Motyl T, Kasterka M, Grzelkowska K, Ostrowski J, Filipecki M, Malicka E, Pioszaj T
Department of Animal Physiology, Faculty of Veterinary Medicine, Warsaw Agricultural University, Poland.
Int J Biochem Cell Biol. 1996 Dec;28(12):1327-35. doi: 10.1016/s1357-2725(96)00083-0.
Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-beta 1 is associated with the inhibition of ornithine decarboxylase (ODC)--the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA)--a potent ODC inducer on antiproliferative and apoptotic effects of TGF-beta 1 in L1210 leukemic cells. Cells were incubated in 2% FCS/RPMI-1640 medium, supplemented with TGF-beta 1 (2 ng/ml). TPA (100 ng/ml) or alpha-difluoromethylornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-3H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determined by RT-PCR and measurement of 14CO2 release from L-1-14C ornithine, respectively. TGF-beta 1 inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-beta 1 administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-beta 1-treated cultures. Administration of TPA simultaneously with TGF-beta 1 significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-beta 1, and prevented its inhibitory action of ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-beta 1-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-beta 1-related growth inhibition of L1210 leukemic cells.
先前的研究表明，暴露于转化生长因子β1（TGF-β1）的白血病细胞的生长抑制和凋亡与鸟氨酸脱羧酶（ODC）（多胺途径的关键酶）的抑制有关。本研究的目的是评估12-O-十四酰佛波醇-13-乙酸酯（TPA）（一种有效的ODC诱导剂）对TGF-β1在L1210白血病细胞中的抗增殖和凋亡作用的影响。细胞在补充有TGF-β1（2 ng/ml）的2%胎牛血清/ RPMI-1640培养基中培养。TPA（100 ng/ml）或α-二氟甲基鸟氨酸（DFMO）（5 mM）。使用[甲基-3H]胸苷、电子显微镜、DNA电泳和台盼蓝排斥法评估细胞增殖、凋亡和坏死。ODC的表达和活性分别通过RT-PCR和测量L-1-14C鸟氨酸释放的14CO2来确定。TGF-β1抑制L1210白血病细胞的增殖并诱导凋亡和坏死性细胞死亡。上述作用分别与在给予TGF-β1后2小时和4小时测量的ODC表达和活性的抑制有关。ODC的不可逆抑制剂DFMO的存在导致DNA凋亡片段化，类似于在TGF-β1处理的培养物中观察到的情况。与TGF-β1同时给予TPA显著降低了TGF-β1的抗增殖、凋亡和坏死作用，并阻止了其对ODC表达和活性的抑制作用。得出以下结论：ODC表达的下调可能是与TGF-β1引起的生长抑制和凋亡相关的早期事件之一；ODC参与TPA对TGF-β1相关的L1210白血病细胞生长抑制的保护作用机制。
