Hypoxia Suppresses Conversion from Proliferative Arrest to Cellular Senescence

Hypoxia suppresses conversion from proliferative arrest to cellular senescence

Olga V. Leontieva, Venkatesh Natarajan, Zoya N. Demidenko, Lyudmila G. Burdelya, Andrei V. Gudkov, and Mikhail V. Blagosklonny1

Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263

Edited* by Arthur B. Pardee, Dana-Farber Cancer Institute, Boston, MA, and approved July 4, 2012 (received for review April 11, 2012) Unlike reversible quiescence, cellular senescence is characterized by Importantly, agents that inhibit mTOR can suppress gerocon- alargeflat cell morphology, β-gal staining and irreversible loss of re- version even though they cause reversible arrest on their own (21– generative (i.e., replicative) potential. Conversion from proliferative 23). As an example, while causing cell cycle arrest, p53 may de- arrest to irreversible senescence, a process named geroconversion, is celerate or suppress geroconversion by inhibiting mTOR (in some driven in part by growth-promoting pathways such as mammalian conditions and cell lines) (21–23). Hypoxia can cause cell cycle target of rapamycin (mTOR). During cell cycle arrest, mTOR converts arrest. Simultaneously, hypoxia decreases cap-dependent trans- reversible arrest into senescence. Inhibitors of mTOR can suppress ger- lation, by inhibiting the mTOR pathway, as well as global trans- oconversion, maintaining quiescence instead. It was shown that hyp- lation by phosphorylating eukaryotic initiation eIF2a and elongation oxia inhibits mTOR. Therefore, we suggest that hypoxia may suppress eEF2 factors (24–36). Given that hypoxia inhibits the mTOR geroconversion. Here we tested this hypothesis. In HT-p21-9 cells, ex- pathway, we suggest that hypoxia should suppress geroconversion pression of inducible p21 caused cell cycle arrest without inhibiting during cell cycle arrest caused by other agents, which otherwise mTOR, leading to senescence. Hypoxia did not prevent p21 induction cause senescence. Here we tested this hypothesis. and proliferative arrest, but instead inhibited the mTOR pathway and geroconversion. Exposure to hypoxia during p21 induction prevented Results senescent morphology and loss of regenerative potential, thus main- Hypoxia Suppresses p21-Induced Senescence. Initially, we investi- taining reversible quiescence so cells could restart proliferation after gated effects of hypoxia on p21-induced senescence in HT-p21-9 – switching p21 off. Suppression of geroconversion was p53- and HIF-1 cells. In these cells, IPTG induces ectopic p21, which causes cell independent, as hypoxia also suppressed geroconversion in cells lack- cycle arrest and senescence (1, 2, 7, 8, 21–23). First, we in- α fi ing functional p53 and HIF-1 . Also, in normal broblasts and retinal vestigated whether hypoxia affects the mTOR pathway. Hypoxia cells, hypoxia inhibited the mTOR pathway and suppressed senes- decreased phosphorylation of S6K (substrate of mTOR) and S6 cence caused by etoposide without affecting DNA damage response, (substrate of S6K; Fig. 1A). In contrast, like rapamycin, hypoxia p53/p21 induction and cell cycle arrest. Also hypoxia suppressed ger- did not affect basal p53 and p21 (Fig. 1A) and did not prevent p21 oconversion in cells treated with nutlin-3a, a nongenotoxic inducer of induction by IPTG (Fig. S1A). As expected, hypoxia did not p53, in cell lines susceptible to nutlin-3a–induced senescence (MEL-10, prevent the proliferative arrest caused by IPTG. In the presence A172,andNKE).Thus,innormalandcancer cell lines, hypoxia sup- of IPTG, cells did not proliferate under hypoxia and normoxia presses geroconversion caused by diverse stimuli. Physiological and (Fig. S1). In agreement with previous results (1, 2, 7, 8), IPTG clinical implications of the present findings are discussed. caused a senescent morphology (Fig. 1B). Hypoxia partially prevented a large flat cell morphology (Fig. 1B)andβ-gal staining oncology | gerontology | biology (Fig. 1 B and C). Most importantly, hypoxia prevented loss of RP: the potential of IPTG-arrested cells to resume proliferation (Fig. ecent evidence emerges that the mammalian target of rapa- 1D and Fig. S1C). When treated with IPTG under normoxia, cells Rmycin (mTOR) pathway is involved in cellular aging (1, 2). lost RP and could not resume proliferation when IPTG was re- Nutrients, cytokines, growth factors, and hormones activate the moved (Fig. 1D). Rapamycin and hypoxia preserved RP during mTOR pathway, which drives cellular mass growth (3, 4). In IPTG-induced arrest. Furthermore, when we treated cells with proliferating cells, cellular growth in size is balanced by cell di- IPTG under different conditions (normoxia, rapamycin, hypoxia, vision. When the cell cycle is arrested and cells thus do not divide, rapamycin plus hypoxia) and, after 3 to 4 d, washed and cultured inappropriate activation of growth-promoting pathways such as them under normoxia, IPTG-pretreated cells could not resume mTOR converts cell cycle arrest into senescence (1, 2). Senescence E fl β proliferation and did not form colonies (Fig. 1 ). Hypoxia and is characterized by a large at cell morphology, -gal staining, and rapamycin prevented loss of RP in IPTG-pretreated cells so that a hypersecretory phenotype (5, 6). In a widely used cellular model, cells formed colonies (Fig. 1E). To exclude that hypoxia abro- induction of ectopic p21 by isopropyl-thio-galactosidase (IPTG) – gated IPTG-induced arrest in some rare cells, which then could arrests HT-p21-9 cells (7, 8). Initially (during 2 3d),thiscondi- form colonies, we also treated cells with IPTG in the presence of tion is reversible: when p21 is switched off, cells resume pro- nocodazole, which eliminates cells not arrested by IPTG (37) liferation (7, 8). While inhibiting the cell cycle, p21 does not (Fig. 1E). Like rapamycin, hypoxia preserved RP in the presence inhibit mTOR, which in turn converts arrest into irreversible se- of nocodazole (Fig. 1E). This indicates that neither rapamycin nescence (1). By day 3, cells become large, flat, and β-gal–positive, and lose regenerative potential (RP): cells cannot resume proliferation when p21 is switched off. The conversion from reversible Author contributions: O.V.L., Z.N.D., A.V.G., and M.V.B. designed research; O.V.L., V.N., arrest to senescence, a process named geroconversion (9), is sup- Z.N.D., L.G.B., and M.V.B. performed research; V.N. and L.G.B. contributed new reagents/ pressed by rapamycin. Thus, rapamycin preserves RP in arrested analytic tools; O.V.L., Z.N.D., A.V.G., and M.V.B. analyzed data; and O.V.L., A.V.G., and cells, so the condition remains reversible: cells can resume pro- M.V.B. wrote the paper. liferation when p21 is switched off. The involvement of mTOR The authors declare no conflict of interest. in geroconversion links cellular senescence to organismal aging. *This Direct Submission article had a prearranged editor. Geroconversion is a cell culture model of cellular aging in the 1To whom correspondence should be addressed. E-mail: [email protected]. organism. In fact, inhibition of the mTOR pathway prolongs life- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. span in diverse organisms, including mammals (10–20). 1073/pnas.1205690109/-/DCSupplemental.

13314–13318 | PNAS | August 14, 2012 | vol. 109 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1205690109 Downloaded by guest on September 26, 2021 1A and Fig. S1A). Next, we determined whether the effect of hypoxia requires the p53 function by using parental HT-p21-9 and HT-p21-9-GSE56 cells, which lack functional p53 (21, 43, 44). As expected, in HT-p21-9-GSE56 cells, IPTG-induced senescence was preventable by rapamycin but not by nutlin-3a (21) (Fig. 2A). Hypoxia preserved RP in both cell lines (Fig. 2A). There was no additive effect of rapamycin and hypoxia (Fig. 2A). Next, we investigated whether HIF-1α was required for suppression of senescence by hypoxia by using siRNA technology to knock HIF-1α out. Hypoxia dramatically induced HIF-1α in control cells, whereas siRNA for HIF-1α prevented this induction (Fig. 2B). Despite lack of HIF-1α induction, hypoxia inhibited S6 phosphorylation (Fig. 2B). This result is in agreement with previous report that regulation of mTOR by hypoxia does not require HIF1α (26). Importantly, hypoxia preserved RP regardless of HIF-1α expression (Fig. 2C).

Hypoxia Suppresses Etoposide-Induced Senescence in Normal Cells. Next, we investigated the effect of hypoxia on senescence in normal cells. To induce senescence, we used low concentrations of etoposide, which has been shown to cause senescence in normal retinal pigment epithelial (RPE) and human tert-immortalized fibroblast WI-38t cells (23, 45). Specifically, cells become large, β-gal–positive and lose RP, so they could not resume proliferation when etoposide was removed. Rapamycin partially preserved RP Fig. 1. Hypoxia suppresses p21-induced senescence. (A) Immunoblot anal- without affecting DNA damage or cell cycle arrest (45). Here we ysis: HT-p21-9 cells were incubated under normoxia (i.e., control, indicated as show that, like rapamycin, hypoxia inhibited S6 phosphorylation “ ” “ ” C ), with 500 nM rapamycin ( R ), under hypoxia (0.2% oxygen, indicated in RPE cells (Fig. 3A). Unlike rapamycin, hypoxia increased as “H”), or under hypoxia plus rapamycin (R+H) for 24 h, and then lysed. Immunoblot was performed with the indicated antibodies. (B) β-gal staining: HT-p21-9 cells were treated with IPTG under normoxia or hypoxia. After 3 d, the cells were stained for β-gal. (Scale bar, 100 μm.) (C) HT-p21-9 cells were incubated with IPTG under normoxia (indicated as “I”), hypoxia (I+H), or with rapamycin (I+R). Percent of β-gal–positive cells was scored by counting microscopically β-gal–positive cells in three different fields for each sample. (D) RP: Cells were treated as described in C. After 3 d, IPTG was washed out and cells were cultivated without IPTG under normoxia for an additional 7 d. RP was determined as fold increase in cell numbers after drugs were washed off (Materials and Methods). (E) Colony formation: HT-p21-9 cells were treated with IPTG under normoxia or hypoxia or in the presence of 500 nM rapamycin (indicated as “R”). After 3 d, IPTG was washed out and colonies were allowed to grow in the fresh drug-free medium under normoxia. After 8 d, colonies were stained. When indicated, the experiment was performed in the presence of nocodazole (+Noc) during treatment with IPTG. (F)HT- p21-9 cells were treated with IPTG alone or with IPTG plus rapamycin in

normoxia or 0.2% O2 hypoxia. After 3 d, cells were washed and allowed to regrow in fresh drug-free medium. Cells were counted on the indicated days after wash (note that, on the day of the last count, cells reached conﬂuence in some wells).

nor hypoxia prevented IPTG-induced arrest. Furthermore, hyp-

oxia by itself caused cell cycle arrest in HT-p21-9 cells (Fig. S2). CELL BIOLOGY Noteworthy for further discussion, there was no additive effect of rapamycin and hypoxia (Fig. 1E). Next, we investigated whether the potential of cells to replicate was not limited to eight to 10 divisions, required to form visible colonies during 7 d of culture after removal of IPTG. Starting at day 8 after removal of IPTG, α– growth curves demonstrated that cells pretreated with IPTG un- Fig. 2. Effects of hypoxia is p53- and HIF-1 independent. (A) HT-p21-9 cells and HT-p21-GSE56 cells (lacking functional p53) were treated with IPTG in der hypoxia fully recovered, whereas cells cultivated with IPTG the presence or absence of 500 nM rapamycin (indicated as “R”)or10μM F under normoxia did not (Fig. 1 ). It is noteworthy that hypoxia Nutlin 3a (Nut) under normoxia or 0.2% O2 hypoxia. After 3 d, cells were did not potentiate the effect of rapamycin (Fig. 1F). washed and incubated in drug-free medium for 7 d. Colonies were stained with crystal violet. (B) HT-p21-9 cells were transfected with siRNA against Suppression of Senescence by Hypoxia Is Not p53- and HIF-1–Dependent. HIF-1α or mock-transfected (with lipofectamine alone). On the next day, cells “ ” Induction of p53 can inhibit the mTOR pathway (38–42). We were split and incubated under normoxia (indicated as N )or0.2%O2 “ ” have shown that induction of p53 inhibited geroconversion in hypoxia ( H ) for 36 h before being lysed. Immunoblotting was performed with indicated antibodies. (C) HT-p21-9 cells transfected with siRNA for HIF- IPTG-treated HT-p21-9 cells (21, 23). Therefore, we investi- 1α or mock-transfected were treated with IPTG under normoxia or 0.2% O2 gated whether the effect of hypoxia was mediated by p53 in- hypoxia. After 3 d, cells were washed and incubated in drug-free medium duction. However, hypoxia did not induce p53 in these cells (Fig. for 9 d. Colonies were stained with crystal violet.

Leontieva et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13315 Downloaded by guest on September 26, 2021 morphology (Fig. 4B) and partially preserved RP in nutlin- treated NKE cells (Fig. 4C). We extended these observations to additional cell lines, demonstrating inhibition of senescent morphology by hypoxia in MEL-10, A172, and TRT-HU1 cells (Figs. S6–S7), in which senescence was caused by nutlin-3a or etoposide. We further investigated the effect of hypoxia on senescence of stem cells. It is known that bone marrow-derived mesenchymal stem cells (MSCs) proliferate more efﬁciently in hypoxic conditions (46). To induce senescence, we treated these cells with nutlin-3a under normoxia or hypoxia. Cells treated under normoxia lost RP, whereas cells treated under hypoxia were able to resume proliferation after removal of nutlin-3a (Fig. S8). We noticed that hypoxia increased p-eEF2 in NKE cells (Fig. 4A) and other cells (Fig. 3A). However, results obtained in MEL- 9 and MEL-10 cells did not support the role of p-eEF2 in the gerosuppressive effect by hypoxia. These two related cell lines responded slightly differently to hypoxia. Although HIF-1α and p-eEF2 was induced in both cell lines, only in MEL-10 did we observe inhibition of the mTOR pathway (p-S6K and pS6; Fig. 5A). In agreement, hypoxia preserved RP only in MEL-10 cells (Fig. 5B). Hypoxia also decreased senescent morphology and β-gal staining caused by nutlin-3a in MEL-10 (Fig. 5C). Thus, hypoxia inhibits mTOR and geroconversion in one cell line (MEL-10), but causes eEF2 phosphorylation in both MEL cell lines (Fig. 5A), indicating that this effect is irrelevant to suppression of senescence. As a second argument, rapamycin decreased (rather than increased) p-eEF2 (Fig. 5A) in MEL-10 and -9 but efﬁciently suppressed senescence in these cells (Fig. 5C), consistent with our previous publication (22). The only similarity in effects of hypoxia and rapamycin on senescence was

Fig. 3. Hypoxia suppresses etoposide-induced senescence in normal RPE cells. (A) Immunoblot analysis: RPE cells were incubated under normoxia (control, indicated as “C”), hypoxia (0.2% oxygen; indicated by “H”), or in the presence of 10 nM rapamycin (“R”) for 24 h, and then lysed. Immunoblot was performed with indicated antibodies. (B) β-Gal staining: RPE cells were

treated with 1 μg/mL etoposide (Et) under normoxia or 0.2% O2 hypoxia or in the presence of 10 nM rapamycin (Et+R) for 3 d and stained for β-gal. (Scale bar, 100 μm.) (C) RP: Cells were treated as described in B. After 3 d, drugs were washed out and cells were cultivated in drug-free medium under normoxia for 7 d to regrow. RP was determined as fold increase in cell numbers after drugs were washed off (Materials and Methods).

phosphorylation of eEF2 (Fig. 3A). Also, hypoxia prevented cell enlargement (Fig. 3B). Importantly, when treated with etoposide under hypoxia, RPE cells partially retained RP and resumed proliferation after removal of etoposide (Fig. 3C). Cells that resumed proliferation fully recovered with almost normal expo- nential growth by day 10 (Fig. S3). Hypoxia also suppressed senescent morphology, β-gal staining, and partially preserved RP in WI38t cells (Fig. S4). It is noteworthy that hypoxia did not decrease levels of DNA damage response caused by etoposide in these cells. Speciﬁcally, etoposide-induced p53, p21, and p-H2AX were not decreased by hypoxia (Fig. S5).

– Hypoxia Suppresses Nutlin-3a Induced Senescence. We next inves- Fig. 4. Hypoxia suppresses nutlin-induced senescence in NKE cells. (A)Im- tigated senescence caused by nongenotoxic induction of p53 by munoblot analysis: NKE cells were treated with 2.5 μM nutlin-3a (indicated nutlin-3a. In several cell lines, nutlin-3a causes cell cycle arrest, as “N”) for 1 h before being placed in 1% O2 (i.e., hypoxia) or left in nor- but, at low concentrations, it does not inhibit mTOR, thereby moxia with or without 10 nM rapamycin (“R”). After 24 h, cells were lysed, leading to senescence (21–23, 37). In normal kidney epithelial and immunoblotting was performed by using the indicated antibodies. (B) β-Gal staining: NKE cells were treated with 2.5 μM nutlin-3a under hypoxia (NKE) cells, nutlin-3a induced p53 and p21 but only slightly β A (1% O2) or in normoxia. After 4 d, cells were stained for -gal. (C) RP: NKE cells affected pS6 (Fig. 4 ). Rapamycin suppressed pS6 without af- μ A were treated with 2.5 M nutlin-3a under 0.2% O2 hypoxia or in normoxia. fecting p53 and p21 induction (Fig. 4 ). Hypoxia did not prevent After 3 d, drugs were washed out, and cells were cultivated in drug-free induction of p53/p21 by nutlin-3a and inhibited pS6 especially in medium for 7 d. RP was determined as fold increase in cell numbers after nutlin-treated cells. Accordingly, hypoxia prevented senescent drugs were washed off (Materials and Methods).

13316 | www.pnas.org/cgi/doi/10.1073/pnas.1205690109 Leontieva et al. Downloaded by guest on September 26, 2021 additional components, including activation of growth-promoting and nutrient-sensing pathways such as mTOR (9). When the cell cycle is arrested, growth-promoting (i.e., anabolic) signaling pathways drive cellular mass growth, as well as compensatory lysosomal hyperactivity with cytoplasmic β-gal staining, hypersecretory phenotype, and permanent loss of proliferative potential (9). Numerous studies demonstrated that hypoxia inhibits the mTOR pathway by multiple mechanisms, depending on experi- mental conditions and cell lines (24–36, 47, 48). We confirmed here that hypoxia deactivated the mTOR pathway in our cellular models of geroconversion. Rapamycin suppressed geroconversion in these cellular models. Like rapamycin, hypoxia prevented irreversible cellular senescence. It was previously shown that hypoxia prevents replicative senescence in MEFs by preventing cell cycle arrest (49). Here we described suppression of geroconversion by hypoxia (a com- pletely unique phenomenon) rather than prevention of cell cycle arrest. In already arrested cells, hypoxia suppressed the conversion of cell cycle arrest into senescence. We caused cell cycle arrest by both DNA damaging (i.e., etoposide) and nondamaging (i.e., ectopic p21 and nutlin-3a) agents. Hypoxia did not prevent H2AX phosphorylation, p53/p21 induction, and cell cycle arrest caused by DNA damage, but instead inhibited the mTOR pathway. In the arrested cells, hypoxia decreased the mTOR activity and senescent phenotype and preserved RP. Most importantly, hypoxia prevented geroconversion during cell cycle arrest caused by ectopic p21 and nutlin-3a, which did not damage DNA. There are several implications of the present findings. Physi- Fig. 5. Hypoxia suppresses nutlin-induced senescence in MEL-10 cells, but ological cellular aging is a conversion of postmitotic cells into not in MEL-9 cells. (A) Immunoblot analysis: Mel-10 and MEL-9 cells were senescent cells. It is noteworthy that levels of oxygen in most incubated under normoxia (indicated by “C”) with or without 10 nM rapa- “ ” “ ” + normal tissues are lower than 1% to 3%. This suggests that low mycin ( R ), 0.2% O2 ( H ) or hypoxia plus rapamycin (R H) for 24 h and levels of oxygen can decelerate premature conversion to senes- lysed. Immunoblotting was performed by using the indicated antibodies. (B) μ cence and extend lifespan. Also, stem cell niches are often ex- RP: Mel10 and Mel9 cells were treated with 5 M nutlin-3a under hypoxia – (0.2% O2) or normoxia. After 3 d, drug was washed out and cells were cul- tremely hypoxic (50 52); perhaps this preserves a quiescent (not tivated in drug-free medium for 7 d. RP was determined as fold increase in senescent) status of stem cells. Finally, hypoxia may play a dual cell numbers after drugs were washed off (Materials and Methods). (C) β-Gal role in aging (53). It suppresses mTOR and geroconversion; staining: MEL-10 cells were treated with 5 μM nutlin-3a under hypoxia or in conversely, hypoxia induces HIF-1, which increases secretion of normoxia. After 3 d, cells were stained for β-gal. mitogens and cytokines. The physiological outcome may be determined by all factors. inhibition of S6K/pS6. We next investigated the involvement of Materials and Methods the LKB1/AMPK pathway, which acts upstream of both mTOR Cell Lines and Reagents. HT-p21-9 cells, derived from HT1080 human fibro- and eEF2 (24). In MEL10 and MEL9 cell lines, hypoxia induced sarcoma cells (American Type Culture Collection), provided by Igor Roninson phosphorylation of AMPK, in parallel with phosphorylation of (University of South Carolina, Charleston, SC), were previously described eEF2 (Fig. 5). However, phosphorylation of AMPK was much (1, 2, 7, 8). HT-p21-9-GSE56 cells, which lack transcriptional function of p53, stronger in MEL-9 cells compared with MEL-10 cells, even were described previously (21, 43, 44). HT-p21-9 cells were cultured in high- though hypoxia did not suppress geroconversion in MEL-9 cells. glucose DMEM without pyruvate supplemented with FC2 serum (HyClone fi Thus, phosphorylation of AMPK/eEF2 was not sufficient for FetalClone II; Thermo Scienti c). In these cells, p21 expression can be turned on or off by using IPTG (7, 8). WI38-Tert cells (WI38 fibroblasts immortalized gerosuppressive effects of hypoxia. Moreover, phosphorylation by Tert) were described previously (22, 23). RPE (2), NKE, MEL9 and MEL10 of AMPK and eEF2 was not necessary, given that rapamycin did melanoma cell lines, and A172 glioblastoma cells were obtained from not cause phosphorylation of AMPK and eEF2 in any cell lines American Type Culture Collection. TRT-HU1 (hTERT-immortalized non- CELL BIOLOGY tested. Furthermore, AMPK and eEF2 were paradoxically de- transformed human urothelial cells) was established and provided by phosphorylated by hypoxia in HT-p21-9 cells (Fig. S9 A and B). Joseph DiDonato (Cleveland Clinic, Cleveland, OH) (54). Cancer cell lines These results are in agreement with previous reports that regu- were cultured in high-glucose DMEM (plus pyruvate) with 10% FBS. RPE lation of mTOR by hypoxia does not correlate with AMPK cells were maintained in MEM plus 10% (vol/vol) FBS, and WI38-Tert and NKE cells were cultured in low-glucose DMEM plus 10% (vol/vol) FBS. Mouse phosphorylation (26) and does not require AMPK or LKB1 (27). MSCs were isolated from bone marrow of mice according to the technical Finally, we did not detect changes in SIRT1 levels under hypoxia protocol from STEMCELL Technologies. MSCs were cultured in complete (Fig. S9), with the exception that hypoxia prevented down- MesenCult medium (mouse) obtained from STEMCELL Technologies. Rapa- regulation of SIRT1 in IPTG-treated HT-p21-9 cells (Fig. S9B). mycin was obtained from LC Laboratories and dissolved in DMSO as 5 mM However, rapamycin did not decrease SIRT1 levels (Fig. S9). solution. IPTG (Invitrogen) was dissolved in water as 50 mg/mL stock solution Thus, the only consistent changes associated with geroconversion and used in cell culture at final concentration of 1.25 to 50 μg/mL. Nutlin 3a, with both rapamycin and hypoxia was inhibition of the S6K/S6 etoposide, and nocodazole were from Sigma-Aldrich, and were dissolved in DMSO. pathway. Discussion Immunoblot Analysis. Whole cell lysates were prepared by using boiling lysis buffer (1% SDS, 10 mM Tris HCl, pH 7.4). Equal amounts of proteins were It is well known that hypoxia induces cell cycle arrest. Cell cycle separated on gradient Criterion polyacrylamide gels (Bio-Rad) and trans- arrest by itself is not yet senescence. Senescence requires ferred to PVDF membranes. The following antibodies were used: rabbit

Leontieva et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13317 Downloaded by guest on September 26, 2021 anti–phospho-S6 (Ser235/236) and mouse anti-S6, mouse anti-phospho RP Assay. Cells were plated at low density, and treated with senescence- p70S6K, rabbit anti–phospho-eEF2, anti-phospho AMPK and anti-SIRT1 from inducing agents (IPTG, etoposide, and nutlin-3a, depending on cell lines) for Cell Signaling; anti-actin antibody from Sigma-Aldrich, and mouse anti- 3 to 4 d under normoxia or hypoxia, with or without rapamycin. Then, cell HIF-1a and anti-p21 from BD Biosciences; and mouse anti-p53 (Ab-6) from numbers were measured (i.e., initial cell numbers) and drugs were washed Oncogene. Secondary anti-rabbit and anti-mouse HRP-conjugated anti- off. Cells were incubated in fresh drug-free medium under normoxia for 5 to bodies were from Cell Signaling. 7 d, and then cell numbers were measured (i.e., final cell numbers). RP was determined as a ratio between final and initial cell numbers as fold increase in SA-β-Gal Staining. β-Gal staining was performed by using a Senescence β-gal cell numbers after drugs were washed off. staining kit (Cell Signaling Technology) according to the manufacturer’s protocol. Cells were incubated at 37 °C until β-gal staining became visible. Colony Formation Assay. Cells were plated at low density, and treated with Development of color was detected under a light microscope. senescence-inducing agents (IPTG, etoposide, or nutlin-3a, depending on cell lines) for 3 to 4 d under normoxia or hypoxia, with or without rapamycin. siRNA Technology. ON-TARGETplus SMART pool siRNA for human HIF-1α was Then, drugs were washed off, and cells were incubated in fresh drug-free purchased from Thermo Scientific. HT-p21-9 cells were transfected with medium under normoxia for 7 to 12 d. Plates were fixed and stained with HIF1a siRNA as described in detail in the legend to Fig. 2. 1.0% crystal violet.

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