SDS
1X Transfer buffer
-24mM Tris
- 192 mM glycine (electrophoresis grade)
- 20% (v/v) Methanol
175