Regulation of Bcl-2 family proteins in antibody-dependent cellular cytotoxicity (ADCC) of small cell lung cancer cells mediated by a bispecific molecule OKT3xbombesin antagonist

Proc Amer Assoc Cancer Res, Volume 46, 2005 2262 Bcl-2 family proteins play a critical role in the regulation of apoptosis. The anti-apoptotic protein Bcl-2 is expressed in 75% of SCLC. We have shown that a bispecific molecule (BsMol) containing OKT3 (anti-CD3 antibody) conjugated with a bombesin antagonist was able to activate T lymphocytes and mediate SCLC cell apoptosis at a low effector to target (E:T) cell ratio. Since most SCLC cell lines do not express caspase 8, an initiator caspase in the death-receptor pathway, we studied whether Bcl-2 family proteins contributed in the process of tumor cell apoptosis by ADCC. Four SCLC cell lines H345, H69, SHP77 and DMS273 were studied. T lymphocytes were separated from healthy donors. SCLC cells were mixed with T lymphocytes at 1:1 ratio in the presence of the BsMol or mouse IgG2a as a negative control. The mixed cells were stained with annexin V, CD45 and propidium iodide (PI), analyzed by flow cytometry from 4h to 72h. Whole cell lysates were prepared at 24, 48, and 72h, and analyzed for expression of Bcl-2, Bad and phosphorylated Bad (Ser112), Bax, Bak, and cleaved poly(ADP-ribose) polymerase (PARP) by Western blot. H345 cells are exquisitely sensitive to ADCC. There is high expression of Bcl-2, Bad and phosphorylated Bad at baseline. Western blot analysis showed phosphorylated Bad decreased significantly at 24h, and became undetectable at 72h in the presence of BsMol, while the expression of Bad remained stable. There was decreased expression of Bcl-2, and increased expression of Bak, Bax, and cleaved PARP at 72h. Flow cytometry analysis showed an increase of annexin V positive SCLC cells from 15% at 24h to 40% at 72h in the presence of the BsMol. Similar results were observed in SCLC cell lines H69 and SHP77. In contrast, DMS 273 cells, which are insensitive to ADCC at low E:T ratio, have barely detectable expression of Bcl-2 at baseline. After 48h, Bcl-2 became easily detectable, then increased significantly at 72h. The expression of cleaved PARP decreased at 72h. While the percentage of annexin V positive cells remained stable from 24h to 72h, the percentage of SCLC cells increased from 26% to 60% at 72h. We conclude that there is a dynamic change in expression of Bcl-2 family proteins in the process of ADCC mediated by the BsMol, leading to apoptosis and SCLC cell death. The increased expression of Bcl-2 in DMS 273 cells during the process may be the underlying mechanism of its resistance of ADCC. Blocking the upregulation of Bcl-2 in combination with immunotherapy may enhance the cytotoxicity of resistant SCLC cells.