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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 1003-1010
Distinct Apoptotic Responses Imparted by c-myc and max
By
Chadd E. Nesbit,
Saijun Fan,
Hong Zhang, and
Edward V. Prochownik
From the Section of Hematology/Oncology, the Department of
Pediatrics, Children's Hospital of Pittsburgh; the Department of
Molecular Genetics and Biochemistry, the University of Pittsburgh
Medical Center; and the University of Pittsburgh Cancer Institute,
Pittsburgh, PA.
 |
ABSTRACT |
The c-myc oncoprotein accelerates programmed cell death (apoptosis)
after growth factor deprivation or pharmacological insult in many cell
lines. We have shown that max, the obligate c-myc heterodimeric partner
protein, also promotes apoptosis after serum withdrawal in NIH3T3
fibroblasts or cytokine deprivation in interleukin-3 (IL-3)-dependent
32D murine myeloid cells. We now show that c-myc- and
max-overexpressing 32D cells differ in the nature of their apoptotic
responses after IL-3 removal or treatment with chemotherapeutic compounds. In the presence of IL-3, c-myc overexpression enhances the
sensitivity of 32D cells to Etoposide (Sigma, St Louis, MO), Adriamycin
(Pharmacia, Columbus, OH), and Camptothecin (Sigma), whereas max
overexpression increases sensitivity only to Camptothecin. Drug
treatment of c-myc-overexpressing cells in the absence of IL-3 did not
alter the spectrum of drug sensitivity other than to additively
accelerate cell death. In contrast, enhanced sensitivity to Adriamycin,
Etoposide, and Taxol (Bristol-Meyers Squibb, Princeton, NJ) was
revealed in max-overexpressing cells concurrently deprived of IL-3.
Differential rates of apoptosis were not strictly correlated with the
ability of the drugs to promote G1 or G2/M arrest. Ectopic expression
of Bcl-2 or Bcl-XL blocked drug-induced apoptosis in both
cell lines. In contrast, whereas Bcl-2 blocked apoptosis in both cell
lines in response to IL-3 withdrawal, Bcl-XL blocked apoptosis in max-overexpressing cells but not in c-myc-overexpressing cells. These results provide mechanistic underpinnings for the idea
that c-myc and max modulate distinct apoptotic pathways.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE c-myc ONCOPROTEIN plays important
roles in cellular transformation, proliferation, and
differentiation.1-4 More recently, a role for c-myc in
programmed cell death (apoptosis) in primary fibroblasts and in
cytokine-dependent hematopoietic cell lines has been documented where,
after the removal of these survival factors, the cells undergo
apoptosis at a greatly accelerated pace.5-8
The molecular mechanisms underlying c-myc-mediated apoptosis in
response to growth factor withdrawal are incompletely understood. The
process seems to be at least partly dependent on the p53 tumor suppressor9,10 and can also be prevented by overexpression of the Bcl-2 oncoprotein.11-13 c-myc-mediated apoptosis
also requires that the protein dimerize with max,7 which,
like c-myc, is a member of the basic-helix-loop-helix-leucine zipper
family.14,15 It is believed that c-myc-max heterodimers
represent the state in which c-myc is able to recognize its specific
DNA binding sites in vivo and activate the expression of adjacent
genes.16,17
max consists of two major protein isoforms of either 151 or 160 amino
acids, respectively.14,15 These length differences are
attributable to a nine-amino acid insertion/deletion between amino
acids 12 and 13. Recently, we have shown that stable ectopic expression
of the longer max isoform (max[L]) results in reduced proliferation,
decreased sensitivity to growth factors, and accelerated apoptosis
after serum deprivation in NIH3T3 fibroblasts or interleukin-3 (IL-3)
withdrawal in the 32D murine myeloid cell line.18 In contrast, overexpression of the shorter isoform results in slightly faster proliferation, increased sensitivity to growth factors, and no
appreciable effect on apoptosis in response to cytokine removal.
The more robust demise of c-myc and max(L)-overexpressing cells in
response to cytokine deprivation raises the question of whether the
biochemical pathways leading to this response are identical or
distinct. In the latter case, it might then be possible to define these
pathways functionally based on whether they can be modulated by other
known proapoptotic or antiapoptotic factors.
In the present study, we have examined the sensitivity of c-myc and
max(L)-overexpressing cells to apoptotic induction by several
pharmacological agents commonly used in cancer chemotherapy. The agents
were chosen based on their differing modes of action, although all
eventually promote cellular death through apoptosis. 32D cells were
used to test the effects of c-myc and max(L) overexpression because
they are a nontumorigenic, euploid cell line that express wild-type p53
protein, a requirement for a normal apoptotic response in several
systems.19,20 We show here that c-myc and max(L) alter the
apoptotic response of 32D cells to pharmacological insult in distinctly
different ways. The ectopic expression of either Bcl-2 or the closely
related protein Bcl-XL was able to prevent apoptosis in
either cell line in response to drug treatment. In contrast, whereas
Bcl-2 was able to block apoptosis in both cell lines after IL-3
withdrawal, Bcl-XL blocked apoptosis only in max(L)-overexpressing cells. Taken together, our results argue that
c-myc and max(L) likely affect different, although overlapping, apoptotic pathways.
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MATERIALS AND METHODS |
Plasmids.
The construction of pSVLneo-max(L) and pSVLneo-c-myc have been
previously described.18 pMEP4-Bcl-2 and the empty pMEP4
parental vector21 were kindly provided by Dr Yusuf Hannun
(Duke University Medical Center, Durham, NC). A full-length human
Bcl-XL cDNA22 in a pBluescript vector was
provided by Dr Craig Thompson (Pritzker School of Medicine, The
University of Chicago, IL). The cDNA was excised with NotI and
EcoRV, made blunt-ended with the Klenow fragment of DNA
polymerase and ligated into the blunt-ended EcoRI site of the
pAPuro expression vector.23
Cell lines.
32D c13 cells19 were obtained from Dr Seth
Corey (Children's Hospital of Pittsburgh). The derivation of cells
overexpressing max(L) (hereafter referred to as 32D-max) and c-myc
(hereafter referred to as 32D-c-myc) has been previously
described.18 Briefly, cells were transfected by
electroporation with NdeI-linearized pSVLneo-max(L) or
pSVLneo-c-myc plasmid DNAs and selected in G-418 (GIBCO-BRL, Grand
Island, NY).24 Pooled G-418-resistant populations of cells
were used to eliminate clonal variability as an explanation for
observed differences in subsequent biological behavior. A population of
control cells was also obtained after stable transfection with the
empty pSVLneo vector and is referred to as 32D-neo. All cell lines were
maintained in RPMI medium (GIBCO-BRL) supplemented with 10% fetal
bovine serum (Hyclone, Logan, UT), 10% conditioned medium from the
IL-3-producing murine WEHI 238 lymphoblastoid cell line, penicillin,
streptomycin, 2 mmol/L glutamine, and 400 µg/mL G-418 (absolute
concentration) (GIBCO-BRL).
To obtain cell lines stably overexpressing Bcl-2, 32D-neo, 32D-max, and
32D-c-myc cells were transfected by electroporation with
BgIII-linearized pMEP4-Bcl-2 and selected in 250 µg/mL
Hygromycin (GIBCO-BRL). Control cell lines, transfected with the empty
pMEP4 vector, were derived in parallel. Cell lines overexpressing
Bcl-XL were obtained by electroporation with
NotI-linearized pAPuro-Bcl-XL and selected in 1 µg/mL Puromycin (Sigma, St Louis, MO). Control cell lines were
obtained by stable transfection with the NotI-linearized pAPuro
parental vector. All cell lines were maintained continuously in the
above stated concentrations of the appropriate antibiotic.
Western blotting.
Western blotting was performed with 50 µg of total cell lysate from
each cell line. Briefly, logarithmically growing cells were pelleted by
centrifugation, washed twice in phosphate-buffered saline (PBS), and
lysed in standard 1× sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) lysis buffer. Protein concentrations were
determined using the Pierce BCA Protein Determination Assay (Pierce,
Rockford, IL) and were then verified by Coomassie blue staining of
electrophoresed aliquots. After SDS-PAGE, proteins were transferred to
Immobilon-P membranes (Millipore, Bedford, MA) using a semi-dry
blotting apparatus (Owl Scientific, Cambridge, MA). All pre-incubations
and incubations with antibodies were performed in PBS-T + 5% nonfat
dry milk.25 Blots were first incubated for 2 hours at room
temperature followed by an overnight incubation at 4°C with a 1:1,000
dilution of rabbit anti-Bcl-2 (#15616E) or Bcl-X (#65186E) antibodies
(Pharmingen, San Diego, CA). After exhaustive washing in PBS-T, the
blots were incubated for 2 hours at room temperature with a 1:1,000
dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) followed by washing with
PBS-T and development of the blots using the "Supersignal"
enhanced chemiluminescence kit (Pierce) according to the supplier's
directions.
Apoptosis studies.
32D cell lines (>95% viability) were seeded at 2 × 105 cells/mL in 6- or 12-well tissue culture plates and
allowed to resume growth for 6 to 8 hours before the addition of
Adriamycin (ADR; Pharmacia, Columbus, OH), Etoposide (VP-16; Sigma),
Camptothecin (CPT; Sigma), Cis-platinum (CDDP; Sigma), Taxol
(Bristol-Meyers Squibb, Princeton, NJ), or Nitrogen mustard (N-Mus;
Merck, West Point, PA) to the indicated final concentrations. All drugs
were dissolved in tissue culture medium as stock solutions 100-fold more concentrated than the highest concentrations used here and were
frozen in small aliquots. In some experiments, IL-3 was withheld to
allow the combined effects of drug treatment and cytokine deprivation to be determined. At various times after the addition of drugs, aliquots of cells were removed and viability was assessed using the
trypan blue exclusion assay. Total DNA was extracted as previously described18 and analyzed by electrophoresis in 2% agarose
gels followed by staining with ethidium bromide.
Cell cycle studies.
Logarithmically growing cells (approximately
5 × 105/mL) were treated with chemotherapeutic agents
for 16 hours. Propidium iodide staining of isolated nuclei was then
performed as previously described.26 Cell cycle analyses
were performed on a Becton Dickinson (Mountain View, CA)
FACSTAR fluorescence-activated cell sorter. For each assay, 2 × 104 cells were analyzed. Quantitation was performed using
single histogram statistics.18
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RESULTS |
c-myc and max differentially affect apoptosis in response to
pharmacological agents.
For this study, we used the 32D myeloid cell line which is diploid,
untransformed, and expresses wild-type p53 protein.19,20 We
tested six structurally unrelated and mechanistically diverse pharmacological compounds commonly used in cancer
chemotherapy.27-29 ADR is a DNA intercalating agent and
inhibitor of topoisomerase II, CPT is a non-DNA binding inhibitor of
topoisomerase I, VP-16 is a nonintercalating topoisomerase II
inhibitor, CDDP is a DNA cross-linking agent, N-Mus is an alkylating
agent, and Taxol is a microtubule inhibitor. Because growth factors
have been previously shown to protect against apoptosis in several
settings,30,31 a potential antiapoptotic role for IL-3 in
c-myc- and max-mediated apoptosis was also investigated.
32D-neo, 32D-c-myc, and 32D-max cells were first incubated continuously
in IL-3-supplemented medium containing different concentrations of
each chemotherapeutic compound. At various times thereafter, the
fraction of apoptotic cells was assessed by trypan blue exclusion. As
shown in Fig 1, each of the tested cell
lines manifested distinct behaviors in response to the drugs. In
comparison to 32D-neo cells, 32D-c-myc cells were more sensitive to
ADR, CPT, and VP-16. Depending on the concentration of drug used and
the times at which apoptosis was assessed, these differences ranged
between 3- and 20-fold. In contrast, 32D-max cells were
indistinguishable from 32D-neo cells in response to all agents except
CPT. In this case, 32D-max and 32D-c-myc cells showed equivalent
degrees of increased sensitivity to the agent. From these studies we
conclude that c-myc overexpression increases the chemotherapeutic
sensitivity of 32D cells to ADR, CPT, and VP-16, whereas max
overexpression increases the sensitivity of 32D cells only to CPT.
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| Fig 1.
Susceptibility of 32 D-neo ( ), 32D-max ( ), and
32D-c-myc ( ) cells to six different antineoplastic agents in the
presence of IL-3. Cells of >95% viability were plated in the
presence of IL-3 and the indicated concentrations of drug as described
in Materials and Methods. At various times thereafter, viability was
determined by trypan blue staining of a 40-µL aliquot. The results
shown here depict survival curves at 40 hours for ADR; 41 hours for
CDDP, CPT, and N-Mus; 16 hours for Taxol; and 48 hours for VP-16. Each
graph is representative of three or more experiments (±1 SE. The
percent values shown have been normalized to those of cells grown in
the absence of any drug for the equivalent length of time. The inserts
show the results of electrophoresis of apoptotic DNAs from cells grown
in the highest concentration of each drug for the times indicated
above. All inserts show DNAs from 32D-neo, 32D-max cells, and 32D-c-myc
(left to right).
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Additional differences among the three cell lines were evident when the
above experiments were performed in the absence of IL-3 (Fig
2). As expected, a more rapid rate of
killing was observed in all cases due to the combined proapoptotic
effects of IL-3 withdrawal and drug treatment. Most importantly, the
removal of IL-3 did not reveal any new differential drug sensitivities
in the 32D-c-myc cell line, which remained more sensitive than 32D-neo cells to killing only by ADR, CPT, and VP-16. In addition, the enhanced
killing afforded by IL-3 depletion was additive rather than
synergistic, with the differences in cell killing between the 32D-neo
and 32D-c-myc cell lines remaining in the 3- to 10-fold range.
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| Fig 2.
32D-neo (), 32D-c-myc (), and 32D-max () cells
were treated as described in Fig 1 except that they were also
maintained in IL-3-depleted medium for the duration of the experiment.
Viability was then determined at 18 hours for ADR, CPT, VP-16, and
CDDP; 14 hours for N-Mus; and 16 hours for Taxol. The results shown here are the average of three experiments ±1 SE. Note that the viability of the cells in the absence of drug is not 100% due to cell
death caused by the absence of IL-3. Viability was again scored by
trypan blue exclusion. Apoptotic DNAs are again depicted as in Fig 1.
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A much different type of behavior was observed in IL-3-deprived
32D-max cells where, in the absence of the cytokine, increased sensitivity to ADR, Taxol, and VP-16 was now observed in cells that
previously were indistinguishable from control 32D-neo cells. A
tendency for 32D-max cells to be more sensitive to CDDP was also seen
but the differences were less than the twofold which we define as
significant.
From the above studies, we conclude that IL-3 does not protect
32D-c-myc cells against killing by any of the agents tested, whereas
the cytokine does protect 32D-max cells against Adriamycin, Taxol, and
VP16-mediated cytotoxicity.
To confirm the results of many other groups that cell death resulting
from IL-3 withdrawal and drug treatment was apoptotic in nature, total
DNAs were extracted from each cell line and examined for evidence of
apoptotic "laddering." As shown in the insets in Figs 1 and 2,
there was, in general, an excellent correlation between cell death
determined by trypan blue exclusion and the degree of DNA
fragmentation.
Cell cycle analyses.
One possible explanation for the observed differential effects on cell
killing (Fig 1) is that c-myc and max altered the ability of 32D cells
to undergo cell cycle arrest after treatment with chemotherapeutic
drugs.32,33 Therefore, we determined the cell cycle
distribution of each of the three cell lines either in the absence of
any drug or after treatment with each of the six previously tested
chemotherapeutic compounds. As seen in Table
1, the overexpression of c-myc and max did,
in some cases, alter the degree to which these agents caused cell cycle
arrest. For example, in comparison with 32D-neo cells, both 32D-c-myc
and 32D-max cells showed a reduced tendency to arrest in G0/G1 after
treatment with ADR and CPT. However, in general there was no
consistency between the ability of an agent to induce arrest at a
particular phase of the cell cycle and the extent of the subsequent
apoptotic response. From these results we conclude that although c-myc
and max were clearly capable of influencing the efficiency of cell
cycle arrest, this did not necessarily correlate with the ability of
the cells to undergo subsequent apoptotic death.
Differential effects of Bcl-2 and Bcl-XL on c-myc- and
max-mediated apoptosis.
Both Bcl-2 and the related protein Bcl-XL exert protective
effects against a wide variety of apoptotic stimuli including cytokine deprivation and chemotherapeutic agents.34-39 In addition,
Bcl-2 overexpression has been shown to protect several different cell types against the accelerated apoptosis mediated by c-myc after cytokine withdrawal.11-13 To investigate the role of each
of these proteins in abrogating apoptosis after IL-3 deprivation or
drug treatment, 32D-neo, 32D-c-myc, and 32D-max cells were transfected with expression vectors for Bcl-2, Bcl-XL, or the
corresponding parental vectors alone. Pools of transfected clones were
again used to ensure that any observed responses were not the result of
clonal variability. Western blotting experiments confirmed that each
protein was expressed at equivalent levels in all three cell lines (Fig
3).
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| Fig 3.
Western blot analysis of 32D cell lysates. (A) 32D-neo,
32D-c-myc, and 32D-max cells were electroporated with either the
linearized pMEP4-Bcl-2 expression vector or the empty pMEP4 parental
vector. Stable clones were selected in hygromycin and pooled. From each cell line 50 µg of total cell lysate was subjected to SDS-PAGE and
Western blotting using an anti-Bcl-2 antibody. (B) 32D-neo, 32D-c-myc,
and 32D-max cell lines were stably transfected with either the
pAPuro-Bcl-XL vector or the pAPuro parental vector. SDS-PAGE and Western blotting were performed as described in (A) except
that an anti-Bcl-X antibody was used.
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We first examined the effects of enforced Bcl-2 and Bcl-XL
expression on apoptosis in response to IL-3 withdrawal. As shown in Fig
4A, overexpression of Bcl-2 conferred
nearly complete protection against cell death in all three cell lines.
In contrast, whereas overexpression of Bcl-XL also
protected 32D-neo and 32D-max cells, it provided minimal protection
(<twofold) against apoptosis in 32D-c-myc cells (Fig 4B). From these
results, we conclude that Bcl-XL exerts a more restrictive
antiapoptotic effect in these cell lines.
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| Fig 4.
Effects of Bcl-2 and Bcl-XL overexpression on
apoptosis in response to IL-3 withdrawal. Bcl-2-overexpressing (A) or
Bcl-XL-overexpressing (B) 32D cell lines, or their
vector-transfected control counterparts, were washed free of IL-3 and
incubated in fresh, IL-3-depleted medium for the times indicated. The
fraction of viable cells at each point was determined as in Figs 1 and
2. The results shown are representative of triplicate experiments ±1
SE.
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The above cell lines were also used to determine whether Bcl-2 and
Bcl-XL could protect 32D cells against apoptosis mediated by chemotherapeutic agents. As shown in Fig
5, both Bcl-2 and Bcl-XL
protected all three cell lines against both ADR and CPT. In some cases,
the protection afforded was complete; for example, Bcl-2 and
Bcl-XL completely spared 32D-max cells. In other cases such
as 32D-c-myc cells exposed to CPT, only exposure to low doses of drug
were associated with a high degree of protection.
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| Fig 5.
Effects of Bcl-2 and Bcl-XL on drug-mediated
apoptosis in 32D cell lines. 32D-neo, 32D-c-myc, and 32D-max cells were
stably transfected with Bcl-2 or Bcl-XL expression plasmids
or with the respective empty parental vectors. The resulting pooled
clones were then tested for their ability to undergo apoptosis in
response to treatment with either ADR or CPT. (A) Effects of ectopic
Bcl-2 expression on the response to ADR treatment. The results shown (±1 SE) are those obtained after a 42-hour exposure to the indicated concentrations of the drug. (B) Effects of ectopic Bcl-2 expression in
response to a 48-hour exposure to the indicated concentrations of CPT.
(C) Effects of ectopic Bcl-XL expression in response to a
48-hour exposure to the indicated concentrations of CPT. The results
shown are representative of three experiments ±1 SE.
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It was possible that the overexpression of Bcl-2 or Bcl-XL
altered the levels of transfected c-myc or max proteins, either by
directly altering their in vivo half-lives, or by affecting the rates
of transcription of their mRNAs. To examine this, we compared the
levels of c-myc and max proteins in 32D-neo, 32D-c-myc, and 32D-max
with the levels in the same cell lines transfected with either Bcl-2 or
Bcl-XL expression vectors. As seen in Fig 6, neither Bcl-2 nor Bcl-XL
significantly affected the levels of c-myc or max proteins. In
comparison to endogenous c-myc, all cell lines derived from 32D-c-myc
expressed 2 to 3 times as much exogenous protein. As expected, the
levels of c-myc in each of the 32D-c-myc cell lines were unaffected by
the removal of IL-3 (Fig 6A). In the case of cultures derived from the
starting 32D-max cell line, all expressed 5 to 10 times more max
protein in comparison to untransfected controls (Fig 6B). The levels of
max in these lines were also unaffected by the coexpression of Bcl-2 or
Bcl-XL.
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| Fig 6.
Levels of c-myc and max proteins in 32D cell lines. (A)
c-myc levels. From the indicated cell lines, 50 µg of protein was resolved by SDS-PAGE, Western blotted, and probed with a polyclonal anti-c-myc antibody.40 Lane 1 shows the level of
endogenous c-myc protein in logarithmically growing 32D-neo cells. Lane
2 shows the disappearance of c-myc in the same cells after a 6-hour deprivation of IL-3. In lanes 3 through 14, the indicated cultures were
deprived of IL-3 for 6 hours to deplete endogenous c-myc stores. Note
the complete absence of endogenous c-myc protein in IL-3-deprived
32D-neo and 32D-max cells. Lanes 4, 7, 10, and 13 show that c-myc
arising from the transfected expression plasmid was expressed at levels
2 to 3 times that of endogenous c-myc and was not downregulated after
IL-3 deprivation. (B) max levels. The indicated cell lines were labeled
with 35S-Translabel as previously
described.18 After lysis in triple detergent
radioimmunoprecipitation buffer, max proteins were immunoprecipitated with a polyclonal rabbit anti-max antibody, resolved by SDS-PAGE, and
subjected to autoradiography. The arrow indicates the position of
endogenous and overexpressed max(L).
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| |
DISCUSSION |
Numerous reports have established that deregulated c-myc expression
promotes apoptosis in various settings, including those resulting from
serum deprivation of primary fibroblasts and the removal of cytokines
from hematopoietic cells.5,6,10 c-myc overexpression has
also been reported to alter the sensitivities of hematopoietic cell
lines to select chemotherapeutic agents.31,41 Recently, we
have shown that overexpression of the 160 amino acid isoform of max,
the obligate c-myc heterodimeric partner, can also promote apoptosis in
NIH 3T3 fibroblasts and in 32D myeloid cells in response to serum or
IL-3 deprivation, respectively.18 The proapoptotic effect
of max is quite strong and rivals or even exceeds that of c-myc.
Despite the similar proapoptotic phenotypes imparted to 32D cells by
c-myc and max, several indirect lines of evidence suggest that they
arise through distinct pathways. For example, although it has been
reported that apoptosis mediated by c-myc requires dimerization with
max,7 the converse does not seem to be true. This is based
on our observations that after the removal of IL-3 from all 32D cell
lines, the levels of endogenous c-myc transcripts and protein rapidly
decline to undetectable levels within 3 to 6 hours, long before
apoptosis becomes evident (Fig 6A) (H.Z. and E.V.P., unpublished
observations).
In the current work, the differential apoptotic responses of 32D-c-myc
and 32D-max cells to chemotherapeutic agents lend further support to
the notion that c-myc and max operate through separate, although
possibly overlapping, biochemical pathways (Fig 2). The ability of IL-3
to protect 32D-max cells against all agents tested (except CPT) is not
seen in the case of 32D-c-myc cells. Instead, these behave much like
control 32D-neo cells on which the effects of drug treatment and IL-3
deprivation are additive. More direct evidence to support the existence
of distinct c-myc and max apoptotic pathways is provided by our
observation that they are differentially affected by the overexpression
of Bcl-2 and Bcl-XL. Whereas Bcl-2 completely protected all
cell lines against the proapoptotic effects of both IL-3 deprivation
and drug treatment, Bcl-XL provided virtually no protection
against IL-3 removal in 32D-c-myc cells, despite protecting them as
well as Bcl-2 against drug-mediated apoptosis.
In 32D-c-myc and 32D-max cells, cellular demise in response to IL-3
withdrawal can be viewed as the sum of two apoptotic events, the first
of which, the "basal" event, is that provided by IL-3 removal
itself (and defined as that occurring in control 32D-neo cells), and
the second of which is that imposed by overexpressed c-myc or max (the
"c-myc" or "max" event). The inability of
Bcl-XL to provide significant protection against apoptosis
after IL-3 removal in 32D-c-myc cells suggests that the overexpression
of c-myc affects the basal event in such a way as to make it
unresponsive to what should otherwise be a fully protective
antiapoptotic stimulus.
Perhaps the most important outcome of this study is that it now
provides us with the ability to order Bcl-2 and Bcl-XL
along the c-myc and max apoptotic pathways. Thus, in the simplest of models, Bcl-2 exerts its effect downstream of both c-myc and max to
protect against apoptosis after either IL-3 withdrawl or drug treatment. Similarly, Bcl-XL seems to function downstream
of c-myc and max to prevent apoptosis in response to cytotoxic drug
treatment. In contrast, Bcl-XL appears to act downstream of
max but upstream of c-myc in cells deprived of IL-3. Although a number
of biochemical and biological properties of Bcl-2 and
Bcl-XL have been described,21,38,39,42-44 it is
not yet clear which of these, if any, is important for the apoptotic
events we have studied here. Nevertheless, this work provides a
framework within which it should be possible to use these and other
modulators of apoptosis to begin to dissect the cell death pathways
used by c-myc, max, and other members of the c-myc oncoprotein family.
| |
FOOTNOTES |
Submitted November 17, 1997;
accepted March 25, 1998.
Supported by NIH Grant No. HL33741 to E.V.P.
Address reprint requests to Edward V. Prochownik, MD, PhD, Section of
Hematology/Oncology, Children's Hospital of Pittsburgh, 3705 Fifth
Ave, Pittsburgh, PA 15213; e-mail: edward_prochownik{at}poplar.chp.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We are grateful to Yusuf Hannun, Dan Johnson, and Craig Thompson for
providing plasmids; to Dan Johnson for advice; and to Don Wojchowski
for reading the manuscript.
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Abstract
Introduction
Abstract