The Role of the Transcription Factor Sp1 in Regulating the Expression of the WAF1/CIP1 Gene in U937 Leukemic Cells

Abstract

The Waf1/Cip1 protein induces cell cycle arrest through inhibition of the activity of cyclin-dependent kinases and proliferating cell nuclear antigen. Expression of the WAF1/CIP1 gene is induced in a p53-dependent manner in response to DNA damage but can also be induced in the absence of p53 by agents such as growth factors, phorbol esters, and okadaic acid. WAF1/CIP1 expression in U937 human leukemic cells is induced by both phorbol ester, a protein kinase C activator, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Both of these agents induce the differentiation of these leukemic cells toward macrophages. We demonstrate that phorbol esters and okadaic acid stimulate transcription from the WAF1/CIP1 promoter in U937 cells. This transcription is mediated by a region of the promoter between ؊154 and ؉16, which contains two binding sites for the transcription factor Sp1. Deletion or mutation of these Sp1 sites reduces WAF1/CIP1 promoter response to phorbol ester and okadaic acid, while a reporter gene under the control of a promoter containing only multiple Sp1 binding sites and a TATA box is induced by phorbol ester and okadaic acid. The WAF1/ CIP1 promoter is also highly induced by exogenous Sp1 in the Sp1-deficient Drosophila Schnieder SL 2 cell line. These results suggest that phorbol ester and okadaic acid activate transcription of the WAF1/CIP1 promoter through a complex of proteins that includes Sp1 and basal transcription factors.Treatment of the human myeloid leukemic cell line U937 with phorbol esters such as phorbol myristate acetate (PMA), 1 an activator of protein kinase C, leads to macrophage/monocyte-like differentiation over a 72-h period (1, 2). This process involves changes in cell-substrate adherence, growth arrest in late G 1 , and increased expression of monocyte markers (3, 4). Similarly, treatment of U937 cells with okadaic acid, a natural product isolated from the black sponge and a potent inhibitor of protein phosphatases 1 and 2A, also induces differentiation of these cells (5), cell cycle arrest, and eventual (72-h) apoptosis (6). Both PMA and okadaic acid induce expression of the cyclindependent kinase inhibitor, WAF1/CIP1 (7,8).WAF1/CIP1 expression is induced by the p53 protein following irradiation of cells (9, 10), but p53-independent expression of WAF1/CIP1 is associated with differentiation of myocytes (11, 12), of HL 60 leukemic cells (13), and of a number of other cells. WAF1/CIP1 is expressed in a number of tissues over the course of murine development, and expression in most tissues is not dependent on the presence of p53 (14). p53-independent expression of the WAF1/CIP1 gene can be induced in cultured cells by a number of agents, including, besides PMA and okadaic acid, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor ␤ (15, 16). Preliminary analysis of the WAF1/CIP1 promoter suggests that the elements mediating response to serum in fibroblasts are located at least 1.9 kb upstream from the transcription start site (14), while...

Unique induction of p21WAF1/CIP1expression by vinorelbine in androgen-independent prostate cancer cells

“…Deletion of Spl-1 and Spl-2 from pWPdel-Bst XI to generate pWPlOl did not result in further reduction of the transcriptional activation (4.5-fold), suggesting that Spl-1 and Spl-2 within the p21 promoter was not essential for vinorelbine-mediated p21 activation. This effect is distinct from the effects of phorbol ester and of okadaic acid (Biggs et al, 1996). However, using the reporter pWPdel-Sma that contains Spl-5 and Spl-6, the expression only achieved approximately a two-fold induction (Po0.01).…”

“…Several important biological modifiers have been shown to activate p21 transcription through different Spl binding sites (Cartel and Tyner, 1999). For example, phorbol ester and okadaic acid induce p21 expression through Spl-1 and Spl-2 sites (Biggs et al, 1996), whereas the Spl-3 site in the promoter of p21 has been shown to be required for p21 induction by transforming growth factor-b, histone deacetylase inhibitors such as TSA and butyrate, lovastatin, nerve growth factor (NGF) as well as calcium (Datto et al, 1995;Nakano et al, 1997;Prowse et al, 1997;Sowa et al, 1997;Lee et al, 1998;Billon et al, 1999). In this report, we demonstrated that Spl-3 and Spl-4 in the promoter of human p21 gene are required for vinorelbinemediated transcriptional restoration of p21 in the p21-deficient Al cells, which may provide a new mechanism in drug-mediated p21 regulation.…”

Ras induces p21Cip1/Waf1 cyclin kinase inhibitor transcriptionally through Sp1-binding sites

“…Besides p53, transcription factor Ap-2 (Zeng et al, 1997), E2F-1/DP-1 complex (Hiyama et al, 1997), E2A (Prabhu et al, 1997), an ets-oncogene related E1AF (Funaoka et al, 1997), C/EBP , RAR (Liu et al, 1996a), VDR (Liu et al, 1996b), STATs (Chin et al, 1996), and Sp1 family (Biggs et al, 1996;Li et al, 1998;Owen et al, 1998;Prowse et al, 1997;Yan and Zi , 1997) are known to stimulate p21 promoter activity. Our results show that Ras induces p21 transcriptionally and that the Ras-responsive region in p21 promoter spans a short region downstream of the nucleotide 7110 relative to the transcription start site.…”

p21WAF1/CIP1 induction by 5-azacytosine nucleosides requires DNA damage

“…To investigate the effect of p53 on p21 WAF1/CIP1 upregulation by DAC, we investigated the effect of DAC (1 mM), MS-275 (1 mM) and phorbol 12-myristate 13-acetate (PMA) (10 and 25 nM) on p21 WAF1/CIP1 expression in ML-1 cells (p53-WT) and HL-60 cells (p53-null) (Wolf and Rotter, 1985). PMA is a protein kinase C activator known to induce p21 WAF1/CIP1 in a p53-independent fashion (Biggs et al, 1996). As seen in Figure 5a, all three agents as well as TSA (300 nM) upregulated p21 WAF1/CIP1 within 48 h in the p53-WT ML-1 cells.…”