title: 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 gene MDM2 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.
authors.affiliation: Eukaryotic Gene Organisation and Expression Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
authors.forename: L A
authors.initials: LA
authors.lastname: Allan
authors.forename: T
authors.initials: T
authors.lastname: Duhig
authors.forename: M
authors.initials: M
authors.lastname: Read
authors.forename: M
authors.initials: M
authors.lastname: Fried
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D002453:Cell Cycle / Q000528:radiation effects; D002460:Cell Line; D018899:CpG Islands; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016193:G1 Phase; D005720:Gamma Rays; D015870:Gene Expression / Q000528:radiation effects; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014170:Transformation, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53