LAK Cell Mediated Apoptosis of Human Bladder Cancer Cells Involves a pH-Dependent Endonuclease System in the Cancer Cell: Possible Mechanism of BCG Therapy

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Abstract
Intravesical bacillus Calmette-Guerin (BCG) is an effective treatment for superficial bladder cancer. However, its mechanism has been only partially elucidated. We studied whether LAK cell killing of human bladder cancer cells occurs via apoptosis (programmed cell death) or necrosis. Fluorescent dye labeled T24 cells were observed to undergo morphologic changes associated with apoptosis in the presence of LAK cells when analyzed under a fluorescence microscope. Furthermore, analysis of the DNA isolated from the cytotoxic assay confirmed that the LAK cell induced death of the T24 cells occurred via apoptosis. By pretreating the LAK cells with antifibronectin antibodies, we were able to significantly inhibit the LAK cell killing of the T24 cells. The percentage of cytotoxicity was reduced from 50% to 13% (p = 0.001), and the apoptotic pattern seen on agarose gel electrophoresis was significantly diminished. There was no significant change in the viability of the LAK cells following treatment with the antibodies. Endonuclease isolation from human bladder cancer T24 cells demonstrated that these cells express a pH-dependent and not a Ca++/Mg++ dependent endonuclease. Significant degradation of a target DNA was observed only in pH 4 to pH 5.6 buffers containing endonuclease from T24 cells and not in pH 6 to pH 8 buffers containing endonuclease from T24 cells. The presence or absence of Ca++/Mg++ in the various pH buffers did not alter the endonuclease activity. Finally, we demonstrated that death of T24 cells can be induced by altering the intracellular pH of the cells to 5.6 or lower with the proton ionophore nigericin. We conclude that LAK cells induce T24 cells to undergo apoptosis and that this process involves the fibronectin molecule present on the LAK cell membrane. Furthermore, the cleavage postulate that, in vivo, LAK cells activated by IL-2 produced by BCG activated CD4+ cells may induce bladder cancer cells to undergo apoptosis. This may partially explain the mechanism whereby BCG achieves its therapeutic effect.

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