CDK2 Is Required By MYC To Induce Apoptosis

[Cell Cycle 5:12, 1342-1347, 15 June 2006]; ©2006 Landes Bioscience

CDK2 Is Required By MYC To Induce Apoptosis Report

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.

Original manuscript submitted: 04/20/06 Revised manuscript submitted: 04/24/06 Manuscript accepted: 04/24/06

MYC regulates the expression of genes responsible for inducing cell growth and proliferation and regulating differentiation, apoptosis, angiogenesis, cellular adhesion and DNA repair.1-4 MYC overexpression causes tumorigenesis by inducing autonomous and unrestrained cellular proliferation and growth, cellular immortalization, genomic destabilization, blocked cellular differentiation, inappropriate angiogenesis and abnormal cellular adhesion.5-21 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.4,22 MYC overexpression also can be inhibited from inducing unrestrained proliferation through cell cycle, which at least in part is mediated through p53.23,24 Whether 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.13,22,26 In particular, MYC activation induces apoptosis in vitro in serum-starved fibroblasts.22 Second, MYC appears to indirectly regulate multiple apoptotic pathways.4,22,27-29 MYC can result in the stimulation of cytochrome C release through the stabilization of the pro-apoptotic protein Bax.30-34 Similarly, MYC stabilizes p19ARF resulting in activation of the p53 pathway.5,27,29,35 MYC-induced apoptosis can be suppressed through the loss of p53 function or expression of BCL2,36 activation of PI3K37 and influence of specific growth factors.13,22,38 MYC may also induce apoptosis through the induction of FADD/FAS.39 MYC 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.43,44 Thus, multiple mechanisms are likely to contribute to MYC’s ability to induce apoptosis in different physiological contexts. Previous studies have suggested that MYC induces apoptosis independent of cell cycle progression.13,45-48 Eilers and colleagues tested this possibility directly by injecting p27 into the nuclei of cells and showing that this failed to prevent MYC from inducing apoptosis.46 However, 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

.D

KEY WORDS

IEN

CE

MYC, CDK2, apoptosis, Cyclin E, p27 ACKNOWLEDGEMENTS

INTRODUCTION

ON

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2859

RIB

*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: dfelsher@ leland.stanford.edu

IST

2Mouse Cancer Genetics Program; National Cancer Institute; Frederick, Maryland USA

OT D

1Division of Oncology; Departments of Medicine and Pathology; Stanford University; Stanford, California USA

UT E

.

Debabrita Deb-Basu1 Eiman Aleem2 Philipp Kaldis2 Dean W. Felsher1

LA

06

NOTE

ND

ES

BIO

SC

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 D.D.).

©

20

Supplementary material can be found at: http://www.landesbioscience.com/journals/cc/ supplement/debbasuCC5-12-sup.pdf

1342

Cell Cycle

2006; Vol. 5 Issue 12

CDK2 is Required by MYC to Induce Apoptosis

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.24 NHF 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.14 To 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.50 Measurement 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-G1 DNA www.landesbioscience.com

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). Twohundred 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 conventional techniques. Bax, Bad, Bcl2, BclXL antibodies 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 performed as described.9 Briefly, 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 manufacturer’s protocol. Nuclei were stained with Propidium Iodide (PI). Histone-3-Phosphate staining of mitotic cells was performed as described.51

RESULTS

CDK2 is required by MYC to induce apoptosis. Upon investigation 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-G1 apoptotic 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

Cell Cycle

1343


ifen induces apoptosis in the Wild-type MEF MYCER cells. 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 activation (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 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 proliferation upon MYC activation in MEFs by suppressing infected with MSCV MYCER GFP retrovirus. GFP positive cells were FACS sorted and MYC-induced apoptosis. MYC expression was induced by addition of 0.1 µM TAM for 48 hours. Wild-type Loss of Cdk2 does not prevent MYC from inducing and Cdk2 KO MEFs expressing MYCER GFP were either not treated or treated with pro-apoptotic signals. MYC has been demonstrated to TAM and stained with 7AAD and Annexin V-PE. The percentage of cells after staining induce apoptosis through the indirect activation of the prois indicated in each quadrant (A). The frequency of Annexin V-PE+/7AAD- positive apoptotic regulators Bax and p535,27,29-35 and suppression cells are summarized (B). (C) Inhibition of Cyclin E/Cdk2 does not inhibit apoptosis of the anti-apoptotic regulators BCL2 and BCLXL.40-42 As 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) expected, in wild-type MEFs, MYC activation resulted in as indicated. To measure apoptosis, the cells were stained with Propidium Iodide (PI) decreased protein levels of BCL2, BCLXL and induction of and analyzed by FACS. The percentage of apoptotic cells was determined from the p53, but no changes in expression of Bad or Bax (Fig. 4). frequency of sub-G1 DNA content. In Cdk2 KO MEFs, basal levels of anti-apoptotic proteins, Bcl2 and BclXL were reduced, and Bim was increased, positive, whereas the Cdk2 KO MEFs grew to high density and only which would be expected to increase apoptosis. Upon MYC induc3% of the cells were trypan blue positive (Fig. 2C and D). Therefore, 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 absence of CDK2 blocks MYC-induced apoptosis and cell death. Addition of Tamoxifen induced apoptosis only in the Wild-type be expected promote apoptosis. In Cdk2 KO MEFs, basal levels of MEF MYCER cells, but not in the Wild-type MEF cells Bcl2 were suppressed relatively to wild-type MEFs. Upon MYC (Supplementary Fig. 1). Thus, MYC overexpression and not tamox- 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 wildtype 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.

1344

Cell Cycle

2006; Vol. 5 Issue 12


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 associated histone kinase activities were measured in Wild-type and Cdk2 KO MYCER GFP cells in presence and absence of MYC activation.

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 NHFs. 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.

DISCUSSION

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 activation. Our results are consistent with the report by Hoang and colleagues that Cyclin A is required by MYC to induce apoptosis,49 but in discordance other reports that suggest that MYC induces apoptosis independent of the cell cycle.13,45-48 There 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 MYCinduced apoptosis in the Rat1A cells.46 MYC has been shown to induce apoptosis through a multitude of www.landesbioscience.com

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.

effects on effectors of apoptosis.4,22,27-29 In particular, MYC has been shown to induce pro-apoptotic regulators Bax and p535,27,29-35 and suppress anti-apoptotic regulators Bcl2 and BclXL.40-42 However, although loss of CDK2 blocked MYC-induced apoptosis, CDK2

Cell Cycle

1345


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 transfected with CDK2 RNAi (E–H). MYC activation was induced by the addition of 0.2 µM 4-hydroxytamoxifen (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 RNAi (H).

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 knockout MEFs, BCL2 expression was suppressed and upon MYC activation 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 signals 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 pathways that mediate apoptosis are intact. Moreover, although known to facilitate apoptosis induced by other stimuli, Cdk2 activation is 1346

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.24 Similarly, 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 apoptosis observed.54 Hence, apoptosis was observed as mechanism of last resort for containing MYC overexpression. Notably, the inhibition of CDK2 by RNAi or p27 induced proliferative 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 transit.50,55-58 Aleem et al., recently showed that Cyclin E binds to and activates Cdc2.59 Our 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

Cell Cycle

2006; Vol. 5 Issue 12


unrecognized essential nodal point that may define whether MYC overexpression induces proliferation versus apoptosis. References 1. Dang CV. c-MYC target genes involved in cell growth, apoptosis, and metabolism. Mol Cell Biol 1999; 19:1-11. 2. Oster SK, Ho CS, Soucie EL, Penn LZ. The myc oncogene: MarvelouslY Complex. Adv Cancer Res 2002; 84:81-154. 3. Eisenman RN. Deconstructing myc. Genes Dev 2001; 15:2023-30. 4. Pelengaris S, Khan M, Evan G. c-MYC: More than just a matter of life and death. Nat Rev Cancer 2002; 10:764-66. 5. Zindy F, Eischen CM, Randle DH, Kamijo T, Cleveland JL, Sherr CJ. Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev 1998; 12:2424-33. 6. Simm A, Halle JP, Adam G. Proliferative and metabolic capacity of rat embryo fibroblasts immortalized by c-myc depends on cellular age at oncogenic transfection. Eur J Cell Biol 1994; 65:121-31. 7. Vafa O, Wade M, Kern S, Beeche M, Pandita TK, Hampton GM, Wahl GM. c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: A mechanism for oncogene-induced genetic instability. Mol Cell 2002; 9:1031-44. 8. Pelengaris S, Littlewood T, Khan M, Elia G, Evan G. Reversible activation of c-Myc in skin: Induction of a complex neoplastic phenotype by a single oncogenic lesion. Mol Cell 1999; 3:565-77. 9. Li Q, Dang CV. c-Myc overexpression uncouples DNA replication from mitosis. Mol Cell Biol 1999; 19:5339-51. 10. Mai S, JHH, Fluri M. c-MYC overexpression associated DHFR gene amplification in hamster, rat, mouse and human cell lines. Oncogene 1996; 12:277-88. 11. Marcu KB, Bossone SA, Patel AJ. myc function and regulation. Annu Rev Biochem 1992; 809-60. 12. Inghirami G, Grignani F, Sternas L, Lombardi L, Knowles DM, Dalla-Favera R. Downregulation of LFA-1 adhesion receptors by C-myc oncogene in human B lymphoblastoid cells. Science 1990; 250:682-6. 13. Harrington EA, Bennett MR, Fanidi A, Evan GI. c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. Embo J 1994; 13:3286-95. 14. Felsher DW, Bishop JM. Transient excess of MYC activity can elicit genomic instability and tumorigenesis. Proc Natl Acad Sci USA 1999; 96:3940-4. 15. Cerni C, Mougneau E, Cuzin F. Transfer of ‘immortalizing’ oncogenes into rat fibroblasts induces both high rates of sister chromatid exchange and appearance of abnormal karyotypes. Exp Cell Res 1987; 168:439-46. 16. Coller HA, Grandori C, Tamayo P, Colbert T, Lander ES, Eisenman RN, Golub TR. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci USA 2000; 97:3260-5. 17. Barr LF, Campbell SE, Bochner BS, Dang CV. Association of the decreased expression of alpha3beta1 integrin with the altered cell: Environmental interactions and enhanced soft agar cloning ability of c-myc-overexpressing small cell lung cancer cells. Cancer Res 1998; 58:5537-45. 18. Chernova OB, Chernov MV, IshizakaY, Agarwal ML, Stark GR. MYC abrogates p53-mediated cell cycle arrest in N-phpsphonacetyl-L-aspartate-treated cells, permitting CAD gene amplification. Mol Cell Biol 1998; 18:536-45. 19. Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White EL, Davis AC, Ihle JN, Cleveland JL. c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression. Genes Dev 2002; 16:2530-43. 20. Ngo CV, Gee M, Akhtar N, Yu D, Volpert O, Auerbach R, Thomas-Tikhonenko A. An in vivo function for the transforming Myc protein: Elicitation of the angiogenic phenotype. Cell Growth Differ 2000; 11:201-10. 21. Janz A, Sevignani C, Kenyon K, Ngo CV, Thomas-Tikhonenko A. Activation of the myc oncoprotein leads to increased turnover of thrombospondin-1 mRNA. Nucleic Acids Res 2000; 28:2268-75. 22. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ, Hancock DC. Induction of apoptosis in fibroblasts by c-myc protein. Cell 1992; 69:119-28. 23. Grandori C, Wu KJ, Fernandez P, Ngouenet C, Grim J, Clurman BE, Moser MJ, Oshima J, Russell DW, Swisshelm K, Frank S, Amati B, Dalla-Favera R, Monnat Jr RJ. Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev 2003; 17:1569-74. 24. Felsher DW, Zetterberg A, Zhu J, Tlsty T, Bishop JM. Overexpression of MYC induces p53-dependent G2 arrest of normal fibroblasts. Proc Natl Academy Sci USA 2000; 97:10544-48. 25. Jain M, Arvanitis C, Chu K, Dewey W, Leonhardt E, Trinh M, Sundberg CD, Bishop JM, Felsher DW. Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science 2002; 297:102-4. 26. Hueber AO, Zornig M, Lyon D, Suda T, Nagata S, Evan GI. Requirement for the CD95 receptor-ligand pathway in c-Myc-induced apoptosis. Science 1997; 278:1305-9. 27. Wagner AJ, Kokontis JM, Hay N. Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Genes and Development 1994; 8:2817-30. 28. Hermeking H, Eick D. Mediation of c-Myc-induced apoptosis by p53. Science 1994; 265:2091-3.

www.landesbioscience.com

29. Wagner AJ, Small MB, Hay N. Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2. Mol Cell Biol 1993; 13:2432-40. 30. Juin P, Hueber AO, Littlewood T, Evan G. c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev 1999; 13:1367-81. 31. Juin P, Hunt A, Littlewood T, Griffiths B, Swigart LB, Korsmeyer S, Evan G. c-Myc functionally cooperates with Bax to induce apoptosis. Mol Cell Biol 2002; 22:6158-69. 32. Soucie EL, Annis MG, Sedivy J, Filmus J, Leber B, Andrews DW, Penn LZ. Myc potentiates apoptosis by stimulating Bax activity at the mitochondria. Mol Cell Biol 2001; 21:4725-36. 33. Shang Y, Baumrucker CR, Green MH. c-Myc is a major mediator of the synergistic growth inhibitory effects of retinoic acid and interferon in breast cancer cells. J Biol Chem 1998; 273:30608-13. 34. Eischen CM, Roussel MF, Korsmeyer SJ, Cleveland JL. Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis. Mol Cell Biol 2001; 21:7653-62. 35. de Stanchina E, McCurrach ME, Zindy F, Shieh SY, Ferbeyre G, Samuelson AV, Prives C, Roussel MF, Sherr CJ, Lowe SW. E1A signaling to p53 involves the p19(ARF) tumor suppressor. Genes Dev 1998; 12:2434-42. 36. Fanidi A, Harrington EA, Evan GI. Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature 1992; 359:554-6. 37. Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J, Evan G. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 1997; 385:544-8. 38. Askew DS, Ashmun RA, Simmons BC, Cleveland JL. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 1991; 6:1915-22. 39. Klefstrom J, Verschuren EW, Evan G. c-Myc augments the apoptotic activity of cytosolic death receptor signaling proteins by engaging the mitochondrial apoptotic pathway. J Biol Chem 2002; 277:43224-32. 40. Eischen CM, Packham G, Nip J, Fee BE, Hiebert SW, Zambetti GP, Cleveland JL. Bcl-2 is an apoptotic target suppressed by both c-Myc and E2F-1. Oncogene 2001; 20:6983-93. 41. Eischen CM, Woo D, Roussel MF, Cleveland JL. Apoptosis triggered by Myc-induced suppression of Bcl-X(L) or Bcl-2 is bypassed during lymphomagenesis. Mol Cell Biol 2001; 21:5063-70. 42. Maclean KH, Keller UB, Rodriguez-Galindo C, Nilsson JA, Cleveland JL. c-Myc augments gamma irradiation-induced apoptosis by suppressing Bcl-XL. Mol Cell Biol 2003; 23:7256-70. 43. Egle A, Harris AW, Bouillet P, Cory S. Bim is a suppressor of Myc-induced mouse B cell leukemia. Proc Natl Acad Sci USA 2004; 101:6164-9. 44. Hemann MT, Bric A, Teruya-Feldstein J, Herbst A, Nilsson JA, Cordon-Cardo C, Cleveland JL, Tansey WP, Lowe SW. Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 2005; 436:807-11. 45. Packham G, Porter CW, Cleveland JL. c-Myc induces apoptosis and cell cycle progression by separable, yet overlapping, pathways. Oncogene 1996; 13:461-9. 46. Rudolph B, Saffrich R, Zwicker J, Henglein B, Muller R, Ansorge W, Eilers M. Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis. Embo J 1996; 15:3065-76. 47. Conzen SD, Gottlob K, Kandel ES, Khanduri P, Wagner AJ, O’Leary M, Hay N. Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc: Transrepression correlates with acceleration of apoptosis. Mol Cell Biol 2000; 20:6008-18. 48. Beier R, Burgin A, Kiermaier A, Fero M, Karsunky H, Saffrich R, Moroy T, Ansorge W, Roberts J, Eilers M. Induction of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth by Myc are genetically separable events. Embo J 2000; 19:5813-23. 49. Hoang AT, Cohen KJ, Barrett JF, Bergstrom DA, Dang CV. Participation of cyclin A in Myc-induced apoptosis. Proc Natl Acad Sci USA 1994; 91:6875-9. 50. Tetsu O, McCormick F. Proliferation of cancer cells despite CDK2 inhibition. Cancer Cell 2003; 3:233-45. 51. Xu B, Kim St, Kastan MB. Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation. Molecular and Cellular Biology 2001; 21:3445-50. 52. Mazumder S, Gong B, Chen Q, Drazba JA, Buchsbaum JC, Almasan A. Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells. Mol Cell Biol 2002; 22:2398-409. 53. Li K, Lin SY, Brunicardi FC, Seu P. Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma. Cancer Res 2003; 63:3593-7. 54. Beer S, Zetterberg A, Ihrie RA, McTaggart RA, Yang Q, Bradon N, Arvanitis C, Attardi LD, Feng S, Ruebner B, Cardiff RD, Felsher DW. Developmental context determines latency of MYC-induced tumorigenesis. PLoS Biol 2004; 2:e332. 55. Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P. Cdk2 knockout mice are viable. Curr Biol 2003; 13:1775-85. 56. Geng Y, Yu Q, Sicinska E, Das M, Schneider JE, Bhattacharya S, Rideout WM, Bronson RT, Gardner H, Sicinski P. Cyclin E ablation in the mouse. Cell 2003; 114:431-43. 57. Parisi T, Beck AR, Rougier N, McNeil T, Lucian L, Werb Z, Amati B. Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells. Embo J 2003; 22:4794-803. 58. Ortega S, Prieto I, Odajima J, Martin A, Dubus P, Sotillo R, Barbero JL, Malumbres M, Barbacid M. Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat Genet 2003; 35:25-31. 59. Aleem E, Kiyokawa H, Kaldis P. Cdc2-cyclin E complexes regulate the G1/S phase transition. Nat Cell Biol 2005; 7:831-6.

Cell Cycle

1347