EXPRESSION OF BCL-2 AND BAX IN TGF-BETA-1-INDUCED APOPTOSIS OF L1210 LEUKEMIC-CELLS

TGF-beta 1 is a multifunctional regulatory peptide (25 kDa) inducing g rowth arrest and apoptosis in many normal and neoplastic cells, In the present study; the involvement of proapoptotic (bar) and antiapoptoti c (bcl-2) genes in the molecular mechanism of TGF-beta 1-induced apopt osis of L1210 leukemic cells was investigated. Bas transcript was meas ured using the RT-PCR method with GAPDH as a ''housekeeping gene'' con trol, whereas Bcl-2 protein was determined using nom cytometry (FITC-c onjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated mouse ant i-IgG(1) antibody as a negative control). Apoptosis was evaluated usin g fluorescence microscopy and flow cytometry after cell staining with DAPI and sulforhodamine or propidium iodide and Hoechst 33342. ROS gen eration was assessed by flow cytometry using the oxidation-sensitive f luorescent marker C-DCDHF-DA. The response of L1210 leukemic cells to TGF-beta 1 was two-directional: I) partial arrest of the cell cycle at G(1)-S transition, and 2) induction of apoptotic cell death, TGF-beta 1 increased the number of leukemic cells with typical morphological f eatures of apoptosis: cell shrinkage, condensation of chromatin, pykno sis and fragmentation of nuclei, followed by secondary necrosis, DIVA cleavage led to a decrease of the nuclear DNA content and the appearan ce of a hypo-diploid peak sub-G(1) in the DNA histogram, The extractio n of low-molecular weight DNA fragments from apoptotic cells showed th at TGF-beta 1-induced apoptosis concerned first of all the cells from G(1) phase. Two phases of intracellular ROS generation in TGF-beta 1-t reated cultures were observed: the first (rapid, 60 min after TGF-beta 1 administration), and the second (slow, occurring between 24 and 48 h of experiment, respectively). The increase of apoptotic cell number in TGF-beta 1-treated cultures (2% FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2, and with a significant d rop of Bcl-2 level in the remaining cells after Wh, The dose-dependent relationship between TGF-beta 1 concentration and Bcl-2 level was non linear and described by power series regression, The lowest Bcl-2 leve l was noted at 1 ng/ml of TGF-beta 1 concentration. The increase of Ba x mRNA:GAPDH mRNA ratio was observed 3 h after TGF-beta 1 (1 ng/mi) ad ministration to both the maintenance (2% FCS/RPMI) and growth promotin g (10 % FCS/RPMI) medium, Regardless of TGF-beta 1 treatment, the quan tity of Bar transcript was dependent on FCS concentration. being highe r in the growth promoting medium. The results of this study indicate t hat bax may function as a primary response gene and together with lowe red Bcl-2 level may determine the induction of apoptotic process in L1 210 leukemic cells exposed to TGF-beta 1.