A Functional Wild-Type p53 Gene Is Expressed in Human Acute... Leukemia Cell Lines

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1998 92: 2977-2979
A Functional Wild-Type p53 Gene Is Expressed in Human Acute Myeloid
Leukemia Cell Lines
Trenna Sutcliffe, Loning Fu, Jacinth Abraham, Homayoun Vaziri and Samuel Benchimol
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1. Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S: Observation of a
single-beam gradient force optical trap for dielectric particles. Optic
Lett 11:288, 1986
2. Ashkin A, Dziedzic JM: Optical trapping and manipulation of
viruses and bacteria. Science 235:1517, 1987
3. Bronkhorst PJ, Streekstra GJ, Grimbergen J, Nijhof EJ, Sixma JJ,
Brakenhoff GJ: A new method to study shape recovery of red blood cells
using multiple optical trapping. Biophys J 69:1666, 1995
4. Huruta RR, Barjas-Castro ML, Saad STO, Costa FF, Cezar CL: A
new method to study mechanical properties of red blood cells using
optical tweezers. Blood 90:6a, 1997 (abstr, suppl 1)
5. Sutera SP, Gardner RA, Boylan CW, Carroll GL, Chang KC,
Marvel JS, Kilo C, Gonen B, Williamson JR: Age related changes in
deformability of human erythrocytes. Blood 65:275, 1985
6. Beutler E, West C: The storage of hard-packed red blood cells in
citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1). Blood
54:280, 1970
7. Card RT, Mohandas N, Perkins HA, Shohet SB: Deformability of
stored red cells. Relationship to degree of packing. Transfusion 22:96,
8. Wolf LC: The membrane and lesions of storage in preserved red
cells. Transfusion 25:185, 1985
A Functional Wild-Type p53 Gene Is Expressed in Human Acute Myeloid Leukemia Cell Lines
To the Editor:
p53 mutations have been detected in about 10% of all acute myeloid
leukemia (AML) patients, mostly in patients with 17p monosomy.1-3
The scarcity of p53 mutations in AML could mean that, in the vast
majority of AML patients, loss of p53 protein function is not required
for the development of this disease. Alternatively, it is possible that
inactivation of the p53 growth regulatory pathway is important and that
this can occur either through disruption of downstream effector
molecules or through epigenetic mechanisms that regulate p53 protein
function. It has been suggested, for example, that inactivation of
wild-type p53 protein in AML occurs through a mechanism involving
conformational change of the protein4,5 or through binding to MDM2
protein.6-8 We have examined the functional status of the wild-type p53
protein expressed in cell lines derived from AML blasts on the basis of
site-specific DNA binding activity, transactivation of p53-responsive
genes, and ability to promote cell cycle arrest in G1 in response to
g-irradiation.9 The first two properties of p53 protein are strongly
associated with its tumor suppressor function.10,11
Nucleotide sequence analysis of the entire p53 coding region in four
p53-expressing AML cell lines (OCI/AML-2, -3, -4, and -5) 12 showed
wild-type sequence. The site-specific DNA binding activity of p53
protein expressed in OCI/AML-3 and OCI/AML-5 cells was examined
using an electrophoretic mobility shift assay (EMSA). Nuclear protein
extracts were prepared from g-irradiated or untreated cells and mixed
with a 32P-labeled double-stranded oligonucleotide containing a p53
binding consensus sequence, p53CON.13 DNA damage increases the
intracellular concentration of p53 protein and is also believed to activate
the latent, sequence-specific DNA binding activity of p53. Whereas
little, if any, DNA binding activity was detected in the nonirradiated
extracts, the formation of a p53:DNA complex was evident when
extracts were prepared from irradiated cells (Fig 1). Inclusion of the
p53-specific monoclonal antibody PAb421 in the binding reaction
resulted in a supershifted p53:DNA complex and served to confirm the
presence of p53 protein in the protein:DNA complex. DNA binding was
not observed when an extract from the p53-negative cell line Lan1 was
used in the EMSA.
Fig 1. DNA binding activity of p53 protein in AML cell lines. Nuclear extracts prepared from untreated or g-irradiated OCI/AML-5 (A) and
OCI/AML-3 (B) cells were incubated with a 32P-labeled double-stranded oligonucleotide containing the p53 consensus sequence (p53CON) with
(1) or without (2) the p53-specific monoclonal antibody PAb421 and analyzed by EMSA. Lan1 cells, which lack p53 protein, were used as a
negative control. The OCI/AML-5 and Lan1 extracts were prepared 3 hours after g-irradiation with a dose of 6 Gy. The OCI/AML-3 extracts were
prepared at the times indicated after g-irradiation with a dose of 2 Gy. The arrow labeled B points to the p53:DNA complex, and the arrow labeled
A points to the supershifted antibody:p53:DNA complex.
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Activation of p21WAF1 gene transcription after g-irradiation depends
on wild-type p53 protein, and the p21WAF1 gene has been proposed to be
a critical downstream effector in the p53-specific pathway of growth
control in mammalian cells.14-17 Northern blot analysis (Fig 2) indicated
that the basal level of p21WAF1 mRNA was ninefold higher in
OCI/AML-5 than in the mutant p53-expressing human erythroleukemia
cell line OCIM2. Furthermore, 3.5 hours after irradiation with 6 Gy,
p21WAF1 mRNA levels increased ninefold in OCI/AML-5 and about
threefold in OCIM2 cells. Irradiated OCI/AML-5 cells contained about
30-fold more p21WAF1 mRNA than did irradiated OCIM2 cells. No
further increase in p21WAF1 mRNA levels was noted at later times after
irradiation. p21WAF1 induction was also observed in irradiated OCI/
AML-3 and OCI/AML-4 cell lines.18 The mRNA levels for GADD45
and MDM2, two other genes known to be transcriptionally regulated by
p53 in response to DNA damage, also increased after g-irradiation of
AML cell lines (data not shown).
OCI/AML-3, OCI/AML-5, and OCIM2 cells were irradiated with a
dose of 6 Gy and cell proliferation was assessed 16 hours later by
propidium iodide staining and flow cytometry. Both OCI/AML-3 and
OCI/AML-5 cells were blocked in the G1 and G2 phases of the cell
cycle with little, if any, cells present in S phase. In contrast, the mutant
p53-expressing OCIM2 cells accumulated in G2 and showed no
evidence of a block in G1 (Fig 3). The failure of OCIM2 cells to arrest in
G1 after g-irradiation suggests that the G1 cell cycle block observed in
irradiated OCI/AML-3 and OCI/AML-5 cells is likely to be dependent
on functional p53 protein. Irradiation-induced G1 arrest was confirmed
by dual-parameter flow cytometry after pulse labeling cells with BrdU
and staining for DNA content with propidium iodide and for BrdU
incorporation with a fluorescein isothiocyanate (FITC)-conjugated
antibody for BrdU. OCI/AML-3 and OCI/AML-5 showed a ninefold
and sixfold increase in the G1:S ratio 16 hours after g-irradiation (6
Gy), respectively. An increase in the G1:S ratio provides a good
indicator of G1 delay.
Our results indicate that p53 function (DNA binding, transactivation, and G1 checkpoint) is not lost during the development
of AML or in the establishment of these AML cell lines. Functional
p53 protein has also been demonstrated in human neuroblastoma,19
non-Hodgkin’s lymphoma,20 and even in certain HPV-positive cancer
Fig 3. Cell cycle changes in AML cells after exposure to g-irradiation (6 Gy). The DNA content was determined by staining the cells
with propidium iodide and the resulting profiles resulting from
propidium iodide fluorescence are shown. For the irradiated cells, the
cell cycle analyses were performed 16 hours after irradiation. OCIM2
cells were used as a control.
cell lines21 that contain wild-type p53 alleles. Hence, loss of p53
function or inactivation of the p53-dependent growth arrest pathway is
not required for the development of certain malignancies, including
Fig 2. Expression of p21WAF1
mRNA in g-irradiated OCI/AML-5
cells. Samples of total RNA (20
mg) prepared from cells at different times after exposure to 6 Gy
of g-irradiation were fractioned
on an agarose-formaldehyde gel,
transferred to a nylon membrane, and hybridized sequentially with 32P-labeled probes for
human p21WAF1 cDNA (A) and 18S
ribosomal RNA (B). OCIM2 cells,
which express mutant p53 protein, were used as a control. Signal intensities were quantitated
on a phosphorimager. The ratio
of the p21WAF1 RNA signal to the
18S ribosomal RNA signal in the
OCIM2 sample (0 hours) was arbitrarily set to 1.0 and the normalized values of p21WAF1 mRNA are
shown at the bottom of (A).
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Supported by grants from the Medical Research Council of Canada
and the National Cancer Institute of Canada.
Trenna Sutcliffe
Loning Fu
Jacinth Abraham
Homayoun Vaziri
Samuel Benchimol
Division of Cellular/Molecular Biology
Ontario Cancer Institute
Princess Margaret Hospital
Department of Medical Biophysics
University of Toronto
Toronto, Ontario, Canada
1. Lai JL, Preudhomme C, Zandecki M, Flactif M, Vanrumbeke M,
Lepelley P, Wattel E, Fenaux P: Myelodysplastic syndromes and acute
myeloid leukemia with 17p deletion. An entity characterized by specific
dysgranulopoiesis and a high incidence of p53 mutation. Leukemia
9:370, 1995
2. Fenaux P, Jonveaux P, Quiquandon I, Lai JL, Pignon JM, LoucheuxLefebvre MH, Bauters F, Berger R, Kerckaert JP: p53 gene mutations in
acute myeloid leukemia with 17p monosomy. Blood 78:1652, 1991
3. Soenen V, Preudhomme C, Rournier C, Daudignon A, Lai JL,
Fenaux P: 17p deletion in acute myeloid leukemia with myelodysplastic
syndrome. Analysis of breakpoints and deleted segments by fluorescence in situ. Blood 91:1008, 1998
4. Zhu YM, Bradbury D, Russell N: Expression of different conformations of p53 in the blast cells of acute myeloblastic leukaemia is
related to in vitro growth characteristics. Br J Cancer 68:851, 1993
5. Zhang W, Hu G, Estey E, Hester J, Deisseroth A: Altered
conformation of the p53 protein in myeloid leukemia cells and
mitogen-stimulated normal blood cells. Oncogene 7:1645, 1992
6. Bueso-Ramos CE, Yang Y, deLeon E, McCowan P, Stass SA,
Albitar M: The human MDM-2 oncogene is overexpressed in leukemias. Blood 82:2617, 1993
7. Watanabe T, Ichikawa A, Saito H, Hotta T: Overexpression of the
MDM2 oncogene in leukemia and lymphoma. Leuk Lymphoma 21:391,
8. Seliger B, Papadileris S, Vogel D, Hess G, Brendel C, Storkel S, Ortel
J, Kolbe K, Huber C, Huhn D, Neubauer A: Analysis of the p53 and MDM-2
gene in acute myeloid leukemia. Eur J Haematol 57:230, 1996
9. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB: Wild-type p53
is a cell cycle checkpoint determinant following irradiation. Proc Natl
Acad Sci USA 89:7491, 1992
10. Pietenpol JA, Tokino T, Thiagalingam S, El-Deiry WS, Kinzler
KW, Vogelstein B: Sequence-specific transcriptional activation is
essential for growth suppression by p53. Proc Natl Acad Sci USA
91:1998, 1994
11. Crook T, Marston NJ, Sara EA, Vousden KH: Transcriptional
activation by p53 correlates with suppression of growth but not transformation. Cell 79:817, 1994
12. Yang GS, Minden MD, McCulloch EA: Influence of schedule on
regulated sensitivity of AML blasts to cytosine arabinoside. Leukemia
7:1012, 1993
13. Funk WD, Pak DT, Karas RH, Wright WE, Shay JW: A
transcriptionally active DNA-binding site for human p53 protein
complexes. Mol Cell Biol 12:2866, 1992
14. Di Leonardo A, Linke SP, Clarkin K, Wahl GM: DNA damage
triggers a prolonged p53-dependent G1 arrest and long-term induction
of Cip1 in normal human fibroblasts. Genes Dev 8:2540, 1994
15. El-Deiry WS, Harper JW, O’Connor PM, Velculescu VE,
Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill DE, Wang Y,
Wiman KG, Mercer WE, Kastan MB, Kohn KW, Elledge SJ, Kinzler
KW, Vogelstein B: WAF1/CIP1 is induced in p53-mediated G1 arrest
and apoptosis. Cancer Res 54:1169, 1994
16. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper
JW, Elledge SJ, Reed SI: p53-dependent inhibition of cyclin-dependent
kinase activities in human fibroblasts during radiation-induced G1
arrest. Cell 76:1013, 1994
17. Slebos RJ, Lee MH, Plunkett BS, Kessis TD, Williams BO, Jacks
T, Hedrick L, Kastan MB, Cho KR: p53-dependent G1 arrest involves
pRB-related proteins and is disrupted by the human papillomavirus 16
E7 oncoprotein. Proc Natl Acad Sci USA 91:5320, 1994
18. Fu L, Benchimol S: Participation of the human p53 38UTR in
translational repression and activation following g-irradiation. EMBO J
16:4117, 1997
19. Goldman SC, Chen CY, Lansing TJ, Gilmer TM, Kastan MB:
The p53 signal transduction pathway is intact in human neuroblastoma
despite cytoplasmic localization. Am J Pathol 148:1381, 1996
20. Maestro R, Gloghini A, Doglioni C, Piccinin S, Vukosavljevic T,
Gasparotto D, Carbone A, Boiocchi M: Human non-Hodgkin’s lymphomas
overexpress a wild-type form of p53 which is a functional transcription
activator of the cyclin-dependent kinase inhibitor p21. Blood 89:2523, 1997
21. Butz K, Shahabeddin L, Geisen C, Spitkovsky D, Ullmann A,
Hoppe-Seyler F: Functional p53 protein in human papillomaviruspositive cancer cells. Oncogene 10:927, 1995
Juvenile Genetic Hemochromatosis Is Clinically and Genetically Distinct
From the Classical HLA-Related Disorder
To the Editor:
Genetic hemochromatosis (GH) is a common HLA-linked recessive
disorder characterized by progressive parenchymal iron loading and the
appearance of clinical manifestations in the fifth decade of life, predominantly in males. HFE has been recently identified as the candidate gene, with
most patients being homozygous for a Cys-282 = Tyr (C282Y) mutation
and others being compound heterozygotes for C282Y and a second mutation,
His-63 = Asp (H63D).1 Homozygosity for C282Y is found in more than
90% of North European patients,2 but in only 64% of severely iron-loaded
Italian individuals.3 This finding may suggest that various genetic iron
overload syndromes exist in addition to the HFE-related one.
Fifteen years ago, we described cases of juvenile GH suggesting that
this was a distinct disease entity.4 In the juvenile condition, males and
females appear to be equally affected. Patients present with hypogonadotropic hypogonadism and, unless proper treatment is started, die early
because of cardiac dysfunction. We now provide further evidence that
the juvenile condition is clinically and genetically distinct from the
classical adult disorder.
The pedigrees of our two Italian families with juvenile GH are shown
in Fig 1. The clinical features of family 1 were reported in 1983,4
whereas family 2 has never been described. Of the four affected individuals,
three presented with hypogonadotropic hypogonadism at 14 to 21 years of
age. The affected male of family 2 presented with cardiac failure at 20 years
of age and died at 21 years of age of congestive cardiomyopathy.