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Letter
Nature Cell Biology - 8, 1291 - 1297 (2006)
Published online: 8 October 2006; | doi:10.1038/ncb1491

Mitogenic signalling and the p16INK4a–Rb pathway cooperate to enforce irreversible cellular senescence

Akiko Takahashi1, Naoko Ohtani1, Kimi Yamakoshi1, Shin-ichi Iida2, Hidetoshi Tahara3, Keiko Nakayama4, Keiichi I. Nakayama5, Toshinori Ide6, Hideyuki Saya2 & Eiji Hara1

1 Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan.

2 Graduate School of Medical Science, Kumamoto University, Kumamoto 860-8556, Japan.

3 Hiroshima University School of Medicine, Hiroshima 734-8551, Japan.

4 Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan.

5 Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.

6 Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure 737-0112, Japan.

Correspondence should be addressed to Eiji Hara hara@genome.tokushima-u.ac.jp

The p16INK4a cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb1, 2, 3, 4, 5 in cellular senescence. Here, we show that the p16INK4a /Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cdelta (PKCdelta) in human senescent cells. Importantly, once activated by ROS, PKCdelta promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS–PKCdelta signalling6, 7, 8. Sustained activation of ROS–PKCdelta signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis9, 10, 11, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16INK4a–Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells.

Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes, such as the induction of apoptotic cell death or senescent cell-cycle arrest1. Thus, both apoptosis and senescence are thought to act as a safe-guard against neoplasia. Unlike apoptotic cells, senescent cells are viable for long periods of time2, 3. It is therefore important to clarify how senescent cell-cycle arrest is enforced in human cells2. Two well established tumour suppressor gene products, pRb and p53, are known to have key roles in senescent cell-cycle arrest3, 4, 5. The activities of pRb and p53 are dramatically increased during cellular senescence and inactivation of these proteins in senescent mouse embryonic fibroblasts (MEFs) results in reversal of the senescent phenotype leading to cell-cycle re-entry12, 13, suggesting that pRb and p53 are required not only for the onset of cellular senescence, but also for the maintenance of the senescence programme in murine cells. However, in human cells, once pRb is fully engaged, particularly by its activator, p16INK4a, senescent growth arrest becomes irreversible and is no longer revoked by subsequent inactivation of pRb and p53 (refs 2, 14, 15). Interestingly, subsequent inactivation of pRb and p53 enables human senescent cells to reinitiate DNA synthesis, but fails to drive a complete cell cycle, suggesting that these cells may be arrested in G2 or M phase of the cell cycle14, 16. However, to date, it is largely unknown how senescent cell-cycle arrest is maintained, even after pRb and p53 are subsequently inactivated in human senescent cells.

To delineate the molecular mechanisms of irreversible cell-cycle arrest in human cell senescence, we used SVts8 cells, a conditionally immortalized human fibroblast cell line that express a temperature-sensitive (ts) mutant of simian virus 40 large T antigen and elevated level of the endogenous telomerase17. SVts8 cells proliferate indefinitely at the permissive temperature (34 °C; Fig. 1a), because large T antigen binds and inactivates both pRb and p53 proteins (Fig. 1b). However, when shifted to the non-permissive temperature (38.5 °C), large T antigen is inactivated and a senescence-like cell-cycle arrest was induced within 5 days (Fig. 1a, b and see Supplementary Information, Fig. S1a). Importantly, once the senescence-like phenotype was induced, subsequent inactivation of pRb and p53 by large T antigen was no longer able to revoke cell-cycle arrest, even though a significant fraction of cells reinitiated DNA synthesis (Fig. 1a–d). These results are consistent with previous data from senescent human primary fibroblasts14, 16, indicating that SVts8 cells are an ideal model system to study irreversible senescent cell-cycle arrest. Interestingly, if Svts8 cells were cultured in low serum (0.2%) medium throughout the incubation at 38.5 °C, a significant fraction of cells reinitiated not only DNA synthesis, but also cell proliferation on shifting the temperature to 34 °C in normal serum (10%) medium (Fig. 1a, c and d). This is not simply because large T antigen was incompletely inactivated in low serum medium at 38.5 °C, as large T antigen was equally dissociated from both pRb and p53, and transcriptional control activities of pRb and p53 were similarly restored at 38.5 °C regardless of serum concentration in culture medium (Fig. 1b and see Supplementary Information, Fig. S1a). Thus, it seems that pRb and/or p53 may require mitogenic signals to initiate a cascade of molecular events underlying irreversible senescent cell-cycle arrest18, 19.

Figure 1. Mitogenic signals cooperate with pRb and/or p53 to induce irreversible cell-cycle arrest.
Figure 1 thumbnail

(a) Time line of temperature shift experiments using SVts8 cells. SVts8 cells were cultured in normal serum (10%) medium at 34 °C. Cells were then incubated at 38.5 °C in normal serum medium or in low serum (0.2%) medium for 5 days. These cells were subsequently incubated at 34 °C in normal serum medium for another 5 days. Representative photographs of the cells in the indicated culture conditions are shown. The scale bars represent 200 mum. (b) Total cell lysates were prepared at indicated times in a (clumn number corresponds to culture condition in Fig. 1a) and were immunoblotted (IB) after immunoprecipitation (IP). The antibodies used are indicated. The total amount of Cdc2, p21 and beta-actin were monitored by direct immunoblotting of the cell lysate. (c) A BrdU incorporation assay was performed at the times indicated in a. The means plusminus s.d. of three independent experiments are shown. (d) SVts8 cells were cultured at 38.5 °C either in normal serum medium (4) or in low serum medium (5) for 5 days. Cells were then cultured at 34 °C in normal serum medium and were subjected to a cell proliferation assay performed in triplicate. The means plusminus s.d. of three independent experiments are shown.



Full FigureFull Figure and legend (35K)
To understand how mitogenic signalling collaborates with pRb and/or p53 to induce irreversible senescent cell-cycle arrest, we focused on the intracellular levels of ROS because ROS are known to be induced by various mitogenic signals20 and are implicated in the onset of cellular senescence21. Intriguingly, the levels of ROS were significantly increased on shifting the temperature to 38.5 °C in normal serum medium (Fig. 2a) and this level remained high even after pRb and p53 were subsequently inactivated by shifting the temperature to 34 °C in SVts8 cells (Fig. 2a). Importantly, induction of ROS was markedly attenuated if SVts8 cells were cultured in low serum medium throughout the incubation at 38.5 °C (Fig. 2a), suggesting that the increased level of ROS may determine the irreversibility of senescent cell-cycle arrest. To test this hypothesis, production of ROS was inhibited by addition of N-acetyl-cysteine (NAC) throughout the incubation at 38.5 °C in normal serum medium (Fig. 2b). Interestingly, NAC treatment enabled SVts8 cells to reinitiate cell proliferation on shifting the temperature to 34 C in normal serum medium (Fig. 2c). Similar results were also observed when diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide diphoshate (NADPH) oxidase, was used instead of NAC (see Supplementary Information, Fig. S1c, d and e).

Figure 2. Involvement of ROS–PKCdelta signalling in irreversible cell-cycle arrest.
Figure 2 thumbnail

(a) SVts8 cells were cultured as described in Fig. 1a (lane number corresponds to culture condition) and relative ROS levels at the indicated times were measured by DCF-DA staining. (b, c) SVts8 cells were cultured at 38.5 °C for 5 days in normal serum medium with NAC at the doses indicated. These cells were subsequently incubated at 34 °C for 5 days in normal serum medium without NAC. Cells were then subjected to analysis of relative ROS levels (b) or to cell proliferation assay performed in triplicate (c). The error bars indicate s.d. (d) SVts8 cells were cultured as described in Fig. 1a and protein expression at the indicated times was examined by western blotting using antibodies indicated. (e, f) SVts8 cells were cultured at 38.5 °C for 5 days in normal serum medium with rottlerin at the doses indicated. These cells were subsequently incubated at 34 °C in normal serum medium without rottlerin for 5 days. Cells were then subjected to analysis of relative ROS levels (e) or to cell proliferation assay performed in triplicate (f). The error bars indicate s.d. (g) Cells at the indicated times (4 and 5) in Fig. 1a were stained with DAPI. The histograms indicate the percentage of polynucleated cells. The error bars indicate s.d. The scale bars represent 50 mum.



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PKCdelta has an established role in activating NADPH-oxidase through phosphorylating p47phox, an essential component of NADPH oxidase7, 8, and the levels of its catalytically active fragment (PKCdelta-CF) were shown to be increased during replicative senescence in human diploid fibroblasts (HDFs)22. This evidence, in conjunction with previous reports showing that PKCdelta acts as a critical downstream mediator of the ROS signalling pathway7, 8, 23, led us to hypothesize that once activated by ROS, PKCdelta itself activates production of ROS through activating NADPH oxidase, thereby establishing a positive-feedback loop to sustain the levels of ROS, even after pRb and p53 were subsequently inactivated in senescent cells. To explore this possibility, the levels of PKCdelta-CF and its kinase activity were measured in SVts8 cells. An intense band with a relative molecular mass of 40,000 (Mr, 40K), which corresponds to PKCdelta-CF, was observed almost exclusively on restoration of pRb and p53 in normal serum medium in SVts8 cells (Fig. 2d). Importantly, the levels of PKCdelta-CF and its kinase activity were even higher when pRb and p53 were subsequently inactivated, and correlated well with the levels of ROS (Fig. 2a, d and see Supplementary Information, Fig. S1f). Furthermore, treatment with rottlerin, a selective PKCdelta inhibitor, throughout the incubation at 38.5 °C in normal serum medium enabled SVts8 cells to reinitiate cell proliferation on shifting the temperature to 34 °C in fresh normal serum medium. Notably, this was accompanied by a substantial reduction in the levels of ROS (Fig. 2e, f), indicating that PKCdelta indeed has a key role in the maintenance of ROS production in senescent cells.

As it has previously been shown that PKCdelta may have the potential to block cytokinesis24, we next examined the morphology and cell-cycle profiles of irreversibly arrested SVts8 cells. A dramatic increase in polynucleated cells was observed when the temperature was shifted to 34 °C after 5 days culture at 38.5 °C in normal serum medium (Fig. 2g and see Supplementary Information, Fig. S2a), indicating that these cells are likely to have severe defects in cytokinesis. Such abnormal phenotypes were not observed if cells were cultured in low serum medium throughout the incubation at 38.5 °C (Fig. 2g and see Supplementary Information, Fig. S2a). Intriguingly, the levels of WARTS were inversely correlated with those of PKCdelta-CF in SVts8 cells (Fig. 2d). Moreover, inhibition of ROS production by NAC treatment throughout the incubation at 38.5 °C resulted in marked recovery of the level of WARTS (Fig. 3a). Conversely, the levels of activated caspase-3, a known activator of PKCdelta6, and PKCdelta-CF were diminished (Fig. 3a). Similar results were also observed when rottlerin was used instead of NAC (Fig. 3b). In contrast, treatment with H2O2 to increase the intracellular levels of ROS caused an activation of caspase-3, a significant induction of PKCdelta activity, a remarkable reduction in WARTS expression and a senescent-like cell-cycle arrest in SVts8 cells (Fig. 3c and see Supplementary Information, Fig. S2b–e). Furthermore, ectopic expression of PKCdelta–CF had similar effects in SVts8 cells at 34 °C (Fig. 3d, e). Taken together, these results strongly suggest that PKCdelta is an upstream regulator of WARTS expression and has positive feedback effects on ROS production in senescent cells.

Figure 3.  Downregulation of WARTS by PKCdelta.
Figure 3 thumbnail

(a) SVts8 cells were cultured at 38.5 °C for 5 days in normal serum medium with NAC at the doses indicated. These cells were subsequently incubated at 34 °C for 5 days in normal serum medium without NAC. Cells were then subjected to western blotting with antibodies indicated. (b) SVts8 cells were cultured at 38.5 °C for 5 days in normal serum medium with Rottlerin at the doses indicated. These cells were subsequently incubated at 34 °C in normal serum medium without Rottlerin for 5 days. Cells were then subjected to western blotting with the antibodies indicated. (c) SVts8 cells cultured at 34 °C were treated with H2O2 as previously described31. Cells were then subjected to western blotting analysis using the antibodies indicated. (d, e) SVts8 cells were infected with retrovirus encoding PKCdelta-CF or control vector at 34 °C. Cells were then subjected to analysis of relative ROS level, western blotting using the antibodies indicated (d) or to cell proliferation assay performed in triplicate at 34 °C in normal serum medium (e). The error bars represent s.d. (f, i) SVts8 cells were infected with retrovirus encoding RNAi against p16INK4a or control26 at 34 °C. Cells were then incubated at 38.5 °C in normal serum medium for 5 days and subsequently incubated at 34 °C in fresh normal serum medium for another 5 days. Cells were then subjected to western blotting using the antibodies indicated (f), cell proliferation assay performed in triplicate (g), analysis of relative ROS levels (h) or to SA-beta-gal assay (i). The error bars represent s.d.



Full FigureFull Figure and legend (44K)
Unlike HDFs, PKCdelta-CF was not induced during replicative senescence in MEFs (data not shown). However, treatment with H2O2 led to a substantial induction in PKCdelta–CF and concomitant reduction of WARTS in wild-type MEFs, but not in MEFs lacking the PKCdelta gene (see Supplementary Information, Fig. S2f)25. These results further support the role of PKCdelta in enforcing stable cell-cycle arrest (see Supplementary Information, Fig. S2g–i). It is noteworthy that the reduction in WARTS was attenuated by the addition of MG132, a proteasome inhibitor (see Supplementary Information, Fig. S3a). However, mutation of the consensus sequence for phosphorylation by PKCdelta (Ser 464 to Ala) did not alter the stability of WARTS in senescent cells (see Supplementary Information, Fig. S3b), suggesting that PKCdelta may regulate the stability of WARTS indirectly through phosphorylating protein(s) controlling the stability of WARTS. It is also worth emphasizing that reduction of the levels of p16INK4a using RNA interference (RNAi)26 diminished activation of ROS–PKCdelta signalling and enabled SVts8 cells to reinitiate cell proliferation on shifting the temperature to 34 °C from 38.5 °C (Fig. 3f–i). Taken together, these data and previous reports showing that p16INK4a can initiate an autonomous senescence programme14, 15, indicate that the p16INK4a–Rb pathway ensures the irreversibility of senescent cell-cycle arrest, at least in part through activating ROS–PKCdelta signalling.

To ascertain the role of the ROS–PKCdelta signalling pathway in primary human cells, we examined whether the ROS–PKCdelta signalling pathway was activated in Ras-induced senescent HDFs27, 28. The levels of ROS and PKCdelta-CF were significantly increased and the level of WARTS was significantly reduced in Ras-induced senescent TIG-3 cells and in Hs68 cells (Fig. 4a, b and see Supplementary Information, Fig. S3c). Importantly, subsequent expression of large T antigen was unable to revoke ROS–PKCdelta signalling or cell-cycle arrest in Ras-induced senescent TIG-3 cells, despite its ability to induce DNA synthesis (see Supplementary Information, Fig. S3d–g), indicating that ROS–PKCdelta signalling pathway does serve as an additional level of security to prevent cell-cycle re-entry in senescent primary human cells. As shown by the experiments in Fig. 3d, overexpression of PKCdelta–CF alone strikingly reduced the level of WARTS and blocked cell proliferation in early passage TIG-3 cells (Fig. 4a, c). Significantly, the arrested cells displayed phenotypic features of cellular senescence (Fig.4d). Moreover, overexpression of PKCdelta-CF itself substantially elevated the levels of ROS and activated caspase-3 in early passage TIG-3 cells (Fig. 4b and see Supplementary Information, Fig. S3h), demonstrating that PKCdelta also has positive feedback effects on ROS production in primary human cells. Interestingly, the levels of ROS induced by overexpression of PKCdelta-CF were remarkably attenuated when cells were treated with DPI (see Supplementary Information, Fig. S3i), suggesting that NADPH oxidase is implicated in this setting. Overexpression of a dominant negative form of WARTS (WARTSKD)9 did not increase the level of ROS or PKCdelta-CF (Fig. 4a, b), but significantly inhibited cell proliferation accompanied by a substantial increase in polynucleated TIG-3 cells (Fig 4e, f), as previously reported9, 10, 11. These results therefore indicate that a reduction in WARTS expression is, at least partly responsible for the cytokinetic block in senescent cells.

Figure 4. p16INK4a–Rb pathway elicits ROS–PKCdelta signalling in primary human diploid fibroblasts.
Figure 4 thumbnail

(a–f) Early passage (40 population doublings level; PDL) TIG-3 cells were infected with retrovirus encoding oncogenic Ras (Ras), p16INK4a, PKCdelta-CF, WARTSKD (ref. 9) or control empty vector. Cells were then subjected to western blotting with antibodies indicated (a), or to analysis of intracellular levels of ROS (b). TIG-3 cells infected with retrovirus encoding PKCdelta-CF or control empty vector were subjected to cell proliferation assay in triplicate (c) and to SA-beta-gal analysis (d). TIG-3 cells infected with retrovirus encoding WARTSKD or control empty vector were subjected for cell proliferation assay performed in triplicate (e). Representative images of the indicated cells are shown (f). (g, h) Early passage (40 PDL) TIG-3 cells were infected with retrovirus encoding RNAi against DP129 or control sequence. After selection with antibiotics, cells were then subjected to western blotting with the antibodies indicated (g) or to analysis of intracellular levels of ROS (h). These assays were performed in triplicate and representative results are shown in g. The error bars represent s.d. The scale bars represent 200 mum in d and 100 mum in f.



Full FigureFull Figure and legend (37K)
To obtain a mechanistic insight into how p16INK4a–Rb-pathway promotes ROS production, we next examined the effects of the reduction of the level of DP1, an essential activator of the E2F transcription factor, by RNA interference (RNAi)29. Interestingly, reduction of the level of DP1 led to a significant increase in the levels of ROS and PKCdelta-CF expression and substantial reduction in WARTS expression in TIG-3 cells (Fig. 4g, h). Notably, the level of manganese superoxide dismutase (MnSOD) expression was increased, whereas those of glutathione peroxidases (GPX) and catalase expression were slightly decreased in DP1-knockdown cells (Fig. 4g). Thus, it is possible that more and more superoxide radicals are converted to H2O2 but are not detoxified to water and oxygen, resulting in accumulation of H2O2, a part of ROS, in DP1-knockdown cells. These results are consistent with a previous report showing that overexpression of MnSOD increases the levels of ROS30. Taken together, our results strongly suggest that the p16INK4a–Rb pathway provokes ROS–PKCdelta signalling through blocking the E2F activity.

To further extend these findings to replicative senescence, ROS–PKCdelta signalling was examined in late passage TIG-3 cells. TIG-3 cells lose their proliferative activity and senesce at around 79 population doublings. These cells were classed as 'early senescent' and were cultured for a further 3 weeks before being classed as 'late senescent'. Although lentivirus mediated large T antigen expression enabled early-senescent cells to reinitiate DNA synthesis and cell proliferation, a similar level of large T antigen expression was unable to stimulate cell proliferation in late-senescent cells (Fig. 5a, b). Importantly, the levels of p16INK4a, ROS and PKCdelta-CF in late-senescent cells were significantly higher than those in early-senescent cells (Fig. 5c, d). Consistent with these results, the level of WARTS was strikingly reduced in late-senescent cells (Fig. 5c) and these levels were unchanged even when large T antigen was subsequently expressed in late-senescent cells (Fig. 5c, d), indicating that ROS–PKCdelta signalling pathway does have a role in replicative senescence. It is also interesting to note that the levels of proteins involved in cytokinesis that we tested were all reduced in late-senescent cells (Fig. 5c). However, the expression levels of these proteins, with the exception of WARTS, returned to the original levels when large T antigen was expressed (Fig. 5c), illustrating the importance of WARTS as a critical downstream target of ROS–PKCdelta signal in late senescence.

Figure 5. Irreversibility of replicative senescence.
Figure 5 thumbnail

(a–d) Young (early passage), early-senescent or late-senescent TIG-3 cells were infected with lentivirus encoding either SV40 large T antigen (LT) or GFP (control). Five days later, these cells were subjected to BrdU incorporation analysis (a), cell proliferation analysis (b), western blotting using the indicated antibodies (c) or to assay for intracellular levels of ROS (d) as described in Fig. 2. These assays were performed in triplicate and representative results are shown in c. The error bars represent s.d. (e) Dual roles for the p16INK4a–Rb pathway in senescent cell-cycle arrest. In proliferating cells, the effects of mitogenic signals in ROS production are counterbalanced by E2F–DP activity. However, when E2F–DP activity is shut down by fully activated pRb, mitogenic signalling, in turn, increases the level of ROS and elicits a positive feedback activation of ROS–PKCdelta signalling pathway. Elevated levels of p16INK4a therefore establish an autonomous activation of ROS–PKCdelta signalling, leading to an irrevocable block to cytokinesis in human senescent cells.



Full FigureFull Figure and legend (43K)
As we were unable to recover the level of WARTS by ectopic expression in senescent cells (see Supplementary Information, Fig. S3b), it is still unclear whether WARTS is the most critical downstream target of the ROS–PKCdelta signalling pathway towards the cytokinetic block in senescent cells. It is possible that PKCdelta may have other targets to enforce the cytokinetic block22. However, overexpression of a dominant negative form of WARTS, on its own, significantly inhibited cell proliferation accompanied by a substantial increase in polynucleated cells (Fig. 4e, f). These results, together with previous reports showing that WARTS is a MEN-kinase required for cytokinesis9, 10, 11, strongly suggest that reduction of WARTS is, at least partly responsible for cytokinetic block in senescent cells. Together, our data reveal a novel function for the p16INK4a–Rb pathway operating in human cell senescence (Fig. 5e). This system may serve as a fail-safe mechanism, especially in case of the accidental inactivation of pRb and p53 in human senescent cells.

Methods
Cells and cell culture.
Normal human diploid fibroblasts TIG-3 cells and HS68 cells were cultured in DME supplemented with 10% fetal bovine serum (FBS) at 37 °C28. SVts8 cells were cultured in DME supplemented with 10% FBS at 34 °C17. Early passage TIG-3 cells (40 population doublings) and Hs68 cells (45 population doublings) were used as young cells. Cell proliferation analysis and SA-betagal assay were performed as previously described29.

Viral infections.
TIG-3 cells and SVts8 cells were rendered sensitive to infection by ecotropic retroviruses as previously described29 and infected with recombinant retroviruses encoding Ras V12 (in pBabe–puro)27, PKCdelta-CF (in pMarX–hygro)6, p16 (in pMarX–hygro)28, WARTSKDK734A(in pMarX–puro)9, p16 shRNA (in pRetrosuper–hygro)26 or DP1 shRNA (in pRetrosuper–puro)29. Pools of drug-resistant cells were analysed 7 days after infection. For lentiviral infection, senescent TIG-3 cells were infected with lentivirus encoding either large T antigen or GFP as previously described14. The infection efficiency (>99%) was monitored by GFP.

Protein analysis.
Immunoblotting and immunoprecipitation were performed as previously described28 with primary antibodies against aurora A (610938; BD Biosciences, San Jose, CA), aurora B (611082; BD), beta-actin (sc-8432; Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3 (9662; Cell Signalling, Beverley, MA), catalase (ab1877; Abcam, Cambridge, UK), cdc2 (#17; Cancer Research UK, London, UK), cyclin A (sc-751; Santa Cruz), cyclin B1 (sc-752; Santa Cruz), DP1 (11834; Abcam), GPX (mo15-3; MBL, Nagoya, Japan), large T antigen (L-19)17, MnSOD (611580; BD), PKCdelta (sc-937; Santa Cruz), PLK1 (06-813; Upstate, Lake Placid, NY), PRC1 (gift from W. Jiang, Burnham Insititute, La Jolla, CA), p16 (Ab-1; Oncogene, Boston, MA), p21 (sc-397; Santa Cruz), p53 (Ab-6; Calbiochem, San Diego, CA), Ras (Ab-4; Oncogene), RB (sc-102; Santa Cruz and 554136; BD), survivin (AF886; R&D Systems, Minneapolis, MN) and WARTS (C-2)9. In some experiments, cells were incubated with 10 muM MG132 (Calbiochem) for 12 h before harvest.

Analysis of intracellular ROS.
To assess the generation of intracellular ROS levels, cells were incubated with 20 muM DCF-DA (Calbiochem) for 20 min at 37 °C. The peak excitation wavelength for oxidized DCF was 488 nm and for emission was 525 nm.

Protein kinase assay.
Immune complex kinase assay was performed as previously described23 with antibody against PKCdelta (sc-937: Santa Cruz). The incubation was carried out for 5 min or 30 min at 30 °C.

Note: Supplementary Information is available on the Nature Cell Biology website.

 Top
Received 22 June 2006; Accepted 30 August 2006; Published online: 8 October 2006.

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Acknowledgements
We thank D. Mann, G. Peters, J. Campisi and U. Kikkawa for helpful comments on the manuscript; R. Agami for providing p16 knockdown construct; D. Beach and G. Hannon for providing pMarX retrovirus vector; W. Jiang and R. Fukunaga for providing anti-PRC1 antibody; and D. Kufe and S. Ohno for PKCdelta cDNA. We also thank A. Hirao for his useful suggestion in analysis of intracellular ROS. This work was supported by grants from Ministry of Education, Science, Sports and Technology of Japan, Ministry of Health, Labour and Welfare of Japan, the Uehara Memorial Foundation, the Sumitomo Foundation, the Nakatomi Foundation, the Sagawa Foundation for Promotion of Cancer Research, the Inamori Foundation and Sankyo Foundation of Life Science (E.H.). A.T. is supported by the Japan Society for the Promotion of Science.

Competing interests statement:  The authors declare that they have no competing financial interests.

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