Methotrexate (MTX) is an effective chemotherapeutic agent in the treatment of a variety of different highly proliferative human tumors. However, sensitivity of normal hematopoietic and gastrointestinal tissue limits the dose of MTX which can be safely administered. MTX acts as a competitive inhibitor of dihydrofolate reductase (DHFR), and one potential way in which the MTX sensitivity of normal tissue could be modified is by the introduction and expression of DHFR sequences encoding a variant enzyme which is less sensitive to MTX. As a way of testing the potential for MTX- resistant DHFR expression in reducing hematopoietic toxicity associated with MTX administration, it is proposed herein to establish lines of transgenic mice, each expressing one of three variant DHFR's (arg22, trp3l, and tyr22) previously demonstrated to confer MTX resistance upon cultured mammalian cells. The recently identified trp3l and tyr22 variants will first be enzymologically characterized to verify their potential metabolic effectiveness. DHFR expression constructs appropriately designed for in vivo expression and analysis will then be established in the germ line of inbred FVB/N mice, screening for expression of transgene message and drug-resistant DHFR activity in different tissues, particularly hematopoietic tissues. Animals positive for expression will be tested for systemic and organ sensitivity to MTX administration, noting possible correlations between expression and drug-resistance in different tissues. Animals expressing MTX(r) DHFR activity in hematopoietic tissues will then be used as donors for syngeneic bone marrow transplant studies to determine if the use of DHFR transgenic marrow renders recipient animals tolerant to administration of higher doses of MTX than recipients receiving control non-transgenic marrow. Finally, normal and DHFR transgenic marrow will be fractionated into cell populations enriched for hematopoietic stem cells vs committed hematopoietic progenitors to determine which of these two populations contributes to survival and drug- resistance in recipient animals. The experiments in this proposal are thus designed to determine quantitatively the extent and conditions under which MTX(r)-DHFR gene expression can be used to reduce the sensitivity of hematopoietic tissues to MTX administration. These results will thus provide an assessment of the potential for application of MTX(r)-DHFR gene transfer to improved tumor chemotherapy.
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