The p21^WAF1/CIP1 Promoter Is Methylated in Rat-1 Cells: Stable Restoration of p53-Dependent p21^WAF1/CIP1 Expression after Transfection of a Genomic Clone Containing the p21^WAF1/CIP1 Gene

Abstract

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible geneMDM2 but not the protein or mRNA of the p53-inducible p21^WAF1/CIP1 gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21^WAF1/CIP1 expression appears to be the result of hypermethylation of the p21^WAF1/CIP1 promoter region, as p21^WAF1/CIP1 protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21^WAF1/CIP1 gene. Stable X-ray-induced p53-dependent p21^WAF1/CIP1 expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21^WAF1/CIP1 gene. The absence of expression of the p21^WAF1/CIP1 gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.

ACKNOWLEDGMENTS

We thank Trevor Littlewood for the p53._302-90 and pBABEPuro plasmids, Xin Lu and Arnold Levine for the p21^WAF1/CIP1 and MDM2 antibodies, respectively, and Parmjit Jat for the rat p21^WAF1/CIP1 cDNA probe. We also thank Peng Yeong Woon, Pieter De Jong, and the HGMP Resource Centre, Hinxton, United Kingdom, for the rat PAC library, Derek Davies and Aaron Rae for the FACS analysis, and Gordon Peters and Martine Lomax for help and advice in preparation of the manuscript.