Induction of apoptosis in AK-5 tumor cells by a serum factor from tumor rejecting animals: cytochrome c release independent of Bcl-2 and caspases

Abstract

The ability to selectively induce apoptosis in tumor cells is the prime goal in cancer immunotherapy and aims at identifying potential molecular targets, regulating this process. Here we show that the sera from the animals which had spontaneously rejected the AK-5 tumor (a rat histiocytoma) had an effective and potent ability to counteract and kill tumor cells by inducing apoptosis, with a high degree of specificity. Apoptosis induced by the serum factor involved the activation of caspases and cytochrome c release to the cytosol. A reduction in mitochondrial transmembrane potential (Dc m ) occurred considerably later than cytochrome c translocation. The antiapoptotic protein Bcl-2 and the pancaspase inhibitor zVADfmk did not prevent cytochrome c release, but completely blocked the reduction in Dc m , DNA fragmentation and apoptosis. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT) pore had no effect on cytochrome c release and apoptosis mediated by serum factor in AK-5 cells, suggesting that apoptosis was independent of MPT. Taken together these results suggest that the serum factor in conjunction with the immune cells may be participating in the efficient rejection of the tumor in syngeneic hosts and Dc m disruption but not cytochrome c release, is a critical and decisive event to trigger apoptotic cell death induced by the serum factor in AK-5 tumor cells.

Activation of NFκB and Ub-proteasome pathway during apoptosis induced by a serum factor is mediated through the upregulation of the 26S proteasome subunits

“…The serum from these tumor rejecting animals has a remarkably specific and potent apoptosis inducing ability towards AK-5/BC-8 cells in in vitro. 21 In order to detect differentially expressed genes during serum factor induced apoptosis, the DD PCR technique was used. The DD PCR gel of BC-8 cells treated with serum factor for 6 h showed numerous differentially expressed bands over untreated control cells but repeated northern blot analysis confirmed a 230 bp fragment, which was upregulated in SF treated cells, to be a true positive ( Figure 1A).…”

“…Animals injected with AK-5 cells s.c. and had rejected the tumor were used as the source of serum factor, previously shown to possess in vitro apoptotic activity towards AK-5 cells. 21 The serum factor is an immunoglobulin like molecule and was purified from tumor challenged animals by ammonium sulphate precipitation followed by fractionation on DEAE-sephadex ion exchange column and purified further on protein G-sepharose affinity column. 21 In the present study, we have used a single cell clone of AK-5 adapted to grow in vitro called BC-8 22 to avoid ambiguity in results due to tumor heterogeneity.…”

“…21 The serum factor is an immunoglobulin like molecule and was purified from tumor challenged animals by ammonium sulphate precipitation followed by fractionation on DEAE-sephadex ion exchange column and purified further on protein G-sepharose affinity column. 21 In the present study, we have used a single cell clone of AK-5 adapted to grow in vitro called BC-8 22 to avoid ambiguity in results due to tumor heterogeneity. BC-8 and P388D1 cells were grown in DMEM and HL-60 cells were cultured in RPMI 1640, both media supplemented with 10% (v/v) heat inactivated fetal calf serum in an atmosphere of 95% air and 5% CO 2 .…”

“…Since earlier work from the lab has shown that apoptosis in BC-8 cells is mediated by a loss of mitochondrial membrane potential by about 9 h and is accompanied by cytochrome C release into the cytosol, 21 we monitored the effect of MG132 on these two parameters. MG132 had no effect on the dissipation of mitochondrial membrane potential associated with apoptosis in BC-8 cells ( Figure 5I).…”

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F0-F1 ATP synthase complex

“…20 We have previously shown the presence of a serum factor (immunoglobulin) in the sera of AK-5 tumor regressing rats that induced apoptosis specifically in AK-5 or BC-8 (a single cell clone of AK-5) cells. 21 Besides reporting differential gene expression, the energy/ATP requirements of the cell during apoptosis and how the cell might cope up with these energy requirements has also been studied. These temporal changes in the F 0 -F 1 ATP synthase subunits genes are comparable over a range of apoptotic stimuli, thus establishing that the F 0 -F 1 ATP synthase is a rather universal player in apoptosis.…”