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Distinct Apoptotic Responses Imparted by c-myc and max
By Chadd E. Nesbit, Saijun Fan, Hong Zhang, and Edward V. Prochownik
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 maxoverexpressing 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 ab-
sence 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 maxoverexpressing 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.
r 1998 by The American Society of Hematology.
T
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.
HE 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 cytokinedependent 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-helixleucine 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
Blood, Vol 92, No 3 (August 1), 1998: pp 1003-1010
MATERIALS AND METHODS
Plasmids. The construction of pSVLneo-max(L) and pSVLneo-cmyc 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
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.
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@
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’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9203-0009$3.00/0
1003
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1004
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 pSVLneomax(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 BgIIIlinearized 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.
NESBIT ET AL
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 phosphatebuffered saline (PBS), and lysed in standard 13 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 1 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 peroxidaseconjugated 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 3 105 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-
Fig 1. Susceptibility of 32 D-neo (h), 32D-max (s), and 32D-c-myc (e) cells to six different antineoplastic agents in the presence of IL-3. Cells of
G95% 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-mL 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
(61 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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APOPTOSIS BY myc AND max
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 3 105/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 3 104 cells were analyzed. Quantitation was
performed using single histogram statistics.18
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 ex-
1005
presses 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 maxmediated 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.
Fig 2. 32D-neo (h), 32D-c-myc (e), and 32D-max (s) 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 61 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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1006
In contrast, 32D-max cells were indistinguishable from 32Dneo 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.
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.
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.
NESBIT ET AL
Table 1. Cell Cycle Profiles After Drug Exposure
% of Cells in Phase
Cell Line
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
32D-neo
32D-c-myc
32D-max
32D-neo
32D-c-myc
32D-max
32D-neo
32D-c-myc
32D-max
32D-neo
32D-c-myc
32D-max
32D-neo
32D-c-myc
32D-max
32D-neo
32D-c-myc
32D-max
32D-neo
32D-c-myc
32D-max
Drug
G0/G1
S
G2/M
None
None
None
ADR
ADR
ADR
CDDP
CDDP
CDDP
CPT
CPT
CPT
N-Mus
N-Mus
N-Mus
Taxol
Taxol
Taxol
VP16
VP16
VP16
48
45
42
31
16
22
22
26
39
32
9
15
39
26
36
8
7
16
38
41
50
30
31
36
14
7
8
35
25
36
13
16
30
22
24
32
15
14
21
22
27
23
22
24
22
55
77
70
43
49
25
55
75
55
39
50
32
77
79
63
40
32
27
The indicated cell lines, in log-phase growth, were exposed for 16
hours to ADR (0.125 µmol/L), CDDP (1 µmol/L), CPT (0.125 µmol/L),
N-Mus (0.1 µmol/L), Taxol (0.5 µmol/L), or VP16 (0.1 µmol/L). Nuclei
were then stained with propidium iodide, and their cell cycle distribution determined by fluorescence-activated cell sorting as previously
described.18 Preliminary experiments indicated that the distributions
shown were reached by 12 hours after drug exposure and did not
change significantly before the onset of detectable apoptosis. None of
the conditions used here induced more than 5% to 10% apoptosis as
determined by the presence of subdiploid nuclear populations and
trypan blue staining.
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).
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).
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APOPTOSIS BY myc AND max
A
1007
B
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 mg 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.
From these results, we conclude that Bcl-XL exerts a more
restrictive antiapoptotic effect in these cell lines.
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.
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).
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 61 SE.
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1008
NESBIT ET AL
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 (61 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 61 SE.
The levels of max in these lines were also unaffected by the
coexpression of Bcl-2 or Bcl-XL.
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
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APOPTOSIS BY myc AND max
A
B
1009
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.
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.
REFERENCES
Fig 6. Levels of c-myc and max proteins in 32D cell lines. (A) c-myc
levels. From the indicated cell lines, 50 mg 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 SDSPAGE, and subjected to autoradiography. The arrow indicates the
position of endogenous and overexpressed max(L).
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-cmyc 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
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1998 92: 1003-1010
Distinct Apoptotic Responses Imparted by c-myc and max
Chadd E. Nesbit, Saijun Fan, Hong Zhang and Edward V. Prochownik
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