REGULATION OF G(1) S TRANSITION AND INDUCTION OF APOPTOSIS IN HL-60 LEUKEMIA-CELLS BY FENRETINIDE (4HPR)/

We previously reported that all-trans retinoic acid (RA) and fenretini de (4HPR) suppress HL-60 leukemia cell growth and cause partial cell a rrest in the G(1)-to-S phase. Moreover, 4HPR but not RA induces apopto sis in HL-60 cells. To investigate further the observed biological eff ects, cyclin D1 and cdk4 expression and the level of phosphorylation o f the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expr ession and Rb phosphorylation were significantly reduced, by 40-75%, a fter 24 hr of treatment with RA or 4HPR; these decreases were either t ransient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In gen eral, more pronounced decreases were seen in the 4HPR-treated cells. E vidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzy me 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 mu M of the CPP32 inhibitor DEVD, but not the ICE inhibitor WAD, significantly reduced t he specific processing of PARP, suggesting that CPP32 is involved in t he mechanism of action of 4HPR. Analysis of 2 lipid-derived second mes sengers, ceramide and diacylglycerol (DAG), as a function of time of t reatment with RA or 4HPR, showed ceramide but not DAG to be significan tly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test furt her whether ceramide may be involved in the signaling cascade that cul minates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B-1, an inhibitor of ceramide synthase, were stu died. Simultaneous treatment of cells with 4HPR and 25-100 mu M fumoni sin BI resulted in a dose-dependent reduction in the elevation in cera mide, the extent of PARP cleavage, and induction of apoptosis. Pretrea tment with DEVD or YVAD, on the other hand, had no effect on the 4HPR- induced increase in ceramide. Int. J. Cancer 78:53-61, 1998. (C) 1998 Wiley-Liss, Inc.