Previous studies suggest that dihydrofolate reductase (DHFR) regulates its own translation. Moreover, intracellular levels of DHFR protein increase following exposure to the antifolate methotrexate (MTX), suggesting that MTX may release the translational inhibition mediated by DHFR [Chu et al. (1993) Biochemistry 32,4756-4760; Ercikan et al. (1993) Adv. Exp. Med. Biol. 338, 537-540]. To further investigate the role of DHFR in translational autoregulation, we have considered the possibility that DHFR directly contacts its cognate mRNA. Binding studies using a series of truncated DHFR mRNAs as probes localized the DHFR/RNA interaction to a 100-base-pair region containing two putative stem-loop structures; initial studies indicated that both of these loop structures are involved in protein binding. Moreover, the binding of MTX to DHFR prevents interaction of the protein with its cognate mRNA, thereby relieving translational autoregulation.

Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex®, BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drugresistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC 50 values 40-and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26-and 97-fold in IC 50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg 50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.Thymidylate synthase (TS, 1 EC 2.1.1.45) catalyzes the de novo biosynthesis of thymidylate, which is necessary for DNA synthesis and repair (1). The mechanism of TS activity involves the reductive methylation of the substrate, 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) by transfer of a methylene group from the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate), to generate 2Ј-deoxythymidine 5Ј-monophosphate (dTMP) and 7,8-dihydrofolate. Human TS has been sequenced (2), purified (3, 4), and crystallized (5). As an attractive target for anti-cancer drug design, since the 1950s, many TS analogues of both the substrate, dUMP, and the cofactor, CH 2 H 4 folate, have been synthesized and tested as potential anti-cancer therapeutics. Until recently, 5-fluorouracil and fluorodeoxyuridine (FdUrd) were the sole TS-targeted drugs approved for clinical application. In vivo, 5-fluorouracil and FdUrd are metabolized to 5-fluoro-2-deoxyuridylate (FdUMP), a compound that subsequently occupies the pyrimidine binding site forming a ternary complex with TS and the folate cofactor, resulting in inhibition of enzyme function. The recent determination of the three-dimensional structure of human TS has allowed the design of highly specific inhibitors, leading to the emergence of novel folate analogues, such as Tomudex (ZD1694), BW1843U89, and Thymitaq (AG337) (Fig. 1) (6). These promising compounds have entered clinical trials in recent years (7).Previous studies h...

These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.