ANTISENSE AS AN APPROACH TO CANCER CHEMOTHERAPY

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The impact of two types of antisense compounds (i.e., oligodeoxyribonucleoside methyl phosphonates, ODNMPs and oligodeoxyribonucleotide-poly-L-lysine conjugates, ODPLCs) which can enter cells, on the expression of drug resistance will be examined. The primary objective is to evaluate the ability of these compounds to suppress the synthesis of dihydrofolate reductase (DHFR) in DHFR overproducing cells. The results obtained will define the limitations and potential of these compounds as anti-tumor agents (alone or in combination with other drugs). Regions of DHFR mRNA sensitive to inhibition by hybridization arrest have been determined in vitro using oligodeoxyribonucleotide (ODs) hybridization techniques in a translational assay. ODNMPs and ODPLCs complementary to these sensitive regions will be synthesized and tested in vitro for their ability to specifically inhibit translation of DHFR mRNA against a background of heterologous substrates included within the same reactions as internal controls. The ability of ODNMPs and ODPLCs to regulate DHFR expression will subsequently be tested in cell culture using MTX-resistant and sensitive cells, alone and in combination with MTX. Effects will be monitored both on a molecular level and in terms of cell viability. The relative importance of sequence, timing of exposure and concentrations required to observe effects will be determined. Radiolabelled ODNMPs, ODPLCs, and ODs will be prepared and utilized to study effects of length and composition on the uptake of these compounds. In a similar fashion, ODs, ODNMPs and ODPLCs will be synthesized to complementary regions of human beta-globin pre- mRNA to determine regions sensitive inhibition of pre-mRNA splicing in vitro. The data obtained from these studies will then be used to study the feasibility of specifically inhibiting splicing in cultured cells which express the human beta-globin gene and in cells which express a human DHFR minigene at high levels.