HYPERSENSITIVITY TO DNA CROSS-LINKING AGENTS ASSOCIATED WITH UP-REGULATION OF GLUCOSE-REGULATED STRESS PROTEIN GRP78

We have shown previously that NAD/poly(ADP-ribose) polymerase-deficien t cells that overexpress M-r 78,000 glucose-regulated stress protein ( GRP78) are resistant to topoisomerase II inhibitors, such as etoposide , m-amsacrine, and doxorubicin. However, these cells have been found t o be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These obse rvations prompted us to examine whether overexpression of GRP78 is ass ociated with modulation of cytotoxicity of clinically useful DNA-cross -linking agents such as melphalan, BCNU, and cisplatin. We up-regulate d GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxygl ucose. Subsequently, these GRP78-overexpressing cells were trypsinized , plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or ci splatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkalin e elution assay. Our results show that the GRP78-overexpressing V79 ce lls are hypersensitive to DNA cross-linking agents compared to the con trol V79 cells. Furthermore, repair of drug-induced DNA cross-links ap pears to be considerably slower in these cells relative to that found in control V79 cells, Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, ( b) up-regulation of GRP78 is associated with potentiation of cytotoxic ity induced by alkylating and platinating agents, and (c) up-regulatio n of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agent s.