Published OnlineFirst November 9, 2010; DOI: 10.1158/0008-5472.CAN-10-0801 Published OnlineFirst on November 9, 2010 as 10.1158/0008-5472.CAN-10-0801 Tumor and Stem Cell Biology Cancer Research Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo Shinji Takeuchi1,4,6, Akiko Takahashi1, Noriko Motoi2, Shin Yoshimoto1, Tomoko Tajima1,3, Kimi Yamakoshi1, Atsushi Hirao5, Shigeru Yanagi3, Kiyoko Fukami3, Yuichi Ishikawa2, Saburo Sone6, Eiji Hara1, and Naoko Ohtani1 Abstract Although the p16INK4a and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16INK4a and p21Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16INK4a and INK4a Waf1/Cip1 p21Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16 and p21 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras- induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either INK4a Waf1/Cip1 p16 or p21 , indicating that p16INK4a and p21Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz (a)anthracene/12-O-tetradecanoylphorbol-13-acetate–induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16INK4a loss. Our findings establish an intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor sup- pression in vivo. Cancer Res; 70(22); 9381–90. ©2010 AACR. Introduction that cellular senescence can be induced by a variety of po- tentially oncogenic stimuli, such as telomere shortening, The p16INK4a and p21Waf1/Cip1 cyclin-dependent kinase DNA damage, oxidative stress, or oncogene expression (CDK) inhibitors are known to play key roles in the onset (11–14), suggesting that cellular senescence is likely to act as of cellular senescence, a state of permanent cell cycle arrest a tumor suppression mechanism in vivo (15, 16). Although in culture (1–6). The simultaneous induction of p21Waf1/Cip1and the roles of p16Ink4a and p21Waf1/Cip1 in cellular senescence p16Ink4a expression cooperatively blocks the activation of both are well documented in various cell culture studies, the in vivo cyclin D kinase (CDK4/6) and cyclin E kinase (CDK2), allowing roles of these CDK inhibitors are poorly understood (17). For INK4a Waf1/Cip1 the accumulation of the dephosphorylated form of the retino- example, mice lacking p16 (18, 19) or p21 (20–22) blastoma tumor suppressor protein (pRb) and thereby causing exhibit only a little predisposition to spontaneous tumor for- permanent cell cycle arrest (7–10). It has become apparent mation. These observations raise the question of whether the results seen in cell culture studies truly reflect the physiologic in vivo Authors' Affiliations: Divisons of 1Cancer Biology and 2Pathology, The roles of these CDK inhibitors . However, it is also possi- Cancer Institute of Japanese Foundation for Cancer Research (JFCR); ble that these weak phenotypes could be due to functional 3School of Life Science, Tokyo University of Pharmacy and Life compensatory effect(s) between p16Ink4a and p21Waf1/Cip1. Sciences, Tokyo, Japan; Divisions of 4Medical Oncology and INK4a 5Molecular Genetics, Cancer Research Institute, Kanazawa University, To directly address the cooperative roles of p16 and Kanazawa,Japan;and6Institute of Health Biosciences, University of p21Waf1/Cip1 in vivo, we generated a compound mouse line INK4a Waf1/Cip1 Tokushima, Tokushima, Japan simply lacking both of the p16 and p21 genes Note: Supplementary data for this article are available at Cancer [double-knockout (DKO) mouse] on a C57BL/6 background, Research Online (http://cancerres.aacrjournals.org/). which is known to be carcinogenesis resistant (23–25), and Corresponding Author: Naoko Ohtani, Division of Cancer Biology, The Cancer Institute of Japanese Foundation for Cancer Research (JFCR), evaluated the roles of these two critical senescence inducers Tokyo 135-8550, Japan. Phone: 81-3-3570-0605; Fax: 81-3-3570- in vitro and in vivo. Intriguingly, DKO mice are significantly 0457; E-mail: [email protected]. more susceptible to chemical carcinogenesis compared with INK4a Waf1/Cip1 doi: 10.1158/0008-5472.CAN-10-0801 mice lacking either p16 or p21 alone. Moreover, ©2010 American Association for Cancer Research. mouse embryonic fibroblasts (MEF) derived from DKO mice www.aacrjournals.org 9381 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst November 9, 2010; DOI: 10.1158/0008-5472.CAN-10-0801 Takeuchi et al. are resistant to the onset and the maintenance of cellular se- into 6-cm-diameter dishes. Cells were maintained for 15 days INK4a nescence in culture. These results indicate that p16 in a 3% O2/5% CO2 culture environment, and the medium plays a critical role in cooperating with p21Waf1/Cip1 in tumor was changed twice a week until the cells were photographed suppression in vivo. and counted. Senescence-associated β-galactosidase assay Materials and Methods Senescence-associated β-galactosidase assay was done as described previously (28). −/− −/− Generation of p16 ;p21 mice −/− p16 mice (C57BL/6) were kindly provided by N.E. Sharp- Analysis of intracellular reactive oxygen species less (University of North Carolina Lineberger Comprehensive To assess the generation of intracellular levels of reactive −/− Cancer Center, Chapel Hill, NC; ref. 19). p21 mice (C57BL/6) oxygen species (ROS), cells were incubated with 20 μmol/L were kindly provided by P. Leder (Harvard Medical School, 2′,7′-dichlorofluorescein diacetate (Calbiochem) for 20 min- −/− −/− Boston, MA; ref. 20). p16 ;p21 DKO mice were generated utes at 37°C. The peak excitation wavelength for oxidized −/− −/− by crossing p16 mice and p21 mice. All animals were 2′,7′-dichlorofluorescein was 488 nm and the emission wave- cared for by using protocols approved by the Committee for length was 525 nm as described previously (6). the Use and Care of Experimental Animals of the Japanese Foundation for Cancer Research. Chemically induced skin tumor formation The chemically induced skin tumor formation analysis was Cell culture done as described previously (29). Briefly, mice of the wild- MEFs were generated from E13.5 embryos of wild-type, type, p16-KO, p21-KO, or DKO genotypes in the resting phase −/− −/− −/− −/− p16 , p21 ,orp16 ;p21 DKO mice as previously de- of the hair cycle (8 weeks old) were shaved and treated with scribed (26). MEFs were grown in DMEM supplemented with 100 μg of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 μL 10% fetal bovine serum in 3% O2/5% CO2 for 2 days, har- of acetone. One week after DMBA treatment, mice were sub- vested, viably frozen, and labeled as passage 0. Serial 3T3 cul- sequently treated twice a week with 12.5 μg of 12-O-tetrade- tivation was done as described (27). Cells were counted in canoylphorbol-13-acetate (TPA) in 100 μL of acetone for 20 triplicate. The number of cells present on the third day weeks. Tumors developed in mice were analyzed for histolo- (N3) was divided by the initial cell number (N0 = 3 × 105) gy at 30 weeks after DMBA treatment. and plotted as growth rate (N3/N0; Supplementary Fig. S1). The increase in the population doubling level (ΔPDL) was Histologic analysis calculated according to the following formula: ΔPDL = log Tissues were fixed in 10% buffered formalin, progressively (Nf/N0)/log 2, where N0 is the initial number of cells (3 × 105) dehydrated through gradients of alcohol, and embedded in and Nf is the final number of cells. paraffin. Samples were sectioned on a microtome at 4-μm thickness, deparaffinized in xylene, rehydrated, and then Western blot analysis stained with H&E for histologic analysis. Proteins (40 μg) were analyzed by Western blotting, as pre- viously described (6). The primary antibodies used were Results p16INK4a (11104 IBL), p21Waf1/Cip1 (sc-6246, Santa Cruz Bio- technology, Inc.), p15INK4b (sc-613, Santa Cruz Biotechnology), p16INK4a;p21Waf1/Cip1 DKO MEFs proliferate without any p53 (1C12, Cell Signaling), p19Arf [ab80 (Abcam) or sc-32748 induction of cellular senescence by serial passaging INK4a Waf1/Cip1 (Santa Cruz Biotechnology)], H-Ras (sc-29, Santa Cruz Compound mice lacking both p16 and p21 on a Biotechnology), pRb (554136, BD Pharmingen), β-actin C57BL/6 background (DKO mice) are fertile and born normally (AC-74, Sigma-Aldrich), and α-tublin (DM-1A, Sigma-Aldrich). in the expected Mendelian ratio. MEFs derived from wild-type INK4a Waf1/Cip1 Secondary antibodies were detected by enhanced chemilumi- mice, p16 knockout (p16-KO) mice, p21 knockout nescence (Amersham). (p21-KO) mice, and DKO mice were subjected to serial passage using the 3T3 protocol under standard culture condi- Retrovirus