We examined the inhibitory effect of two saponin preparations from Red ginseng, 20(R)- and 20(S)-ginsenoside-Rg3, in comparison with that of ginsenoside-Rb2, on lung metastasis produced by two highly metastatic tumor cells, B16-BL6 melanoma and colon 26-M3.1 carcinoma, in syngeneic mice. In an in vitro analysis, both saponin preparations showed a significant inhibition of adhesion to fibronectin (FN) and laminin (LM) by B16-BL6 melanoma. Similarly, they significantly inhibited the invasion of B16-BL6 cells into the reconstituted basement membrane (Matrigel)/FN in a dose-dependent manner. In an experimental metastasis model using B16-BL6 melanoma, consecutive intravenous (i.v.) administrations of 100 micrograms/mouse of 20(R)- or 20(S)-ginsenoside-Rg3 1, 2, 3 and 4 d after tumor inoculation led to a significant decrease in lung metastasis. The inhibitory effect of i.v. administration of both ginseng saponins on the tumor metastasis of B16-BL6 melanoma was also recognized in a low dose of 10 micrograms/mouse. The oral administration (p.o.) of both saponins (100-1000 micrograms/mouse) induced a significant decrease in lung metastasis of B16-BL6 melanoma. Moreover, both ginseng saponins were effective in inhibiting of lung metastasis produced by colon 26-M3.1 carcinoma. When 20(R)- or 20(S)-ginsenoside-Rg3 was orally administered consecutively after tumor inoculation in a spontaneous metastasis model using B16-BL6 melanoma, both of them significantly inhibited lung metastasis. In the experiment involving neovasculization by tumor cells in vivo, both mice groups given each saponin preparation after tumor inoculation exhibited a significant decrease in the number of blood vessels oriented toward the tumor mass, with no repression of tumor size. These findings suggest that both ginseng saponins, 20(R)- and 20(S)-ginsenoside-Rg3, possess an ability to inhibit the lung metastasis of tumor cells, and the mechanism of their antimetastatic effect is related to inhibition of the adhesion and invasion of tumor cells, and also to anti-angiogenesis activity.

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G 1 , S, and G 2 /M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G 2 /M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G 2 /M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G 2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.The MCM protein family, presently with six members, was originally identified from its involvement in the initiation of DNA replication at autonomously replicating sequences in budding yeast (1-7) and later found to be conserved through eukaryotes (8 -16). Although definite functions of the MCM proteins remain largely unknown, they have been implicated in the regulatory machinery allowing DNA to replicate only once during the S phase (reviewed in Refs. 17 and 18).In mammalian cells, MCM proteins are present in the nuclei in two different forms, one extractable by nonionic detergents and the other tightly associated with chromatin, which is resistant to such extraction. The level of mammalian MCM does not greatly vary during the cell cycle, but the chromatin-bound form gradually becomes dissociated with progression through the S phase (19 -23). It is now assumed that the bound form is associated with prereplicative chromatin and released at the time of replication; the soluble form existing abundantly in G 2 nuclei is considered inactive and no longer capable of binding to chromatin. At least in budding yeast, the chromatin regions to which MCM7 binds during the G 1 phase contain the replication origins (24). However, details of the mode of MCM-chromatin binding remain unclear. In budding yeast and the Xenopus egg extract system, it has been shown that MCM-chromatin binding is regulated through multiple mechanisms, while MCM binds to chromatin depending on CDC6 and the origin recognition complex (24 -27), where the binding is negatively regulated by both S phase and mitotic CDKs (24, 28 -30). However, there is so far no direct evidence as to wheth...

The small GTPase Rho and one of its targets, Rhoassociated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can speci®cally recognize distinct phosphorylated sites on glial ®brillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate ®lament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of antiRho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/ moesin (ERM) proteins and Ser 19 -phosphorylated myosin light chain. These results raise the possibility that Rhokinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

No abstract