Vacuolar-proton-ATPase is involved in the response of cancer cells to ionizing radiation and is a new target for radiosensitization

Purpose: We have recently described a novel response of human cancer cells to radiation consisting of accumulation of acidic vesicular organelles (AVO) (Cancer Research, 61:439-444, 2001). Acidification of AVO was inhibited by bafilomycin A1 - a specific inhibitor of vacuolar-proton-ATPase. To examine the role of AVO function in the cellular response to radiation, we determined the effect of specific inhibitors of v-H+-ATPase (bafilomycin and concanamycin) on the clonogenic survival of various cancer cell lines. In addition, Northern analysis of mRNA encoding v-H+-ATPase was performed to elucidate possible mechanisms of post-radiation v-H+-ATPase regulation. Materials and Methods: Total RNA was isolated from human cancer cell lines including MCF-7 (breast), LoVo (colon), and LNCaP (prostate) cells 48 hours post-irradiation, and Northern analyses were performed. RNA was separated on a denaturing formaldehyde gel and blotted to a nylon membrane. Probes made from cDNAs to v-H+-ATPase were radioactively labeled, hybridized to the membranes, and exposed to X-ray film for determination of mRNA expression levels. Clonogenic survival assays were performed with MCF-7 and LoVo cell lines. Cells were plated at 30 cells/cm2, and irradiated 24 hours post-plating. Bafilomycin or concanamycin were added to culture media in various concentrations at the time of irradiation. The media was changed after a 24-hour incubation, and colonies were stained with crystal violet for counting 7 days post-irradiation. Results: Radiation-induced accumulation of acidic vesicular organelles in MCF-7, LoVo and LNCaP cells was associated with a two-fold increase in the steady-state level of mRNA for subunit c of v-H+-ATPase. Bafilomycin and concanamycin increased clonogenic cell death after irradiation in a dose-dependent manner. At low concentrations (bafilomycin 2nM, concanamycin 2pM) these agents acted as radiosensitizers, without significant toxicity to unirradiated cells. At 2nM, bafilomycin reduced the clonogenic survival of MCF-7 cells from 0.70 to 0.51 following 2 Gy (p=0.01) and from 0.27 to 0.17 following 4 Gy (p<0.01). Concanamycin (2 pM) reduced the clonogenic survival following exposure to 3 Gy from 0.45 to 0.28 (p=0.015). LoVo cells exhibited a surviving fraction of 0.42 after 3 Gy, versus 0.21 with bafilomycin (p<0.01). Conclusion: These results suggest that formation of acidic vesicular organelles after irradiation serves to protect cells against radiation damage, and that interference with their acidification can decrease clonogenic cell survival post-radiation. Low concentrations of v-H+-ATPase inhibitors such as bafilomycin A1 and concanamycin sensitize cancer cells to radiation without affecting non-irradiated cells. The vacuolar-proton-ATPase can therefore provide a new target for modulation of the radiosensitivity of human epithelial cancer cells