In an additional control, we observed the presence of in vitro-modified 3XFlag-tagged TS in a lysate of RKO cells transfected so as to express 3xFlag-tagged TS

In an additional control, we observed the presence of in vitro-modified 3XFlag-tagged TS in a lysate of RKO cells transfected so as to express 3xFlag-tagged TS. preclinical analytic studies or make practical the individual tailoring of dosing. Keywords: Ternary complex, thymidylate synthase, drug adduct, drug adduct-specific antibody, ternary complex-specific antibody, FTS INTRODUCTION TS BIX 01294 catalyses the reductive methylation of 2-deoxyuridine-5-monophosphate (dUMP) to 2-deoxythymidine-5-monophosphate (dTMP) with provision of a carbon donated by 5, 10-methylene tetrahydrofolate (DMTHF) [1, 2]. dTMP is then converted to dTTP for use in DNA synthesis. As a necessary component of DNA replication, TS is an attractive target for cancer treatment. The anti-metabolite drug 5FU, a fluoropyrimidine, and fluoropyrimidine analogues are used to inhibit TS in cancer treatment [3]. Intracellularly, 5FU is converted to active metabolites fluorodeoxyuridine (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine triphosphate (FUTP). FdUMP competes with dUMP and, covalently with DMTHF, binds TS to form BIX 01294 a ternary complex (5FU-modified TS, TS-F) [1], terminating its activity. The ternary complex consists of a covalent bond between Cys198 of TS and C-6 of FdUMP and covalent bonds of the methylene group to both C-5 of FdUMP and N-5 of folate. Graded inhibition of TS results in degrees of inhibition of DNA synthesis. FdUTP can, in place of dTTP, incorporate into DNA and result in DNA damage directly by mis-incorporation or indirectly by stimulating DNA repair [4-6]. FUTP, in place of UTP, incorporates into, and damages or impairs function of, RNA [7-9]. Fluoropyrimidines are an essential component of colorectal cancer chemotherapy [10], are also used to treat other gastrointestinal cancers, breast cancer, and head and neck cancers, and are often included in combination chemotherapeutic regimens. Despite large numbers of 5FU-related clinical studies [11], there has been a little done to individually tailor fluoropyrimidine dosage for cancer therapy. The separate quantification of native unmodified TS (TS-N) and TS-F after treatment could be used to optimize dosing and tumor responses. Drake et.al, used immunoblots (IB) to quantify total TS and TS-F [12]. Quantification of total TS, TS-N and TS-F was also done using radiochemicals [13-15]. These methods are tedious at best, however. To work toward a more facile quantification, we developed a monoclonal antibody by using TS-F as the immunizing antigen. By IB, the antibody specifically recognized TS-F from 5FU-treated cell lysates and from 5FU-treated BIX 01294 cancer xenograft tissues. A plausible moderate-term future goal would be to quantify separately TS-N and TS-F in tissues by developing an assay that used a nonspecific anti-TS antibody and a specific anti-TS-F antibody, so as to permit clinical monitoring of fluoropyrimidine cellular activity, expressed as measured ratio of TS-F to the remaining TS-N. RESULTS Verifying the method of TS modification in vitro It is known that cellular TS-F migrates slower than TS-N in denaturing protein gels, by IB [16]. By IB using anti-TS antibody (TS106), we also observed cellular TS-F migrating slower than TS-N in the in vitro-modified RKO cell lysate (Figure ?(Figure1A).1A). Results were compared with a lysate of 5FU-treated RKO cells, in which TS-F migrates slower than TS-N. Open in a separate window Rabbit Polyclonal to ARMX3 Figure 1 TS modification in vitro(A) RKO cells were treated with 5FU in culture, and an RKO cell lysate was modified in vitro using FdUMP and DMTHF. IB analysis was done using TS106. (B) Purified rGST-TS and rTS were modified in vitro and analyzed after separation by denaturing gel and Coomassie staining. (C) IB analysis of in vitro-modified rTS, rGST-TS, and 3xFlag-tagged TS in an RKO cell lysate, using TS106. We produced rTS and modified it in vitro to form rTS-F. In Coomassie-stained denaturing protein gels, we observed rTS-F migrating slower than un-modified rTS (rTS-N) (Figure ?(Figure1B).1B). This verified our in vitro-modification of rTS to rTS-F. We BIX 01294 also observed in vitro modified rGST-TS-F migrating slower than unmodified rGST-TS. By IB using TS106, we observed slower migration of rTS-F than rTS-N and, similarly, of rGST-TS-F than rGST-TS (Figure ?(Figure1C).1C). In an additional control, we observed the presence of in vitro-modified 3XFlag-tagged TS in a lysate of RKO cells transfected BIX 01294 so as to express 3xFlag-tagged TS. After these confirmations, the purified rTS-F was used to immunize animals for antibody.