p53-dependent induction of WAF1 by cold shock in human ... - Nature

Oncogene (1998) 16, 1507 ± 1511  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

SHORT REPORT

p53-dependent induction of WAF1 by cold shock in human glioblastoma cells Takeo Ohnishi, Xinjiang Wang, Ken Ohnishi and Akihisa Takahashi Department of Biology, Nara Medical University, Kashihara, Nara 634, Japan.

Induction of WAF1 expression was investigated in human glioblastoma cell lines diering in p53 gene statuses after cold shock treatment. Accumulation of both wild-type (wt) and mutant p53 (mp53) was induced by cold shock at 48C for 60 min, however, WAF1 accumulation was induced by cold shock in A-172 cells carrying the wtp53 but not in T98G cells carrying the mp53. Inactivation of wtp53 by a dominant negative p53 mutant (p53Trp248) abolished cold shock-induced WAF1 expression in A-172 transfectant cells. Furthermore, no WAF1 expression was induced by cold shock in p53de®cient human osteosarcoma Saos-2 cells. Northern blot analysis showed that the WAF1 but not p53 gene was activated by cold shock only in A-172 cells. These ®ndings suggest that WAF1 expression is cold shockinducible in human glioblastoma cells, and that this induction may be due to signal transduction mediated by p53 in response to non-genotoxic stress, cold shock. Keywords: p53; WAF1; signal transduction; cold shock; non-genotoxic stress

Advert environmental conditions evoke stress responses in organisms, a classic one of which is the induction of heat shock protein (hsp) genes. Similarly to heat shock, cold shock also induces cellular stress response characterized by a speci®c pattern of gene expression. Cold shock-induced proteins (Csps) have been well studied in Escherichia coli (Goldstein et al., 1990; Jones and Inouye, 1994; Jones et al., 1996). In contrast to hsps which are protein chaperones to help to refold the non-native proteins, prevent their aggregation or direct protein degradation (review in Becker and Craig, 1994), Csps have been shown to be RNA chaperones to prevent the formation of secondary structures in RNA at low temperature (Jones et al., 1996; Jiang et al., 1997). To our knowledge, cold shock proteins have not yet been found in human cells, thus, the eects of cold shock on mammalian cells are poorly documented. On the other hand, the p53-dependent stress response that leads to cell growth arrest and/or cell death in mammalian cells (Kastan et al., 1992; Lowe et al., 1993) has attracted much interest of research in recent years. A cyclindependent inhibitor, WAF1p21/CIP1/sdi1 was later found to

Correspondence: T Ohnishi, Department of Biology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634, Japan Received 5 March 1996; revised 15 October 1997; accepted 15 October 1997

be a major mediator for p53-dependent G1 arrest (ElDeiry et al., 1993, Deng et al., 1995). We thought it of interest to determine whether the cold shock could evoke the p53-mediated signaling in mammalian cells. Here, we show that cold shock, as a novel nongenotoxic stress, also induces WAF1 gene expression in a p53-dependent manner in human glioblastoma cells, which strongly supports an idea that p53 is a multifunctional stress protein (Wang and Ohnishi, 1997). To examine the eects of cold shock on activation of the WAF1 gene, we ®rst examined WAF1 accumulation after cold shock by Western blot analysis. Two human glioblastoma A-172 and T98G cells diering in the p53 statuses were used in this experiment. A-172 cells bearing the wtp53 gene (Matsumoto et al., 1994) are competent in both activating the expression of a p53-dependent reporter gene and WAF1 (Jung et al., 1995; Ohnishi et al., 1996) and T98G cells have homologous mp53 with a single G to A transition in the third position of codon 237 that resulted in a missense mutant of Met to Ile (Ullrich et al., 1992) and have lost the ability of nucleus translocation (unpublished data) but remained competent in p53-independent WAF1 induction (Aoki et al., 1997). Brie¯y, A-172 and T98G cells were plated (56105/25 cm2 ¯ask) 12 h before treatment at dierent temperatures. Cells were returned to 378C and cultured for 10 h for gene expression after treatment at 20, 15 or 48C for 60 min. Total protein was prepared and subjected to Western blot analysis. As shown in Figure 1a, WAF1 accumulation was induced in A-172 cells bearing wtp53 and found to be inversely correlated with the treatment temperature (1.9-, 1.9- and 3.7-fold for 20, 15 and 48C, respectively, as compared to the control cells cultured at 378C). In contrast, in T98G cells bearing mp53, the basal level of WAF1 was very low and no apparent accumulation was observed after temperature shift. On the other hand, in A-172 cells, a similar pattern of wtp53 accumulation was also observed concomitantly with WAF1 accumulation. These results indicated that cold shock at 48C for 60 min acted as eective stress to induce signi®cant WAF1 and p53 accumulation in A-172 cells. To con®rm the dependence of WAF1 accumulation on cold shock, proteins were extracted from cells coldshocked at 48C for dierent periods followed by culturing at 378C for 10 h. As shown in Figure 1b, although cold shock at 48C even for as short as 15 min induced apparent WAF1 accumulation, cold shock at 48C for 60-min induced maximal WAF1 accumulation in A-172 cells. Then, we examined the time course of p53 and WAF1 accumulation after cold shock at 48C for 60 min. As shown in Figure 1c and 1d, p53

Cold shock induces WAF1 expression T Ohnishi et al

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wtp53 accompanied with an increase of wtp53 but not in T98G cells bearing mp53. To demonstrate the p53-dependence of WAF1 induction by cold shock, plasmids pC53-248 (provided by B Vogelstein, Johns Hopkins Oncology Center, MD, USA) that contains a mp53 gene which encodes a dominant negative mp53 (p53Trp248) (Kern et al., 1992), and pCMV-Neo, the control vector, were stably transfected into A-172 cells. Transfection of this mutant p53 into A-172 cells has been shown to block the WAF1 accumulation by heat shock or a protein

accumulation in A-172 cells occurred earlier than WAF1, reaching a peak at 10 h (3.5-fold) after cold shock, decreasing thereafter. Signi®cant WAF1 accumulation occurred in A-172 cells 6 h (31-fold) after cold shock, followed by a steady-state increase until 36 h (64-fold). In contrast to A-172 cells, no WAF1 accumulation was observed in T98G cells during the observation period, although there was a slight accumulation of mp53 10 h after cold shock (Figure 1c). These results demonstrated that WAF1 accumulation was cold shock-inducible in A-172 cells bearing

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Figure 1 WAF1 and p53 accumulation by cold shock. (a) Temperature- and p53-depedencies of WAF1 and p53 accumulation. Cells were cultured at 378C for 10 h after treatment at the indicated temperatures for 60 min and then lysed. The extracted proteins were subjected to Western blot analysis with anti-p53 monoclonal antibody (PAb1801) or monoclonal anti-human WAF1 antibody (EA 10) (Oncogene Science Inc., Uniondale, NY, USA). A BLAST1 system (DuPont/Biotechnology Systems NEN, Boston, MA, USA) was employed to enhance the speci®c signals. (b) Eects of dierent treatment times at 48C on WAF1 accumulation in A-172 cells. Western blot analysis of WAF1 was done with whole cell lysates from the cells cultured at 378C for 10 h after cold shock at 48C for the indicated periods. (c) Time course of p53 accumulation after cold shock at 48C for 60 min. Upper photographs, Western blot analysis of p53 with PAb1801 from a 15% SDS ± PAGE gel; Lower graph, Relative amounts of p53 at dierent time points after cold shock at 48C for 60 min normalised to the p53 level in non-treated control cells. &, A-172; &, T98G. N, the control sample cultured at 378C. (d) Time course of WAF1 accumulation after cold shock at 48C for 60 min. Western blot analysis of WAF1 was performed with the same samples as in C. Symbols are the same as in C. PC, positive control, A-172 cells cold-shocked at 48C for 60 min then cultured at 378C for 10 h


kinase inhibitor, H-7 (Wang et al., 1997), indicating the dominance of this p53 mutant over the endogeneous wtp53 in A-172 cells. These cells were cold-shocked at 48C for 60 min and then subjected to Western blot analysis for WAF1 expression and p53 accumulation. As shown in Figure 2a, signi®cant accumulation of wtp53 in A-172/neo occurred 10 h after cold shock, whereas more signi®cant accumulation of mp53 occurred in A-172/Trp248, beginning from 3 h after cold shock. The accumulation of mp53 in A-172/ Trp248 (Figure 2a) after cold shock is consistent with the observation in T98G cells (Figure 1c). However, the accumulated dominant negative p53 mutant did not lead to WAF1 accumulation in A-172/Trp248 (Figure 2b) which is also similar to the result with T98G cells. In contrast, WAF1 accumulation occurred in A-172/ neo 3 h after cold shock, although it was attenuated as compared with parental A-172 cells (Figure 1d). Two clones for each transfectant were examined and similar results were obtained. These results suggest that inactivation of the wtp53 by this dominant mutant abolishes the WAF1 accumulation after cold shock, although the reasons why our several clones of A-172/

Trp248 transfectant cells exhibited a higher basal expression of WAF1 as compared with parental cells are unknown. To further con®rm this p53-dependence, a p53-null human osteosarcoma Saos-2 cell line (Diller et al., 1990) (provided by JCRB, Setagaya, Tokyo, Japan) was also examined. As shown in Figure 2b, no p53 was detectable and no WAF1 accumulation was observed in Saos-2 cells within a period of 36 h after cold shock at 48C for 60 min. Collectively, these results support the conclusion that cold shock induction of WAF1 expression is a p53-dependent response. To con®rm that cold shock-induced WAF1 accumulation was the consequence of activation of WAF1 expression, Northern blot analysis was carried out. Brie¯y, total RNA was prepared after culturing the cold-shocked cells at 378C for 3, 6 or 10 h, then subjected to electrophoresis (20 mg RNA/well) on a 1% agarose gel containing 17% formaldehyde. The blots were probed for WAF1 mRNA with a 2.1 kbp BamHI fragment of the WAF1/sdil cDNA of plasmid 2D10-5 (provided by Dr A Noda, Meiji Cell Technology

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Figure 2 Abolishment of cold-shock-induced WAF1 expression by a dominant negative mutant p53 in A-172 cells and the absence of WAF1 expression after cold shock in p53-null Saos-2 cells. (a) The plasmid pC53-248 that contains a mp53 gene which encodes a dominant negative mp53 (p53Trp248) and the control vector pCMV-Neo were transfected into A-172 cells. The stable transfectants A-172/neo and A-172/Trp248 were selected (Wang et al., 1997), subjected to cold shock at 48C for 60 min and then cultured at 378C. Whole lysates were prepared at 3, 6 and 10 h after cold shock and subjected to immunoblotting with monoclonal antibodies against p53 (DO-1) or WAF1 (EA 10) (Oncogene Science Inc., Uniondale, NY, USA). The bars indicate the doublet p53 bands of dierentially charged species in A-172 cells (Ullrich et al., 1992). The arrowhead indicates the WAF1 band. N, the non-treated control. (b) Saos-2 cells were cold shock at 48C for 60 min and then cultured at 378C. Whole lysates were prepared after culturing the cold-shocked cells at 378C for the indicated time and subjected to Western blot analysis for p53 and WAF1. PC, the positive control, A-172 cells cultured at 378C for 36 h after cold shock at 48C for 60 min. Symbols are same as in a

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Figure 3 Induction of WAF1 expression by cold shock. (a) A172 or T98G cells were cold-shocked at 48C for 60 min, then cultured at 378C for the indicated periods. Then, total RNA was prepared and 20 mg RNA was used for Northern blot analysis. Upper photograph, Northern blot with a WAF1 cDNA probe; lower photograph, ethidium bromide staining of 28S rRNA. (b) 10 mg total RNA of the same samples as in a was used for Northern blot analysis of p53 mRNA. Upper photograph, Northern blot with a p53 cDNA probe; lower photograph, ethidium bromide staining of 28S rRNA

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Center, Odawara, Japan) as a probe. Ethidium bromide staining of 28S rRNA was used as an internal control for equal loading. As shown in Figure 3a, an obvious increase in WAF1 mRNA was observed in A-172 cells 3 h after cold shock (2.8-fold compared to non-cold-shocked control). This elevated level further increased at 6 and 10 h (9.6- and 8.9-fold, respectively). In contrast, the WAF1 mRNA level in T98G cells was very low, and was not increased after cold shock. The same RNA samples were used for detection of p53 gene expression after cold shock. For p53 mRNA, a 1.6 kbp XbaI/XhoI fragment of the p53 cDNA of plasmid pCMVNc9 (provided by Dr M Oren, Weizmann Institute of Science, Rehovot, Israel) was used as a probe. As shown in Figure 3b, p53 mRNA level remained unchanged in both A-172 and T98G cells after cold shock. These Northern blotting results clearly showed that cold shock activated WAF1 gene expression only in A-172 cells which may in turn caused WAF1 protein accumulation in these cells. In contrast, p53 mRNA was not aected by cold shock, suggesting that cold-shock-induced p53 accumulation is due to post-transcriptional events evoked by cold shock stress, probably through similar mechanisms used in U.V. or radiation-induced stress response. p53 exerts its biological functions by inducing downstream mediator genes such as WAF1, Bax and gadd45 or through interactions with other regulatory proteins, all of which are involved either in cell cycle control, apoptosis or DNA repair (reviewed in Gottlieb and Oren, 1996). Thus, p53-centered signal transduction represents a very important mechanism in the cellular responses to variety of stresses and become known as an integrator of stress response signals (Jack and Weinberg, 1996; Hall et al., 1996; Wang and Ohnishi, 1997). In the present study, we have shown that cold shock induces WAF1 gene expression and protein accumulation in a p53-dependent manner. This response of the p53 pathway to cold shock is much slower than that to g-rays using the same system, in the latter case, p53 accumulation reaches a peak 1.5 h and WAF1 accumulation reaches a peak 6 h after 3 Gy grays in A-172 cells (data not shown). We consider this dierence may be due to the dierent types of cellular damage induced by dierent stresses because cold

shock damages cellular membranes by inducing lipid phase transition (Drobnis et al., 1993), which may activate signal transduction pathways dierent from that triggered by g-ray-induced DNA damage. Cold shock-induced WAF1 may be one of the mediators of the protective mechanisms for organisms against various stresses, because WAF1 can act as an inducer of G1 arrest before DNA replication through modi®cation of retinoblastoma protein and E2F functions or as a suppressor for p53-dependent apoptosis (Polyak et al., 1996). This notion is supported by the evidences that cold shock stress enhanced the survivals of mouse skin and cultured keratinocytes against genotoxic stress (Ota et al., 1996). On the other hand, cold shock activation of the p53 pathway may preferentially lead to apoptosis in speci®c cell types (Kruman et al., 1992; Gregory and Milner, 1994), through which organisms can eliminate the coldshock-damaged cells. Thus, activation of the p53 pathway by cold shock may be an important response to maintain homeostasis. Taking our data from using stress factors such as heat shock (Ohnishi et al., 1996), low pH (Ohtsubo et al., 1997), a protein kinase inhibitor, H-7 (Wang et al., 1997) and cold shock, WAF1 seems to be a general stress-responsive protein inducible by both genotoxic and non-genotoxic cellular stresses, which further emphasizes the importance of p53-mediated signal transduction in cellular stress response for maintenance of genomic stability and cell homeostasis. Abbreviations wtp53, wild-type p53; mp53, mutant p53; DMEM, Dulbecco's Modi®ed Eagle's Medium; HRP, Horseradish peroxidase; BLAST, Blotting Ampli®cation System; PBS, phosphate-buered saline; SDS, sodium dodecyl sulfate. Acknowledgements We are grateful to Drs A Noda, M Oren and B Vogelstein for providing plasmids 2D10-5, pCMVNc9, pC53-248 and pCMV-Neo. This work was supported, in part, by grants from the Ministry of Education, Science, Sports and Culture, Japan and `Space Utilization Frontiers Joint Research Projects' promoted by NASDA.

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