The overall goal of the proposed research is to elucidate the role of extracellular and intracellular pH in determining the toxicity of chemotherapeutic drugs to cancer cells in vitro and in vivo. Results from our laboratory and from others indicate that intracellular and extracellular pH may play an important role in determining cellular response to the antitumor drug, mitomycin C, in vitro. Therefore, the effects of altering the intracellular and extracellular pH microenvironment on the cytotoxicity and molecular damage produced by mitomycin C, chlorozotocin, and 1,3-bis-(2-chloroethyl)-1-nitrosourea will be studied in EMT6 mouse mammary carcinoma cells in vitro. Intracellular pH will be measured using flow cytometry to detect a pH sensitive fluorescent dye. Cytotoxicity will be determined using colony formation assays and molecular damage will be assessed using alkaline elution methods to detect DNA single strand breaks, DNA-DNA crosslinking, and DNA-protein crosslinking. BALB/c KaRw mice bearing EMT6 tumors will be used to assess tumor pH in vivo and to determine the effects of pH on antitumor drug efficacy in vivo. The effects of regional variations in tumor pH on tumor cell sensitivity to drug will be determined using colony formation assays and alkaline elution measurements of drug- induced DNA damage. Because cellular regulation of pH is controlled by two ion exchangers, a Na+/H+ exchanger and a C1- /HCO3- exchanger, the drugs which inhibit these exchangers will be used to manipulate pH and alter drug toxicity. The knowledge obtained from these studies may enable the design of therapeutic regimens which will be effective against tumor cells situated in low pH environments.
