Motyl T, Grzelkowska K, Zimowska W, Skierski J, Wareski P, Płoszaj T, Trzeciak L
Department of Animal Physiology, Faculty of Veterinary Medicine, Warsaw Agricultural University, Poland.
Eur J Cell Biol. 1998 Apr;75(4):367-74. doi: 10.1016/s0171-9335(98)80070-8.
TGF-beta1 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 antiapoptotic (bcl-2) genes in the molecular mechanism of TGF-beta1-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-IgG1 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-beta1 was two-directional: 1) partial arrest of the cell cycle at G1-S transition, and 2) induction of apoptotic cell death. TGF-beta1 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-G1 in the DNA histogram. The extraction of low-molecular weight DNA fragments from apoptotic cells showed that TGF-beta1-induced apoptosis concerned first of all the cells from G1 phase. Two phases of intracellular ROS generation in TGF-beta1-treated cultures were observed: the first (rapid, 60 min after TGF-beta1 administration), and the second (slow, occurring between 24 and 48h of experiment, respectively). The increase of apoptotic cell number in TGF-beta1-treated cultures (2% FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2, and with a significant drop of Bcl-2 level in the remaining cells after 24 h. The dose-dependent relationship between TGF-beta1 concentration and Bcl-2 level was nonlinear and described by power series regression. The lowest Bcl-2 level was noted at 1 ng/ml of TGF-beta1 concentration. The increase of Bax mRNA:GAPDH mRNA ratio was observed 3h after TGF-beta1 (1 ng/ml) administration to both the maintenance (2% FCS/RPMI) and growth promoting (10% FCS/RPMI) medium. Regardless of TGF-beta1 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 L1210 leukemic cells exposed to TGF-beta1.
转化生长因子β1（TGF-β1）是一种多功能调节肽（25 kDa），可诱导许多正常细胞和肿瘤细胞生长停滞并凋亡。在本研究中，研究了促凋亡基因（bax）和抗凋亡基因（bcl-2）在TGF-β1诱导L1210白血病细胞凋亡分子机制中的作用。使用以甘油醛-3-磷酸脱氢酶（GAPDH）作为“管家基因”对照的逆转录聚合酶链反应（RT-PCR）方法测量Bax转录本，而使用流式细胞术（异硫氰酸荧光素（FITC）偶联的抗Bcl-2单克隆抗体和FITC偶联的小鼠抗IgG1抗体作为阴性对照）测定Bcl-2蛋白。在用4',6-二脒基-2-苯基吲哚（DAPI）和磺基罗丹明或碘化丙啶以及Hoechst 33342对细胞染色后，使用荧光显微镜和流式细胞术评估细胞凋亡。使用氧化敏感荧光标记C-二氯二氢荧光素二乙酸酯（C-DCDHF-DA）通过流式细胞术评估活性氧（ROS）的产生。L1210白血病细胞对TGF-β1的反应是双向的：1）细胞周期在G1期向S期转变时部分停滞，以及2）诱导凋亡性细胞死亡。TGF-β1增加了具有典型凋亡形态特征的白血病细胞数量：细胞皱缩、染色质凝聚、核固缩和核碎裂，随后发生继发性坏死。DNA裂解导致核DNA含量降低，并在DNA直方图中出现亚G1期的亚二倍体峰。从凋亡细胞中提取低分子量DNA片段表明，TGF-β1诱导的凋亡首先涉及G1期的细胞。在TGF-β1处理的培养物中观察到细胞内ROS产生的两个阶段：第一个阶段（快速，在给予TGF-β1后60分钟），以及第二个阶段（缓慢，分别发生在实验的24至48小时之间）。在TGF-β1处理的培养物（2%胎牛血清（FCS）/ RPMI 1640）中凋亡细胞数量的增加与表达bcl-2的细胞数量减少相关，并且在24小时后剩余细胞中的Bcl-2水平显著下降。TGF-β1浓度与Bcl-2水平之间的剂量依赖关系是非线性的，并通过幂级数回归描述。在TGF-β1浓度为1 ng/ml时观察到最低的Bcl-2水平。在向维持培养基（2% FCS/RPMI）和促生长培养基（10% FCS/RPMI）中给予TGF-β1（1 ng/ml）3小时后，观察到Bax mRNA:GAPDH mRNA比值增加。无论是否进行TGF-β1处理，Bax转录本的量均取决于FCS浓度，在促生长培养基中更高。本研究结果表明，bax可能作为初级反应基因发挥作用，并且与降低的Bcl-2水平一起可能决定暴露于TGF-β1的L1210白血病细胞中凋亡过程的诱导。
