DiPietrantonio A M, Hsieh T C, Olson S C, Wu J M
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595, USA.
Int J Cancer. 1998 Sep 25;78(1):53-61. doi: 10.1002/(sici)1097-0215(19980925)78:1<53::aid-ijc10>3.0.co;2-6.
We previously reported that all-trans retinoic acid (RA) and fenretinide (4HPR) suppress HL-60 leukemia cell growth and cause partial cell arrest in the G1-to-S phase. Moreover, 4HPR but not RA induces apoptosis in HL-60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40-75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-ribose) polymerase (PARP) to an 89-kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3'-ends of DNA. Overnight pretreatment with 0.5-5.0 microM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid-derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B1, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25-100 microM fumonisin B1 resulted in a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR-induced increase in ceramide.
我们之前报道过，全反式维甲酸（RA）和芬维A胺（4HPR）可抑制HL-60白血病细胞的生长，并使细胞在G1期至S期出现部分阻滞。此外，4HPR而非RA可诱导HL-60细胞凋亡。为了进一步研究观察到的生物学效应，我们评估了细胞周期蛋白D1和细胞周期蛋白依赖性激酶4（cdk4）的表达以及视网膜母细胞瘤蛋白Rb的磷酸化水平。用RA或4HPR处理24小时后，细胞周期蛋白D1和cdk4的表达以及Rb的磷酸化显著降低了40%-75%；这些降低要么是短暂的，例如cdk4仅在24小时时降低，要么持续72小时。一般来说，在4HPR处理的细胞中观察到更明显的降低。4HPR诱导凋亡的证据来自：（1）聚（ADP-核糖）聚合酶（PARP）裂解为89 kDa的截短产物；（2）琼脂糖凝胶电泳上出现DNA梯带；（3）地高辛标记核苷酸原位掺入DNA游离3'-末端的水平更高。用0.5-5.0 microM的CPP32抑制剂DEVD进行过夜预处理，而非ICE抑制剂YVAD，可显著减少PARP的特异性加工，这表明CPP32参与了4HPR的作用机制。分析两种脂质衍生的第二信使神经酰胺和二酰基甘油（DAG）在RA或4HPR处理后的时间变化，结果显示4HPR在处理3小时时可使神经酰胺显著（尽管是短暂的）增加2倍，而DAG无此变化。为了进一步测试神经酰胺是否可能参与导致4HPR处理的HL-60细胞凋亡诱导的信号级联反应，我们研究了神经酰胺合酶抑制剂伏马菌素B1的作用。用4HPR和25-100 microM伏马菌素B1同时处理细胞，可导致神经酰胺升高、PARP裂解程度和凋亡诱导呈剂量依赖性降低。另一方面，用DEVD或YVAD进行预处理对4HPR诱导的神经酰胺增加没有影响。
