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Molecular and Cellular Biology, February 1999, p. 1582-1594, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

Edward Chu,1,* Sitki M. Copur,2 Jingfang Ju,1 Tian-men Chen,1 Samir Khleif,2 Donna M. Voeller,2 Nobuyuki Mizunuma,2 Mahendra Patel,2 Gladys F. Maley,3 Frank Maley,3 and Carmen J. Allegra2

Department of Medicine and Pharmacology, Yale Cancer Center and VA CT Cancer Center, Yale University School of Medicine, New Haven, Connecticut 065201; Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, Maryland 20889-51052; and Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 122013

Received 15 June 1998/Returned for modification 18 August 1998/Accepted 22 October 1998

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

* Corresponding author. Mailing address: VA CT Healthcare System, Cancer Center-111D, 950 Campbell Ave., West Haven, CT 06516. Phone: (203) 937-3421. Fax: (203) 937-3803.

Molecular and Cellular Biology, February 1999, p. 1582-1594, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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