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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4344-4352
CBF -SMMHC, Expressed in M4eo Acute Myeloid Leukemia,
Reduces p53 Induction and Slows Apoptosis in Hematopoietic Cells
Exposed to DNA-Damaging Agents
By
Martin Britos-Bray,
Manuel Ramirez,
Wangsen Cao,
Xinping Wang,
P.
Paul Liu,
Curt I. Civin, and
Alan D. Friedman
From The Johns Hopkins Oncology Center, Division of Pediatric
Oncology, Baltimore, MD; and the National Human Genome Research
Institute, Bethesda, MD.
 |
ABSTRACT |
CBF -SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as a
result of inv(16), but how it contributes to leukemogenesis is
unknown. p53 mutations are rare in de novo AML, but they
are common in many malignancies. Expression of CBF-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation or
etoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3
cells expressing a CBF-SMMHC variant that does not interfere with
DNA binding by CBF, indicating that a CBF genetic target regulates p53
induction. The p53 gene may be regulated by CBF, because p53 mRNA
levels were reduced by CBF-SMMHC. Reduced p53 induction was not
caused by slowed cell proliferation, a consequence of CBF-SMMHC
expression, because p53 was induced similarly in control cultures and
in cultures propagated in 10-fold less interleukin-3 (IL-3).
CBF-SMMHC did not slow apoptosis resulting from IL-3 withdrawal,
where p53 induction is minimal, but slowed apoptosis in Ba/F3 cells
exposed to 10 Gy of ionizing radiation or 3 to 8 µg/mL etoposide,
providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosis
was temporary, because all the cells exposed to these doses ultimately
died, and clonal survival assays performed using 0.04 µg/mL etoposide
did not show protection by CBF-SMMHC. p21 levels were increased in
cells subjected to DNA damage, regardless of CBF-SMMHC expression
and attenuated p53 induction. Bcl-2, bcl-xL,
bcl-xS, and bax levels were unaffected by CBF-SMMHC. Attenuated p53 induction may contribute to leukemogenesis by
CBF-SMMHC by slowing apoptosis via a p21-independent mechanism.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CBF-SMMHC IS expressed in the blasts
of patients with the French-American-British M4eo subtype of acute
myeloid leukemia (AML) as a consequence of the inv(16)(p13q22) or
the t(16;16)(p13q22) chromosomal abnormalities.1-4 In
CBF-SMMHC the CBF transcription factor is fused to the tail
domain of a smooth muscle myosin heavy chain (SMMHC).
Core binding factor (CBF) is a family of heterodimeric transcription
factors containing a common subunit, CBF, and one of three
CBF subunits, CBF1, AML1 (CBF2), or CBF3.5-9
CBF does not bind DNA directly, but increases the affinities of the CBF subunits for DNA.7,8 The CBF subunits contain a
Runt-homology domain required for DNA binding and for
heterodimerization.6,10 Mice lacking CBF or AML1 fail to
develop definitive hematopoiesis.11-14
The tail domain of SMMHC is a coiled-coil dimerization domain that can
multimerize into large filaments.15 CBF-SMMHC can sequester CBF subunits into nonfunctional
complexes.16,17 Mice in which the CBF-SMMHC cDNA has
been "knocked-in" to its endogenous locus fail to develop
definitive hematopoiesis or leukemia and die in utero.18
This phenotype is identical to that of CBF- or AML1-null mice,
providing additional evidence that CBF-SMMHC acts by inhibiting CBF
functions.
AML1-ETO, expressed from the t(8;21) chromosome in AML, and TEL-AML1,
expressed from the t(12;21) chromosome in B-lineage acute lymphocytic
leukemias, are each capable of repressing transcriptional activation by
CBF,19-21 and AML1-ETO knock-in mice do not develop definitive hematopoiesis or leukemia.22,23 The similarity
between these mice, mice expressing CBF-SMMHC, and mice lacking
CBF or AML1 supports the hypothesis that all of the CBF oncoproteins act to inhibit CBF functions.
We expressed CBF-SMMHC inducibly from the sheep metallothionein (MT)
promoter in two interleukin-3 (IL-3)-dependent hematopoietic cell
lines, Ba/F3 B-lymphoid cells and 32D cl3 myeloid cells. Exposure of
these lines to zinc chloride induced CBF-SMMHC levels more than
10-fold, to levels similar to endogenous CBF. As a result, cell
generation time increased 1.7-fold, with a 4-fold increase in the ratio
of G1 to S phase cells and evident hypophosphorylation of the
Retinoblastoma (Rb) protein.24 Also, 32D cl3 granulocytic differentiation was not inhibited, even though CBF regulates genes encoding myeloid differentiation markers, such as myeloperoxidase and
neutrophil elastase.25 We speculated that in M4eo AML
initial genetic hits occur which allow higher-level expression of
CBF-SMMHC and thus, inhibition of differentiation.
We now provide data that support an alternative model for how
CBF-SMMHC contributes to leukemogenesis. p53 is widely mutated in
human malignancies and yet is rarely altered in de novo
AML.26 p53 is induced in response to DNA strand
breaks,27-29 leading to cell cycle arrest or
apoptosis.28,30-34 Thus, normal cells are afforded an
opportunity to repair the damage or to die, whereas cells harboring a
p53 mutation may be more susceptible to developing additional genetic
alterations, leading to malignant progression. When cells
expressing CBF-SMMHC were treated with ionizing radiation (IR) or VP-16, p53 induction was markedly reduced.
Similarly, CBF-SMMHC did not affect the rate of apoptosis resulting
from IL-3 withdrawal, but did prolong the survival of Ba/F3 cells
exposed to 10 Gy of IR or 3 to 8 µg/mL of VP-16. Reduced p53
induction and slowed apoptosis did not result from slowed proliferation alone, because these effects were not observed when the growth rate of
Ba/F3 cells was slowed by transfer to media containing 10-fold-reduced
IL-3. Also, p53 induction was normal in Ba/F3 cells expressing a mutant
version of CBF-SMMHC that cannot interact with CBF subunits,
indicating that a CBF genetic target regulates p53 induction. Reduced
p53 induction may be caused in part by direct inhibition of p53 gene
transcription, because p53 mRNA levels were reduced by CBF-SMMHC.
Attenuated p53 induction and slowed apoptosis may contribute to
leukemogenesis by CBF-SMMHC.
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MATERIALS AND METHODS |
Cell culture and transduction.
Ba/F3 cell lines were maintained in RPMI 1640 supplemented with 10%
(vol/vol) heat-inactivated fetal bovine serum (HI-FBS), 1 ng/mL murine
IL-3 (R & D Systems, Minneapolis, MN), and penicillin-streptomycin (P/S). Zinc chloride was added to 100 µmol/L final concentration when
indicated. Ba/F3 lines expressing CBF-SMMHC or containing the empty
MTCB6 vector were maintained in 1.2 mg/mL G418. To remove IL-3, cells
were washed twice with phosphate-buffered saline (PBS) and then
resuspended in RPMI 1640 with HI-FBS and P/S.
DNA-damaging agents.
Cells were exposed to gamma irradiation using a Gammacell 40 irradiator
(Atomic Energy of Canada, Ottawa, Canada). VP-16 (Sigma, St Louis, MO)
was kept as 10 mg/mL stock in dimethyl sulfoxide. Once added, VP-16 was
not removed from the cultures. Cells cultured with zinc for 18 hours
before exposure to DNA-damaging agents were also cultured with zinc
chloride during, and if necessary, after, these treatments.
Cell death and apoptosis analysis, clonogenic assay, and cell cycle
analysis.
At designated times after exposure to IR or during exposure to VP-16, 5 × 105 cells were washed with PBS and stained with
propidium iodide (PI) and fluorescein isothiocyanate (FITC)-Annexin V
using the ApoAlert Kit (Clonetech, Palo Alto, CA). The cells were
resuspended in 200 µL of binding buffer, and 5 µL of FITC-Annexin V
and 5 µL of 50 µg/mL PI were then added. After incubation at room
temperature for 10 minutes the cells were subject to
fluorescence-activated cell sorter (FACS) analysis with a
FACSort apparatus (Becton Dickinson, Mountain View, CA).
For clonogenic assays, cells were diluted to 0.3 to 2 cells/mL, based
on the results of preliminary experiments, so that colonies would be
present in less than 50% but more than 10% of the wells. After
dilution, zinc and/or VP-16 was added as indicated and each group of cells was cultured in two 96-well dishes. Colony yields were
scored 8 days later.
Viable cell counting was performed by enumerating cells which exclude
trypan blue dye using a hemocytometer. Cell cycle analysis was
performed after a 30-minute bromo-deoxy-uridine (BrdU)
pulse using FITC-anti-BrdU/PI staining and FACScan analysis as
described.24
Western blotting.
Total cellular protein extracts were subjected to Western blotting as
described,35 except that 5% nonfat dried milk was included
with both the primary and secondary antisera. Polyclonal rabbit
antiserum raised against a CBF-GST fusion protein was used at a
ratio of 1:1,000.36 p53 (Ab-3) and p21WAF1/CIP1
(Ab-5) rabbit antisera (Oncogene Research, Cambridge, MA) were used at
ratios of 1:200, 1:100, and 1:200, respectively. Bcl-2 (N-19),
bcl-xL/S (L-19), and bax (N-20) antisera (Santa Cruz
Biotechnology, Santa Cruz, CA) were used at a ratio of 1:100. The bands
were visualized with an electrochemiluminescence kit
(Amersham, Arlington Heights, IL). Filters were stripped as
described.24
Northern blotting.
Poly-A-containing RNA was isolated using a Mini RiboSep ULTRA kit
(Collaborative Biomedical Products, Bedford, MA). Five micrograms of
mRNA was subjected to Northern blotting with the murine p53 and mdm2
cDNAs (kindly provided by C. Canman and M. Kastan, St Jude Children's Research Hospital) and the human actin cDNA
as described.25
Statistical methods.
The Student's t-test was used to compare survival and
apoptosis between experimental groups.
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RESULTS |
CBF-SMMHC reduces p53 induction by DNA damage.
For these studies we used a pool of vector-transfected Ba/F3 cells
(MTCB6) and two subclones expressing CBF-SMMHC from the MT promoter
(MTINV-2 and MTINV-3).24 Ba/F3 lines were chosen in lieu of
32D cl3 lines for lines for these experiments because the latter were
difficult to maintain, perhaps due to excess MT promoter leakiness.
Parental Ba/F3 cells respond to IR by inducing p53 and undergoing
either a G1 arrest in IL-3 or accelerated apoptosis in the absence of
IL-3.31 To test the effect of CBF-SMMHC on p53 induction
by IR, MTCB6, MTINV-2, or MTINV-3 cells were cultured for 18 hours in the presence or absence of zinc. These six cultures were
then split into two, and one culture from each pair was irradiated to
10 Gy. Cultures initially treated with zinc were continued in zinc
after irradiation. Ninety minutes later total cellular extracts were
prepared and subjected to Western blotting for p53, p21, and
CBF-SMMHC. The blot was stained with Fast Green (Sigma, St Louis,
MO) as a loading control (Fig
1A). In the absence of irradiation,
exposure to zinc did not lead to increased p53 levels in either MTINV
line, indicating that CBF-SMMHC alone cannot induce p53. IR induced
p53 levels in the MTCB6 cells, and induction was similar in the
presence or absence of zinc. p53 was induced similarly by IR in the
MTINV-2 and MTINV-3 cells in the absence of zinc. However, when
CBF-SMMHC was expressed at high levels as a result of zinc
treatment, p53 induction by IR was reduced threefold to fourfold.
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| Fig 1.
CBF-SMMHC inhibits p53 induction but induces
p21WAF1/CIP1 in Ba/F3 cells exposed to IR or VP-16. (A)
MTCB6 cells (vector-transfected) and MTINV-2 or MTINV-3 cells
(expressing CBF-SMMHC from the MT promoter) were cultured ± 100 µmol/L zinc chloride for 18 hours. Half of the cells from each of
these four cultures were then irradiated to 10 Gy. Cells exposed
initially to zinc were continued in zinc after irradiation. Total
cellular protein extracts were prepared 90 minutes later and
electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel.
The proteins were then transferred to a nitrocellulose filter, which
was stained with Fast Green to control protein loaded (bottom panel)
and subjected to Western blotting with antisera specific for murine
p53, p21WAF1/CIP1, and CBF. The latter antisera detected
CBF-SMMHC, which was induced by zinc in the MTINV-2 and MTINV-3
cells. (B) MTCB6 and MTINV-3 cells were cultured ± zinc for 18 hours.
The cells were then exposed to 8 µg/mL VP-16 for 150 minutes in the
presence or absence of IL-3. Total cellular extracts were then prepared
and subjected to Western blotting for p53, p21, and CBF, with CBF
levels serving as a control for protein loading.
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IR alone induced p21 in each of these lines. p21 levels remained high
90 minutes post-IR in the MTCB6 or MTINV cultures exposed to zinc (Fig
1A), and were similarly elevated 3 hours or 4.5 hours post-IR
as well (data not shown). Thus, CBF-SMMHC attenuated p53 induction,
but not p21 induction, by IR.
To assess the effect of CBF-SMMHC on p53 and p21 induction by VP-16,
we exposed MTCB6 or MTINV-3 cells to 8 µg/mL VP-16 for 150 minutes, in the presence or absence of IL-3. We used
150 minutes because p53 induction was delayed in response to VP-16
compared with IR (data not shown). Cells were cultured ± zinc, but
all cultures were exposed to VP-16. CBF levels were also analyzed as
a loading control (Fig 1B). Again, zinc did not affect the degree of
p53 induction in MTCB6 cells. However, induction of p53 by VP-16 was
reduced approximately threefold in MTINV-3 cells expressing
CBF-SMMHC as a result of exposure to zinc, both in the presence and
in the absence of IL-3. Induction of p21 by VP-16 was similar in MTCB6
cells regardless of the presence of zinc, and p21 levels were higher in
MTINV-3 cells exposed to VP-16 when CBF-SMMHC expression was
induced. As indicated by the CBF levels, paired samples, ± zinc,
had nearly identical protein content.
CBF-SMMHC slows apoptosis induced by DNA damage.
CBF-SMMHC did not induce apoptosis in Ba/F3 cells cultured in
IL-3.24 We sought to determine whether CBF-SMMHC alters the rate of apoptosis induced by IL-3 withdrawal. To assess survival, cells were stained with PI and subjected to FACScan analysis. On
average, 94% of parental, MTCB6, or MTINV Ba/F3 cells excluded PI, and
therefore were detected as viable by this assay (data not shown). The
effect of zinc on the survival of MTINV-3 cells after IL-3 withdrawal
was determined (Fig 2). Induction of
CBF-SMMHC did not affect the proportion of MTINV-3 cells alive 6, 18, 24, or 42 hours after IL-3 withdrawal in duplicate experiments. At 6 hours survival was 88%, and at 18 hours survival was 39%. Also, removal of both IL-3 and serum for 6 hours reduced MTINV-3 cell survival to 78% (±5%) and induction of CBF-SMMHC further reduced their survival, to 57% (±6%) (data not shown). Thus, CBF-SMMHC did not slow the rate of cell death resulting from serum and/or IL-3 withdrawal.
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| Fig 2.
CBF-SMMHC does not slow the rate of cell death
resulting from IL-3 withdrawal in Ba/F3 cells. MTINV-3 cells were
cultured ± zinc chloride for 18 hours. They were then washed three
times with PBS and cultured without IL-3. Cells initially exposed to
zinc were continued in zinc. At 0, 6, 18, 24, and 42 hours an aliquot
of cells from each culture was analyzed for the proportion of surviving
cells by PI staining and FACScan analysis. Mean results from duplicate
experiments are shown. Standard errors (SEs) were smaller than the data
points depicted.
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We next sought to determine whether CBF-SMMHC affected the rate of
cell death induced by IR or VP-16. Parental Ba/F3, MTCB6, MTINV-2, and
MTINV-3 cells were cultured in the presence or absence of zinc for 18 hours and then exposed to 10 Gy of IR. The cells were then continued in
culture in the presence or absence of IL-3 for 6 hours. Cultures
pretreated with zinc for 18 hours were continued in zinc after
irradiation. The cells were stained with both PI and FITC-Annexin V
before FACScan analysis, so that the proportion of viable cells and the
proportion of cells undergoing apoptosis could be measured
simultaneously. An example of data obtained for cells cultured without
IL-3 after irradiation is shown in Fig 3A.
The cluster of cells containing approximately 30 U or less of PI were
considered to be alive. For all three lines, 10 Gy reduced the
proportion of viable cells at 6 hours to about 20%, whereas IL-3
withdrawal alone only reduced viability to 88% at 6 hours (Fig
2). The survival of MTCB6 cells was not affected by zinc, whereas
the survival of both the MTINV-2 and MTINV-3 cells was increased about
3-fold when CBF-SMMHC was induced with zinc. These increases in
survival were reflected in the proportion of cells that were negative
for Annexin V binding. Because Annexin V binding is an early marker for
apoptosis,37,38 this result indicates that cell death from
irradiation is a result of apoptosis and that induction of CBF-SMMHC
with zinc protected the cells from apoptosis. The results of the PI
exclusion assay from three such experiments are shown in the left panel
of Fig 3B. In the absence of IL-3, CBF-SMMHC increased survival of
MTINV-2 and MTINV-3 cells irradiated to 10 Gy 2.5-fold, compared with
parental Ba/F3 or MTCB6 cells (P < .05). Results with
Annexin V paralleled these survival data (data not shown).
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| Fig 3.
CBF-SMMHC slows apoptosis in Ba/F3 cells exposed to IR
or VP-16. (A) MTCB6, MTINV-2, or MTINV-3 cells were cultured in the
absence or presence of zinc for 18 hours. The cells were then removed
from IL-3 and irradiated to 10 Gy, and 6 hours later the cells stained
with PI and FITC-Annexin V and subjected to FACScan analysis. After
irradiation, cells initially in zinc were continued in zinc. Data from
a typical experiment are shown. The cluster of cells that excluded PI
and was determined to be alive is boxed in the upper left panel. (B)
The proportion of parental Ba/F3, MTCB6, MTINV-2, or MTINV-3 cells
surviving 10 Gy or IR followed by culture in the absence of IL-3 for 6 hours, surviving 8 µg/mL VP-16 for 6 hours in the absence of IL-3, or
surviving 3 µg/mL VP-16 for 18 hours in the presence of IL-3 are
shown. Ratios of the proportion of cells surviving in the presence of
zinc to the proportion of cells surviving in the absence of zinc were
calculated for each cell line in each experiment. Results shown are the
mean and SE from three experiments.
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In the presence of IL-3, 10 Gy of IR did not induce significant cell
death or apoptosis. Ba/F3 cells underwent a transient G1/S arrest and
then resumed cycling when exposed to a similar dose of IR in
IL-3.31 We also treated the cells with 50 Gy of IR in the
presence of IL-3. At this dose, approximately 50% of the cells were
alive 18 hours later, and induction of CBF-SMMHC did not confer
protection from cell death (data not shown).
A similar approach was taken to determine whether CBF-SMMHC
protected cells from VP-16. In preliminary experiments with control cells, doses and exposure times for VP-16 that allowed approximately 20% survival were identified. VP-16 was used at 3 µg/mL for 18 hours
in the presence of IL-3 or at 8 µg/mL for 6 hours in the absence of
IL-3. The results of three experiments are diagramed in the center and
right panels of Fig 3B. PI exclusion results again correlated with
FITC-Annexin V binding (data not shown). CBF-SMMHC protected Ba/F3
cells 1.8-fold from apoptosis and cell death occurring as consequence
of VP-16 exposure, both in the presence and absence of IL-3. Protection
from VP-16 at these doses was transient, because by 48 hours none of
the cells were viable (data not shown).
To determine whether CBF-SMMHC could prevent apoptosis and therefore
increase clonogenic survival, we plated MTCB6 or MTINV-3 cells at low
cell densities in 96-well dishes in the presence or absence of 0.04 µg/mL VP-16. The use of a dose of VP-16 in this range was necessary
to allow Ba/F3 cells to survive and develop into colonies. Cells from
each group were cultured ± zinc. The proportion of plated cells which
gave rise to colonies 8 days later was enumerated (Fig
4). The cloning efficiency of MTCB6 cells
in the absence of zinc or VP-16 was 100%, and the cloning efficiency
of MTINV-3 cells was 70%. Addition of zinc alone did not affect the
colony yield from MTCB6 cells, but reduced the colony yield from
MTINV-3 cells threefold, to 25%. This decrease was likely caused by a
growth inhibitory effect of CBF-SMMHC. VP-16 reduced the colony
yield from MTCB6 cells twofold, and zinc did not alter this effect.
VP-16 alone also reduced the colony yield from MTINV-3 cells twofold,
to 30% of the plated cells. Treatment of MTINV-3 cells with both zinc
and VP-16 led to a colony yield of only 5%, actually lower than would
have been predicted from a threefold reduction because of CBF-SMMHC
and a twofold reduction because of VP-16. Thus, CBF-SMMHC did not
prevent cell death resulting from VP-16 in this clonogenic assay.
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| Fig 4.
CBF-SMMHC did not increase the clonogenic survival of
Ba/F3 cells. MTCB6 or MTINV-3 cells were cultured ± zinc for 18 hours. VP-16 was then added to 0.04 µg/mL to an aliquot of each cell
line. Culture was then continued in the same conditions in two 96-well
dishes per group. Cells were plated at 0.3 to 2 cells/mL, based on the
results of preliminary experiments, so that between 10% and 50% of
the wells would develop a colony. The number of colonies were scored 8 days later, and the proportion of initially plated cells that formed
colonies is shown for each cell line and culture condition.
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Slowed proliferation does not reduce p53 induction or slow apoptosis
resulting from VP-16 exposure.
CBF-SMMHC increased the generation time of Ba/F3 cells from 9.6 hours to 14.4 hours and increased the ratio of G1 phase cells to S
phase cells from 0.8 to 3.5.24 We sought to mimic this effect by culturing the cells in lower IL-3 concentrations (Fig 5A). We found that after 48 hours of
culture in 0.1 ng/mL IL-3, the generation time of Ba/F3 cells increased
from 9.6 hours to 16.5 hours, and the G1/S ratio increased from 0.6 to
3.1, without evident apoptosis. We exposed parental Ba/F3 cells
cultured in either 1.0 or 0.1 ng/mL IL-3 to 8 µg/mL VP-16 for 6 hours
and assessed survival by PI exclusion. Slowed proliferation during G1
did not protect the cells from VP-16-mediated cell death. We also
assessed p53 induction in cells cultured similarly, ± VP-16, for 150 minutes (Fig 5B). Slowed proliferation during G1 did not attenuate p53
induction by VP-16. Equal loading was evident from the levels of a
cross-reactive band.
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| Fig 5.
Ba/F3 cells cultured in reduced IL-3 had a slowed G1 to S
transition, but did not have reduced p53 induction or increased
survival in response to VP-16. (A) Parental Ba/F3 cells were cultured
in 1.0 ng/mL IL-3, which allows optimum growth, or in 0.1 ng/mL IL-3.
After 48 hours, cell generation time was determined by enumerating the
number of viable cells present in the cultures on subsequent days. To
determine the ratio of the proportion of cells in G1 phase to the
proportion of cells in S phase (G1/S), the cells were exposed to 30 µmol/L BrdU for 30 minutes, fixed, stained with anti-BrdU-FITC and
PI, and subjected to FACScan analysis. The proportion of cells
surviving exposure to 8 µg/mL VP-16 for 6 hours was determined as in
Fig 3. (B) Ba/F3 cells cultured in 1.0 ng/mL or 0.1 ng/mL IL-3 for 48 hours were continued in cultured ± 8 µg/mL VP-16 for 150 minutes.
Total cellular protein extracts were then obtained and subjected to
Western blot analysis for p53.
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CBF-SMMHC does not alter bcl-2,
bcl-xL, bcl-xS, or bax levels.
Apoptosis is regulated in part by a balance between antiapoptotic bcl-2
family members such as bcl-2 and bcl-xL and proapoptotic family members such as bax and bcl-xS.39 The
bax gene is directly activated by p53.40 The same extracts
used to assess the affect of VP-16 on p53 and p21 levels in Fig 1C were
analyzed for expression of bcl-2, bcl-xL,
bcl-xS, and bax (Fig 6). The
levels of these proteins were unaffected by zinc, in MTCB6 cells, or by
induction of CBF-SMMHC, in MTINV-3 cells. Therefore, slowed
apoptosis in CBF-SMMHC-expressing cells exposed to VP-16 is not
caused by altered expression of bcl-2, bcl-xL,
bcl-xS, or bax.
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| Fig 6.
CBF-SMMHC does not affect bcl-2, bcl-xL,
bcl-xS, or bax expression in Ba/F3 cells exposed to VP-16.
The same MTCB6 and MTINV-3 extracts described in Fig 1C were subjected
to Western blotting for bcl-2, bcl-xL, bcl-xS,
and bax.
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A CBF-SMMHC variant that cannot bind CBF does not
attenuate p53 induction.
CBF( 2-11)-SMMHC, which lacks amino acids 2-11 of the CBF
segment of CBF-SMMHC, does not interact with CBF subunits,
inhibit CBF DNA binding, or inhibit Ba/F3 cell
proliferation.17,41 MTINV (2-11) cells,
which express this CBF-SMMHC variant from the MT promoter, were
cultured in the presence or absence of zinc for 18 hours. An aliquot
from each culture was then irradiated 10 Gy. Induction of p53 90 minutes post-IR and of CBF(2-11)-SMMHC by zinc was then assessed
by Western blotting (Fig 7A).
Induction of p53 was robust, even when expression of
CBF(2-11)-SMMHC was induced. This result indicates that
CBF-SMMHC attenuates p53 induction in Ba/F3 cells by interfering
with activation of a critical gene or set of genes by CBF.
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| Fig 7.
CBF-SMMHC must interact with CBF and
inhibit CBF transactivation to attenuate p53 induction, and reduces p53
mRNA expression in Ba/F3 cells. (A) MTINV( 2-11) Ba/F3 cells,
which express a CBF-SMMHC variant that cannot bind CBF, were
cultured ± 100 µmol/L zinc chloride for 18 hours. Half of the cells
from each of these cultures were then irradiated to 10 Gy. Cells
exposed initially to zinc were continued in zinc after irradiation.
Total cellular protein extracts were prepared 90 minutes later and
subjected to Western blotting with antisera specific for murine p53 and
CBF-SMMHC. Fast Green staining of the blot is shown as a control for
protein loading. (B) MTCB6 and MTINV-3 cells were cultured ± zinc for
18 hours. Poly-A-containing mRNA was then prepared from each culture.
Five micrograms of each sample was subjected to electrophoresis on a
1% formaldehyde-agarose gel, transferred to a nylon membrane, and
probed sequentially for p53, mdm2, and actin.
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CBF-SMMHC reduced p53 mRNA levels.
If CBF activates the p53 gene, then CBF-SMMHC would be predicted to
reduce p53 mRNA levels. Alternatively, if increased mdm2 protein levels
account for decreased p53 induction by DNA damage, then CBF-SMMHC
might increase mdm2 mRNA levels. To test these possibilities, MTCB6 and
MTINV-3 cells were cultured for 18 hours in the presence or absence of
zinc. Poly-A-containing mRNA was then isolated from each culture, and
5 µg of each sample was subjected to Northern blotting for p53, mdm2,
and actin (Fig 7B). Zinc alone did not affect p53 or mdm2 mRNA levels
in CB6 cells. However, in MTINV-3 cells induction of CBF-SMMHC with
zinc reduced the level of p53 mRNA relative to actin mRNA approximately
fourfold, but did not alter the level mdm2 mRNA. Similarly, in MTINV-3
cells treated with VP-16, induction of CBF-SMMHC reduced p53 mRNA
levels approximately twofold (data not shown).
| |
DISCUSSION |
Our results suggest that CBF-SMMHC contributes to leukemogenesis by
attenuating the p53 response to DNA damage. Several studies indicate
that apoptosis as a consequence of DNA damage in hematopoietic cells
requires induction of p53. p53 is required for apoptosis of thymocytes
exposed to IR or VP-16.32,33 Expression of exogenous p53 in
the M1 myeloid leukemia line induces apoptosis.42 Also, inhibition of p53 induction, by expression of the viral E6 protein, inhibits apoptosis in Ba/F3 cells exposed to IR and withdrawn from
IL-3.31
Because p53 mutations are rare in de novo AMLs,26 reduced
induction of p53 by CBF-SMMHC might serve as an alternative
mechanism to interfere with a p53-dependent apoptotic pathway. Indeed,
reduced p53 induction resulting from CBF-SMMHC expression correlated with slowed apoptosis in Ba/F3 cells treated with IR or VP-16. Slowed
apoptosis might enable a rare cell to express additional genetic
changes, survive, and progress towards transformation. Also, in
cooperation with other mutations already present in preleukemic cells,
reduced levels of p53 resulting from CBF-SMMHC expression might
directly stimulate survival. Reduced p53 levels stimulated the survival
of solid tumor cells subjected to hypoxia.30 Perhaps a
similar selective advantage can contribute to leukemogenesis in vivo
independent of DNA damage.
The doses of IR or VP-16 we used to induce p53 represent a greater
degree of DNA damage than most leukemic precursors are likely subjected
to in vivo. p53 was not induced in Ba/F3 cells exposed to 0.04 µg/mL
VP-16 for 1.5, 3, 4.5, or 24 hours (data not shown). Perhaps p53 is
induced more effectively by physiological levels of DNA damage in
preleukemic cells.
Increased expression of p21WAF1/CIP1 occurred despite
reduced p53 expression in cells treated with IR or VP-16. In some
experiments, induction of CBF-SMMHC led to even higher levels of p21
(Fig 1B), as reported previously.24 p21 protects colon
carcinoma cells from apoptosis.34 However, expression of
the viral E7 protein in Ba/F3 cells, which mimics p21 overexpression by
inactivating Rb and releasing E2F proteins, does not sensitize them to
IR in the presence or absence of IL-3.43 This finding
suggests that the reduced radio- and chemo-sensitivity of
CBF-SMMHC-expressing Ba/F3 cells results from attenuated p53
induction and not from increased p21 expression. Critically testing
whether decreased p53 induction is the primary cause of slowed
apoptosis after DNA damage in CBF-SMMHC-expressing cells would
require coexpression of exogenous p53, in a manner dependent on DNA
damage.
CBF-SMMHC decreases the proportion of Ba/F3 cells in S phase
1.7-fold,24 raising the possibility that protection from
apoptosis was caused by decreased exposure to DNA damage during S
phase. However, IR damages DNA in all cell cycle phases, and exposure to VP-16 was for 18 hours in the presence of IL-3, allowing sufficient time for all cells to enter S phase.
CBF-SMMHC increases the proportion of cells in G1 phase 1.7-fold,
but does not affect the proportion of cells in G2.24 A
lengthened G1 might allow increased time for DNA repair before S phase.
However, DNA repair processes are rapid, and time spent in the G2 phase
may be a more critical determinant of radiation and VP-16
sensitivity.44-46 Also, we found that slowing the G1 to S
transition in Ba/F3 cells by reducing IL-3 in the culture media did not
attenuate p53 induction or stimulate survival when the cells where
exposed to VP-16.
The clinical observation that about 60% of patients with AMLs
associated with inv(16) or t(8;21) are cured by conventional chemotherapy, whereas only about 30% of the remaining AML patients are
cured47 suggests that CBF-SMMHC and AML1-ETO might
sensitize hematopoietic cells to chemotherapy. Yet we observed
protection from IR and VP-16, and from Ara-C and daunomycin as well
(data not shown). However, the protection observed was modest and was only compared with that obtained using control Ba/F3 lines. Ba/F3 cells
expressing bcr-abl(p210) were more resistant than MTINV lines to IR or
VP-16 (M.B., X.W., and A.D.F., unpublished observation, August 1997).
Similarly, subsets of AML that are generally refractory to chemotherapy
may have stronger protective mechanisms than AMLs expressing
CBF-SMMHC or AML1-ETO.
We have begun to investigate the mechanism of reduced p53 induction as
a consequence of CBF-SMMHC expression. A CBF-SMMHC variant that
cannot interact with CBF subunits did not attenuate p53 induction
(Fig 6A) and did not confer protection from IR or VP-16 (M.B. and
A.D.F., unpublished observation, August 1997). These findings suggest
that CBF-SMMHC reduces p53 induction by reducing the expression of a
gene regulated by CBF. Consistent with this model, Ba/F3 cells
expressing AML1-ETO also had reduced p53 induction in response to IR
(M.B. and A.D.F., unpublished observation, May 1998). If direct binding
of p53 by CBF-SMMHC is required to decrease p53 expression, then
cells expressing CBF(2-11)-SMMHC might have been expected to have
attenuated p53 induction. Our Northern blot data suggest that reduction
of p53 mRNA levels partly accounts for attenuated p53 induction in the
presence of CBF-SMMHC. Additional experiments will be required to
determine whether the p53 gene is regulated by CBF. Canonical CBF
consensus-binding sites, 5 -ACC(A/G)CA-3, are present at bps 656 and
1912 of the murine p53 intron 1 (GenBank #U10088) and at bp 1348 of the
human intron 1 (GenBank #X54156), but not in the murine or human p53
promoter regions between bps  420 and +1.48 Even if CBF
does not regulate the p53 gene, CBF oncoproteins might bind these or
other sites and repress its transcription.
p53 protein levels are controlled by several mechanisms, including
amino-terminal phosphorylation and mdm2-mediated
degradation.49-51 Our future experiments will also
elucidate the effect of CBF-SMMHC on these pathways and will
identify the genetic targets of CBF and CBF-SMMHC responsible
for the phenotypes of hematopoietic cells expressing
CBF-SMMHC. Therapies designed to inhibit the effect of
CBF-SMMHC on p53 induction and apoptosis might prove useful for
leukemias expressing CBF oncoproteins.
| |
ACKNOWLEDGMENT |
We thank C. Canman and M. Kastan for Ba/F3 cells, for the p53 and mdm2
cDNA, and for helpful discussions.
| |
FOOTNOTES |
Submitted March 25, 1998;
accepted August 5, 1998.
Supported in part by National Institutes of Health Grant No. HL51388
(A.D.F.). A.D.F. is a Leukemia Society Scholar, and P.P.L. is a
Leukemia Society Special Fellow.
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
1734 solely to indicate this fact.
Address reprint requests to Alan D. Friedman, MD, Johns Hopkins
Oncology Center, Room 3-109, 600 N Wolfe St, Baltimore, MD 21287.
| |
REFERENCES |
1.
Arthur DC, Bloomfield CD:
Partial deletion of the long arm of chromosome 16 and bone marrow eosinophilia in acute nonlymphocytic leukemia, a new association.
Blood
61:994, 1983[Abstract/Free Full Text]
2.
Le Beau MM, Larson RA, Bitter MA, Vardiman JW, Golomb HM, Rowley JD:
Association of inversion of chromosome 16 with abnormal marrow eosinophils in acute myeloid leukemia.
N Engl J Med
309:630, 1983[Abstract]
3.
Liu PP, Tarle SA, Hajre A, Claxton DF, Marlton P, Freedman M, Siciliano MJ, Collins FS:
Fusion between transcription factor CBF/PEBP2 and a myosin heavy chain in acute myeloid leukemia.
Science
261:1041, 1993[Abstract/Free Full Text]
4.
Liu PP, Wijmenga C, Hajra A, Blake TB, Kelley CA, Adelstein RS, Bagg A, Rector J, Cotelingam J, Willman CL, Collins FS:
Identification of the chimeric protein product of the CBFB-MYH11 fusion gene in inv(16) leukemia cells.
Genes Chromosomes Cancer
16:77, 1996[Medline]
[Order article via Infotrieve]
5.
Bae SC, Yamaguchi-Iwai Y, Ogawa E, Maruyama M, Inuzuka M, Kagoshima H, Shigesada K, Satake M, Ito Y:
Isolation of PEBP2B cDNA representing the mouse homolog of human acute myeloid leukemia gene, AML1.
Oncogene
8:809, 1993[Medline]
[Order article via Infotrieve]
6.
Ogawa E, Maruyama M, Kagoshima H, Inuzuka M, Lu J, Satake M, Shigesada K, Ito Y:
PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene.
Proc Natl Acad Sci USA
90:6859, 1993[Abstract/Free Full Text]
7.
Ogawa E, Inuzuka M, Maruyamna M, Satake M, Naito-Fujimoto M, Ito Y, Shigesada K:
Molecular cloning and characterization of PEBP2, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2.
Virology
194:314, 1993[Medline]
[Order article via Infotrieve]
8.
Wang S, Wang Q, Crute BE, Melnikova IN, Keller SR, Speck NA:
Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.
Mol Cell Biol
13:3324, 1993[Abstract/Free Full Text]
9.
Levanon D, Negreanu V, Bernstein Y, Bar-Am I, Avivi L, Groner Y:
AML1, 2 and 3. The human members of the runt domain gene-family.
Genomics
23:425, 1994[Medline]
[Order article via Infotrieve]
10.
Meyers S, Downing JR, Hiebert SW:
Identification of AML1 and the (8;21) translocation protein AML1-ETO as sequence specific DNA binding proteins: The runt homology domain is required for DNA binding and protein-protein interactions.
Mol Cell Biol
13:6336, 1993[Abstract/Free Full Text]
11.
Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR:
AML-1, the target of multiple chromosomal translocations in human leukemia, is essential for normal murine fetal liver hematopoiesis.
Cell
84:321, 1996[Medline]
[Order article via Infotrieve]
12.
Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH, Speck NA:
Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.
Proc Natl Acad Sci USA
93:3444, 1996[Abstract/Free Full Text]
13.
Wang Q, Stacy T, Miller JD, Lewis AF, Gu T-L, Huang X, Bushweller JH, Bories J-C, Alt FW, Ryan G, Liu PP, Wynshaw-Boris A, Binder M, Marin-Padilla M, Sharpe AH, Speck NA:
The CBF subunit is essential for CBF2 (AML1) function in vivo.
Cell
87:697, 1996[Medline]
[Order article via Infotrieve]
14.
Sasaki K, Yagi H, Bronson RT, Tominaga K, Matsunashi T, Deguchi K, Tani Y, Kishimoto T, Komori T:
Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor .
Proc Natl Acad Sci USA
93:12359, 1996[Abstract/Free Full Text]
15.
Adelstein RS, Sellers JR:
Myosin structure and function, in
Barany M
(ed):
Biochemistry of Smooth Muscle Contraction. New York, NY, Academic, 1996, p 3.
16.
Wijmenga C, Gregory PE, Hajra A, Schrock E, Ried T, Eils T, Liu PP, Collins FS:
CBF-SMMHC chimeric protein involved in AML forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.
Proc Natl Acad Sci USA
93:1630, 1996[Abstract/Free Full Text]
17. Adya N, Stacy T, Speck NA, Liu PP: The leukemic protein
CBF-SMMHC sequesters CBF2 into cytoskeletal filaments and
aggregates. Mol Cell Biol (in press)
18.
Castilla LH, Wijmenga C, Stacy T, Speck NA, Eckhaus M, Marin-Padilla M, Collins FS, Wynshaw-Boris A, Liu PP:
Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knocked-in leukemia gene CBFB-MYH11.
Cell
87:687, 1996[Medline]
[Order article via Infotrieve]
19.
Meyers S, Lenny N, Hiebert SW:
The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation.
Mol Cell Biol
15:1974, 1995[Abstract]
20.
Frank R, Zhang J, Uchida H, Meyers S, Hiebert SW, Nimer SD:
The AML1/ETO fusion protein blocks transactivation of the GM-CSF promoter by AML1B.
Oncogene
11:2667, 1995[Medline]
[Order article via Infotrieve]
21.
Hiebert SW, Sun W, Davis JN, Golub T, Shurtleff S, Buijs A, Downing JR, Grosveld G, Roussel MF, Gilliland DG, Lenny N, Meyers S:
The t(12;21) translocation converts AML1-1B from an activator to a repressor of transcription.
Mol Cell Biol
16:1349, 1996[Abstract]
22.
Yergeau DA, Hetherington CJ, Wang Q, Zhang P, Sharpe AH, Binder M, Marin-Padilla M, Tenen DG, Speck NA, Zhang D-E:
Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML1-ETO fusion gene.
Nat Genet
15:303, 1997[Medline]
[Order article via Infotrieve]
23.
Okuda T, Cai Z, Yang S, Lenny N, Lyu C-J, van Deursen JMA, Harada H, Downing JR:
Expression of a knocked-in AML1-ETO leukemia gene inhibits the establishment of normal definitive hematopoiesis and directly generates dysplastic hematopoietic progenitors.
Blood
91:3134, 1998[Abstract/Free Full Text]
24.
Cao W, Britos-Bray M, Claxton DF, Kelley CA, Speck NA, Liu PP, Friedman AD:
CBF-SMMHC, expressed in M4eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells.
Oncogene
15:1315, 1997[Medline]
[Order article via Infotrieve]
25.
Nuchprayoon I, Meyers S, Scott LM, Suzow J, Hiebert S, Friedman AD:
PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2/CBF oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells.
Mol Cell Biol
14:5558, 1994[Abstract/Free Full Text]
26.
Imamura J, Miyoshi I, Koeffler P:
p53 in hematologic malignancies.
Blood
84:2412, 1994[Free Full Text]
27.
Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW:
Participation of p53 protein in the cellular response to DNA damage.
Cancer Res
51:6304, 1991[Abstract/Free Full Text]
28.
Lowe SW, Ruley HE, Jacks T, Housman DE:
p53-dependent apoptosis modulates the cytotoxicity of anticancer agents.
Cell
74:957, 1993[Medline]
[Order article via Infotrieve]
29.
Nelson WG, Kastan MB:
DNA strand breaks: The DNA template alterations that trigger p53-dependent DNA damage response pathways.
Mol Cell Biol
14:1815, 1994[Abstract/Free Full Text]
30.
Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, Giaccia AJ:
Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors.
Nature
379:88, 1996[Medline]
[Order article via Infotrieve]
31.
Canman CE, Gilmer TM, Coutts SB, Kastan MB:
Growth factor modulation of p53-mediated growth arrest versus apoptosis.
Genes Dev
9:600, 1995[Abstract/Free Full Text]
32.
Lowe SW, Schmitt SW, Smith SW, Osborne BA, Jacks T:
p53 is required for radiation-induced apoptosis in mouse thymocytes.
Nature
362:847, 1993[Medline]
[Order article via Infotrieve]
33.
Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML, Wylie AH:
Thymocyte apoptosis induced by p53-dependent and independent pathways.
Nature
362:849, 1993[Medline]
[Order article via Infotrieve]
34.
Polyak K, Waldman T, He T-C, Kinzler KW, Vogelstein B:
Genetic determinants of p53-induced apoptosis and growth arrest.
Genes Dev
10:1945, 1996[Abstract/Free Full Text]
35.
Scott LM, Civin CI, Rorth P, Friedman AD:
A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.
Blood
80:1725, 1992[Abstract/Free Full Text]
36.
Cameron S, Taylor DS, TePas EC, Speck NA, Mathey-Prevot B:
Identification of a critical regulatory site in the human interleukin-3 promoter by in vivo footprinting.
Blood
83:2851, 1993[Abstract/Free Full Text]
37.
Martin SJ, Finucane DM, Amarante-Mendes GP, O'Brien GA, Green DR:
Phosphatidylserine externalization during CD95-induced apoptosis of cells and cytoplasts required ICE/CED-3 protease activity.
J Biol Chem
271:28753, 1996[Abstract/Free Full Text]
38.
Vanags DM, Porn-Ares MI, Coppola S, Burgress DH, Orrenius S:
Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis.
J Biol Chem
271:31075, 1996[Abstract/Free Full Text]
39.
Reed JC, Miyashita T, Takayama S, Wang H-G, Sato T, Krajewski S, Aime-Sempe C, Bodrug S, Kitada S, Hanada M:
BCL-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to chemotherapy.
J Cell Biochem
60:23, 1996[Medline]
[Order article via Infotrieve]
40.
Miyashita T, Reed JC:
Tumor suppressor p53 is a direct transcriptional activator of the human bax gene.
Cell
80:293, 1995[Medline]
[Order article via Infotrieve]
41. Cao W, Adya N, Britos-Bray M, Liu PP, Friedman AD: The core
binding factor interaction domain and the smooth muscle
myosin heavy chain segment of CBF-SMMHC are both required to
slow cell proliferation. J Biol Chem (in press)
42.
Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, Oren M:
Wild-type p53 induces apoptosis of myeloid leukemia cells that is inhibited by interleukin-6.
Nature
352:345, 1991[Medline]
[Order article via Infotrieve]
43.
Canman CE, Kastan MB:
Small contribution of G1 checkpoint control manipulation to modulation of p53-mediated apoptosis.
Oncogene
16:957, 1998[Medline]
[Order article via Infotrieve]
44.
Palayoor ST, Macklis RM, Bump EA, Coleman N:
Modulation of radiation-induced apoptosis and G2/M block in murine T-lymphoma cells.
Radiat Res
141:235, 1995[Medline]
[Order article via Infotrieve]
45.
Nishii K, Kabarowski JHS, Gibbons DL, Griffiths SD, Titley I, Wiedemann LM, Greaves MF:
tsBCR-ABL kinase activation confers increased resistance to genotoxic damage via cell cycle block.
Oncogene
13:2225, 1996[Medline]
[Order article via Infotrieve]
46.
Bedi A, Barber JP, Bedi GC, El-Diery WS, Sidransky D, Vala MS, Akhtar AJ, Hilton J, Jones RJ:
BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: A mechanism of resistance to multiple anticancer agents.
Blood
86:1148, 1995[Abstract/Free Full Text]
47.
Samuels BL, Larson RA, Le Beau MM, Daly KM, Bitter MA, Vardiman JW, Barker CM, Rowley JD, Golomb HM:
Specific chromosomal abnormalities in AML correlate with drug susceptibility in vivo.
Leukemia
2:79, 1988[Medline]
[Order article via Infotrieve]
48.
Bienz-Tadmor B, Zakut-Houri R, Libresco S, Givol D, Oren M:
The 5 region of the p53 gene: Evolutionary conservation and evidence for a negative regulatory element.
EMBO J
4:3209, 1985[Medline]
[Order article via Infotrieve]
49.
Haupt Y, Ruth M, Kazaz A, Oren M:
Mdm2 promotes the rapid degradation of p53.
Nature
387:296, 1997[Medline]
[Order article via Infotrieve]
50.
Kubbutat MH, Jones SN, Vousden KH:
Regulation of p53 stability by Mdm2.
Nature
387:299, 1997[Medline]
[Order article via Infotrieve]
51.
Siliciano JD, Canman CE, Taya T, Sakaguchi K, Appella E, Kastan MB:
DNA damage induces phosphorylation of the amino terminus of p53.
Genes Dev
11:3471, 1997[Abstract/Free Full Text]
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Abstract
Introduction