The uptake of nucleosides by mammalian cells is complex and mediated by multiple transport proteins that differ in their substrate specificity and sensitivity to inhibitors. There are two major groups of nucleoside transporters. First, equilibrative (e) also known as facilitated diffusion mechanism and second, concentrative (c). Equilibrative is further divided into two. This is based on the sensitivity to NBMPR, so es is sensitive and ei is insensitive. All concentrative type transporters are insensitive (ci) to NBMPR (1 micromolar) and depending on substrate specificity are divided into 3, hence cif accepts formycin B, cit, thymidine, and cib, accepts broad range of nucleosides. The basic hypothesis of this project has been that selective chemotherapy using nucleoside transport inhibitors in combination with antimetabolites can be developed based on the nucleoside transport properties of the tumor and the dose-limiting normal tissues. Studies during the previous funding period have defined nucleoside transport properties of tumor cell lines and critical normal cells of the bone marrow and intestine, and have identified differences that might be exploited by combining inhibitors of nucleoside transport with inhibitors of de novo thymidylate synthesis. The applicant's group and others have also identified changes in nucleoside transport in myeloid leukemia cells during differentiation that might be exploited to increase the activity of purine nucleoside analogs against this leukemia. Thus, the continuation of this project will test the expanded hypothesis that there are both intrinsic differences in nucleoside transport between normal tissues and some tumors (Aim 3), and differences that can be induced in some tumors by differentiating agents, that can be exploited for therapeutic purposes. This hypothesis will be tested through in vitro and in vivo studies of the modulation of thymidylate synthase inhibitors by nucleoside transport inhibitors in rhabdomyosarcoma (Aims 1 and 2); and in vitro studies of the modulation of purine nucleoside analog (specifically, 2-chlorodeoxyadenosine, 2-CdA) toxicity by differentiating agents in myeloid leukemia cells (Aim 4). The recent cloning of some of the nucleoside transporters will permit these studies to be carried to the molecular level, and the development of molecular reagents and assays (Aim 5) needed to determine the nucleoside transporter phenotype of normal tissues and tumors from small samples. It is anticipated that the in vitro and animal studies, and the development of the molecular reagents will provide a sound basis for the translation of these therapeutic approaches from the laboratory to the clinic.
