CDK2 is required by MYC to induce apoptosis.
ABSTRACT Depending upon the cellular and physiologic context, the overexpression of the MYC proto-oncogene results in rapid cell growth, proliferation, induction of apoptosis and/or proliferative arrest. What determines the precise consequences upon MYC activation is not clear. We have found that cyclin-dependent kinase 2 (CDK2) is required by MYC to induce apoptosis. MYC-induced apoptosis was suppressed in mouse embryonic fibroblasts (MEF) knocked out for Cdk2 or normal human fibroblasts (NHF) upon expression of the CDK2 inhibitor p27 or treated with RNAi directed at CDK2. Knockout of Cdk2 did not prevent MYC from inducing p53 and Bim. The inhibition of CDK2 did not prevent apoptosis induced by the DNA damaging agent etoposide. Our results surprisingly suggest that CDK2 defines whether MYC induction causes apoptosis.
- [Show abstract] [Hide abstract]
ABSTRACT: The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC's diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer.Cold Spring Harbor Perspectives in Medicine 06/2014; 4(6). · 7.56 Impact Factor
Article: Urban legends: pemphigus vulgaris.[Show abstract] [Hide abstract]
ABSTRACT: Pemphigus vulgaris (PV) is the most common type of pemphigus. PV pathogenesis is still debated, and treatment remains challenging. We investigated five controversial topics: (1) What are the target antigens in PV? (2) Do desmogleins adequately address PV pathophysiology? (3) How does acantholysis occur in PV? (4) Is PV still a lethal disease? (5) What is the role of rituximab (RTX) in PV treatment? Results from extensive literature searches suggested the following: (1) Target antigens of PV include a variety of molecules and receptors that are not physically compartmentalized within the epidermis. (2) PV is caused by a variety of autoantibodies to keratinocyte self-antigens, which concur to cause blistering by acting synergistically. (3) The concept of apoptolysis distinguishes the unique mechanism of autoantibody-induced keratinocyte damage in PV from other known forms of cell death. (4) PV remains potentially life-threatening largely because of treatment side effects, but it is uncertain which therapies carry the highest likelihood of lethal risk. (5) RTX is a very promising treatment option in patients with widespread recalcitrant or life-threatening PV. RTX's cost is an issue, its long-term side effects are still unknown, and randomized controlled trials are needed to establish the optimal dosing regimen.Oral Diseases 12/2011; 18(5):442-58. · 2.40 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The goal of contemporary research in pemphigus vulgaris and pemphigus foliaceus is to achieve and maintain clinical remission without corticosteroids. Recent advances of knowledge on pemphigus autoimmunity scrutinize old dogmas, resolve controversies, and open novel perspectives for treatment. Elucidation of intimate mechanisms of keratinocyte detachment and death in pemphigus has challenged the monopathogenic explanation of disease immunopathology. Over 50 organ-specific and non-organ-specific antigens can be targeted by pemphigus autoimmunity, including desmosomal cadherins and other adhesion molecules, PERP cholinergic and other cell membrane (CM) receptors, and mitochondrial proteins. The initial insult is sustained by the autoantibodies to the cell membrane receptor antigens triggering the intracellular signaling by Src, epidermal growth factor receptor kinase, protein kinases A and C, phospholipase C, mTOR, p38 MAPK, JNK, other tyrosine kinases, and calmodulin that cause basal cell shrinkage and ripping desmosomes off the CM. Autoantibodies synergize with effectors of apoptotic and oncotic pathways, serine proteases, and inflammatory cytokines to overcome the natural resistance and activate the cell death program in keratinocytes. The process of keratinocyte shrinkage/detachment and death via apoptosis/oncosis has been termed apoptolysis to emphasize that it is triggered by the same signal effectors and mediated by the same cell death enzymes. The natural course of pemphigus has improved due to a substantial progress in developing of the steroid-sparing therapies combining the immunosuppressive and direct anti-acantholytic effects. Further elucidation of the molecular mechanisms mediating immune dysregulation and apoptolysis in pemphigus should improve our understanding of disease pathogenesis and facilitate development of steroid-free treatment of patients.Autoimmunity 09/2011; 45(1):7-35. · 2.75 Impact Factor
©2006 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
progression.13,45-48Eilers and colleagues tested this possibility directly by injecting p27
into the nuclei of cells and showing that this failed to prevent MYC from inducing apop-
tosis.46However, not all reports confirm that MYC induces apoptosis independent of the
cell cycle. Most notably, Cyclin A is required by MYC to induce apoptosis.49
Here, we describe that the suppression of Cyclin E/CDK2 through the cell cycle
inhibitor p27, RNAi directed against CDK2, or cells knocked out for CDK2 all suppressed
the ability of MYC, to induce apoptosis. These results demonstrate that Cyclin E/CDK2
[Cell Cycle 5:12, 1342-1347, 15 June 2006]; ©2006 Landes Bioscience
1342 Cell Cycle2006; Vol. 5 Issue 12
Dean W. Felsher1
1Division of Oncology; Departments of Medicine and Pathology; Stanford University;
Stanford, California USA
2Mouse Cancer Genetics Program; National Cancer Institute; Frederick, Maryland USA
*Correspondence to: Dean W. Felsher; Division of Oncology; Departments of
Medicine and Pathology; 269 Campus Drive; Stanford University; Stanford,
California 94305-5151 USA; Tel.: 650.498.5269; Fax: 650.725.1429; Email:
Original manuscript submitted: 04/20/06
Revised manuscript submitted:04/24/06
Manuscript accepted: 04/24/06
Previously published online as a CellCycleE-publication:
MYC, CDK2, apoptosis, Cyclin E, p27
We thank the members of the Felsher laboratory
for their many helpful suggestions. We thank
Dr. Osamu Tetsu (UCSF) for providing the
CDK2 RNAi target sequence and transfection
protocol. We also thank Drs. W.J. Nelson and
Soichiro Yamada for help performing time-lapse
video microscopy. This work was supported by
the National Cancer Institute Grants 1R01
CA89305-01A1, 3RO1 CA89305-0351, 1RO1
CA105102 (to D.F.) and T32 CA09151 (to
Supplementary material can be found at:
CDK2 Is Required By MYC To Induce Apoptosis
Depending upon the cellular and physiologic context, the overexpression of the MYC
proto-oncogene results in rapid cell growth, proliferation, induction of apoptosis and/or
proliferative arrest. What determines the precise consequences upon MYC activation is
not clear. We have found that cyclin-dependent kinase 2 (CDK2) is required by MYC to
induce apoptosis. MYC-induced apoptosis was suppressed in mouse embryonic fibrob-
lasts (MEF) knocked out for Cdk2 or normal human fibroblasts (NHF) upon expression of
the CDK2 inhibitor p27 or treated with RNAi directed at CDK2. Knockout of Cdk2 did
not prevent MYC from inducing p53 and Bim. The inhibition of CDK2 did not prevent
apoptosis induced by the DNA damaging agent etoposide. Our results surprisingly sug-
gest that CDK2 defines whether MYC induction causes apoptosis.
MYC regulates the expression of genes responsible for inducing cell growth and prolif-
eration and regulating differentiation, apoptosis, angiogenesis, cellular adhesion and DNA
repair.1-4MYC overexpression causes tumorigenesis by inducing autonomous and
unrestrained cellular proliferation and growth, cellular immortalization, genomic destabi-
lization, blocked cellular differentiation, inappropriate angiogenesis and abnormal cellular
MYC overexpression is restrained from causing tumorigenesis by at least two different
molecular mechanisms. Under some circumstances, MYC activation induces apoptosis
which serves as a barrier to otherwise unchecked cellular proliferation.4,22MYC over-
expression also can be inhibited from inducing unrestrained proliferation through cell
cycle, which at least in part is mediated through p53.23,24Whether MYC induces apoptosis
or proliferation may be determined by the specific differentiative context of a cell that
epigenetically regulates the particular gene expression program elicited.25
There are many clues to the mechanism by which MYC induces apoptosis. First, MYC
induces apoptosis in specific contexts.13,22,26In particular, MYC activation induces apop-
tosis in vitro in serum-starved fibroblasts.22Second, MYC appears to indirectly regulate
multiple apoptotic pathways.4,22,27-29MYC can result in the stimulation of cytochrome C
release through the stabilization of the pro-apoptotic protein Bax.30-34Similarly, MYC
stabilizes p19ARF resulting in activation of the p53 pathway.5,27,29,35MYC-induced
apoptosis can be suppressed through the loss of p53 function or expression of BCL2,36
activation of PI3K37and influence of specific growth factors.13,22,38MYC may also induce
apoptosis through the induction of FADD/FAS.39MYC has been reported to induce
apoptosis through suppression of the anti-apoptotic regulators BCL2 and BCLXL.40-42
The pro-apoptotic protein Bim was recently shown to be increased in B cells overexpressing
MYC and Bim was found to contribute to MYC-induced apoptosis.43,44Thus, multiple
mechanisms are likely to contribute to MYC’s ability to induce apoptosis in different
Previous studies have suggested that MYC induces apoptosis independent of cell cycle
www.landesbioscience.com Cell Cycle1343
plays a surprisingly critical role in regulating whether MYC induces
proliferation versus apoptosis.
MATERIALS AND METHODS
Cell lines. All cells were cultured in DMEM without phenol red
supplemented with 10% FBS and Penicillin/Streptomycin. MYC
was introduced into the Wild-type MEFs and CDK2 knocked out
(Cdk2 KO) MEFs through retroviral infection with the MSCV
MYCER GFP virus. MYCER was cloned into the EcoRI site of the
MSCV IRES GFP retroviral vector purchased from Clontech.
Retroviruses were prepared by transfecting MSCV MYCERIRES
GFP construct using lipofectamine in a Phoenix ecotropic packaging
line, generously provided by Dr. Garry Nolan (Stanford University).
FACS sorted MYCER GFP expressing cells were utilized. MYC
expression was induced by addition of 4-hydroxy-tamoxifen (TAM)
for 24 or 48 hours.
The generation of Normal Human Fibroblasts (NHFs) that
express MYCER was described previously.24NHF MYCER cells
were treated with NHF MYCER cells were treated with
4-hydroxy-tamoxifen (TAM) at concentrations indicated to induce
MYC activity, as we have previously described.14To introduce p27
expression in the NHF MYCER cells, these cells were infected with
a retrovirus derived from pBabe Neo p27, generously provided by
Dr. Bruno Amati (European Institute of Oncology, Milan, Italy).
Retroviruses were prepared by transfecting this plasmid construct
using lipofectamine in a Phoenix amphotropic packaging line. The
frequency of cells productively infected by the p27 retrovirus was
determined by immunofluorescent staining for p27 protein expression
using an anti-p27 primary antibody, sc-1641 (Santa Cruz, CA), and
Alexa conjugated secondary antibody (Molecular probes, OR).
Staining of nuclei was done by DAPI (Vector laboratories, CA).
RNAi transfection. NHF MYCER cells were transfected with
cdk2 small interfering (si) RNA using Oligofectamine (Invitrogen, CA)
exactly as described.50Measurement of S, M phase and apoptosis
was done 48 hours after transfection.
Apoptosis assays. To perform Annexin V-PE/7AAD staining
Wild-type and Cdk2 KO MYCER GFP MEFs were induced with
0.1 µM TAM for 48 hours. Cells were washed and stained with
Phycoerythrin (PE) labeled Annexin V (Annexin V-PE) and
7-amino-actinomycin D (7AAD) (Beckton Dickinson, CA). 5000
cells were analyzed by flow cytometry and the Annexin V-PE
-/7AAD-, Annexin V-PE+/7AAD-, and Annexin V-PE+/7AAD+and
Annexin V-PE-/7AAD+populations were enumerated. The two
populations of Annexin V-PE+/7AAD-, Annexin V-PE+/7AAD+and
Annexin V-PE-/7AAD+, Annexin V-PE+/7AAD+have been found to
correspond to early and late apoptotic cells, and both late apoptotic
and necrotic cells, respectively.
Time-lapse video microscopy was performed using an Intelligent
Imaging Innovations imaging system with a Zeiss Axiovert 200M
including 175 Watt Xenon light source with a dual galvanometric
filter changer, a Coolsnap interline CCD camera, an x-y motorized
stage, and a Plan-Neofluor 40X 0.75NA objective with GFP and
DIC optics. Cells were mounted in a closed live-cell imaging chamber
with DMEM phenol red free media supplemented with 25 mM
HEPES, and placed on the microscope stage with a custom stage
heater to maintain the temperature at 37˚C. Images of Wild-type
and Cdk2 KO MYCER GFP MEFs induced with 0.5 µM TAM for
24 hours were acquired every 10 minutes for 12 hours.
Apoptosis was also measured by the frequency of Trypan blue
negative versus positive cells and the frequency of sub-G1DNA
content as measured by flow cytometric analysis (FACS) as described.9
Briefly, the cells were trypsinized, centrifuged, washed twice with
PBS and fixed in cold 70% ethanol. Staining with Propidium Iodide
was performed and the cells were then analyzed by flow cytometry.
H1 histone kinase assay. To measure Cyclin/CDK activity,
lysates were prepared from MEFs after MYC induction using an
immunoprecipitation buffer containing 50 mM Tris HCl pH 8.0,
150 mM NaCl, 1% Triton X-100, 1 mM DTT, 10 mM NaF, 0.5 mM
Sodium Vanadate, 100 µg/ml PMSF and a 1X protease inhibitor
cocktail (Calbiochem, CA). The protein concentration was measured
using the BCA kit from Pierce Biotechnology (Rockford, IL). Two-
hundred grams of lysate was incubated with Protein-A Sepharose
beads and antibody for Cyclin E or CDK2 for 2 hours at 4˚C. The
beads were washed four times with lysis buffer and twice with kinase
buffer and used for kinase assay. Kinase assays were performed using
a kinase assay buffer containing 50 mM Tris HCl pH 7.5, 10 mM
MgCl2, 1 mM DTT, 15 µg histone, 30 µM ATP and 3 µCi γP32
ATP. The reaction was stopped after 30 minutes by heating with 2X
lammli loading buffer for 5 min at 95˚C. The immunoprecipitated
products were separated by SDS-PAGE and autoradiography was
performed. Antibodies against Cyclin E and CDK2 antibodies were
purchased from Santa Cruz Biotechnology,.
Western analysis. Western analysis was performed using conven-
tional techniques. Bax, Bad, Bcl2, BclXLantibodies were purchased
from Pharmingen; CA. Bim antibody was purchased form Stressgen
(Canada). The α-tubulin and p53 antibodies were purchased from
Calbiochem, CA and Vector Laboratories, CA respectively.
DNA content and cell cycle analysis. BrdU labeling was per-
formed as described.9Briefly, actively growing cells were pulsed with
10 µM BrdU for 1 hour. Staining with anti-BrdU FITC and
Propidium Iodide was performed as described using the anti-BrdU
FITC antibody manufactured from Beckton Dickinson (San Jose, CA).
BrdU immunofluorescence was performed according to the manu-
facturer’s protocol. Nuclei were stained with Propidium Iodide (PI).
Histone-3-Phosphate staining of mitotic cells was performed as
CDK2 is required by MYC to induce apoptosis. Upon investi-
gation of the mechanisms by which MYC overexpression perturbs
cell cycle regulation, we serendipitously observed that the inhibition
of CDK2 function impaired apoptosis. We utilized Cdk2 Wild-type
or knockout (KO) mouse embryonic fibroblasts (MEFs) that had
been stably infected with a retrovirus containing MYCER and GFP.
First, by FACS for Annexin V-PE and 7AAD staining (Fig. 1A
and B), that would detect apoptotic and necrotic cells respectively,
we found that upon MYC activation, 48% of the Wild-type MEFs,
while only 10% of the Cdk2 KO cells stained positive for Annexin
V-PE, but negative for 7AAD. Second, the fraction of sub-G1apop-
totic cells upon MYC induction as measured by FACS analysis of
propidium iodide stained cells in wild-type cells was 64%, whereas
in the Cdk2 KO cells was 15%, similar to the background (Fig. 1C).
However, loss of CDK2 did not block apoptosis caused by treatment
with etoposide (Fig. 1C). Third, by time-lapse video microscopy
(Figs. 2A and B), we found that upon MYC induction in wild-type
MEFs, 59% of cells underwent apoptosis, however in Cdk2
knockout MEFs only 8% of cells underwent apoptosis (Fig. 2A and
B). Moreover, after 24 hours of MYC induction, wild-type MEFs
grew to a low cell density and 60% of the cells were trypan blue
CDK2 is Required by MYC to Induce Apoptosis
CDK2 is Required by MYC to Induce Apoptosis
1344 Cell Cycle2006; Vol. 5 Issue 12
positive, whereas the Cdk2 KO MEFs grew to high density and only
3% of the cells were trypan blue positive (Fig. 2C and D). Therefore,
absence of CDK2 blocks MYC-induced apoptosis and cell death.
Addition of Tamoxifen induced apoptosis only in the Wild-type
MEF MYCER cells, but not in the Wild-type MEF cells
(Supplementary Fig. 1). Thus, MYC overexpression and not tamox-
ifen induces apoptosis in the Wild-type MEF MYCER
We speculated that that the loss of Cdk2 by blocking
MYC-induced apoptosis may influence cellular proliferation.
As expected, MYC induction alone resulted in a modest
decrease of cellular proliferation in the wild-type MEFs most
likely due to increased apoptosis (Fig. 3A). As predicted,
the loss of Cdk2 resulted in a further increase in cellular
proliferation upon MYC induction (Fig. 3A), that was not
associated with significant changes in the frequency of cells
in S phase, as measured by BRDU incorporation (Fig. 3B).
We considered that is was possible that MYC was inducing
Cyclin E associated histone kinase activity independent of
Cdk2. To address this possibility, we confirmed that H1
associated kinase activity associated with Cyclin E or Cdk2
was absent in the Cdk2 KO cells regardless of MYC activa-
tion (Fig. 3C). Thus, MYC does not appear to induce
Cyclin E associated activity independent of Cdk2. Instead,
the loss of Cdk2 most likely results in increased cellular
proliferation upon MYC activation in MEFs by suppressing
Loss of Cdk2 does not prevent MYC from inducing
pro-apoptotic signals. MYC has been demonstrated to
induce apoptosis through the indirect activation of the pro-
apoptotic regulators Bax and p535,27,29-35and suppression
of the anti-apoptotic regulators BCL2 and BCLXL.40-42As
expected, in wild-type MEFs, MYC activation resulted in
decreased protein levels of BCL2, BCLXL and induction of
p53, but no changes in expression of Bad or Bax (Fig. 4).
In Cdk2 KO MEFs, basal levels of anti-apoptotic proteins,
Bcl2 and BclXL were reduced, and Bim was increased,
which would be expected to increase apoptosis. Upon MYC induc-
tion in Cdk2 KO MEFs, we observed further reduction of levels of
BclXL, further increase in levels of Bim and p53, all of which would
be expected promote apoptosis. In Cdk2 KO MEFs, basal levels of
Bcl2 were suppressed relatively to wild-type MEFs. Upon MYC
induction, Bcl2 was induced (Fig. 4). Thus, MYC activation in
Cdk2 KO cells generally results in enhanced induction of
pro-apoptotic protein expression.
MYC requires CDK2 to induce apoptosis, but not cell cycle
in normal human cells. To determine if MYC requires CDK2 to
induce apoptosis and/or cell cycle transit in NHFs, NHF
MYCER cells were infected with retroviral vectors containing p27
to inhibit Cyclin E/CDK2 or RNAi directly specifically at CDK2.
We confirmed that cells infected with retrovirus containing p27,
but not a control empty retroviral vector expressed robust levels
Figure 2. Time-lapsed video microscopy of MYC activation in Cdk2 wild-
type and KO MEFs. Wild-type and Cdk2 KO MYCER GFP MEFs 24 hours
after MYC induction with TAM is indicated (A). The consequences of
MYC activation for 28.5 hours in the Wild-type and Cdk2 KO MYCER
GFP MEFs are shown (B). While the Cdk2 cells remain proliferative (ii),
a significant number of the Wild-type MEFs undergo apoptosis (i). The
Wild-type MEFs become rounded after MYC activation for 26 hours, with
evidence of blebbing of the plasma membrane, shrinkage after 27.3
hours and fragmentation into apoptotic bodies after 28.5 hours. The
Cdk2 KO MYCER cells do not undergo apoptosis 26, 27.3 or 28.5 hours
after MYC activation. The pixel size of the images are 0.322
micron/pixel. (C) Morphology of Wild-type and Cdk2 KO MYCER GFP
MEFs by phase microscopy is shown in the absence and presence of
MYC activation for 24 hours with 0.1 µM TAM. (D) Percentage of dead
cells was determined by trypan blue staining.
Figure 1. Absence of Cdk2 inhibits MYC-induced apoptosis. Annexin V-PE/7AAD
(A and B) and Propidium Iodide staining (C). Wild-type and Cdk2 KO MEFs were
infected with MSCV MYCER GFP retrovirus. GFP positive cells were FACS sorted and
MYC expression was induced by addition of 0.1 µM TAM for 48 hours. Wild-type
and Cdk2 KO MEFs expressing MYCER GFP were either not treated or treated with
TAM and stained with 7AAD and Annexin V-PE. The percentage of cells after staining
is indicated in each quadrant (A). The frequency of Annexin V-PE+/7AAD-positive
cells are summarized (B). (C) Inhibition of Cyclin E/Cdk2 does not inhibit apoptosis
mediated by MYC but not etoposide. Wild-type and CdkK2 KO cells were treated
with TAM (0.1 µM) to induce MYC. Cells were treated with Etoposide (ET) (10 µM)
as indicated. To measure apoptosis, the cells were stained with Propidium Iodide (PI)
and analyzed by FACS. The percentage of apoptotic cells was determined from the
frequency of sub-G1DNA content.
www.landesbioscience.com Cell Cycle1345
effects on effectors of apoptosis.4,22,27-29In particular, MYC has been
shown to induce pro-apoptotic regulators Bax and p535,27,29-35and
suppress anti-apoptotic regulators Bcl2 and BclXL.40-42However,
although loss of CDK2 blocked MYC-induced apoptosis, CDK2
CDK2 is Required by MYC to Induce Apoptosis
of p27 as detected by immunofluorescence (Fig. 5A).
Similarly, by Western analysis we confirmed that RNAi to
CDK2 suppressed CDK2 protein expression by 80%
(Supplementary Fig. 2).
The inhibition of Cyclin E/CDK2 activation through
p27 or RNAi directed at CDK2 induced cell cycle arrest in
NHFs as measured by the frequency of cells in S phase, by
BrdU incorporation, or mitotic index by Histone-3
Phosphate (H3P) staining (Fig. 5B, C, E, F and G). MYC
activation alone induced a modest increase in S phase and
mitotic index. When we activated MYC and inhibited
Cyclin E/CDK2 through p27 or RNAi, MYC could still
induce DNA synthesis and mitosis. Thus, MYC can induce
cell cycle transit independent of Cyclin E/CDK2 activity in
We found that MYC-induced robust apoptosis/cell death in
NHFs cells grown in vitro as expected (Fig. 5D and H,
Supplementary Fig. 3). When Cyclin E/CDK2 was inhibited by p27
overexpression or transfection with CDK2 RNAi, MYC-induced
cell death was inhibited, as measured by Trypan blue (Fig. 5D and
H), and this was associated with decreased apoptosis, as measured by
Annexin V staining (Supplementary Fig. 3). Importantly, a control
RNAi had no effect on apoptosis induced by MYC (Supplementary
Fig. 3). Therefore, MYC requires CDK2 to induce apoptosis, but
not DNA synthesis or mitosis in NHFs.
Here, we have described evidence supporting the role of the cell
cycle regulatory gene product, CDK2, as a nodal point that appears to
be required for apoptosis induced by MYC activation. The inhibition
of CDK2 by p27 expression, using RNAi directed against CDK2 or
the knock-out of CDK2, suppressed MYC-induced apoptosis in
NHFs and MEFs, as measured by measuring viable cells by Trypan
blue staining, FACS/PI analysis, Annexin staining and/or time-lapse
video microscopy. Moreover, we demonstrated that MYC could
induce cell cycle transit, albeit attenuated, independent of CDK2
activity. Combined, our results suggest that MYC induces apoptosis
through a cell cycle dependent mechanism requiring CDK2 activa-
Our results are consistent with the report by Hoang and colleagues
that Cyclin A is required by MYC to induce apoptosis,49but in
discordance other reports that suggest that MYC induces apoptosis
independent of the cell cycle.13,45-48There are several possible
explanations. In contrast to these prior studies, we were able to confirm
our findings using a genetic approach in NHFs, using RNAi to target
CDK2, and in MEFs knocked out for Cdk2. Since it is possible to
over-ride the ability of p27 to suppress MYC induced apoptosis, the
differences in the level of suppression of Cdk2 may account for the
observation that microinjection of p27 failed to suppress MYC-
induced apoptosis in the Rat1A cells.46
MYC has been shown to induce apoptosis through a multitude of
Figure 3. MYC induces increased cellular proliferation independent
of Cdk2 activity in MEFs. MYC was induced with TAM (0.1µM) in
Wild-type and Cdk2 KO MYCER GFP MEFs. MYC induces S phase
entry independent of Cdk2 as determined by cell proliferation (A)
and BRDU incorporation (B). Wild-type and Cdk2 KO MYCER GFP
MEFs were either untreated or induced for MYC activation with
TAM for 3 days (A) or 24 hours (B). C) Cyclin E and Cdk2 associ-
ated histone kinase activities were measured in Wild-type and Cdk2
KO MYCER GFP cells in presence and absence of MYC activation.
Figure 4. Analysis of apoptosis related proteins upon MYC activation.
Western analysis of p53, BCLXL, BCL2, Bax, Bad, Bim and tubulin was
performed in the Wild-type and Cdk2 KO MEFs in presence and absence
of MYC activation with 0.1 µM TAM for 24 hrs. Each experiment was
repeated three times and a representative experiment is shown.
CDK2 is Required by MYC to Induce Apoptosis
1346Cell Cycle2006; Vol. 5 Issue 12
generally promoted the ability of MYC to induce pro-apoptotic
signals. In Cdk2 knockout MEFs, MYC activation still induced
pro-apoptotic gene products Bim and p53 and still suppressed
expression of anti-apoptotic gene product BCLXL. In Cdk2 knock-
out MEFs, BCL2 expression was suppressed and upon MYC activa-
tion BCL2 expression was now induced. The suppression of BCL2
expression could contribute to the decreased apoptosis upon MYC
activation in Cdk2. However, in context of the prop-apoptotic sig-
nals of the induction p53 and Bim and the suppression of BCLXL,
the induction of BCL2 alone would seem less likely to be sufficient
to abrogate MYC induced apoptosis.
Our results are consistent with the possibility that MYC induces
apoptosis through a mechanism coupled with cell cycle transit.
Inhibition of Cdk2 did not block apoptosis induced by the DNA
damage inducing agent etoposide suggesting that the effector path-
ways that mediate apoptosis are intact. Moreover, although known
to facilitate apoptosis induced by other stimuli, Cdk2 activation is
not thought to directly induce apoptosis.52,53
Thus, Cdk2 is not likely to directly mediate
MYC’s induction of apoptosis. Rather, Cdk2
activity appears to be required to permit
MYC to induce apoptosis. Consistent with
this interpretation, our accompanying paper
presents results that suggest that inhibition of
Cyclin E/Cdk2 prevents MYC overexpression
from inducing inappropriate DNA replication
(Deb Basu et al.).
Hence, MYC might induce apoptosis
under circumstances when cells are actively
proliferating, but would not induce apoptosis
in cells that are quiescent, arrested or capable
of undergoing cell cycle arrest. Indeed, we
have described that MYC overexpression in
NHFs induces a p53-dependent proliferative
arrest and not apoptosis.24Similarly, in vivo,
MYC overexpression in vivo in adult murine
hepatocytes induced a proliferative arrest, but
in neonatal hepatocytes that were proliferating
or in proliferating liver tumor cells, was apop-
tosis observed.54Hence, apoptosis was
observed as mechanism of last resort for con-
taining MYC overexpression.
Notably, the inhibition of CDK2 by RNAi or p27 induced pro-
liferative arrest of NHFs, but failed to prevent MYC activation from
inducing cell cycle transit, albeit it now occurred at an attenuated
rate. Our results are discordance with many recent publications that
confirm that CDK2 activity is not required for cell cycle tran-
sit.50,55-58Aleem et al., recently showed that Cyclin E binds to and
activates Cdc2.59Our results may reflect that there are differences in
the role of CDK2 in cell cycle transit in mouse versus human cells,
embryonic fibroblasts versus foreskin fibroblasts. Finally, there may
be differences between the chronic knockout versus the acute loss of
Cdk2, but this needs to be experimentally validated. MYC activation
may be able to at least partially bypass the requirement of CDK2
activity for cell cycle transit.
We conclude that MYC’s ability to induce apoptosis may be
coupled to the regulation of the cell cycle and cellular proliferation.
CDK2, a cell cycle regulatory gene product, may serve as a previously
Figure 5. MYC requires CDK2 to induce apoptosis,
but not cell cycle entry in Normal Human
Fibroblasts. NHF MYCER cells were retrovirally
infected by the pBabeneop27 virus (A–D) or trans-
fected with CDK2 RNAi (E–H). MYC activation was
induced by the addition of 0.2 µM 4-hydroxy-
tamoxifen (TAM). Successful infection of the NHF
MYCER cells with the pBabeneop27 retrovirus was
confirmed by immunofluorescence (A). MYC
induces S phase (B, E and F) and M phase (C and
G) independent of CDK2. The percentage of S
phase was determined by measuring BrdU positive
foci and M phase was determined by staining the
cells with the mitotic marker Histone-3-phosphate.
CDK2 is required for MYC-induced apoptosis (D
and H). Adherent and floating cells were collected
and stained with Trypan blue to determine the
percentage of dead cells after p27 overexpression
by retroviral infection (D) or inhibition of CDK2 by
www.landesbioscience.comCell Cycle 1347
unrecognized essential nodal point that may define whether MYC
overexpression induces proliferation versus apoptosis.
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CDK2 is Required by MYC to Induce Apoptosis