The long term goals of this study are to develop and to improve the use of folate analogs for cancer treatment. Two areas of interrelated investigation are proposed for the next five years. (1) To study the mechanisms that regulate the content of dihydrofolate reductase (DHFR) in human cells. Our hypothesis is that there are several intracellular mechanisms which could regulate the level of DHFR in a selective manner, and that DHFR could autoregulate its own synthesis, directly or indirectly. This autoregulation could be altered by inhibitors through their interaction with DHFR. To test this hypothesis, several specific questions are addressed: (a) Will human DHFR recognize and interact with a unique sequence at the 5' flanking region of its DHFR gene, and if it does what effect will DHFR have on the transcription of DHFR gene? (b) Will human DHFR recognize some unique RNA species in cells? (c) What effect will DHFR have on the translational efficiency of the three cytoplasmic DHFR mRNA species from human cells? (d) What effect will methotrexate (MTX) or folate metabolites (including polyglutamate forms) have on the possible interaction of DHFR and its related DNA or RNA? (e) What is the effect of MTX on the content of DHFR related RNA in cells? (2) To study the mechanisms involved in the development of MTX resistance due to overproduction of DHFR. The hypothesis is that the DNA from dead cells may facilitate the overproduction of DHFR in other cells. The physiological state of DNA in other cells may also play a key role. We intend to work on a more defined system initially by (a) examining the effect of DNA or nuclei isolated from parental human KB cell line and its different DHFR overproducing cell (OPC) lines on the frequency of isolating DHFR or UMP synthetase OPC from KB Parental cells; (b) isolating the gene(s) which could influence the frequency of generating DHFR OPC; (c) studying the effect of nuclei from cells treated with DNA interacting anticancer compounds such as VP 16 in promoting the frequency of isolating DHFR OPC from KB cells; (d) investigating the effect of nuclei in promoting DHFR OPC clones of KB cells treated with DNA damaging compounds at sublethal dosages. The information gathered will be important not only in development of new protocols or new drugs for cancer treatment, but also in the understanding of basic mechanisms of enzyme and gene regulation.
