Transduced P16ink4a Peptides Inhibit Hypophosphorylation of the Retinoblastoma Protein and Cell Cycle Progression Prior to Activation of Cdk2 1 Complexes in Late G1

[CANCER RESEARCH 59, 2577–2580, June 1, 1999] Advances in Brief

Transduced p16INK4a Peptides Inhibit Hypophosphorylation of the Retinoblastoma Protein and Cell Cycle Progression Prior to Activation of Cdk2 1 Complexes in Late G1

David R. Gius,2 Sergei A. Ezhevsky, Michelle Becker-Hapak, Hikaru Nagahara, Michael C. Wei, and Steven F. Dowdy3 Howard Hughes Medical Institute and Departments of Pathology and Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Abstract involved in phosphorylating pRb and the requirement for G1 cell cycle progression are presently poorly understood. Because of inherent Progression of cells through the G phase of the cell cycle requires 1 limitations of cellular manipulation by methods such as transfection cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and and viral vector infection, such as lag time for gene expression and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained low efficiencies, these experiments cannot address the critical issue of the temporal activity required by cyclin D:Cdk4/6 complexes for pRb an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hy- phosphorylation and G1 cell cycle progression. Therefore, to precisely pophosphorylated pRb and elicited an early G1 cell cycle arrest, provided investigate the kinetic requirement for early G1 phase cell cycle cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 progression by cyclin D:Cdk4/6 complexes, we transduced p16INK4a activity is required for early G1 phase cell cycle progression up to, but not peptidyl mimetics directly into ϳ100% of cells and assayed for pRb beyond, activation of cyclin E:Cdk2 complexes at the restriction point and phosphorylation and kinetics of cell cycle progression. Transduction is thus nonredundant with cyclin E:Cdk2 in late G . 1 of p16 mimetics results in rapid (Ͼ20 min) inactivation of Cdk4/6 Introduction complexes and thus allows for an analysis of Cdk4/6 kinetic require- ments. We report here that cyclin D:Cdk4/6 complexes perform the

Progression of eukaryotic cells from the early and mid G1 phase of activating hypophosphorylation of pRb in early and mid G1 which is the cell cycle through the G1 restriction point, into late G1 phase, nonredundant with the inactivating hyperphosphorylation of pRb by through the G -S phase transition and then into S phase requires the 1 cyclin E:Cdk2 complexes in late G1. In addition, Cdk4/6 activity is concerted activities of several members of the Cdk4 family, i.e., cyclin required for early G1 phase cell cycle progression up to, but not D:Cdk4/6, cyclin E:Cdk2, and cyclin A:Cdk2 complexes (1–3). One beyond, activation of cyclin E:Cdk2 complexes at the restriction point important substrate of G cyclin:Cdk complexes is the product of the 1 and into late G1 phase. retinoblastoma tumor suppressor gene, pRb, a negative regulator of early G1 phase cell cycle progression, which contains 16 putative Cdk Materials and Methods consensus phosphorylation sites (1). pRb exists in two general phosphorylated forms in G1. In early and mid G1, pRb is present as an Cell Culture and Flow Cytometry Analysis. Human HaCaT keratino- active, hypophosphorylated form that associates with cellular tran- cytes were maintained as described (6). For G1 cell cycle arrest, HaCaT cells scription factors and contains a ϳ1:1 molar ratio of phosphate to pRb were contact inhibited by plating at high density (6 ϫ 106/10-cm dish) for 36–40 h in 10% FBS, trypsinized, and replated at low density (1.5 ϫ 105/well (4–6). At the late G1 restriction point, pRb becomes initially inactivated by hyperphosphorylation and contains a ϳ10:1 molar ratio of of a 6-well dish) and assayed for cell cycle position at various time points up phosphate to pRb, resulting in the release of transcription factors (1, to 30 h postreplating or treated with TAT-p16 peptides at 5, 10, and 15 h postreplating. DNA content FACS analysis was performed as described (6). 4–6). Thus, pRb appears to be both activated and inactivated in a Labeling and Immunoprecipitations. G -arrested, contact-inhibited nonredundant fashion by use of Cdk phosphorylation sites coupled to 1 HaCaT cells were pretreated with TAT-p16 peptides for 1 h and then labeled phosphate molar ratios. in the presence of TAT-p16 peptides for 4 h with 3–5 mCi of [32P]orthophos- In human oncogenesis, there is a strong selection for genetic alter- phate (ICN Biomedicals) per 10-cm dish or with 250 ␮Ci [35S]methionine INK4a ation of one or more members of the p16 , cyclin D:Cdk4/6, and (NEN) as described (6). Cellular lysates were prepared, and pRb was immu- pRb pathway involved in regulating G1 cell cycle progression (7). noprecipitated by addition of G99-549 anti-pRb antibodies (PharMingen) that However, the timing and duration of cyclin D:Cdk4/6 complexes recognize only the fast migrating, un- and hypophosphorylated forms of pRb as described (6). Immune complexes were collected on protein A-Sepharose Received 3/24/99; accepted 4/16/99. (Pharmacia), washed three times, resolved by SDS-PAGE, transferred to The costs of publication of this article were defrayed in part by the payment of page nitrocellulose filters, and analyzed by phosphorimaging (Molecular Dynam- charges. This article must therefore be hereby marked advertisement in accordance with ics). After PhosphorImager analysis, nitrocellulose filters were immunoblotted 18 U.S.C. Section 1734 solely to indicate this fact. with anti-pRb antibodies as described (6). 1 D. R. G. was supported by an ASTRO fellowship. S. A. E. was supported by an National Cancer Institute Training Grant CA09547-13. M. C. W. was supported by an Kinase Assay. Rabbit anti-Cdk2 (Santa Cruz Biotechnology) immunopre- NIH MSTP Training Grant GM07200. S. F. D. is an Assistant Investigator of the Howard cipitates were washed three times with ELB, followed by washes two times Hughes Medical Institute. 2 with kinase buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2,1mM DTT, and Present address: Radiation Oncology Center, Mallinckrodt Institute of Radiology, ␮ ␮ ␮ Washington University School of Medicine, St. Louis, MO. 1 M unlabeled ATP] and suspended in 25 l of kinase buffer plus 100 Ci 3 To whom requests for reprints should be addressed, at Howard Hughes Medical of [␥-32P]ATP (Amersham; 6000 Ci/mmol) plus 2 ␮g of histone H1 (Sigma) Institute, Washington University School of Medicine, 4940 Parkview Place, Campus Box substrate. Reactions were incubated for 30 min at 30°C, stopped by addition of 8022, St. Louis, MO 63110. Phone: (314) 362-1722; Fax: (314) 362-1802; E-mail: 2ϫ SDS buffer, separated on SDS-PAGE, and analyzed by phosphorimaging [email protected]. 4 The abbreviations used are: Cdk, cyclin-dependent kinase; pRb, retinoblastoma (Molecular Dynamics). Equal amounts of rabbit antimouse antibodies were protein; FACS, fluorescence-activated cell sorter. used as negative controls. 2577

was capable of binding to and inactivating Cdk4/6 in vitro.We

synthesized 32-mer peptides that consisted of an NH2-terminal TAT protein transduction domain (11 amino acids; Ref. 8), followed by a glycine residue for free bond rotation and a COOH-terminal 20-mer of either functional wild-type p16INK4a or charge-matched control sequences (Fig. 1A). To monitor transduction into cells, TAT-p16 peptides were labeled with FITC and added to the media of cells. Flow cytometry analysis (FACS) of treated cells demonstrated that TAT-p16-FITC peptides transduced rapidly into ϳ100% of cells, achieving maximum intracellular concentrations in Ͻ20 min (Fig. 1B). Confocal microscopy analysis confirmed transduction of TAT-p16-FITC peptides into ϳ100% cells and revealed an intracellular location of the transduced peptide and not mere attachment to the cellular membrane (data not shown; Ref. 8). Thus, consistent with our TAT fusion proteins pub- lished previously (6, 8, 10, 11), TAT-p16 peptides transduce into ϳ100% of cells in a rapid, concentration-dependent fashion.

To functionally test the ability of TAT-p16 peptides to elicit a G1 phase cell cycle arrest, we used human HaCaT keratinocytes as a model cell culture system because of their sensitivity to p16INK4a- mediated cell cycle arrest (6). Asynchronous HaCaT keratinocytes were treated with 10, 50, or 100 ␮M TAT-p16 wild-type or control peptides for 30 h and then analyzed for cell cycle position by DNA content and FACS analysis (Fig. 1C). Treatment of HaCaT cells with

TAT-p16 wild-type peptide resulted in a significant G1 phase cell cycle arrest, whereas the TAT-control peptide had minimal effects on

cell cycle position. Cell cycle arrest prior to the late G1 restriction point has been shown to result in loss of hyperphosphorylated forms

of pRb (4, 6, 12, 13). Consistent with an early G1 cell cycle arrest, anti-pRb immunoblot analysis of asynchronous HaCaT cells treated with TAT-p16 peptides showed loss of hyperphosphorylated pRb (data not shown). Taken together, these observations demonstrate that TAT-p16 wild-type peptides rapidly transduce into ϳ100% of cells Fig. 1. Characterization of TAT-p16 peptides. A, structure of TAT-p16 wild-type (WT) and mutant (MUT) peptides represented in single-letter amino acid code. B, FACS and retain the previously associated properties of p16 to elicit an early analysis of FITC-labeled TAT-p16 peptide 20 min after addition to cells. Note narrow G1 phase cell cycle arrest. peak width of transduced cells. Control is FITC labeling reaction in the absence of Transduction of p16 Peptide into Cells Inhibits pRb Hypophos- peptide. C, asynchronous HaCaT keratinocytes were transduced with increasing concentrations of TAT-p16 wild-type (WT) or mutant (MUT) peptides for 30 h and analyzed for phorylation. On denaturing SDS-PAGE immunoblots, unphospho- cell cycle position by propidium iodide staining for DNA content by FACS. rylated and hypophosphorylated pRb comigrate (6) and hence, are indistinguishable. Therefore, to analyze the influence of accumulated TAT-p16 Peptides. Thirty-two-mer TAT-p16 peptides were synthesized

so that each contained an NH2-terminal 11-mer TAT protein transduction domain (single-letter code, YGRKKRRQRRR; Ref. 8) followed by a glycine residue and either a 20-mer wild-type p16 sequence (WT, DAAREGFLATLV- VLHRAGAR; Ref. 9) or a charge-match control sequence (MUT, ARGRAL- TAHVDRLGEFVAAL). After synthesis and purification, peptides were re- suspended in water. FITC-labeled TAT-p16 peptides were generated by fluorescein labeling (Pierce), followed by gel filtration on a S-12 column attached to an fast protein liquid chromatography (Pharmacia).

Results Transduction of p16 Peptidyl Mimetics into Cells. To focus on

the question of exactly when during the progression of G1 phase of the cell cycle cyclin D:Cdk4/6 complexes are required, we chose to introduce peptidyl mimetics of p16INK4a, a negative regulator of Cdk4/6 (2), by the rapid method of protein transduction (8, 10). Treatment of cells with peptides and proteins containing the protein transduction domain from HIV TAT protein results in a rapid transduction into ϳ100% of cells in a given population (primary or Fig. 2. Transduction of TAT-p16 peptide results in loss of pRb hypophosphorylation. ␮ transformed cells) in a receptorless fashion (6, 8, 10, 11). In addition, G1 arrested, contact-inhibited HaCaT cells were treated with either 100 M TAT-p16 wild-type (WT) or mutant (MUT) peptides for 1 h and then [32P]orthophosphate labeled because of its concentration dependency, TAT-mediated transduction for4hinthepresence of TAT-p16 peptides. pRb was immunoprecipitated, transferred to results in a near equivocal intracellular concentration of the trans- a filter, analyzed by phosphorimaging for 32P content (A), and then the same filter was probed by anti-pRb immunoblot analysis to normalize for pRb protein levels (B). Control duced protein from cell to cell in the population. Previously, Fahraeus (ctrl) cells were untreated. Note the loss of pRb hypophosphorylation, appearance of et al. (9) demonstrated that a 20-amino acid peptidyl mimetic of p16 unphosphorylated pRb, and their comigration on SDS-PAGE. 2578

in early G1 by plating at high density for 36 h in 10% serum. Cells were then released from arrest by replating at low density and analyzed for pRb phosphorylation status and Cdk2 kinase activity at various time points (Fig. 3A). Immunoprecipitation of pRb from [32P]orthophosphate-labeled cellular lysates and immunoblot analysis showed that pRb was hypophosphorylated at replating (Fig. 2, t ϭ 0). pRb remained hypophosphorylated at 5 and 10 h postreplating and initially became inactivated by hyperphosphorylation at 15 h postreplating (Fig. 3A, top panel). Anti-Cdk2 immunoprecipitation- kinase analysis first detected Cdk2 activity, likely cyclin E:Cdk2 complexes, at 15 h postreplating (Fig. 3A, bottom panel). On the basis

of DNA FACS analysis, replated HaCaT cells progress from late G1 phase into S phase at Ͼ22 h (data not shown). Thus, inactivation of pRb by hyperphosphorylation and activation of Cdk2 complexes

occur concomitantly in late G1.

We next treated G1 arrested, contact-inhibited HaCaT cells that

were then released from G1 arrest by replating at low density and treated with 10, 50, or 100 ␮M TAT-p16 wild-type peptide at 5, 10, and 15 h postreplating and analyzed for cell cycle position at 30 h postreplating by DNA content and FACS analysis (Fig. 3B). Trans- duction of TAT-p16 wild-type peptides into the synchronized cells

were capable of eliciting a G1 cell cycle arrest when transduced at 5 and 10 h postreplating. However, TAT-p16 wild-type peptides were

unable to effect a G1 arrest when transduced into cells at 15 h postreplating, consistent with the appearance of active Cdk2 complexes and hyperphosphorylated pRb. Taken together, these observations directly demonstrate, for the first time, that cyclin D:Cdk4/6

complexes are required for early and mid G1 phase cell cycle progression up to, but not beyond, the point of Cdk2 activation and

transition through the restriction point into late G1. Fig. 3. Kinetics of TAT-p16-mediated G1 arrest. A, contact-arrested HaCaT cells were released from the G1 block by replating at low density, followed for pRb phosphorylation status by anti-pRb immunoblot analysis (top panel) and for Cdk2 activity by anti-Cdk2 immunoprecipitation-kinase analysis using histone H1 as a substrate (bottom panel). Hyperphosphorylated pRb first appears at 15 h postreplating, concomitant with activation of Cdk2 complexes. B, contact-arrested and released HaCaT cells from above (A) were transduced with increasing concentrations of TAT-p16 wild-type peptides at 5, 10, and 15, h postreplating and then analyzed for cell cycle progression by FACS analysis for DNA content. Cells treated at 5 and 10 h retain the ability to be arrested by TAT-p16 peptides; however, at 15 h, TAT-p16 peptides are unable to effect a G1 arrest, consistent with activation of Cdk2 complexes. Control cells were released from G1 arrest but untreated with peptides.

TAT-p16 peptides and hence, resultant inactivation of Cdk4/6 complexes on pRb hypophosphorylation, G1 arrested contact-inhibited HaCaT cells present in 10% FBS, containing only hypophosphorylated pRb and no hyperphosphorylated pRb, were treated with 100 ␮M TAT-p16 wild-type or mutant peptides for 1 h, followed by the addition of [32P]orthophosphate for 4 h. pRb was immunoprecipitated from cellular lysates with anti-pRb antibodies, resolved by SDS- 32 PAGE, transferred to a filter, and analyzed for pRb PO4 content (Fig. 2A). The same filter was then normalized for pRb protein levels by anti-pRb immunoblot analysis (Fig. 2B). Treatment with TAT-p16 wild-type peptides resulted in a marked loss of pRb hypophosphorylation and the appearance of unphosphorylated pRb (Lane 2). In contrast, cells treated with TAT-p16 control peptides retained hypophosphorylated pRb, as did untreated control cells (Lanes 1 and 3). These observations suggest that in cells containing physiological Fig. 4. Model of cyclin:Cdk complex kinetics in regulating pRb phosphorylation and G1 phase cell cycle progression. In G0, p130, a pRb-related pocket protein, binds E2Fs concentrations of cyclin D:Cdk4/6 complexes, pRb is only hypophos- (30). Newly synthesized pRb is unphosphorylated and becomes activated (capable of phorylated in vivo and not hyperphosphorylated. binding transcription factors) when hypophosphorylated by cyclin D:Cdk4/6 complexes in TAT-p16 Peptides Arrest Cells before Activation of Cdk2 Com- early G1. The initial inactivating hyperphosphorylation of pRb occurs by activation of cyclin E:Cdk2 complexes at the restriction point, resulting in release of transcription plexes in Late G1. To directly ascertain the kinetic requirement of factors, such as E2Fs, from pRb that result in transcriptional activation. Thus, the functioning of cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes are both nonredundant and cyclin D:Cdk4/6 activity in promoting G1 phase cell cycle progression, we sought to transduce TAT-p16 peptides into synchronized diametrically opposed. After degradation of cyclin E from Cdk2 in S phase, cyclin A:Cdk2 complexes maintain pRb hyperphosphorylation into late G2 phase, followed by HaCaT cells at various time points. HaCaT cells were contact arrested cyclin B:CDC2 in M phase. 2579

Discussion ulation. Future experiments directed at characterizing specific pRb isoforms associated with specific transcription factors in cells that Advancement from the early G1 phase of the cell cycle through the express subsets of cyclin D1, D2, D3:Cdk4, and Cdk6 complexes restriction point and into late G1 phase requires the activity of several should help to understand the complex regulatory events that occur in cyclin:Cdk complexes and hyperphosphorylation of pRb at the restric- early G and at the restriction point. tion point (1–3, 6, 11, 12, 14). The requirement for both cyclin 1

D:Cdk4/6 and cyclin E:Cdk2 activity for G1 phase cell cycle progres- Acknowledgments sion has been shown previously (15–17). However, the kinetic requirement for cyclin D:Cdk4/6 activity remained unclear. By use of We thank D. Lane and R. Fahraeus (University of Dundee) for input on p16 the protein transduction methodology (8), we were able to rapidly peptide sequences, M. Dustin (Washington University) for confocal micros- introduce the p16INK4a-negative regulator of Cdk4/6 into ϳ100% of copy, C. Turck (University of California at San Francisco) for peptide synthesis, and all of the members of the Dowdy lab for critical input. cells in the population in a concentration-dependent fashion. This method presents a superior technique to perform both biological and References in vivo biochemical assays on the entire cellular population in precise 1. Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell, 81: 323–330, timing intervals. In addition, cellular manipulation by protein trans- 1995. duction avoids problems associated with transfection of a limited 2. Sherr, C. J. Cancer cell cycles. Science (Washington DC), 274: 1672–1677, 1996. percentage of the cells and unregulated overexpression of cyclins that 3. Sherr, C. J., and Roberts, J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev., 9: 1149–1163, 1995. can lead to nonphysiological conditions within the cell. 4. Mittnacht, S., and Weinberg, R. A. G1/S phosphorylation of the retinoblastoma We show here that introduction of a p16INK4a peptidyl mimetic into protein is associated with an altered affinity for the nuclear compartment. Cell, 65: 381–393, 1991. synchronized keratinocytes by protein transduction results in a G1 5. Mittnacht, S., Lees, J. A., Desai, D., Harlow, E., Morgan, D. O., and Weinberg, R. A. phase cell cycle arrest, provided that Cdk2 complexes have not Distinct sub-populations of the retinoblastoma protein show a distinct pattern of become activated. In addition, inactivation of Cdk4/6-containing com- phosphorylation. EMBO J., 13: 118–127, 1994. 6. Ezhevsky, S. A., Nagahara, H., Vocero-Akbani, A., Gius, D., Wei, M. C., and Dowdy, plexes results in the loss of pRb hypophosphorylation and the appear- S. F. Hypo-phosphorylation of the retinoblastoma protein by cyclin D:cdk4/6 com- ance of the unphosphorylated form of pRb. Thus, cyclin D:Cdk4/6 plexes results in active pRb. Proc. Natl. Acad. Sci. USA, 94: 10699–10704, 1997. activity, presumably for hypophosphorylation of pRb (and likely other 7. Hall, M., and Peters, G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv. Cancer Res., 68: 67–108, 1996. substrates), is required to progress to the restriction point but not 8. Nagahara, H., Vocero-Akbani, A., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A., and Dowdy, S. F. Transduction of full length through it. Transition across the late G1 restriction point appears to TAT fusion proteins into mammalian cells: p27Kip1 mediates cell migration. Nat. require activation of Cdk2 complexes, likely cyclin E:Cdk2 com- Med., 4: 1449–1452, 1998. plexes, that inactivate pRb by hyperphosphorylation, causing release 9. Fahraeus, R., Paramio, J. M., Ball, K. L., Lain, S., and Lane, D. P. Inhibition of pRb of transcription factors from pRb (Fig. 4). These observations are phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16CDKN2/INK4A. Curr. Biol., 6: 84–91, 1996. entirely consistent with previous reports demonstrating the constitu- 10. Vocero-Akbani, A., Vander Heyden, N., Lissy, N. L., Ratner, L., and Dowdy, S. F. tive expression and activity of cyclin D:Cdk4/6 complexes in early G1 Killing HIV infected cells by direct transduction of an HIV protease-activated of cycling cells (6, 11, 18–20). In addition, work by Ikeda et al. (21) caspase-3 protein. Nat. Med., 5: 29–33, 1999. 11. Lissy, N. A., Van Dyk, L., Becker-Hapak, M., Mendler, J. H., Vocero-Akbani, A., demonstrated that in G0, E2F transcription factors are bound by a and Dowdy, S. F. TCR-antigen induced cell death (AID) occurs from a late G1 phase pRb-related pocket protein, p130. Thus, as the cell progresses from a cell cycle check point. Immunity, 8: 57–65, 1998. 12. DeCaprio, J. A., Furukawa, T., Ajchenbaum, F., Griffin, J. D., and Livingston, D. M. G1 arrested state into a G0 state containing inactive Cdk4/6 com- The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple plexes, pRb becomes dephosphorylated, allowing p130 access to bind stages during cell cycle entry and progression. Proc. Natl. Acad. Sci. USA, 89: and regulate this family of transcription factors. 1795–1798, 1992. 13. Lee, W. H., Shew, J. Y., Hong, F. D., Sery, T. W., Donoso, L. A., Young, L. J., The data presented here demonstrate that the roles of cyclin Bookstein, R., and Lee, E. Y. The retinoblastoma susceptibility gene encodes a D:Cdk4/6 and cyclin E:Cdk2 complexes in advancing early and late nuclear phosphoprotein associated with DNA binding activity. Nature (Lond.), 329: G phase, respectively, are nonredundant and in fact diametrically 642–645, 1987. 1 14. Ohtsubo, M., Theodoras, A. M., Schumacher, J., Roberts, J. M., and Pagano, M. opposed with respect to pRb regulation; cyclin D:Cdk4/6 complexes Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol. activate pRb, whereas cyclin E:Cdk2 complexes inactivate pRb. pRb Cell. Biol., 15: 2612–2624, 1995. 15. Koh, J., Enders, G. H., Dynlacht, B. D., and Harlow, E. 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USA, 92: 6289–6293, 1995. individually contain a combination of one or perhaps two phosphates. 18. Dowdy, S. F., Van Dyk, L., and Schreiber, G. H. Cell cycle synchronization by Indeed, two-dimensional isoelectric focusing of hypophosphorylated elutriation. In: K. Adolph (ed.), Human Genome Methods, pp. 121–132. Boca Raton, Ͼ FL: CRC Press, 1997. pRb shows the presence of 12 hypophosphorylated pRb isoforms in 19. Won, K. A., Xiong, Y., Beach, D., and Gilman, M. Z. Growth-regulated expression keratinocytes and human peripheral blood lymphocytes.5 The gener- of D-type cyclin genes in human diploid fibroblasts. Proc. Natl. Acad. Sci. USA, 89: ation of multiple isoforms of active hypophosphorylated pRb may 9910–9914, 1992. 20. Ajchenbaum, F., Ando, K., DeCaprio, J. A., and Griffin, J. D. Independent regulation serve to target subsets of hypophosphorylated pRb isoforms to spe- of human D-type cyclin gene expression during G1 phase in primary human T cific transcription factors and, hence, result in promoter-specific reg- lymphocytes. J. Biol. Chem., 268: 4113–4119, 1993. 21. Ikeda, M. A., Jakoi, L., and Nevins, J. R. A unique role for the pRb protein in controlling E2F accumulation during cell growth and differentiation. Proc. Natl. 5 S. A. Ezhevsky, M. Becker-Hapak, and S. F. Dowdy, unpublished observation. Acad. Sci. USA, 93: 3215–3220, 1996.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. Transduced p16INK4a Peptides Inhibit Hypophosphorylation of the Retinoblastoma Protein and Cell Cycle Progression Prior to Activation of Cdk2 Complexes in Late G 1

David R. Gius, Sergei A. Ezhevsky, Michelle Becker-Hapak, et al.

Cancer Res 1999;59:2577-2580.

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