- Email: [email protected]
TPA activates p21WAF-1 promoter in human T-cells through its second most upstream Sp1 site
BBRC Biochemical and Biophysical Research Communications 304 (2003) 696–700 www.elsevier.com/locate/ybbrc
TPA activates p21WAF-1 promoter in human T-cells through its second most upstream Sp1 site Y. Schavinsky-Khrapunsky,a M. Huleihel,b M. Aboud,a,* and A. Torgemana a
Department of Microbiology and Immunology, Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel b Institute for Applied Biosciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel Received 3 March 2003
Abstract The p21WAF-1 promoter contains binding sites for a number of transcription factors which mediate its activation by a variety of external signals. Moreover, it has been reported that the transcription factors involved in p21WAF-1 activation by certain signaling factors, like the phorbol ester TPA, may vary in different cell types. We were interested in elucidating the mechanism of p21WAF-1 activation by TPA in human T-cells, since this activation could explain the antagonistic effect of PKC on apoptosis induction in these cells noted in our previous studies. Using the Jurkat human T-cells we found that TPA activated p21WAF-1 expression by a PKC-dependent mechanism and that out of six Sp1 binding sites residing in its promoter the second most upstream one was critically essential for this activation. Since p21WAF-1 is known to inhibit the onset of apoptosis, its PKC-dependent activation may likely account for the PKC antagonistic effect on apoptosis induction in these cells. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: p21WAF-1 ; Transcription factors; TPA; PKC; Sp1; CDK; CDK-inhibitors; Cell-cycle; Human T-cells; Jurkat cells
The cyclin-dependent kinase (CDK) inhibitor p21WAF-1 is a central element of the cell growth control. It exists in complexes containing cyclins, CDKs, and the proliferating nuclear antigen (PCNA) [1]. Under physiological conditions, it recruits these complexes to promote the cell cycle progression, whereas under stress conditions, which elevate its level [2,3], it acts to arrest the cell cycle [4] and to facilitate DNA damage repair [5]. p21WAF-1 expression is affected by divers external signals like cytokines [6,7], growth factors [8,9], phorbol esters [10–12], and others [2,3]. These factors exert their effect through various transcription factors which interact with their recognition sites residing in the p21WAF-1 promoter [13]. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is one of the external factors that has been shown to activate p21WAF-1 expression [13]. TPA is a potent protein kinase C (PKC) activator and most of its biological effects are exerted through PKC-associated pathways [14,15]. There are at least 12 different PKC * Corresponding author. Fax: +972-8-647-7626. E-mail address: [email protected] (M. Aboud).
isoforms that are classified into three groups; the conventional PKCs which require calcium and diacylglycerol (DAG) for their activation, the novel PKCs which are calcium-independent but DAG-dependent, and the atypical PKCs which are both calcium- and DAG-independent. Only the conventional and novel PKCs can be activated by TPA [14]. Extended exposure to TPA leads, in certain cell types, to a downregulation of these PKCs [16,17]. In previous studies we have demonstrated such a TPA-induced downregulation of the TPA-responsive PKCs in Jurkat cells (human T-cell line), which consequently become depleted of PKC activity after 24 h of exposure to TPA [18,19]. We have also shown that TPA induces apoptosis in these cells [19], but the onset of this programmed cell death has been noted to start only when the cells become depleted of the TPA-responsive PKCs, or if their activity is blocked by specific inhibitors [18–20]. It is, therefore, evident not only that this apoptotic effect does not need these PKCs but is rather antagonized by their activities. Notably, in this context, PKC activity has been found in certain cell lines as
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00650-8
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700
capable of enhancing p21WAF-1 expression [10–12,21]. On the other hand, elevated level of p21WAF-1 is known to inhibit the onset of apoptosis [22–28]. On this ground it was of interest to find out whether the TPA-activated PKC could elevate p21WAF-1 expression also in Jurkat cells, since such elevation might, at least partially, disclose the mechanism of its anti apoptotic effect in these cells. In addition, since the mode of p21WAF-1 stimulation by PKC has been shown to vary between deferent cell lines [10–12,21], the second goal of the present study was to elucidate the mode of its stimulation by TPA in these particular human T-cells. The present study demonstrates that TPA can, indeed, stimulate p21WAF-1 expression in these cells and that it exerts this effect at the transcriptional level by a PKCdependent mechanism, involving the Sp1 transcription factor. There are six Sp1 binding sites on the p21WAF-1 promoter which are located near its transcriptional start point [29–32]. Our data show that this TPA effect is mediated by the second most upstream Sp1 site that we designate as Sp1(2) (see Fig. 3A for schematic mapping of these and other sites residing in this promoter).
Materials and methods Cells. The Jurkat T-cell line was used throughout this study. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, glutamine, and antibiotics. Plasmids and transient transfection assay. The plasmids expressing luciferase by the w.t. p21WAF-1 promoter (p21-Luc) or p21WAF-1 promoters with different mutations, each inactivating a different Sp1 binding site (Sp1mut1p21-Luc, etc.) or through the )215/+8 and )143/ +8 fragments of the p21WAF-1 promoter ()215/+8 p21-Luc and )143/ +8 p21-Luc, respectively) and the plasmid pc53-SN3 expressing w.t. p53 through the hCMV promoter, were kindly provided by Dimitris Kardassis (University of Crete Medical School, Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology of Hellas, Herakolion, GR-71110, Greece). The pZLPKC-a plasmid expressing constitutively active PKC-a through the MoMuLV LTR [33,34] was provided by Eta Livneh from our department.
697
Antibodies and Western blot analysis. Mouse monoclonal antibodies against p21WAF-1 and Sp1, and anti mouse IgG rabbit antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA, whereas mouse anti human actin monoclonal antibodies were purchased from ICN Biomedicals, Ohio, USA. The level of p21WAF-1 and Sp1 proteins was estimated by Western blot analysis of whole cell extracts (100 lg protein), as previously described [18]. To assess equal loading of the tested samples, the filters were stripped of the first detecting antibodies and reacted with anti human actin antibodies. Treatment of cells with TPA and PKC-specific inhibitor. TPA (Sigma Chemicals Israel, Holon, Israel), dissolved in DMSO (Sigma), was added to cell cultures at a final concentration of 50 nM. Control cells received the same volume of DMSO. When indicated, the PKC-specific inhibitor bisindolylmaleimide GF 109203X (BI, Sigma) [35] was added to the cultures, at a final concentration of 2 lM, one hour before TPA, to ensure its penetration into the cells before PKC activation. Unless otherwise specified, these treatments were carried out for 6 h after TPA addition, since our previous studies [18,20] have indicated that during this period the level of the TPA-responsive PKCs, as well as their activity, are still high in these cells. Electrophoretic mobility shift assay. To demonstrate a PKC-dependent nuclear protein binding to the Sp1(2) site of the p21WAF-1 promoter, we prepared two oligonucleotides spanning from position )124 to position )101 of this promoter, which included the Sp1(1) and Sp1(2) sites in the following combinations: oligo A contained mutant Sp1(1) (mut 1) and wild type (w.t.) Sp1(2) and oligo B, which served as a control, contained w.t. Sp1(1) and mutant Sp1(2) (mut 2) (see Fig. 3A for their detailed sequences). These oligos were P32 -end labeled, reacted with nuclear extracts (20 lg protein) of Jurkat cells treated with TPA alone or together with BI, or of untreated cells. Protein binding to these oligos was determined by Electrophoretic mobility shift assay (EMSA) and the bound protein was identified by supershift analysis with the indicated mononuclear antibodies, as previously described [40].
Results and discussion PKC-dependent elevation of p21WAF -1 protein by TPA To determine whether TPA affects p21WAF-1 expression in Jurkat cells, we measured, first, the level of p21WAF-1 protein in their whole cell extract after 6 h of TPA treatment in the absence and presence of BI. The Western
Fig. 1. PKC-dependent stimulation of p21WAF-1 expression by TPA. Jurkat cells were treated with TPA for 6 h in the absence or presence of the PKC inhibitor BI and untreated cells served as control. Then (A) the level of the p21WAF-1 protein was measured in their whole cell extract (100 lg protein) by Western blot analysis with anti p21WAF-1 monoclonal antibodies. Equal loading of the various extract samples was assessed by stripping the blot from the initial detecting antibodies and reacting it with anti human actin antibodies, or (B) they were transfected with p21-Luc reporter construct (10 lg). (C) Untreated Jurkat cells were transfected with either p21-Luc alone, or with p21-Luc + PKC-a-expressing plasmid. Transfection efficiency in panels B and C was assessed by adding 1 lg MSV-LTR-b-gal plasmid to each transfection mixture. The enzymatic activities were measured 48 h after transfection. The Luc activity was normalized according to the b-gal activity and the results represent the average of triplicates SD.
698
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700
blot analysis, illustrated in Fig. 1A, shows that p21WAF-1 protein was undetectable in the untreated control cells, whereas its level was profoundly elevated in the TPAtreated cells, but the presence of BI during the TPAtreatment prevented this elevation. Together, these data suggest that the elevation of p21WAF-1 protein by TPA in these cells was dependent on the TPA-induced PKC activity. PKC-dependent transcriptional activation of p21WAF -1 promoter by TPA To further elucidate the mechanism of the TPA effect on p21WAF-1 , we examined the expression of the p21-Luc reporter construct in Jurkat cells pre-treated for 6 h with TPA alone or in the presence of BI. Fig. 1B shows that TPA alone markedly stimulates the expression of the p21WAF-1 promoter, whereas the presence of BI diminishes this effect. This observation indicates that TPA exerts it stimulatory effect on p21WAF-1 expression at the transcriptional level and further supports the notion that this effect depends on PKC activity. Effect of PKC-expressing vector on p21WAF -1 expression To provide additional and more direct evidence for the role of PKC in the transcriptional activation of p21WAF-1 by TPA in Jurkat cells, these cells were transfected with p21-Luc alone or together with the pZL-PKC-a plasmid, which expresses a constitutively active form of PKC-a. This PKC was chosen as a representative of the TPA-responsive isoforms. Fig. 1C shows that pZL-PKC-a profoundly stimulated the expression of the p21WAF-1 promoter. Addition of BI to the cell culture shortly after transfection prevented this stimulation, thus confirming that the stimulation of p21WAF-1 by the pZL-PKC-a vector was, indeed, mediated by the enzymatic activity of the encoded PKC.
Identification of the site on the p21WAF -1 promoter which mediates its TPA-induced activation p21WAF-1 is one of the direct downstream targets of p53, which activate p21WAF-1 by interacting with two p53 binding sites residing on the p21WAF-1 promoter [13,36] (see Fig. 2A for their location). In preliminary experiments we ruled out the involvement of these sites in the TPA-induced activation of p21WAF-1 promoter by showing that mutants of p21WAF-1 promoter, lacking these two p53 sites, were readily stimulated by TPA (data not shown). Sp1 is another transcription factor that attracted a wide interest because of its pivotal role in mediating the activation of p21WAF-1 by a variety of external signals [13,29–32,37–39]. There are six Sp1 binding sites near the transcription starting point of this promoter [29–32] (see Fig. 3A). In preliminary experiments we noted that mutants of p21WAF-1 promoter, lacking all other transcription factor-binding sites, except these six Sp1 sites, could still be effectively activated by TPA (data not shown). Therefore, the next experiment was conducted to identify which of these six Sp1 sites specifically mediated the TPA stimulatory effect on p21WAF-1 . For this purpose, we examined the effect of TPA on a series of p21WAF 1 mutant promoters, each carrying a mutation that inactivated a different Sp1 site (see Fig. 3A for these mutations). This experiment showed that only the mutation ‘‘mut 2,’’ which was introduced in the second most upstream Sp1 site [i.e. Sp1(2)], diminished the promoter response to TPA (Fig. 3B). This mutant promoter could still be effectively activated by co-transfection with the p53-expressing plasmid pc53-SN3, indicating that the transcriptional function of this promoter was not totally destroyed by mut 2. It is, therefore, evident that this effect of TPA is exclusively mediated by this particular Sp1 site, mostly like via PKC-dependent binding of Sp1 protein to this site.
Fig. 2. Evidence for the role of Sp1(2) site in p21WAF-1 activation by TPA. (A) Schematic illustration of the p21WAF-1 promoter with the two p53 sites and the six Sp1 sites and the mutations introduced in each one of them. Nucleotide positions from the transcriptional start point, designated as 0, are marked by numbers in several points along this promoter. (B) Reporter p21WAF-1 -Luc constructs, each carrying the indicated mutation that inactivates a different Sp1 site (as shown in A), were transfected into TPA-treated and untreated Jurkat cells. The MSV-b-gal plasmid was included in each transfection mixture for assessing the transfection efficiency. The Luc activity was normalized according to the b-gal activity.
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700
699
Conclusive comments
Fig. 3. EMSA and supershift analysis of the PKC-dependent nuclear protein binding to Sp1(2) site. Nuclear extracts, prepared from Jurkat cells treated with TPA in the absence or presence of BI or from untreated cells, were reacted with oligo A or oligo B and subjected to EMSA. Where indicated, supershift analysis was performed with the indicated monoclonal antibodies in order to identify the protein bound to the Sp1(2) site.
We have previously demonstrated that TPA induces apoptosis in Jurkat cells, but noted that the process of this programmed death can start only when the TPAinduced PKC activity is depleted from the cells by extended TPA treatment or blocked by a specific inhibitor [40]. Moreover, we have observed that the TPA-activated PKC can also protect these cells from apoptosis induction by the DNA-damaging agent cisplatin (unpublished data). Thus, while PKC is known to exert anti apoptotic effects in certain cell types and pro-apoptotic effects in others [41], these data indicate that in Jurkat cells PKC acts rather as an anti-apoptotic factor. Our observation in the present study, that PKC elevates the level of p21WAF-1 protein in Jurkat cells, may provide, at least, a partial explanation for its anti-apoptotic effect in these cells, since elevated levels of p21WAF-1 have been shown by other laboratories to delay the onset of apoptosis [22–28]. While clarifying the mechanism of the TPA-induced elevation of p21WAF-1 protein level in these cells we noted that TPA exerts its effect on p21WAF-1 expression at the transcriptional level, by enhancing the binding of Sp1 protein to the Sp1(2) site in the p21WAF-1 promoter through a PKC-dependent mechanism. The mechanism by which PKC induces this specific binding is unclear yet and requires further investigation.
PKC-dependent Sp1 binding to Sp1(2) site In an attempt to provide a more direct evidence for this putative PKC-dependent Sp1 binding to Sp1(2) site, we used two oligonucleotides spanning from position )124 to position )101 of the p21WAF-1 promoter: oligo A containing mutant Sp1(1) (mut 1) and wild type (w.t.) Sp1(2) and oligo B containing w.t. Sp1(1) and mutant Sp1(2) (mut 2) (see Fig. 3A for their detailed sequence). These oligos were reacted with nuclear extracts (20 lg protein) of Jurkat cells treated with TPA alone or together with BI and with extract of untreated control cells and then subjected to EMSA. Fig. 3 shows that the extract of the TPA-treated cells formed 3 complexes on oligo A, which were represented by bands I, II, and III, whereas only bands II and III were detected with oligo B. Furthermore, band I was also undetectable when oligo A was reacted with extracts of the cells treated with TPA+BI and of the untreated cells. Together these data indicate that band I represents a complex formed on the Sp1(2) site and that the formation of this complex depends on PKC activity. Finally, the supershift analysis presented in Fig. 3 confirms the presence of the Sp1 protein in complex I by demonstrating that this complex is supershifted by anti Sp1 monoclonal antibodies but not by the anti p21WAF-1 monoclonal antibodies used as irrelevant antibodies for negative control. We presently do not know the significance of the other two bands.
Acknowledgments This study was supported by grants from the Association of International Cancer Research (AICR), the Israel Science Foundation of The Israeli National Academy of Sciences and Humanities, the Israeli Cancer Association, the Israel Cancer Research Fund (ICRF), and the Chief Scientist Office of the Israeli Health Ministry.
References [1] H. Zhang, Y. Xiong, D. Beach, Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes, Mol. Biol. Cell. 4 (1993) 897–906. [2] K. Macleod, N. Sherry, G. Hannon, D. Beach, T. Tokino, K. Kinzler, B. Vogelstein, T. Jacks, p53-dependent and independent expression of p21 during cell growth, differentiation and DNA damage, Genes Dev. 9 (1995) 935–944. [3] H. Namaba, T. Hara, T. Tukazaki, K. Migita, N. Ishikawa, K. Ito, S. Nagataki, S. Yamashita, Radiation-induced G1 arrest is selectively mediated by the p53-WAF1/Cip1 pathway in human thyroid cells, Cancer Res. 55 (1995) 2075–3080. [4] H. Zhang, G. Hannon, D. Beach, p21-containing cyclin kinases exist in both active and inactive states, Genes Dev. 8 (1994) 1750– 1758. [5] J. LaBaer, M.D. Garrett, L.F. Stevenson, J.M. Slingerland, New functional activities of the p21 family of CDK inhibitors, Genes Dev. 11 (1997) 847–862. [6] T. Bellido, C.A. OÕbrien, P.K. Roberson, S.C. Manolagas, Transcriptional activation of the p21(WAF1/CIP1/CDI1) gene
700
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700 by interleukin-6 type cytokines. A prerequisite for their prodifferentiating and anti-apoptotic effects on human osteoblastic cells, J. Biol. Chem. 273 (1998) 21137–21144. A.C. Hobeika, P.S. Subramaniam, H.M. Johnson, IFNa induces the expression of the cyclin-dependent kinase inhibitor p21 in human prostate cancer cells, Oncogene 14 (1997) 1165– 1170. J.M. Li, M.B. Datto, X. Shen, Y. Hu, X. Wang, Sp1, but not Sp3 functions to mediate promoter activation by TGF-b through canonical Sp1 binding sites, Nucleic Acids Res. 26 (1998) 2449– 2456. N. Billon, L.A. van Grunsven, B.B. Rudkin, The CDK inhibitor p21WAF1/CIP1 is induced through p300-dependent mechanism during NGF-mediated neural differentiation of PC12 cells, Oncogene 13 (1996) 2047–2054. J. Zezula, V. Sexel, C. Hutter, A. Karel, W. Schutz, M. Freissmut, The cyclin-dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells, J. Biol. Chem. 272 (1997) 29967–29974. E.C. Dempsey, A.C. Newton, D. Mochly-Rosen, A.P. Fields, M.E. Reyland, P.A. Insel, R.O. Messing, Protein kinase C isozymes and the regulation of diverse cell responses, Am. J. Physiol. Lung Cell Mol. Physiol. 279 (2000) L429–L438. J.R. Biggs, A.S. Kraft, The role of the Smad3 protein in phorbol ester-induced promoter expression, J. Biol. Chem. 274 (1999) 36987–36994. A.L. Gartel, A.L. Tyner, Transcriptional regulation of the p21((WAF1/CIP1)) gene, Exp. Cell Res. 246 (1999) 280–289. H. Mellor, P.J. Parker, The extended protein kinase C superfamily, Biochem. J. 332 (1998) 281–292. D. Ron, M.G. Kazanietz, New insights into the regulation of protein kinase C and novel phorbol ester receptors, FASEB J. 13 (1999) 1658–1676. Z. Lu, D. Liu, A. Hornia, W. Devonish, M. Pagano, D.A. Foster, Activation of protein kinase C triggers its ubiquitination and degradation, Mol. Cell. Biol. 18 (1998) 839–845. S. Isonishi, K. Ohkawa, T. Tanaka, S.B. Howell, Depletion of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances platinum drug sensitivity in human ovarian carcinoma cells, Br. J. Cancer 82 (2000) 34–38. N. Mor-Vaknin, A. Torgeman, D. Galron, M. Lochelt, R.M. Flugel, M. Aboud, The long terminal repeats of human immunodeficiency virus type-1 and human T-cell leukemia virus type-I are activated by 12-O-tetradecanoylphorbol-13-acetate through different pathways, Virology 232 (1997) 337–344. A. Torgeman, Z. Ben-Aroya, A. Grunspan, E. Zelin, E. Butovsky, M. Hallak, M. Lochelt, R.M. Flugel, E. Livneh, M. Wolfson, I. Kedar, M. Aboud, Activation of HTLV-I long terminal repeat by stress-inducing agents and protection of HTLV-I infected T-cells from apoptosis by the viral Tax protein, Exp. Cell Res. 271 (2001) 169–179. A. Torgeman, N. Mor-Vaknin, E. Zelin, Z. Ben-Aroya, M. Lochelt, R.M. Flugel, M. Aboud, Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C, Virology 281 (2001) 10–20. J.R. Biggs, J.E. Kudlow, A.S. Kraft, The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells, J. Biol. Chem. 271 (1996) 901–906. J.L.M. Gervais, P. Seth, H. Zhang, Cleavage of CDK inhibitor p21Cip1=Waf1 by caspases is an early event during DNA damageinduced apoptosis, J. Biol. Chem. 273 (1998) 19207–19212. M. Gorospe, C. Cirielli, X. Wang, P. Seth, M.C. Capogrossi, N.J. Holbrook, p21Waf1=Cip1 protects against p53-mediated apoptosis of human melanoma cells, Oncogene 14 (1997) 929–935. M. Gorospe, X. Wang, K.Z. Guyton, N.J. Holbrook, Protective role of p21Waf1=Cip1 against prostaglandin A2-mediated apoptosis
[25] [26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
of human colorectal carcinomam cells, Mol. Cell. Biol. 16 (1996) 6654–6660. M. Gorospe, X. Wang, N.J. Holbrook, Functional role of p21 during cellular response to stress, Gene Expr. 7 (1999) 377–385. M. Gorospe, X. Wang, N.J. Hollbrook, p53-dependent elevation of p21Waf1 expression by UV light is mediated through mRNA stabilization and involves a vanadate-sensitive regulatory system, Mol. Cell. Biol. 18 (1998) 1400–1407. Y.H. Jin, K.J. Yoo, Y.H. Lee, S.K. Lee, Caspase 3-mediated cleavage of p21WAF1=CIP1 associated with cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells, J. Biol. Chem. 275 (2000) 30256–30263. Y. Zhang, N. Fujita, T. Tsuruo, Caspase-mediated cleavage of p21Waf1=Cip1 converts cancer cells from growth arrest to undergoing apoptosis, Oncogene 18 (1999) 1131–1138. G. Koutsodontis, A. Moustakas, D. Kardassis, The role of Sp1 family members, the proximal GC-Rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WA-1=Cip1 , Biochemistry 41 (2002) 12771–12784. G. Koutsodontis, I. Tentes, P. Papakosta, A. Moustakas, D. Kardassis, Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21WAF1=Cip1 gene by the p53 tumor suppressor protein, J. Biol. Chem. 276 (2001) 29116–29125. S. Lu, G. Jenster, D.E. Epner, Androgen induction of cyclindependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex, Mol. Endocrinol. 14 (2000) 753–760. A. Moustakas, D. Kardassis, Regulation of the human p21WAF1=Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members, Proc. Natl. Acad. Sci. USA 95 (1998) 6733–6738. E. Eldar, Y. Zisman, A. Ullrich, E. Livneh, Overexpression of protein kinase C a-subtype in Swiss/3T3 fibroblasts causes loss of both high and low affinity receptor numbers for epidermal growth factor, J. Biol. Chem. 265 (1990) 13290–13296. E. Livneh, T. Shimon, E. Bechor, Y. Doki, I. Schieren, I.B. Weinstein, Linking protein kinase C to cell cycle: ecotopic expression of PKC in NIH3T3 cells alters the expression of cyclins and Cdk inhibitors and induces adipogenesis, Oncogene 12 (1996) 1545–1555. D. Toullec, P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Loriolle, The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C, J. Biol. Chem. 266 (1991) 15771–15781. W.S. El-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator of p53 tumor suppression, Cell 75 (1993) 817–825. D. Kardassis, P. Papakosta, K. Pardali, A. Moustakas, c-Jun transactivates the promoter of the human p21WAF1=Cip1 gene by acting as a superactivator of the ubiquitous transcription factor Sp1, J. Biol. Chem. 274 (1999) 29572–29581. G.I. Owen, J.K. Richer, L. Tung, G. Takimoto, K.B. Horwitz, Progesterone regulates transcription of the p21WAF1 cyclin dependent kinase inhibitor gene through Sp1 and CBP/p300, J. Biol. Chem. 273 (1998) 10696–10701. K. Pardali, A. Kurisaki, A. Moren, P. ten Dijke, D. Kardassis, A. Moustakas, Role of Smad proteins and transcription factor Sp1 in p21Waf1/Cip1 regulation by transforming growth factorb, J. Biol. Chem. 275 (2000) 29244–29256. A. Torgeman, N. Mor-Vaknin, M. Aboud, Sp1 is involved in protein kinase C independent activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate, Virology 254 (1999) 279–287. R.A. Franklin, J.A. McCubrey, Kinases: positive and negative regulators of apoptosis, Leukemia 14 (2000) 2019–2034.
TPA activates p21WAF-1 promoter in human T-cells through its second most upstream Sp1 site Y. Schavinsky-Khrapunsky,a M. Huleihel,b M. Aboud,a,* and A. Torgemana a
Department of Microbiology and Immunology, Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel b Institute for Applied Biosciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel Received 3 March 2003
Abstract The p21WAF-1 promoter contains binding sites for a number of transcription factors which mediate its activation by a variety of external signals. Moreover, it has been reported that the transcription factors involved in p21WAF-1 activation by certain signaling factors, like the phorbol ester TPA, may vary in different cell types. We were interested in elucidating the mechanism of p21WAF-1 activation by TPA in human T-cells, since this activation could explain the antagonistic effect of PKC on apoptosis induction in these cells noted in our previous studies. Using the Jurkat human T-cells we found that TPA activated p21WAF-1 expression by a PKC-dependent mechanism and that out of six Sp1 binding sites residing in its promoter the second most upstream one was critically essential for this activation. Since p21WAF-1 is known to inhibit the onset of apoptosis, its PKC-dependent activation may likely account for the PKC antagonistic effect on apoptosis induction in these cells. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: p21WAF-1 ; Transcription factors; TPA; PKC; Sp1; CDK; CDK-inhibitors; Cell-cycle; Human T-cells; Jurkat cells
The cyclin-dependent kinase (CDK) inhibitor p21WAF-1 is a central element of the cell growth control. It exists in complexes containing cyclins, CDKs, and the proliferating nuclear antigen (PCNA) [1]. Under physiological conditions, it recruits these complexes to promote the cell cycle progression, whereas under stress conditions, which elevate its level [2,3], it acts to arrest the cell cycle [4] and to facilitate DNA damage repair [5]. p21WAF-1 expression is affected by divers external signals like cytokines [6,7], growth factors [8,9], phorbol esters [10–12], and others [2,3]. These factors exert their effect through various transcription factors which interact with their recognition sites residing in the p21WAF-1 promoter [13]. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is one of the external factors that has been shown to activate p21WAF-1 expression [13]. TPA is a potent protein kinase C (PKC) activator and most of its biological effects are exerted through PKC-associated pathways [14,15]. There are at least 12 different PKC * Corresponding author. Fax: +972-8-647-7626. E-mail address: [email protected] (M. Aboud).
isoforms that are classified into three groups; the conventional PKCs which require calcium and diacylglycerol (DAG) for their activation, the novel PKCs which are calcium-independent but DAG-dependent, and the atypical PKCs which are both calcium- and DAG-independent. Only the conventional and novel PKCs can be activated by TPA [14]. Extended exposure to TPA leads, in certain cell types, to a downregulation of these PKCs [16,17]. In previous studies we have demonstrated such a TPA-induced downregulation of the TPA-responsive PKCs in Jurkat cells (human T-cell line), which consequently become depleted of PKC activity after 24 h of exposure to TPA [18,19]. We have also shown that TPA induces apoptosis in these cells [19], but the onset of this programmed cell death has been noted to start only when the cells become depleted of the TPA-responsive PKCs, or if their activity is blocked by specific inhibitors [18–20]. It is, therefore, evident not only that this apoptotic effect does not need these PKCs but is rather antagonized by their activities. Notably, in this context, PKC activity has been found in certain cell lines as
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00650-8
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700
capable of enhancing p21WAF-1 expression [10–12,21]. On the other hand, elevated level of p21WAF-1 is known to inhibit the onset of apoptosis [22–28]. On this ground it was of interest to find out whether the TPA-activated PKC could elevate p21WAF-1 expression also in Jurkat cells, since such elevation might, at least partially, disclose the mechanism of its anti apoptotic effect in these cells. In addition, since the mode of p21WAF-1 stimulation by PKC has been shown to vary between deferent cell lines [10–12,21], the second goal of the present study was to elucidate the mode of its stimulation by TPA in these particular human T-cells. The present study demonstrates that TPA can, indeed, stimulate p21WAF-1 expression in these cells and that it exerts this effect at the transcriptional level by a PKCdependent mechanism, involving the Sp1 transcription factor. There are six Sp1 binding sites on the p21WAF-1 promoter which are located near its transcriptional start point [29–32]. Our data show that this TPA effect is mediated by the second most upstream Sp1 site that we designate as Sp1(2) (see Fig. 3A for schematic mapping of these and other sites residing in this promoter).
Materials and methods Cells. The Jurkat T-cell line was used throughout this study. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, glutamine, and antibiotics. Plasmids and transient transfection assay. The plasmids expressing luciferase by the w.t. p21WAF-1 promoter (p21-Luc) or p21WAF-1 promoters with different mutations, each inactivating a different Sp1 binding site (Sp1mut1p21-Luc, etc.) or through the )215/+8 and )143/ +8 fragments of the p21WAF-1 promoter ()215/+8 p21-Luc and )143/ +8 p21-Luc, respectively) and the plasmid pc53-SN3 expressing w.t. p53 through the hCMV promoter, were kindly provided by Dimitris Kardassis (University of Crete Medical School, Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology of Hellas, Herakolion, GR-71110, Greece). The pZLPKC-a plasmid expressing constitutively active PKC-a through the MoMuLV LTR [33,34] was provided by Eta Livneh from our department.
697
Antibodies and Western blot analysis. Mouse monoclonal antibodies against p21WAF-1 and Sp1, and anti mouse IgG rabbit antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA, whereas mouse anti human actin monoclonal antibodies were purchased from ICN Biomedicals, Ohio, USA. The level of p21WAF-1 and Sp1 proteins was estimated by Western blot analysis of whole cell extracts (100 lg protein), as previously described [18]. To assess equal loading of the tested samples, the filters were stripped of the first detecting antibodies and reacted with anti human actin antibodies. Treatment of cells with TPA and PKC-specific inhibitor. TPA (Sigma Chemicals Israel, Holon, Israel), dissolved in DMSO (Sigma), was added to cell cultures at a final concentration of 50 nM. Control cells received the same volume of DMSO. When indicated, the PKC-specific inhibitor bisindolylmaleimide GF 109203X (BI, Sigma) [35] was added to the cultures, at a final concentration of 2 lM, one hour before TPA, to ensure its penetration into the cells before PKC activation. Unless otherwise specified, these treatments were carried out for 6 h after TPA addition, since our previous studies [18,20] have indicated that during this period the level of the TPA-responsive PKCs, as well as their activity, are still high in these cells. Electrophoretic mobility shift assay. To demonstrate a PKC-dependent nuclear protein binding to the Sp1(2) site of the p21WAF-1 promoter, we prepared two oligonucleotides spanning from position )124 to position )101 of this promoter, which included the Sp1(1) and Sp1(2) sites in the following combinations: oligo A contained mutant Sp1(1) (mut 1) and wild type (w.t.) Sp1(2) and oligo B, which served as a control, contained w.t. Sp1(1) and mutant Sp1(2) (mut 2) (see Fig. 3A for their detailed sequences). These oligos were P32 -end labeled, reacted with nuclear extracts (20 lg protein) of Jurkat cells treated with TPA alone or together with BI, or of untreated cells. Protein binding to these oligos was determined by Electrophoretic mobility shift assay (EMSA) and the bound protein was identified by supershift analysis with the indicated mononuclear antibodies, as previously described [40].
Results and discussion PKC-dependent elevation of p21WAF -1 protein by TPA To determine whether TPA affects p21WAF-1 expression in Jurkat cells, we measured, first, the level of p21WAF-1 protein in their whole cell extract after 6 h of TPA treatment in the absence and presence of BI. The Western
Fig. 1. PKC-dependent stimulation of p21WAF-1 expression by TPA. Jurkat cells were treated with TPA for 6 h in the absence or presence of the PKC inhibitor BI and untreated cells served as control. Then (A) the level of the p21WAF-1 protein was measured in their whole cell extract (100 lg protein) by Western blot analysis with anti p21WAF-1 monoclonal antibodies. Equal loading of the various extract samples was assessed by stripping the blot from the initial detecting antibodies and reacting it with anti human actin antibodies, or (B) they were transfected with p21-Luc reporter construct (10 lg). (C) Untreated Jurkat cells were transfected with either p21-Luc alone, or with p21-Luc + PKC-a-expressing plasmid. Transfection efficiency in panels B and C was assessed by adding 1 lg MSV-LTR-b-gal plasmid to each transfection mixture. The enzymatic activities were measured 48 h after transfection. The Luc activity was normalized according to the b-gal activity and the results represent the average of triplicates SD.
698
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700
blot analysis, illustrated in Fig. 1A, shows that p21WAF-1 protein was undetectable in the untreated control cells, whereas its level was profoundly elevated in the TPAtreated cells, but the presence of BI during the TPAtreatment prevented this elevation. Together, these data suggest that the elevation of p21WAF-1 protein by TPA in these cells was dependent on the TPA-induced PKC activity. PKC-dependent transcriptional activation of p21WAF -1 promoter by TPA To further elucidate the mechanism of the TPA effect on p21WAF-1 , we examined the expression of the p21-Luc reporter construct in Jurkat cells pre-treated for 6 h with TPA alone or in the presence of BI. Fig. 1B shows that TPA alone markedly stimulates the expression of the p21WAF-1 promoter, whereas the presence of BI diminishes this effect. This observation indicates that TPA exerts it stimulatory effect on p21WAF-1 expression at the transcriptional level and further supports the notion that this effect depends on PKC activity. Effect of PKC-expressing vector on p21WAF -1 expression To provide additional and more direct evidence for the role of PKC in the transcriptional activation of p21WAF-1 by TPA in Jurkat cells, these cells were transfected with p21-Luc alone or together with the pZL-PKC-a plasmid, which expresses a constitutively active form of PKC-a. This PKC was chosen as a representative of the TPA-responsive isoforms. Fig. 1C shows that pZL-PKC-a profoundly stimulated the expression of the p21WAF-1 promoter. Addition of BI to the cell culture shortly after transfection prevented this stimulation, thus confirming that the stimulation of p21WAF-1 by the pZL-PKC-a vector was, indeed, mediated by the enzymatic activity of the encoded PKC.
Identification of the site on the p21WAF -1 promoter which mediates its TPA-induced activation p21WAF-1 is one of the direct downstream targets of p53, which activate p21WAF-1 by interacting with two p53 binding sites residing on the p21WAF-1 promoter [13,36] (see Fig. 2A for their location). In preliminary experiments we ruled out the involvement of these sites in the TPA-induced activation of p21WAF-1 promoter by showing that mutants of p21WAF-1 promoter, lacking these two p53 sites, were readily stimulated by TPA (data not shown). Sp1 is another transcription factor that attracted a wide interest because of its pivotal role in mediating the activation of p21WAF-1 by a variety of external signals [13,29–32,37–39]. There are six Sp1 binding sites near the transcription starting point of this promoter [29–32] (see Fig. 3A). In preliminary experiments we noted that mutants of p21WAF-1 promoter, lacking all other transcription factor-binding sites, except these six Sp1 sites, could still be effectively activated by TPA (data not shown). Therefore, the next experiment was conducted to identify which of these six Sp1 sites specifically mediated the TPA stimulatory effect on p21WAF-1 . For this purpose, we examined the effect of TPA on a series of p21WAF 1 mutant promoters, each carrying a mutation that inactivated a different Sp1 site (see Fig. 3A for these mutations). This experiment showed that only the mutation ‘‘mut 2,’’ which was introduced in the second most upstream Sp1 site [i.e. Sp1(2)], diminished the promoter response to TPA (Fig. 3B). This mutant promoter could still be effectively activated by co-transfection with the p53-expressing plasmid pc53-SN3, indicating that the transcriptional function of this promoter was not totally destroyed by mut 2. It is, therefore, evident that this effect of TPA is exclusively mediated by this particular Sp1 site, mostly like via PKC-dependent binding of Sp1 protein to this site.
Fig. 2. Evidence for the role of Sp1(2) site in p21WAF-1 activation by TPA. (A) Schematic illustration of the p21WAF-1 promoter with the two p53 sites and the six Sp1 sites and the mutations introduced in each one of them. Nucleotide positions from the transcriptional start point, designated as 0, are marked by numbers in several points along this promoter. (B) Reporter p21WAF-1 -Luc constructs, each carrying the indicated mutation that inactivates a different Sp1 site (as shown in A), were transfected into TPA-treated and untreated Jurkat cells. The MSV-b-gal plasmid was included in each transfection mixture for assessing the transfection efficiency. The Luc activity was normalized according to the b-gal activity.
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700
699
Conclusive comments
Fig. 3. EMSA and supershift analysis of the PKC-dependent nuclear protein binding to Sp1(2) site. Nuclear extracts, prepared from Jurkat cells treated with TPA in the absence or presence of BI or from untreated cells, were reacted with oligo A or oligo B and subjected to EMSA. Where indicated, supershift analysis was performed with the indicated monoclonal antibodies in order to identify the protein bound to the Sp1(2) site.
We have previously demonstrated that TPA induces apoptosis in Jurkat cells, but noted that the process of this programmed death can start only when the TPAinduced PKC activity is depleted from the cells by extended TPA treatment or blocked by a specific inhibitor [40]. Moreover, we have observed that the TPA-activated PKC can also protect these cells from apoptosis induction by the DNA-damaging agent cisplatin (unpublished data). Thus, while PKC is known to exert anti apoptotic effects in certain cell types and pro-apoptotic effects in others [41], these data indicate that in Jurkat cells PKC acts rather as an anti-apoptotic factor. Our observation in the present study, that PKC elevates the level of p21WAF-1 protein in Jurkat cells, may provide, at least, a partial explanation for its anti-apoptotic effect in these cells, since elevated levels of p21WAF-1 have been shown by other laboratories to delay the onset of apoptosis [22–28]. While clarifying the mechanism of the TPA-induced elevation of p21WAF-1 protein level in these cells we noted that TPA exerts its effect on p21WAF-1 expression at the transcriptional level, by enhancing the binding of Sp1 protein to the Sp1(2) site in the p21WAF-1 promoter through a PKC-dependent mechanism. The mechanism by which PKC induces this specific binding is unclear yet and requires further investigation.
PKC-dependent Sp1 binding to Sp1(2) site In an attempt to provide a more direct evidence for this putative PKC-dependent Sp1 binding to Sp1(2) site, we used two oligonucleotides spanning from position )124 to position )101 of the p21WAF-1 promoter: oligo A containing mutant Sp1(1) (mut 1) and wild type (w.t.) Sp1(2) and oligo B containing w.t. Sp1(1) and mutant Sp1(2) (mut 2) (see Fig. 3A for their detailed sequence). These oligos were reacted with nuclear extracts (20 lg protein) of Jurkat cells treated with TPA alone or together with BI and with extract of untreated control cells and then subjected to EMSA. Fig. 3 shows that the extract of the TPA-treated cells formed 3 complexes on oligo A, which were represented by bands I, II, and III, whereas only bands II and III were detected with oligo B. Furthermore, band I was also undetectable when oligo A was reacted with extracts of the cells treated with TPA+BI and of the untreated cells. Together these data indicate that band I represents a complex formed on the Sp1(2) site and that the formation of this complex depends on PKC activity. Finally, the supershift analysis presented in Fig. 3 confirms the presence of the Sp1 protein in complex I by demonstrating that this complex is supershifted by anti Sp1 monoclonal antibodies but not by the anti p21WAF-1 monoclonal antibodies used as irrelevant antibodies for negative control. We presently do not know the significance of the other two bands.
Acknowledgments This study was supported by grants from the Association of International Cancer Research (AICR), the Israel Science Foundation of The Israeli National Academy of Sciences and Humanities, the Israeli Cancer Association, the Israel Cancer Research Fund (ICRF), and the Chief Scientist Office of the Israeli Health Ministry.
References [1] H. Zhang, Y. Xiong, D. Beach, Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes, Mol. Biol. Cell. 4 (1993) 897–906. [2] K. Macleod, N. Sherry, G. Hannon, D. Beach, T. Tokino, K. Kinzler, B. Vogelstein, T. Jacks, p53-dependent and independent expression of p21 during cell growth, differentiation and DNA damage, Genes Dev. 9 (1995) 935–944. [3] H. Namaba, T. Hara, T. Tukazaki, K. Migita, N. Ishikawa, K. Ito, S. Nagataki, S. Yamashita, Radiation-induced G1 arrest is selectively mediated by the p53-WAF1/Cip1 pathway in human thyroid cells, Cancer Res. 55 (1995) 2075–3080. [4] H. Zhang, G. Hannon, D. Beach, p21-containing cyclin kinases exist in both active and inactive states, Genes Dev. 8 (1994) 1750– 1758. [5] J. LaBaer, M.D. Garrett, L.F. Stevenson, J.M. Slingerland, New functional activities of the p21 family of CDK inhibitors, Genes Dev. 11 (1997) 847–862. [6] T. Bellido, C.A. OÕbrien, P.K. Roberson, S.C. Manolagas, Transcriptional activation of the p21(WAF1/CIP1/CDI1) gene
700
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
Y. Schavinsky-Khrapunsky et al. / Biochemical and Biophysical Research Communications 304 (2003) 696–700 by interleukin-6 type cytokines. A prerequisite for their prodifferentiating and anti-apoptotic effects on human osteoblastic cells, J. Biol. Chem. 273 (1998) 21137–21144. A.C. Hobeika, P.S. Subramaniam, H.M. Johnson, IFNa induces the expression of the cyclin-dependent kinase inhibitor p21 in human prostate cancer cells, Oncogene 14 (1997) 1165– 1170. J.M. Li, M.B. Datto, X. Shen, Y. Hu, X. Wang, Sp1, but not Sp3 functions to mediate promoter activation by TGF-b through canonical Sp1 binding sites, Nucleic Acids Res. 26 (1998) 2449– 2456. N. Billon, L.A. van Grunsven, B.B. Rudkin, The CDK inhibitor p21WAF1/CIP1 is induced through p300-dependent mechanism during NGF-mediated neural differentiation of PC12 cells, Oncogene 13 (1996) 2047–2054. J. Zezula, V. Sexel, C. Hutter, A. Karel, W. Schutz, M. Freissmut, The cyclin-dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells, J. Biol. Chem. 272 (1997) 29967–29974. E.C. Dempsey, A.C. Newton, D. Mochly-Rosen, A.P. Fields, M.E. Reyland, P.A. Insel, R.O. Messing, Protein kinase C isozymes and the regulation of diverse cell responses, Am. J. Physiol. Lung Cell Mol. Physiol. 279 (2000) L429–L438. J.R. Biggs, A.S. Kraft, The role of the Smad3 protein in phorbol ester-induced promoter expression, J. Biol. Chem. 274 (1999) 36987–36994. A.L. Gartel, A.L. Tyner, Transcriptional regulation of the p21((WAF1/CIP1)) gene, Exp. Cell Res. 246 (1999) 280–289. H. Mellor, P.J. Parker, The extended protein kinase C superfamily, Biochem. J. 332 (1998) 281–292. D. Ron, M.G. Kazanietz, New insights into the regulation of protein kinase C and novel phorbol ester receptors, FASEB J. 13 (1999) 1658–1676. Z. Lu, D. Liu, A. Hornia, W. Devonish, M. Pagano, D.A. Foster, Activation of protein kinase C triggers its ubiquitination and degradation, Mol. Cell. Biol. 18 (1998) 839–845. S. Isonishi, K. Ohkawa, T. Tanaka, S.B. Howell, Depletion of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances platinum drug sensitivity in human ovarian carcinoma cells, Br. J. Cancer 82 (2000) 34–38. N. Mor-Vaknin, A. Torgeman, D. Galron, M. Lochelt, R.M. Flugel, M. Aboud, The long terminal repeats of human immunodeficiency virus type-1 and human T-cell leukemia virus type-I are activated by 12-O-tetradecanoylphorbol-13-acetate through different pathways, Virology 232 (1997) 337–344. A. Torgeman, Z. Ben-Aroya, A. Grunspan, E. Zelin, E. Butovsky, M. Hallak, M. Lochelt, R.M. Flugel, E. Livneh, M. Wolfson, I. Kedar, M. Aboud, Activation of HTLV-I long terminal repeat by stress-inducing agents and protection of HTLV-I infected T-cells from apoptosis by the viral Tax protein, Exp. Cell Res. 271 (2001) 169–179. A. Torgeman, N. Mor-Vaknin, E. Zelin, Z. Ben-Aroya, M. Lochelt, R.M. Flugel, M. Aboud, Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C, Virology 281 (2001) 10–20. J.R. Biggs, J.E. Kudlow, A.S. Kraft, The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells, J. Biol. Chem. 271 (1996) 901–906. J.L.M. Gervais, P. Seth, H. Zhang, Cleavage of CDK inhibitor p21Cip1=Waf1 by caspases is an early event during DNA damageinduced apoptosis, J. Biol. Chem. 273 (1998) 19207–19212. M. Gorospe, C. Cirielli, X. Wang, P. Seth, M.C. Capogrossi, N.J. Holbrook, p21Waf1=Cip1 protects against p53-mediated apoptosis of human melanoma cells, Oncogene 14 (1997) 929–935. M. Gorospe, X. Wang, K.Z. Guyton, N.J. Holbrook, Protective role of p21Waf1=Cip1 against prostaglandin A2-mediated apoptosis
[25] [26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
of human colorectal carcinomam cells, Mol. Cell. Biol. 16 (1996) 6654–6660. M. Gorospe, X. Wang, N.J. Holbrook, Functional role of p21 during cellular response to stress, Gene Expr. 7 (1999) 377–385. M. Gorospe, X. Wang, N.J. Hollbrook, p53-dependent elevation of p21Waf1 expression by UV light is mediated through mRNA stabilization and involves a vanadate-sensitive regulatory system, Mol. Cell. Biol. 18 (1998) 1400–1407. Y.H. Jin, K.J. Yoo, Y.H. Lee, S.K. Lee, Caspase 3-mediated cleavage of p21WAF1=CIP1 associated with cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells, J. Biol. Chem. 275 (2000) 30256–30263. Y. Zhang, N. Fujita, T. Tsuruo, Caspase-mediated cleavage of p21Waf1=Cip1 converts cancer cells from growth arrest to undergoing apoptosis, Oncogene 18 (1999) 1131–1138. G. Koutsodontis, A. Moustakas, D. Kardassis, The role of Sp1 family members, the proximal GC-Rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WA-1=Cip1 , Biochemistry 41 (2002) 12771–12784. G. Koutsodontis, I. Tentes, P. Papakosta, A. Moustakas, D. Kardassis, Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21WAF1=Cip1 gene by the p53 tumor suppressor protein, J. Biol. Chem. 276 (2001) 29116–29125. S. Lu, G. Jenster, D.E. Epner, Androgen induction of cyclindependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex, Mol. Endocrinol. 14 (2000) 753–760. A. Moustakas, D. Kardassis, Regulation of the human p21WAF1=Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members, Proc. Natl. Acad. Sci. USA 95 (1998) 6733–6738. E. Eldar, Y. Zisman, A. Ullrich, E. Livneh, Overexpression of protein kinase C a-subtype in Swiss/3T3 fibroblasts causes loss of both high and low affinity receptor numbers for epidermal growth factor, J. Biol. Chem. 265 (1990) 13290–13296. E. Livneh, T. Shimon, E. Bechor, Y. Doki, I. Schieren, I.B. Weinstein, Linking protein kinase C to cell cycle: ecotopic expression of PKC in NIH3T3 cells alters the expression of cyclins and Cdk inhibitors and induces adipogenesis, Oncogene 12 (1996) 1545–1555. D. Toullec, P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Loriolle, The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C, J. Biol. Chem. 266 (1991) 15771–15781. W.S. El-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator of p53 tumor suppression, Cell 75 (1993) 817–825. D. Kardassis, P. Papakosta, K. Pardali, A. Moustakas, c-Jun transactivates the promoter of the human p21WAF1=Cip1 gene by acting as a superactivator of the ubiquitous transcription factor Sp1, J. Biol. Chem. 274 (1999) 29572–29581. G.I. Owen, J.K. Richer, L. Tung, G. Takimoto, K.B. Horwitz, Progesterone regulates transcription of the p21WAF1 cyclin dependent kinase inhibitor gene through Sp1 and CBP/p300, J. Biol. Chem. 273 (1998) 10696–10701. K. Pardali, A. Kurisaki, A. Moren, P. ten Dijke, D. Kardassis, A. Moustakas, Role of Smad proteins and transcription factor Sp1 in p21Waf1/Cip1 regulation by transforming growth factorb, J. Biol. Chem. 275 (2000) 29244–29256. A. Torgeman, N. Mor-Vaknin, M. Aboud, Sp1 is involved in protein kinase C independent activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate, Virology 254 (1999) 279–287. R.A. Franklin, J.A. McCubrey, Kinases: positive and negative regulators of apoptosis, Leukemia 14 (2000) 2019–2034.