1. No category

From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
bcl-2 Proto-oncogene Expression in Normal and Neoplastic
Human Myeloid Cells
By Domenico Delia, Antonella Aiello, Davide Soligo, Enrico Fontanella, Cecilia Melani, Francesco Pezzella,
Marco A. Pierotti, and Giuseppe Della Porta
The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2
proto-oncogene in normal and neoplastic myeloid cells.
Using a recently developed monoclonal antibody (MoAb) t o
the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated.
By two-color flow cytometric analysis, bcl-2 was evidenced
in CD33' and CD33+/CD34+myeloid cells as well as in the
more primitive CD33-/CD34+ population. The leukemic cell
lines HL-60, KGI, GM-1, and K562 were bcl-2 positive together with ll of 14 acute myeloid leukemias (AML) and
three of three chronic myeloid leukemias (CML) in blast
crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic
compartment was found in refractory anemia with excess of
blasts (RAEB) and in transformation (RAEB-t). Western blots
of myeloid leukemias and control lymphocytes extracts
evidenced an anti-bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KGI cell lines,
both positive for the bcl-2 protein, exhibited the appropriate
size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that
the bcl-2 gene is operative in myeloid cells and that the
anti-bcl-2 MoAb identifies its product and not a crossreactive epitope. Induction of HL-60 differentiation toward
the monocytic and granulocytic pathways was accompanied
by a marked decrease in bcl-2 mRNA and protein levels;
bivariate flow cytometric analysis showed that the fraction
becoming bcl-2 negative was in the G1 phase of the cell cycle.
These data establish that the bcl-2 proto-oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner,
0 1992by The American Society of Hematology.
T
on the expression of bcl-2 proto-oncogene in normal and
malignant myeloid cells, including a population of CD34'/
CD33- hematopoietic progenitor cells.
HE GENE bcl-2 is involved in the t(14;18) chromosomal translocation present in more than 70% follicular B-cell lymphomas,',' where the fusion of the 3' nontranslated region of bcl-2 with the joining (JH) segment of Ig
heavy chain gene results in a chimeric transcript encoding a
normal bcl-2 product.* The inappropriately elevated mRNA
levels and the lack of point mutations of the coding region3
support the view that the deregulation of the gene which
leads to quantitative differences in the levels of bcl-2
protein4 is the major factor of transformation.
Normal bcl-2 transcripts have been detected in activated
but not in resting T- and B-cell lymphocyte^^.^ and in a
variety of T-, B-, and pre-B lymphoblastoid cell lines
negative for the t( 14;18) chromosomal breakpoint.'.' Moreover, immunohistochemical analysis using recently produced monoclonal antibodies (MoAbs) specific for the
human bcl-2 protein7a8have shown that medullary but not
cortical thymocytes and mantle zone but not germinal
center B cells of normal lymph nodes are bcl-2 positive.
Resting blood T and B lymphocytes also express the
protein, despite the reported undetectable levels of mRNA.'
Together, these findings indicate that bcl-2 is actively
regulated during normal lymphopoiesis.
The bcl-2 gene encodes a major nonglycosylated protein
of 26 Kd lacking leader or kinase d0mains.4~~~'~
Subcellular
fractionation studies have recently established that bcl-2 is
an inner mitochondrial membrane protein," the first example of a proto-oncogene with such localization.
At present, while the biochemical function of bcl-2
protein is unknown and the reported in vitro GTP-binding
activity" has not been c~nfirmed,""~
its physiologic function
in blocking programmed cell death or apoptosis is well
established. For example, B cells from hyperplastic lymph
nodes of bcl-2-Ig transgenic mice possess in vitro a significantly longer survival than normal B cells'4; in addition,
deregulated bcl-2 prolongs the survival of growth-factordeprived hematopoietic cell
In this report we present biochemical and molecular data
Blood, Vol79, No 5 (March I), 1992: pp 1291-1298
MATERIALS AND METHODS
Cells, biopsies, and induction of differentiation. The leukemic
cell lines KG1 (myeloblastic),HL-60 (promyelocytic),GM-1 (monoblastic), K562 (erythromyeloid), Raji (B lymphoblastic), and the
colon carcinoma cell line HT-29, all mycoplasma free, were
maintained in RPMI-1640 (Bioproducts Inc, Walkersville, MD)
supplemented with 10% heat-inactivated fetal calf serum (Imine
Scientific, Santa Ana, CA), 2 mmol/L glutamine, 100 U/mL
penicillin, and 100 Fg/mL streptomycin (RPMI-C) in a 5% CO,
humidified incubator at 37°C.
Bone marrow aspirates and trephine biopsies from patients with
various myeloproliferative disorders were obtained for diagnosis;
the latter were fixed in either 10% buffered formalin or Bouin's
solution, decalcified overnight in nitric acid or in 10% Titriplex
(Merck, Darmstadt, Germany), and embedded in paraffin. Blood
samples containing elevated numbers of CD34-positive hematopoietic progenitor cells were obtained from nonhematologic cancer
patients undergoing autologous bone marrow transplantation and
receiving recombinant human granulocyte-macrophage colonystimulating factor (GM-CSF) after myeloablative chemotherapy.'8
Spleen lymphocyteswere obtained by mechanical disaggregation of
uninvolved biopsies of patients with nonhematologic tumors and
undergoing splenectomy for therapeutic purposes. Bone marrow,
From Istituto Nazionale per Lo Studio e La Cura Dei Tumori,
Milan; Istituto di Scienze Mediche, University of Milan, Italy; and John
Radcliffe Hospital, Headington, w o r d , UK
Submitted July IS, 1991; accepted October 22, 1991.
Supported by the Associazione Italiana Ricerca Cancro.
Address reprint requests to Domenico Delia, PhD, Istituto Nazionale Tumori, Via G Venezian I , 20133 Milano, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I992 by The American Society of Hematology.
0006-4971I92 17905-0022$3.0010
1291
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
bcl-2 IN MYELOID CELLS
Fig 2. Two-color flow cytometric analysis of bcl-2 protein in
CD33’ and CD34’ populations.
The analyses were performed on
paraformaldehyde-fixed/Tritontreated cells labeled with the
specified MoAbs and with isotype-specific, FITC-and PE-conjugated second antibodies. For
each fluorescence histogram (log
scale), the horizontaland vertical
markers (set up on negative controls) separate the reactive from
unreactive cells. Analysis on bone
marrow (A) and on blood cells of
an rhGM-CSF-treated patient (B)
are shown. The size distribution
(forward v 90”light scattering) of
the latter sample is depicted in
top left histogram together with
bitmaps 1 and 2, which encompass the small- and large-size
cells, respectively; in this specimen most of CD34’ cells were
found in bitmap 1.
1293
-4.
d.
a
a
€9
c3
0
0
10”
10’
lo2
bcl-2
90LS
CD34
lo3
CD33
blood, and spleen suspensions were enriched in mononuclear cells
by centrifugation(30 minutes at 4oog) on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Erythrocyte-depleted bone marrows were
prepared by treatment with an erythrocyte lysing solution (Coulter
Electronics,Hialeah, FL). Granulocytic and monocytic differentiation of HL-60 was achieved by culturing the cell line in the
presence of 1.2% dimethyl sulfoxide (DMSO) (Sigma Chemical
Co,St Louis, MO) and 10ng/mLof 12-0-tetradecanoylphorbol-13acetate (TPA) (Sigma), respectively. DMSO was added every 72
hours.
MoAbs, cell permeabilizaiion, immunostaining procedures, and
flow cytometric anabsis. The mouse MoAb to bcl-2 protein used in
this study (clone 124, IgG2a isotype) has been recently described7
and proven suitable for immunostaining of cryostat and paraffinembedded tissue sections and for Western blotting analysis; it was
raised against a synthetic peptide corresponding to amino acids 41
to 54 of the bcl-2 protein.
MoAbs to CD33 (MY9, IgG2b i~otype)’~
and CD34 (QBEND10,
IgG1)” were obtained from Coulter Immunology (Hialeah, FL)
and N. Bradley (Quantum Biosystems, Cambridge, UK), respectively. The MoAbs specific for HLA-DR (MA6, IgM), CDllb
(clone 44,IgGl), and CDllc (clone 3.9, IgG1)” were obtained
from the Fourth International Workshop on Human Leucocyte
Antigens.
The fixation/permeation protocol for optimal immunodetection
of bcl-2 internal molecule will be described in detail elsewhere.
Briefly, a pellet of lo7 cells was resuspended in 1 mL of 2%
ioo
10’
io2 lo3
HLA-DR
100
lo’
102
103
CD33
paraformaldehyde in phosphate-buffered saline (PBS) and kept
for 10 minutes at 4°C; 100 pL of 0.05% Triton XlOO (Sigma) in PBS
was then added and 10 minutes later the cells were washed twice in
PBS plus 1% bovine serum albumin (BSA) and used for labeling.
Single- and two-color indirect immunofluorescence (IF) staining of
fixed or viable cells was performed at 4°C on 96-well microtiter
plates as describedz2;the cells were preincubated for 10 minutes
with 1% human heat-inactivated AB-serum to prevent binding of
the antibodies to Fc receptors.
For the single-color IF the cells were incubated (30 minutes)
with saturating doses of the specified MoAb, washed four times in
RPMI-C, and incubated again (30 minutes) with a 1:30 dilution of
fluorescein (FITC)- or phycoerythrin (PE)-conjugated antimouse
Ig antibodies purchased from Technogenetics(Trezzano SN,Italy;
cat. 3900-2) and Southern BiotechnologyAssociates Inc (Birmingham, AL; cat. 1010-09), respectively.
For the two-color IF, combinations of primary MoAbs (or
irrelevant mouse Igs for the negative controls) of different isotypes
were simultaneously added to the cells; their binding was shown by
FITC- and PE-conjugated goat antibodies specific for the mouse
IgGl isotype (cat. 1070-02; 1070-09), IgG2a (cat. 1080-02; 108009), IgG2b (cat. 1090-01; 1090-09), or IgM (1020-02; 1020-09)
purchased from Southern Biotechnology.
Immunocytochemical staining of air-dried paraformaldehydefixed cytospin preparations was performed by the alkalineantialkaline phosphatase (APAAP)techniqueU using a commercial kit (Dakopatts, Glostrup, Denmark); three APAAP repeats
Fig 1. Immunocytochemical reactivity of anti-bcl-2 MoAb with normal bone marrow cells. Cytospin preparation of Ficoll-enriched bone
marrow aspirate, fixed with 2% paraformaldehyde and permeabilized with 0.001% Triton X100, immunostained by the APAAP methods and
counter-stained with Gill’s Hematoxylin. In this preparation three myeloid blasts, two late erythroblasts, and a lymphocyte are intensely stained
for bcl-2 whereas more mature myeloid cells are negative; some perinuclear staining is also evident (original magnification x 1.000).
Fig 3. lmmunolocalization of bcl-2 protein in normal and pathologic bone marrow biopsies. Sections of paraffin-embedded bone marrow
biopsies were deparaffinized and immunostained by the APAAP technique as described in Materials and Methods. (A) Normal bone marrow
biopsy showing scattered lymphocytes and two aggregates of blasts (arrows) stained for bcl-2 (original magnification x 400). (B) An RAEB
showing a large nodule of bcl-2 reactive cells (original magnification x 400); inset (original magnification x 25). (C) An RAEBt with clusters of
bcl-2 blasts (original magnification x 400). Fig 8. Immunocytochemical reactivity of anti-bcl-2 MoAb with normal and DMSO-treated HL-60
cells. APAAP-immunostained cytospin preparations of untreated (A) and DMSO-treated (6 days) (B) cells. Note the marked reduction in bcl-2
staining after DMSO treatment; in particular, cells with a mort? mature chromatin pattern and nuclear indentations are bcl-2 negative whereas
two blasts exhibit cytoplasmic positivity (original magnification x 1,OOO).
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
DELIA ET AL
1294
allowed optimal amplification of the reaction. Sections from
paraffin-embedded bone marrow specimens were deparaffinized in
xylene and graded ethanols, incubated with PBS containing 5 %
decomplemented human A B serum, and immunostained by the
APAAP method; normal mouse serum and an MoAb specific for
the leukocyte common antigen CD45 (Dako) were used as negative
and positive controls of the APAAP reaction, respectively. Antibcl-2 reactive cells were quantified at 400 X magnification by
means of an ocular grid in more than 20 randomly selected fields
(equivalent to 2 mm' of tissue).
Double-color staining for bcl-2 (green) and DNA (red) was
performed on cells that had been previously paraformaldehydefixedlTriton-permeabilizedand indirectly labeled for bcl-2 using
an FITC-tagged secondary antibody; the cells were then incubated
(30 minutes at room temperature) with 1 mglmL RNAse A
(Sigma) in PBS and finally in 1 mL of 10 WglmL propidium iodide
(Sigma) in PBS.
Flow cytometric analysis of single- and two-color stained samples
was performed on an EPICS-C instrument (Coulter Electronics,
Hialeah, FL) equipped with a 5-W argon-ion laser set at 488 nm
and 5 0 mW output power. Whenever required, color overlaps
were eliminated by electronic compensation.
Westemblot analysis. Lysates were prepared by solubilizing 10'
cells in 2 mL of a 50 mmol/L Tris buffer solution (TBS) p H 7.5
containing0.5% NP40,1% antagosin. 0.001% phenylmethylsulphonyl fluoride (PMSF); after ultracentrifugation, the supernatants
were recovered, boiled for 5 minutes in the presence of 5 %
B-mercaptwthanol and loaded (50 FLlslot) on 12.5% sodium
dodecyl sulfate (SDS)-polyacrylamide gel, electrophoresed, and
blotted onto BA8S nitrocellulose membranes (Schleicher & Schuell,
Dassel, Germany). After neutralization with 5 % BSA in TBS, the
membranes were incubated (20 hours at 4°C) with the anti-bcl-2
MoAb (5 )rg/mL) or with an equal amount of normal mouse IgG
(for the negative control) diluted in TBS containing 5 % BSA and
0.01% NaN3, hence washed extensively with TBS with or without
0.05% Triton XIOO, incubated for 1 hour at room temperature
(RT) with '"I-labeled goat antimouse Ig (5 x 10" cpm in 20 mL)
(Amersham, Amersham, UK), washed again, and exposed to
autoradiographic film.
Northem blots. Poly-A enriched mRNA from treated o r untreated cell lines was extracted by a kit (cat. 27-9254-01) purchased
from Pharmacia LKB (Uppsala, Sweden). Northern blots were
performed as described"; mRNA samples (5 kgllane) were electrophoresed in I % agaroselformaldehyde, transferred onto Duralon
UV nylon membranes (Stratagene, La Jolla, CA), and autocrosslinked. Prehybridizations, hybridizations, and washes under stringent conditions were performed as described." The bcl-2 probe
was a 1.5-kb Hindlll-EcoRI insert from pFLl obtained from M.
Cleary.' One hundred nanograms of the fragment, radiolabeled to
a specific activity of 2 to 5 x 10" cpm/pg with "P by the random
primer DNA labeling procedure, was used per hybridization.
Table 1. Reactivity of the Anti-bcl-2 MoAb With Myeloid Disorders
No. of
Cases
3
1
3
1
FAB
Diagnosis
RA
RAEB
RAEB
RAEB
1
1
RAEB-t
2
RAEB-t
3
AML"
AMLt
CML"
RAEB-t
bcl-2'
Cells
+
+
++
+++
+
++
+++
Distribution
Clusters
Clusters
Clusters
Clusters and
diffuse
Clusters
Clusters
Clusters and
diffuse
11
1
3
CML
CML-BC
3
ET
2
PV
6
-
+++
+++
+++
++
Diffuse
Rare nodules
Diffuse
Clusters
The studies were performed on bone marrow sections and aspirates
the former were labeled by the APAAP method, the latter by
immunofluorescence and examined byflow cytometry ( t 7 of 11 cases).
A semiquantitative estimate of the number of bcl-2' cells was based on
a comparison with normal bone marrow biopsies as follows: +, up to
20% stained cells; + +, up to 30% stained cells; + + +, more than 30%
stained cells: -,similar to normal controls.
Abbreviations: RA, refractoly anemia; RAEB, refractory anemia with
excess of blasts; RAEB-t, RAEB in transformation; AML, acute myeloid
leukemia: CML, chronic myeloid leukemia: CML-BC, CML in blast crisis;
ET, essential thrombocythemia; PV, polycythemia vera.
(*);
labeled cells on erythrocyte-dcpleted samples; however, on
Ficoll-separatcd samples (and thus depletcd of most of the
granulocytes) 78% 2 8.6% wcrc found positivc. In the
latter samples the CD33+ myeloid cells were 28.5% 2
21.1% and the CD34' cells 5.3% 2 3.5%. Dual-color IF
analysis showed that 89.5% 2 3.6% of the CD33' and
75.7% 2 21.2% of the CD34' fractions were bcl-2 positive
(Fig 2A). Coexpression of bcl-2 and CD34 was also found in
peripheral blood samples from patients trcated with recombinant human (rh) GM-CSF and presenting elevated numc
RESULTS
Detection of bcl-2 protein in myeloid cells. bcl-2 positive
myeloid cells were found in normal bone marrows as shown
by the immunostaining of cytospin preparations (Fig 1);
70% of myeloblasts, 83% of promyelocytes, 40% of myelocytes, 12.5% of metamyelocytes, 12.5% of polymorphonuclear cells, and 60% of monoblasts were bcl-2+, whereas
monocytes were totally negative. Although the localization
of the protein was prcvalcntly cytoplasmic, some perinuclear staining was observed.
The immunofluorescence flow cytometric analysis of
bcl-2 in normal bone marrows cvidcnccd 18% 2 5.3% of
26 kDFig 4. lmmunoblot analysis of bcl-2 protein in myeloid cells.
Lysates from equal numbers of cells (2.5 x los) were run under
reducing conditions on SDS-polyacrylamidegel electrophoresis (SDSPAGE), transblotted to nitrocellulose, reacted with the anti-bcl-2
MoAb and with '%goat antimouse, and finally exposed to autoradiographic film. Spleen lymphocytes and HT-29 cell line were included as
positive and negative controls. AML is a fresh acute myeloblastic
leukemia.
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
bcl-2 IN MYELOID CELLS
1295
A
HL60
..tu
[I
kb
kb
-
9.5
7.54.4-
2.42.4-
1.4Fig 5. bcl-2 mRNA in KG1 and HL-60 cells. Equal
amounts of poly-A enriched mRNA (5 pg/lane) were
size-fractionated in agarose, blotted onto nylon filter,
hybridized with UP-labeledDNA probe pFLl specific
for bcl-2, and exposed t o autoradiographic film with
intensifying screen. Poly-A RNA from the B-lymphoblastoid cell line Raji and the colon carcinoma HT29
were included as positive and negative controls,
respectively. Exposure times were 60 hours (A) and 9
days (6).
1.4-
0.3
-
0.3-
cont. 2d 5d 5d
TPA
2d
DMSO
The myeloid leukemic cell lines HL-60, KG1, GMI, and
K562 were found, by IF and immunocytochemistry, positive
for bcl-2; the mean fluorescence staining intensity values
indicated KGl as having thc highest amount of protein per
cell (not shown).
Westem and Northem blot analysis. To verify that, on
myeloid cells, the binding of bcl-2 MoAb was to the bcl-2
gene product rather than to a cross-reactive epitope,
further studies at protein and mRNA level were performed.
On Western blots of myeloid leukemia lysatcs the antibcl-2 MoAb identified a protein of identical size (26 Kd) to
that of spleen lymphocyte lysates (used here as positive
control) and corresponding to bcl-2 (Fig 4). The 26-Kd
protein was also dcmonstrated in the early myelo/
erythroblastic cell line K562; this finding, which suggests
that the bcl-2 gene may be expressed by erythroid progenitors, is supported by thc reactivity of the anti-bcl-2 MoAb
bers of CD34' circulating progenitor cells; onc of these
cases was CD33-, HLA-DR' (Fig 2b).
Immunohistochemical staining of paraffin-embedded normal bone marrow biopsy sections evidenced 14.5% (range
10% to 16%) of anti-bcl-2 reactive nucleated elements,
most of which were lymphoid cells and myeloid blasts (Fig
3A).
On fresh leukemias the anti-bcl-2 MoAb rcacted with 11
of 14 AML, 1 of 7 CML, and 3 of 3 CML in blast crisis
(Table 1); no correlation between bcl-2 positivity and
French-American-British (FAB) classification was found.
Analysis of myelodysplastic syndromes evidcnced a significant increase in bcl-2' blasts in most RAEB and RAEB-t;
in some cases clustcrs or nodules of positive cells were
observed (Fig 3, B and C). The number of bcl-2' cells were
also augmented in polycythemia vera but not in essential
thrombocythemia.
Table 2. bcl-2 Protein Modulation in Chemically Induced HL-60 Cell Maturation
Davs of Treatment
TPA
bcl-2 O h
i
CD11b
CDllc
DMSO
bcl-2 O h
I
CD11b
CDllc
Morphology'
CG
IN
0
1
2
3
4
5
97
118
<1
<1
55
99
90
64
52
92
91
86
29
90
89
88
23
91
81
78
30
89
89
63
97
118
<1
<1
89
113
1
1
90
116
16
1
89
120
14
4
89
118
13
7
114
9
11
1.5
2
24
10
27
10
84
6
7
8
74
110
12
7
69
107
10
5
65
112
6
4
6
29
45
33
40
28
For each specified marker, the percentages of positive cells after background subtraction are shown. In addition, the mean fluorescence intensity
of bcl-2 positive cells, measured on a log scale, is indicated.
*Morphologic analysis of DMSO-treatedcells was performed on May-GrBnwald/Giemsastained cytospin preparations; the percentage of cells
bearing cytoplasmic granules (CG) and indented nuclei (IN) is reported.
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
DELIA ET AL
1296
with normal bone marrow erythroblasts (see Fig 1). Differences in the intensity of the bands indicated that each cell
line presented variable amounts of bcl-2 protein that were,
among the myeloid leukemias, highest in KG1, albeit lower
than in spleen lymphocytes.
Results of Northern blot hybridizations are shown in Fig
5. The bcl-2 specific probe pFLl detected a major mRNA
transcript in HL-60 and KG1 of similar size (approximately
7.5 kb) to that found in the B-lymphoblastoid cell line Raji.
Strikingly, the KG1 cell line expressed at least 50-fold more
mRNA than Raji and this difference did not correspond to
a similar augmentation in protein level.
Altogether, the concordance between the immunobiochemical and molecular results indicates that myeloid cells
express the bcl-2 proto-oncogene, whose product is recognized by the anti-bcl-2 MoAb.
bcl-2 on in vitro differentiating cells. The pattern of bcl-2
staining in normal bone marrow (BM) myeloid cells is in
accord with a differentiation-regulated expression of the
bcl-2 gene. The bcl-2 positive promyelocytic cell line HL-60,
which can be induced to differentiate along either the
granulocytic or monocytic pathway when treated in vitro
with specific chemicals,25provides a suitable model to study
this relationship.
Therefore, we have cultured the HL-60 cells in the
presence of 1.25% DMSO and 10 ng/mL TPA and monitored at various intervals of time the levels of bcl-2 protein,
in relation to other differentiation markers, morphology,
and proliferation.
The results are listed in Table 2. It can be seen that the
DMSO-induced granulocytic maturation was accompanied
by a progressive decrease in bcl-2 positivity, from greater
than 95% in the untreated control to 65% by day 8 (Fig 6).
Similar downregulation of the 26-Kd protein was confirmed
by Western blot analysis (Fig 7). Parallel morphologic
changes (eg, increased numbers of cytoplasmic granules
and indented nuclei) as well as reduction of the proliferative rate (Fig 6) were observed. As expected, C D l l b and
C D l l c became detectable on a small fraction of cells.z6
Dual-color flow cytometric analysis (Fig 6) showed that
most of the bcl-2 negative cells were in the G1 phase of the
cell cycle. These cells were, in addition, characterized by a
granulocytic morphology (Fig 8).
The TPA-triggered monocytic/macrophagic differentiation resulted in a rapid decrease in bcl-2 expression (Fig 6);
within 24 hours the bcl-2 positivity dropped to 55% and by
day 5 to about 30%. These changes were also observed on
Western blots (Fig 7). The TPA-induced maturation was
evidenced by the strong upregulation of C D l l b and C D l l c
molecules?6The majority of bcl-2 negative cells were in the
G1 phase of the cell cycle.
Downregulation of bcl-2 gene was detected on Northern
blots (Fig 5); the very faint bands present at day 2 became
undetectable by day 5, despite measurable amounts of
protein. This finding may actually suggest that the protein
has a long half-life.
GI G2-M
G1 G2-M
t i
DAYS
1 - 1
J/b
d.
,
I
a
n
3
+
I
lo0
10'
io2
bcl-2
lo3
DNA
DNA
Fig 6. Relationship between bcl-2 and cell cycle in differentiating
HL-60 cells. The left and middle histograms were obtained from the
flow cytometric analysis of single-color labeled samples, whereas
those on the right were from the analysis of bel-2 (ordinate, green)/
DNA (abscissa, red) double-labeled samples; the horizontal window,
set up on a negative control, defines the threshold between bcl-2
reactive and unreactive cells.
DISCUSSION
The bcl-2 proto-oncogene, rearranged and deregulated
in B-cell lymphomas carrying the t(14;18) translocation, is
expressed in normal and neoplastic cells of the lymphoid
lineage negative for the t( 14;18) chromosomal abnormality12,5.6and the levels of transcription appear to correlate
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
bcl-2 IN MYELOID CELLS
Fig 7. lmmunoblot analysis of bel-2 in differentiating HL-60 cells. Lysatesfromequal numbers of cells
(2.5 x lov)were electrophoresedunder reducingconditions. Spleen lymphocytes and the HT-29 cell line
were used as positive and negative controls, respectively. Note that both TPA- and DMSO-induced differentiation pathways are accompanied by a timedependent reduction of bcl-2 protein levels; this
change occurs more rapidly with TPA.
1297
26 k D
with proliferation and differentiation stage: This relationship has been recently further elucidated by immunohistochemical studies of lymphoid tissues using a newly developed MoAb to bcl-2 gene product,7.Rshowing strong levels
of bcl-2 protein in many normal blood T and B lymphocytes,
mantle-zone but not germinal center B cells, in medullary
but not cortical thymocytes, and undetectable levels in
proliferating lymphoid cells.
AIthough studies regarding the involvement of bcl-2 in
human hematopoietic cells have been mostly limited to the
lymphoid lineage, bcl-2 is not lineage restricted; indeed, the
presence of minimal amounts of transcripts in a monoblastoid cell line2.‘ and the recent immunodetection of the
protein in normal myeloid cells2’ suggest that this protooncogene may also play a role in myeloid cell differentiation.
In this report we have provided novel data on the
expression of bcl-2 in normal and malignant cells of the
myeloid lineage. We based this evidence partly on analysis
using an anti-bcl-2 MoAb whose specificity for the 26-Kd
protein was confirmed by Western blot of a variety of
myloblastic leukemias (including a fresh case). Additionally, we have shown by Northern blot analysis the presence
of normal-size bcl-2 transcripts of 7.5 Kb in HL-60 and KG1
cell lines.
To perform quantitative and multiparameter flow cytometric measurements of bcl-2 on single cells, we developed
an appropriate fixation/permeabilizationprocedure for the
detection of the protein, localized primarily in the inner
mitochondrial and, to a lesser extent, on the perinuclear
membrane.”
The bcl-2 protein levels among normal myeloid cells are
inversely related to maturation; thus, a large fraction of
myeloblastsand promyelocytesare bcl-2’ whereas metamyelocytes and polymorphonuclear cells are mostly bcl-2
negative, and monocytes totally negative. These findings
have been substantiated by two-color flow cytometric analysis showing a large percentage of normal bone marrow
bcl-2+cells expressing CD33, a marker for myeloid cells19as
well as CD34, a marker for myeloid progenitor cells
including stem cells.’” Coexpression of bcl-2 and CD34
molecules has been further documented on circulating
CD34+ hematopoietic progenitor cells present in blood
samples of rhGM-CSF-treated patients. In particular, the
bcl-2 protein has been found on CD34+/CD33-/HLA-DR+
cells characterized by low right angle and low forward light
Contr.
2 4 6
3 6 9
TPA
DMSO
scattering properties; these features are consistent with a
progenitor of most of the colony-formingcells in long-term
marrow cultures.”-m However, further work is needed to
determine whether the hematopoietic stem cell is bcl-2’. In
view of the fact that bcl-2 protein confers stress resistance
to heat shock, ethanol, methotrexate and serum deprivation,I6 and survival,"^" it is attractive to hypothesize that
bcl-2 on early hematopoietic progenitor cells, eg, CD33-/
CD34’ (?stem cells), favors their long-lived capacity and
confers resistance to various external insults.
Overall, the pattern of bcl-2 distribution in normal bone
marrow myeloid cells appears concordant with the in vitro
findings on HL-60, whose differentiation toward the granulocytic and monocytic pathways results in a downregulation
of bcl-2 transcription and protein levels. Correlations with
cell growth and morphology have shown that the bcl-2
negative cells are mostly in G1 phase of the cell cycle and
present features of mature cells.
We have assessed the expression of bcl-2 molecule on
fresh myeloproliferative disorders; given that the anti-bcl-2
MoAb reacts with paraffin-embedded tissues, the study was
extended to bone marrow biopsies. The results have shown
that greater than 70% of AML are bcl-2 positive, whereas
most CML are bcl-2 negative.
Increased numbers of bcl-2 positive blasts have been
found in myelodysplastic syndromes. No clear-cut correlation between FAB classification and bcl-2 positivity was
observed. Overall, it appears that the expression of bcl-2 in
myeloid disorders reflects that found in their normal cell
counterparts.
The expression of bcl-2 in myeloid leukemias raises the
question of its potential prognostic value. In fact, because
many anticancer agents also operate through the activation
of programmed cell death3’and bcl-2 levels are positively
associated with resistance to apoptosis,I6 the possibility
exists that bcl-2 positive leukemias are less responsive to
chemotherapy than those that are negative. This speculation is now open to investigation.
In conclusion, the data presented here provide the basis
for future studies on the role of bcl-2 in myeloid cell
commitment, maturation, and survival.
ACKNOWLEDGMENT
The artwork was by Mario M i n i . We thank Prof A.M. Gianni
for providing blood specimens of rhGM-CSF treated patients, and
Prof D.Y. Mason for helpful advice and critical suggestions.
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
DELIA ET AL
1298
REFERENCES
1. Bakhshi A, Jensen JP, Goldman P, Wright AJ, McBride OW,
Epstein AL, Korsmeyer SJ: Cloning the chromosomal breakpoint
of t(14;18) of human lymphomas: Clustering around JH on
chromosome 14 and near a transcriptional unit on 18. Cell 412399,
1985
2. Cleary ML, Smith SD, Sklar J: Cloning and structural analysis
of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulintranscript
resulting from the t(14;18) translocation.Cell 47:19,1986
3. Hua C, Raffeld M, KO HS, Fast P, Bakhshi A, Cossman J:
Mechanisms of bcl-2 activation in human follicular lymphomas.
Oncogene 5:233,1990
4. Chen-Levy Z, Nourse J, Cleary M L The Bcl-2 candidate
proto-oncogene product is a 24-kilodalton integral membrane
protein highly expressed in lymphoid cell lines and lymphomas
carrying the t(14;18) translocation. Mol Cell Biol9:701,1989
5. Reed JC, Tsujimoto Y, Alpers JA, Croce CM, Nowell P C
Regulation of bcl-2 proto-oncogene expression during normal
human lymphocyte proliferation. Science 236:1295,1987
6. Graninger WB, Set0 M, Boutain B, Goldman P, Korsmeyer
SJ Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal
and neoplastic cells. J Clin Invest 801512,1987
7. Pezzella F, Tse AGD, Cordell JL, Pulford KAF, Gatter KC,
Mason D Y Expression of the bcl-2 oncogene protein is not specific
for the 14;18 chromosomal translocation. Am J Pathol 137225,
1990
8. Zutter M, Hockenbery D, Silverman GA, Korsmeyer SJ:
Immunolocalization of the Bcl-2 protein within hematopoietic
neoplasms. Blood 781062,1991
9. Tsujimoto Y, Croce C M Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human
follicular lymphoma. Proc Natl Acad Sci USA 835214,1986
10. Tsujimoto Y, Ikegaki N, Croce CM: Characterizationof the
protein product of bcl-2, the gene involved in follicular lymphomas.
Oncogene 23,1987
11. Hockenbery D, Nunez G, Milliman C, Schreiber RD,
Korsmeyer SJ: Bcl-2 is an inner membrane protein that blocks
programmed cell death. Nature 348:334,1990
12. Haldar S, Beatty C, Tsujimoto Y,Croce CM: The bcl-2 gene
encodes a novel G protein. Nature 342195,1989
13. Monica K, Chen-Levy Z, Cleary M L Small G proteins are
expressed ubiquitously in lymphoid cells and do not correspond to
Bcl-2. Nature 346189,1990
14. McDonnell TJ, Deane N, Platt FM, Nunez G, Jaeger U,
McKearn JP, Korsmeyer SJ Bcl-2-immunoglobulin transgenic
mice demonstrate extended B cell survival and follicularlymphoproliferation. Cell 5779,1989
15. Vaux DL, Cory S, Adams S: Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B
cells. Nature 335:440,1988
16. Tsujimoto Y Stress resistance conferred by high level of
bcl-2a protein in human B lymphoblastoid cell. Oncogene 4:1331,
1989
17. Nunez G, London L, Hockenbery D, Alexander M, McKeam JP, Korsmeyer SJ: Deregulated bcl-2 gene expression
selectively prolongs survival of growth factor-deprivedhemopoietic
cell lines. J Immunol1443602,1990
18. Siena S, Bregni M, Brando B, Belli N, Ravagnani F, Gandola
L, Stem AC, Lansdorp PM, Bonadonna G, Gianni AM: Flow
cytometry estimation of circulating hematopoietic progenitors for
autologous transplantation in cancer patients. Blood 77400,1991
19. Griffin JD, Linch D, Sabbath P, Larcom P, Schlossman S F
A monoclonal antibody reactive with normal and leukemic myeloid
progenitor cells. Leuk Res 8521,1984
20. Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan
P, Delia D, Sutherland DR, Baker MA, Greaves M F Expression
of the CD34 gene in vascular endothelial cells. Blood 75:2417,1990
21. Schmidt R E Non lineagehatural killer section report: New
and previously defined clusters, in Knapp W, Dorken B, Gilks WR,
Rieber EP, Schmidt RE, Stein H, von dem Borne AEGKr (eds):
Leucocyte Typing IV. Oxford, UK, Oxford University, 1989, p 517
22. Aiello A, Delia D, Fontanella E, Giardini R, Rilke F, Della
Porta G: Expression of differentiation and adhesion molecules in
sporadic Burkitt's lymphomas. Hematol Oncol8:229,1990
23. Cordell J, Falini B, Erber WN, Ghosh A, Abdulaziz Z,
MacDonald S, Pulford K, Stein H, Mason DY: Immunoenzymatic
labeling of monoclonal antibodies using complexes of alkaline
phosphatase and monoclonal anti-alkaline phosphatase (APAAF'
complexes). J Histochem Cytochem 32219,1984
24. Sambrook J, Fritsh EF, Maniatis T: Molecular Cloning. A
Laboratory Manual (ed 2). Cold Spring Harbor, NY,Cold Spring
Harbor Laboratory, 1989
25. Lubbert M, Koeffler P H Leukemic cell lines as a paradigm
for myeloid differentiation. Cancer Rev 1033,1988
26. Ferrero D, Pessano S, Pagliardi GL, Rovera G: Induction of
differentiation of human myeloid leukemias: Surface changes
probed with monoclonal antibodies. Blood 61:171,1983
27. HockenberyDM, Zutter M, Hickey W, Nahm M, Korsmeyer
SJ: BCL2 protein is topographicallyrestricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 88:6961,1991
28. Brandt J, Baird N, Lu L, Srour E, Hoffman R: Characterization of human hematopoietic progenitor cell capable of forming
blast cell containing colonies in-vitro. J Clin Invest 821017,1988
29. Andrews RG, Singer JW, Bernstein ID: Precursors of
colony-forming cells in humans can be distinguished from colonyformingcells by the expressionof the CD33 and CD34 antigens and
light scatter properties. J Exp Med 1691721,1989
30. Sutherland HJ,Eaves CJ,Dragowska W, Lansdorp PM:
Characterization and partial purification of human marrow cells
capable of initiating long-term hematopoiesis in vitro. Blood
741563,1989
31. Eastman A Activationof programmed cell death by anticancer agents: Cisplatin as a model system. Cancer Cells 2:275,1990
From www.bloodjournal.org by guest on February 2, 2015. For personal use only.
1992 79: 1291-1298
bcl-2 proto-oncogene expression in normal and neoplastic human
myeloid cells
D Delia, A Aiello, D Soligo, E Fontanella, C Melani, F Pezzella, MA Pierotti and G Della Porta
Updated information and services can be found at:
http://www.bloodjournal.org/content/79/5/1291.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.