Regulation of cell proliferation by induction of p21/WAF1 in rat bladder carcinoma cells using the Cre–loxP system
Introduction
p21/WAF1 is a cyclin-dependent kinase inhibitory protein [1] that regulates the cell cycle, potentially functioning as a tumor suppressor [2]. It is transcriptionally induced by p53 [2], the function of which is missing in a high percentage of human tumors [3], [4]. Many reports have documented alterations in the p53 gene in bladder carcinomas [5], [6], [7], [8]. In the absence of a functional p53 protein, expression of p21/WAF1 is often undetectable or present at only a very low level, and immunocytochemical studies have demonstrated a global decrease of p21/WAF1 expression in human tumors compared with normal tissues [9]. Moreover, loss of p21/WAF1 expression has been reported to be an important prognostic predictor in several human malignancies, including bladder carcinomas [3], [4], [10], [11]. Since it has been shown that p21/WAF1 expression can also be mediated through p53-independent pathways [7], [12], [13], [14], the possibility arises of overexpression applied for treatment of bladder carcinomas regardless of p53 alterations.
In this study we detected p53 gene mutations by performing PCR-SSCP analysis and direct sequencing in BC31 bladder carcinoma cells, established from a rat bladder carcinoma as previously reported [15]. Then, we induced expression of p21/WAF1 with the Cre–loxP system, which is able to regulate gene expression by on/off switching [16], [17]. Activation of the transgene flanked by a pair of loxP sites can be controlled on/off by using the Cre-producing recombinant adenovirus vector. This methodology is particularly useful in cases where there is difficulty in obtaining stable clones because of inhibition of cell proliferation by a targeted gene such as p21/WAF1.
Section snippets
BC31 cell culture
The rat BC31 cell line, established from a chemically-induced rat bladder transitional cell carcinoma [15], [18] was maintained in culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum under 5% CO2/95% air at 37 °C in a humidified incubator.
DNA Extraction, PCR-SSCP and direct sequencing
DNA samples were extracted by using Sepa Gene (Sanko Junyaku Co., Ltd, Tokyo). Primers were synthesized to amplify exons 5, 6, 7 and 8 according to published sequences [19]. The primer sequences were 5′-GATTCTTTCTCCTCTCCTAC-3′
p53 gene mutations in BC31 cells
We examined exons 5–8 for p53 gene mutations by PCR-SSCP and confirmed the results by direct sequencing of both strands. BC31 had mutations in codon 165 (exon 5, CAA→TAA) and codon 273 (exon 8, TGT→TGG). No wild type of p53 gene was detected in BC31.
p21/WAF1 protein induction with the Cre–loxP system in BC31 cells
We confirmed induction of p21/WAF1 expression by Cre treatment in the transfectants by Western blot analysis (Fig. 2). Levels of p21/WAF1 were found to be overexpressed in some transfectants treated with Cre compared to levels in the non-treated
Discussion
In the present study, we identified p53 mutations in BC31 cells and demonstrated that the cyclin dependent kinase inhibitor p21/WAF1 plays a very important role in reducing cell proliferation. p21/WAF1 is thought to regulate cell cycle events by binding directly to cyclin-dependent kinases and thus causing G1 arrest. p27/KIP1 is another cyclin-dependent kinase inhibitor and an association has been reported between loss of p21/WAF1 and p27/KIP1 expression and progression in a number of tumor
Acknowledgements
We would like to thank to Dr Izumi Saito for providing the Cre loxP gene expression switching vector (pCALNAL5) and the Cre recombinase producing adenovirus vector (AdexCre). This study was supported by a Grant-in-Aid for Cancer Research from the Society for Promotion of Pathology (Nagoya, Japan).
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