Regulation of p 53 expression by thymidylate synthase ( translational regulation y gene expression )

Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (1) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as g-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression. Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by 5,10methylenetetrahydrofolate to form dTMP and dihydrofolate (1, 2). Because the TS-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis, this enzyme has been an important target for cancer chemotherapy for nearly 40 years (3–6). In addition to its critical role in enzyme catalysis, TS functions as an RNA binding protein (7–10). Studies from this laboratory have demonstrated that translation of human TS mRNA is regulated by its own protein product via a negative autoregulatory mechanism whereby the binding of TS protein to at least two distinct sequences on its own TS mRNA results in translational repression (7, 8). More recently, studies have documented that TS, in addition to directly interacting with its own TS mRNA, is capable of binding to several cellular RNA species (11–13). An immunoprecipitation–RNA–random (r)PCR method was developed to isolate cellular RNA sequences that formed ribonucleoprotein (RNP) complexes with TS in human colon cancer H630 cells (13). With this approach, nine different cellular mRNAs including those corresponding to the myc family and the p53 tumor suppressor gene were identified. Previous studies had shown that TS binds in vitro to the C-terminal coding region of c-myc mRNA. Further investigation revealed that this RNA–protein interaction results in translational repression of c-myc mRNA (12). These studies suggested that TS may be involved in the coordinate regulation of expression andyor function of a host of cellular genes. Although recent work has identified a direct interaction between human TS protein and p53 mRNA (13), the in vivo biological significance of this RNA–protein interaction remains to be characterized. The p53 tumor suppressor plays an essential role for preserving the integrity of the genome and for maintaining regulation of cell cycle progression (14–17). Several investigators have shown that the levels of p53 are acutely increased in both normal and malignant cells in response to DNA-damaging agents (18–21). Although definitive mechanistic studies remain to be performed, the induced expression of p53 after DNA damage appears to be regulated at least in part, by translational and post-translational regulatory processes. The importance of translational regulatory mechanisms underlying the expression of p53 has been recently supported by studies that suggest that the expression of murine p53 is controlled by a negative autoregulatory feedback process (22, 23). Mosner et al. (23) demonstrated that the p53 protein end product binds to the 59 untranslated region of its corresponding p53 mRNA and, in so doing, effectively represses translation. A more recent report by Fu et al. (24) confirmed that the expression of human p53 was also controlled at the translational level, presumably, through an autoregulatory feedback loop. However, in contrast to the murine system where p53 protein interacts with the 59 untranslated region of its corresponding mRNA sequence, human p53 protein binds to a sequence contained within the 39 untranslated region of the human message (24, 25). Because the biosynthesis of p53 is regulated, in part, at the translational level and a TS–RNP complex composed of TS protein and a sequence corresponding to human p53 mRNA was identified in human colon cancer cells, we decided to further investigate the biological role of the interaction between TS protein and p53 mRNA. For these studies, we transfected the respective full-length human TS and Escherichia coli TS cDNAs under the control of a constitutive cytomegalovirus promoter into HCT-C cells that express wild-type p53 and nonfunctional TS protein. In this report, we show that the overexpression of human and E. coli TS in the HCT-C cell line results in marked suppression of synthesis of p53 protein with no associated change in p53 mRNA levels. Our findings demonstrate that TS protein binds directly to the p53 mRNA resulting in translational repression and identifies a mechanism by which p53 expression is controlled. MATERIALS AND METHODS Cell Culture. The human colon cancer HCT-8 and HCT-C cell lines were a gift from S. Berger (University of South Carolina, Columbia) and Y. Rustum (Roswell Park Memorial Cancer Institute, Buffalo, NY). The HCT-C line is a mutant subline of the HCT-8 parental cell line, and it expresses a Ser3 Leu mutation at amino acid position 216 of the human TS protein (26). Enzyme kinetic analysis has shown that this mutant TS protein is nearly completely inactive (27). Cells were maintained in RPMI 1640 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas. org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: TS, thymidylate synthase; RNP, ribonucleoprotein; RT-PCR, reverse transcription-coupled PCR; DHFR, dihydrofolate reductase. ‡To whom reprint requests should be addressed at: VA Connecticut Healthcare System, Cancer Center, 111-D, 950 Campbell Avenue, West Haven, CT 06516.