4--demethylamino-4--hydroxystaurosporine and 7-oxostaurosporine

We have established a bioassay system using a mouse cdc2 mutant cell line, tsFT210, to detect inhibitors of the mammalian cell cycle. When cultured at the high temperature, restrictive temperature at 39.4 degrees C, tsFT210 cells can be arrested at G2 phase and are large in size. Four hours after release from G2 arrest, the cells entered into the G1 phase. At this time, G1 phase cells were easily discriminated from the G2/M-cells by their size under microscopic observation. The cell-morphology-based bioassay utilizing tsFT210 cells is very simple and sensitive for detecting cdc2 kinase inhibitors and also G2/M-phase inhibitors of the mammalian cell cycle. To demonstrate the merits of this bioassay, the effects of protein kinase inhibitors isolated from actinomycetes were investigated. RK-286C and RK-1409, which are structurally related to staurosporine, inhibited cell cycle progression at the G2 phase in both G2-synchronized and nonsynchronized cultures of tsFT210 cells. Another kinase inhibitor, sangivamycin, inhibited cell cycle progression at the G2 phase of cells released from temperature arrest but did not inhibit that of the exponentially growing cells. Using the bioassay system, we carried out screening of the cell cycle inhibitors from the microbial metabolites and have discovered several new inhibitors, including novel compounds such as tryprostatins A, B and acetophthalidin. Thus, this bioassay allowed for the detection of cell cycle inhibitors and provided a convenient and useful method for the screening of new inhibitors from the microbial metabolites.

Topics: Actinomycetales; Alkaloids; Animals; Biological Assay; CDC2 Protein Kinase; Cell Cycle; Mice; Staurosporine; Tumor Cells, Cultured

A novel inhibitor of protein kinase C was found in the fermentation of a soil actinomycete, strain RK-1409. According to the taxonomic studies, the producing strain was designated as Streptomyces platensis subsp. malvinus RK-1409. The protein kinase C inhibitor, RK-1409 (7-oxostaurosporine) inhibited the morphological change of a human chronic erythroleukemia cell, K-562, induced by phorbol 12,13-dibutyrate (PDBu) at the concentration of 10 ng/ml. The concentration of 3 ng/ml inhibited the activity of protein kinase C in vitro. RK-1409 inhibited the cell cycle progression at G2 phase of K-562 cells.

Topics: Alkaloids; Carbazoles; G2 Phase; Indole Alkaloids; Protein Kinase C; Staurosporine; Streptomyces