Since serum is a source of hormones, growth factors, cytokines and antioxidants being a survival factor for cell culture its withdrawal from the culture medium induces apoptosis in a variety of cell types. In the present study the effect of FCS - deprivation on Bcl-2 protein level, Bax transcript and apoptosis of L1210 leukaemic cells was examined. The possibility of the existence of cellular origin "death factors" was also investigated. A simultaneous flow cytometric analysis of Bcl-2 protein using FITC-conjugated Mo anti-Bcl-2 antibody and the percentage of apoptotic cells by measurement of sub-Gl region in the DNA (stained with DAPI) histogram was performed. Bax transcript was measured using the RT-PCR method with GAPDH as a reference gene. ROS generation was assessed by flow cytometry using the oxidation - sensitive fluorescent marker C-DCDHF-DA. FCS deprivation induced a massive apoptosis of leukaemic cells, reaching 40% of the cell population after 24h. Typical features of apoptosis such as cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis (putrosis) were observed using fluorescence microscopy. An increase in intracellular ROS generation by 100% after 24h of serum deprivation was also observed. The apoptotic effect of FCS - deficiency was accompanied by a 32.7% decrease in the number of cells containing Bcl-2 protein (cells with specific anti-Bcl-2 fluorescence), thereby a negative relationship (r=0.98, P<0.01) between the extent of apoptosis and bcl-2 expression was calculated. Since serum deficiency eliminated primarily the cells with the lowest Bcl-2 level, the average Bcl-2 level in the population of surviving cells was even higher than in the control cultures, growing in optimal conditions (10% FCS/RPMI). The conditioned medium (CM), obtained from 24h FCS - deprived L1210 leukaemic cells, contained >10 kDa molecules, which were able to down- regulate Bcl-2, up-regulate Bax, and induce apoptosis. This effect was more visible when >10 kDa CM filtrate was administered to poorly-fed (2% FCS/RPMI) than better-fed (10% FCS/RPMI) cultures. It is concluded that: 1) the susceptibility of L1210 leukaemic cells to serum deprivation - induced apoptosis is dependent on Bcl-2 protein level, and 2) serum - deprived leukaemic cells release >10 kDa molecule/s being inductor/s of programmed cell death, acting through the regulation of Bax/Bcl-2 rheostat.
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