Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-express ion of the M-r 78,000 glucose-regulated stress protein (GRP78) and the subs equent development of resistance to inhibitors of topoisomerase II. These p henomena also occur in V79-derived cell lines that are deficient in poly(AD P-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitiv ity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bi s (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have a lso previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearin g L1210 tumors. These observations prompted us to examine whether 6-AN pre- treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitizati on to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6 -AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immu noblotting. To determine sensitivity after GRP78 up-regulation, the cells w ere washed and grown for 41/2 h in growth medium devoid of 6-AN, before bei ng treated with DNA cross-linking agents. The 48 h time period allowed p(AD PR) metabolism to return to normal while GRP78 levels remained elevated, th us allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, c ell sensitivity was determined by clonogenic survival assay or a fluorescen ce-based cytotoxicity assay. Based on changes in ICS, values, 6-AN caused a n increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-ex pression of GRP78 in human tumor cell lines is associated with increased se nsitivity to clinically useful chemotherapy agents. This sensitization occu rred in three different tumor cell lines, each bearing a separate genetic d efect associated with altered sensitivity. (C) 1999 Academic Press.