title: The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C.
abstract: A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Box G-J1, Brown University, Providence, RI 02912, USA. zhiyong_han@brown.edu
authors.forename: Z
authors.initials: Z
authors.lastname: Han
authors.forename: P
authors.initials: P
authors.lastname: Pantazis
authors.forename: T S
authors.initials: TS
authors.lastname: Lange
authors.forename: J H
authors.initials: JH
authors.lastname: Wyche
authors.forename: E A
authors.initials: EA
authors.lastname: Hendrickson
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D008301:Maleimides / Q000737:chemistry; D015394:Molecular Structure; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D019311:Staurosporine / Q000031:analogs & derivatives; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D019253:Proto-Oncogene Proteins c-bcl-2; D011493:Protein Kinase C; D019311:Staurosporine; C088060:bisindolylmaleimide; C064758:Ro 31-8220