Expression of bcl-2 and bax in TGF-β1-induced apoptosis of L1210 leukemic cells

TGF-β1 is a multifunctional regulatory peptide (25 kDa) inducing growth arrest and apoptosis in many normal and neoPlastic cells. In the present study, the involvement of proapoptotic ( bax ) and antiapototic ( bcl-2 ) genes in the molecular mechanism of TGF-β1-induced apoptosis of L1210 leukemic cells was investigated. Bax transcript was measured using the RT-PCR method with GAPDH as a “housekeeping gene” control, whereas bcl-2 protein was determined using flow cytometry (FITC-conjugated monoclonal anti- bcl-2 antibody and FITC-conjugated mouse anti-IgG 1 antibody as a negative control). Apoptosis was evaluated using fluorescence microscopy and flow cytometry after cell staining with DAPI and sulforhodamine or propidium iodide and Hoechst 33342. ROS generation was assessed by flow cytometry using the oxidation-sensitive fluorescent marker C-DCDHF-DA. The response of L1210 leukemic cells to TGF-β1 was two-directional: 1) Partial arrest of the cell cycle at G 1 -S transition, and 2) induction of apoptotic cell death. TGF-β1 increased the number of leukemic cells with typical morphological features of apoptosis: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis. DNA cleavage led to a decrease of the nuclear DNA content and the appearance of a hypodiploid peak sub-G 1 in the DNA histogram. The extraction of lowmolecular weight DNA fragments from apoptotic cells showed that TGF-βl-induced apoptosis concerned first of all the cells from G t phase. Twophases of intracellular ROS generation in TGF-βl-treated cultures were observed: the first (rapid, 60 min after TGF-β1 administration), and the second (slow, occurring between 24 and 48h of experiment, respectively). The increase of apoptotic cell number in TGF-β1 treated cultures (2 % FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2 , and with a significant drop of BcI-2 level in the remaining cells after 24h. The dose-dependent relationship between TGF-βl concentration and bcl-2 level was nonlinear and described by power series regression. The lowest BcI-2 level was noted at lng/ml of TGF-βl concentration. The increase of Bax mRNA:GAPDH mRNA ratio was observed 3h after TGF-βl (1 ng/ ml) administration to both the maintenance (2 % FCS/RPMI) and growth promoting (10% FCS/RPMI) medium. Regardless of TGF-β1 treatment, the quantity of Bax transcript was dependent on FCS concentration, being higher in the growth promoting medium. The results of this study indicate that bax may function as a primary response gene and together with lowered bcl-2 level may determine the induction of apoptotic process in LU10 leukemic cells exposed to TGF-βl.