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

• Published in: Molecular and cellular biology Vol. 20; no. 4; pp. 1291 - 1298
• Main Authors: Allan, LA, Duhig, T, Read, M, Fried, M
• Format: Journal Article
• Language: English
• Published: 01.02.2000
• Subjects: CIP1 gene; p21 protein; WAF1 gene
• ISSN: 0270-7306
• DOI: 10.1128/MCB.20.4.1291-1298.2000

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 gene MDM2 but not the protein or mRNA of the p53- inducible p21 super(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21 super(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21 super(WAF1/CIP1) promoter region, as p21 super(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 super(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21 super(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 super(WAF1/CIP1) gene. The absence of expression of the p21 super(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.