Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [114]-124).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [135]-144).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 186-200).

The activity of dihydrofolate reductase, a key enzyme in the de novo biosynthesis of thymidylate, purines, and some amino acids, had previously been found to be increased in a methotrexate-resistant mouse leukemia (L5178Y) cell line as compared to the activity in the parental methotrexate-sensitive cell line. The increased activity was composed of two forms of the enzyme, one of which, form 2, was highly insensitive to inhibition by methotrexate. Both forms were purified to near homogeneity and using the antibodies prepared against them, it could be demonstrated that the two forms are antigenically distinct. The increased dihydrofolate reductase activity present in the methotrexate resistant cells resulted from an overproduction of both forms of the enzyme due to the presence of abundant mRNA coding for these enzymes. An increase in the dihydrofolate reductase gene copy number could be demonstrated in the resistant cells. mRNA coding for form 1 and form 2 enzymes was greatly enriched by polysome immunoprecipitation and complimentary DNA (cDNA) was synthesized in vitro from these enriched mRNA molecules. Evidence was found for the presence of methotrexate-insensitive forms of dihydroflate reductase in the blast cells of three out of eight acute myelogenous leukemia patients, and in two (distinct from the above) of the eight patients the activity was significantly increased. In contrast to the overproduction of dihydrofolate reductase protein in the methotrexate-resistant mouse cell line, increased enzyme activity in a methotrexate-resistant human promyelocytic leukemia (HL-60) cell line could not be correlated with an increase in the enzyme protein. Furthermore, the amounts of dihydrofolate reductase mRNA and gene-dosages were similar in the parental metho-trexate-sensitive and methotrexate-resistant cells. The enzyme from the resistant cells differed significantly in some of its physical and kinetic properties from that present in the parental cells. An increase in dihydrofolate reductase activity resulting from a modification of the enzyme rather than gene amplification has not to date been reported in the literature and may present a novel mechanism of resistance to methotrexate. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate