Mechanisms of Selective Actions of Antiviral Agents

University Of Miami School Of Medicine, Coral Gables FL

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Abstract

DESCRIPTION (provided by applicant): The long-term objective of this research is to explore host cell factors that influence chemotherapeutic effects of anti-HIV agents. During the previous grant period dideoxycytidine (ddC) and arabinosyl-cytosine (araC) resistant H9-ddC and H9-araC subclones were generated which had reduced deoxycytidine kinase (dCK) activity, ddCTP and araCTP formation. These cells also had reduced thymidine kinase (TK) activity, TK2 mRNA, drug influx and nucleoside transport sites. Consequently they were collaterally resistant to AZT, d4T, and FdUR (5-fluoro-2'-deoxyuridine). The investigators have propagated H9 cells in the presence of 10 and 100 uM 3TC (here after called H9-3TC cells). Consistent to araC and ddC resistant cells, H9-3TC cells had reduced ddC and 3TC incorporations. However, in contrast to ddC and araC resistant cells, H9-3TC cells incorporated less AZT and FdUR. These observations suggest that 3TC, ddC, and araC (all analogs of dC) induce cellular resistance but by different mechanisms and affect several unknown cellular factors involved in their metabolism and in the metabolism of other unrelated drugs. The specific aims of this proposal are to: (i) explore mechanisms of 3TC resistance and compare them with ddC and araC resistance; (ii) characterize molecular mechanisms alterations in dCK and TK expression and genes involved in 3TC, ddC, araC, and AZT metabolic pathway in the resistant cells; (iii) examine the sensitivities of HIV-1 to ddC, 3TC, and AZT in the resistant cells and determine whether multiple drug resistance is limited to 119 cells; and (iv) explore the biochemical and molecular basis of reduced nucleoside transport. These aims will be achieved by studying the biochemical and molecular mechanisms of the resistant cells. The effect of resistance on gene expression in the resistant cells will be followed by DNA micro-array analysis. DNA hypermethylation and mutations in the dCK and TK genes will be investigated using Southern blotting and sequencing respectively. HIV sensitivity to 3TC, ddC, and AZT in the resistant cells will be examined by p24 analysis. Understanding of these factors will help design strategies to improve effectiveness of the antiviral agents.