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Molecular and Cellular Biology, February 2000, p. 1291-1298, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The p21WAF1/CIP1 Promoter Is Methylated in Rat-1 Cells: Stable Restoration of p53-Dependent p21WAF1/CIP1 Expression after Transfection of a Genomic Clone Containing the p21WAF1/CIP1 Gene

Lindsey A. Allan,dagger Trevor Duhig, Moira Read, and Mike Fried*

Eukaryotic Gene Organisation and Expression Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom

Received 1 October 1999/Returned for modification 2 November 1999/Accepted 8 November 1999

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 p21WAF1/CIP1 gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21WAF1/CIP1 expression appears to be the result of hypermethylation of the p21WAF1/CIP1 promoter region, as p21WAF1/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 p21WAF1/CIP1 gene. Stable X-ray-induced p53-dependent p21WAF1/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 p21WAF1/CIP1 gene. The absence of expression of the p21WAF1/CIP1 gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.

* Corresponding author. Mailing address: Eukaryotic Gene Organisation and Expression Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Phone: 44-171-269-3297. Fax: 44-171-269-3093. E-mail: fried{at}icrf.icnet.uk.

dagger Present address: Biomedical Research Centre, Ninewells Hospital & Medical School, Dundee DD1 9SY, United Kingdom.

Molecular and Cellular Biology, February 2000, p. 1291-1298, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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