WAF 1 / CIP 1 Transcriptional Repressor of p 21 A Novel Evolutionarily Conserved Element Is a General

The effective induction of p21 (p21) expression in p53-negative cancer cells is an important avenue in cancer management. We investigated the ability of various common chemotherapeutic drugs to induce p21 expression in p53-negative cancer cells and showed that the induction of p21 expression by oxaliplatin is caused by the derepression of a previously unrecognized negative regulatory element with a Sp1/Sp3 palindrome sequence core at 216 to 236 of the p21 proximal promoter. Electrophoretic mobility shift and antibody super-shift assays confirmed the specific binding of Sp1/Sp3, and showed that the oxaliplatin-mediated derepression of p21 transcription was associated with an increased Sp1/Sp3 phosphorylation and binding affinity to the oxaliplatin-responsive element. A search of the ENCODE database for vertebrate-conserved genomic elements identified the Sp1/Sp3 palindrome element as the only vertebrateconserved element within the 500-bp proximal p21 promoter region, indicating its fundamental importance. In in vivo competition assays, transfected synthetic Sp1/Sp3 palindrome elements derepressed the cotransfected or endogenous p21 promoter in a dosage-dependent manner. This derepression was not seen in oxaliplatin-treated cells, suggesting that the exogenous Sp1/Sp3 palindrome and oxaliplatin had the same downstream signaling target. Taken together, our results revealed, for the first time, this evolutionarily conserved Sp1/Sp3 palindrome element in the proximal p21 promoter that serves as a regulatory repressor to maintain p21 basal level expression. Cancer Res; 72(23); 6236–46. 2012 AACR. Introduction The tumor suppression function of p21 (CDKN1, p21) has been shown not only in mouse models of p21 deficiency (1–3) but also in human cancers under certain conditions (4–6). However, the expression of p21 is often repressed in p53-negative cancer cells and the identification of chemotherapeutic drugs that effectively induce p21 expression in p53-negative cancer cells represents an important element in cancer management (4, 7–8). p21 expression is primarily regulated at the transcriptional level with p53-dependent and -independent activation. Both regulatory pathways are critical in cellular housekeeping and environmental response and are expected to be conserved during evolution (9, 10). Therefore, promoter–reporter assays are broadly accepted as an indication of p21 expression and are used to identify responsive cis-elements for mechanism studies. Indeed, p53 activates p21 transcription through 2 conserved p53-responsive elements on the p21 promoter (11–13). p53independent p21 regulation is achieved through various transcription factors, including Sp1/Sp3, located in the well-studied p21 proximal promoter region. Sp1/Sp3 is a potent activator for the p21 promoter activity, and the 6 classic binding sites within the 119 to 50 bp on the p21 promoter have been found to be responsive to transcriptional activation or repression effects of various agents (13). To complement experimental assays that identify specific functional elements, the functional significance of regulatory sequences are often further assessed by examining evolutionary conservation across multiple species, because the existence of consensus protein factor-binding sequences may not indicate functional promoter sequences and many of these predicted sites do not have function in vivo (14–16). Although the evolutionarily conserved sequences may not fully overlap with experimentally identified functional elements, the existence of highly conserved sequences within an experimentally identified functional element suggests that its fundamental functions are under evolutionary constraint (17). In addition, many of the bona fide functional elements may not show up in the conservation map because of the limitation in calculation methods and species/functional specificity (18–20). Further confirmation of the functional element is usually done by more precise experimental methods including single nucleotide mutations in various testing systems (21, 22). Authors' Affiliations: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; and Mount Sinai School of Medicine, New York, New York W. Xu and Q. Zhu contributed equally to this work. Corresponding Author: Dawei Li, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China. Phone: 86-021-34204744; Fax: 86-21-34205436; E-mail: daweili@sjtu.edu. cn doi: 10.1158/0008-5472.CAN-12-1236 2012 American Association for Cancer Research. Cancer Research Cancer Res; 72(23) December 1, 2012 6236 American Association for Cancer Research Copyright © 2012 on January 11, 2013 cancerres.aacrjournals. org Downloaded from Published OnlineFirst October 12, 2012; DOI:10.1158/0008-5472.CAN-12-1236