LAK CELL-MEDIATED APOPTOSIS OF HUMAN BLADDER-CANCER CELLS INVOLVES A PH-DEPENDENT ENDONUCLEASE SYSTEM IN THE CANCER CELL - POSSIBLE MECHANISM OF BCG THERAPY

Intravesical bacillus Calmette-Guerin (BCG) is an effective treatment for superficial bladder cancer. However, its mechanism has been only p artially elucidated. We studied whether LAK cell killing of human blad der cancer cells occurs via apoptosis (programmed cell death) or necro sis. Fluorescent dye labeled T24 cells were observed to undergo morpho logic changes associated with apoptosis in the presence of LAK cells w hen 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 signif icantly inhibit the LAK cell killing of the T24 cells. The percentage of cytotoxicity was reduced from 50% to 13% (p = 0.001), and the apopt otic pattern seen on agarose gel electrophoresis was significantly dim inished. There was no significant change in the viability of the LAK c ells following treatment with the antibodies. Endonuclease isolation f rom human bladder cancer T24 cells demonstrated that these cells expre ss a pH-dependent and not a Ca++/Mg++ dependent endonuclease. Signific ant degradation of a target DNA was observed only in pH 4 to pH 5.6 bu ffers containing endonuclease from T24 cells and not in pH 6 to pH 8 b uffers 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 ind uced by altering the intracellular pH of the cells to 5.6 or lower wit h the proton ionophore nigericin. We conclude that LAK cells induce T2 4 cells to undergo apoptosis and that this process involves the fibron ectin molecule present on the LAK cell membrane. Furthermore, the clea vage of the T24 cellular DNA may occur via a pH-dependent endonuclease present in these cells. We postulate that, in vivo, LAK cells activat ed by IL-2 produced by BCG activated CD4(+) cells may induce bladder c ancer cells. to undergo apoptosis. This may partially explain the mech anism whereby BCG achieves its therapeutic effect.