Meinhardt G, Roth J, Hass R
Medizinische Klinik Innenstadt, Department of Hematology/Oncology, Ludwig-Maximilians-University, Ziemssenstrasse 1, 80336 Munich, Germany.
Cell Death Differ. 2000 Sep;7(9):795-803. doi: 10.1038/sj.cdd.4400709.
Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.
在人U937髓系白血病细胞中，用5 nM 12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）激活蛋白激酶C（PKC）72小时，与诱导黏附相关，随后是单核细胞分化和G0/G1细胞周期停滞。在本研究中，我们证明，除了这些效应外，约25%的U937细胞在TPA处理后积聚在凋亡的亚G1期。在整个培养时间过程中均可检测到这些凋亡悬浮细胞的出现，且与0.5至500 nM之间的TPA浓度无关。用离心淘析同步化的细胞进行的实验表明，G1期细胞对TPA介导的凋亡具有主要易感性。虽然贴壁细胞表达包括整合素CD11c在内的分化标志物，但在TPA处理的悬浮部分中这种效应不太明显。此外，先前的研究表明分化的U937细胞存在细胞周期停滞。因此，TPA处理激活PKC与贴壁群体中细胞周期蛋白依赖性激酶/细胞周期蛋白抑制剂p21WAF/CIP/sdi - 1的显著表达以及随后的G0/G1细胞周期停滞相关。相反，悬浮细胞在TPA刺激后未能诱导出显著水平的p21WAF/CIP/sdi - 1。免疫印迹实验表明，促凋亡因子Bax、Bad和Bak在对照U937细胞以及TPA处理的贴壁或悬浮细胞中的表达均无差异。然而，抗凋亡因子包括Bcl - 2、Bcl - xL和Mcl - 1在贴壁群体中显著诱导，而在悬浮细胞中未检测到诱导。在此背景下，在TPA处理前用半胱天冬酶 - 3/半胱天冬酶 - 7特异性四肽抑制剂DEVD孵育可分别防止细胞在亚G1期积聚，表明这些半胱天冬酶参与其中。综上所述，这些数据表明PKC激活可以传递不同的信号通路，如诱导黏附并伴随单核细胞分化和生长停滞，或诱导半胱天冬酶介导的凋亡并伴随无法黏附和分化。
