The overall goal of this project is the identification of the major mechanisms which account for the development of resistance to VP-16 (Etoposide), a topoisomerase II directed antineoplastic drug, through GRP78 dependent pathways. Our preliminary studies with poly(ADP-ribose) deficient cell lines that overexpress GRP78 clearly demonstrate a very good association between induction of GRP78 and development of resistance to VP-16. We will investigate and determine the mechanisms of association of GRP78 induction and VP-16 resistance by examining the following questions. 1) We will identify the specific components of poly(ADP-ribose) synthesis system responsible for induction of GRP78 and its association with VP-16 resistance using specific approaches to interfere with selected components of the poly(ADP-ribose) synthesis system. 2) Since VP-16 is a cell cycle specific agent we will determine whether overexpression of GRP78 is affecting VP-16 sensitivity by altering cell cycle distribution. 3) To elucidate the relationship between induction of GRP78 and VP-16 resistance from a mechanistic standpoint we will determine whether induction of GRP78 interferes with normal functioning of topoisomerase II by interfering with its catalytic activity, phosphorylation pattern or the topoisomerase II-mediated DNA cleavage/religation in presence and absence of VP-16. 4) We will also identify the consequences of GRP78 overexpression by determining whether the overexpression of GRP78 confers resistance to VP-16 induced mutation and SCE by preventing the cells from undergoing VP-16-induced chromosomal deletions and rearrangements. 4) Furthermore, as an application of our finding, we will simulate in vivo situation of chemotherapy in tissue culture cells to determine whether a decrease in NAD level due to activation of poly(ADP-ribose) polymerase by a selected DNA damaging chemotherapeutic agents including VP-16 results in the induction of GRP78 and subsequent development of resistance to VP-16. To achieve this goal we plan to study the model systems that include a) Chinese hamster ovary cells C-.1 that overexpress GRP78 because of the expression of a stably integrated GRP78 expression vector, b) two groups of poly(ADP-ribose) deficient cell lines developed in our lab from V79 Chinese hamster cell line that also overexpress GRP78, and 3) a Hela cell derivative that specifically inhibits poly(ADP-ribose) polymerase activity by expressing antisense transcripts of poly(ADP-ribose) polymerase. The health relatedness of this study is to gain more insight into the mechanisms of VP-16 resistance so that therapeutic approaches could be developed to defeat the specific mechanisms identified.
