Abstract
Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three- to five-fold more sensitive (IC50 0.1 mM) to HU than GJIC-deficient derivatives (IC50 0.3 - 0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% - 80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding Gjb1 increased HU toxicity (IC500.1 mM). The effects were not due to connexin expression per se or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% - 20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.
Highlights
Gap junctions are plasma membrane channels that connect the interiors of neighboring cells and permit the direct cell-to-cell diffusion of molecules and ions smaller than ~1 kDa
We found that HU was more toxic in gap junctional intercellular communication (GJIC)-positive cells and HU resistant cells were killed when co-cultured with HU-sensitive cells in a GJICdependent manner
Several GJIC-deficient derivatives of WB-F344 [16] and known as WB-aB1, WB-bA2, WB-cD6, and WB-dA2 were used here. These cells do not exhibit GJIC by scrape-loading/dye transfer (SL/DT) assay, but express connexin 43 (Cx43) in amounts comparable to WB-F344 cells; this protein is localized to the plasma membrane (Figures 1(a)-(c))
Summary
Gap junctions are plasma membrane channels that connect the interiors of neighboring cells and permit the direct cell-to-cell diffusion of molecules and ions smaller than ~1 kDa. Cytotoxicity was directly correlated with GJIC whereas reduced cell death occurred in GJIC-coupled cells [5] The mechanisms for these effects are poorly understood. Injured cells may impact naive cells through poorly understood bystander effects whereby toxic factors and death-inducing signal molecules such as calcium ion and free radicals may move from injured cells to neighbors through gap junction channels. Ribonucleotide reductase (RR) inhibitors are an important class of anti-cancer drugs, but how GJIC affects their cytotoxicity has not been well studied. This enzyme catalyzes the de novo conversion of ribonucleotide diphosphate to deoxyribonucleoside diphosphate and is the rate-limiting step in DNA synthesis [6]. We found that HU was more toxic in GJIC-positive cells and HU resistant cells were killed when co-cultured with HU-sensitive cells in a GJICdependent manner
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