The MGSA/GRO protein is endogenously expressed in almost 70% of the melanoma cell lines and tumors, but not in normal melanocytes. We have previously demonstrated that over-expression of human MGSA/GROa, b or g in immortalized murine melanocytes (melan-a cells) enables these cells to form tumors in SCID and nude mice. To examine the possibility that the MGSA/GRO eect on melanocyte transformation requires expression of other genes, dierential display was performed. One of the mRNA's identi®ed in the screen as overexpressed in MGSA/GRO transformed melan-a clones was the newly described M-Ras or R-Ras3 gene, a member of the Ras gene superfamily. Over-expression of MGSA/GRO upregulates M-Ras expression at both the mRNA and protein levels, and this induction requires an intact glutamine-leucine-arginine (ELR)-motif in the MGSA/ GRO protein. Western blot examination of Ras expression revealed that K-and N-Ras proteins are also elevated in MGSA/GRO-expressing melan-a clones, leading to an overall increase in the amount of activated Ras. MGSA/GRO-expressing melan-a clones exhibited enhanced AP-1 activity. The eects of MGSA/GRO on AP-1 activation could be mimicked by over-expression of wild-type M-Ras or a constitutively activated M-Ras mutant in control melan-a cells as monitored by an AP-1-luciferase reporter, while expression of a dominant negative M-Ras blocked AP-1-luciferase activity in MGSA/GRO-transformed melan-a clones. In the in vitro transformation assay, over-expression of M-Ras mimicked the eects of MGSA/GRO by inducing cellular transformation in control melan-a cells, while over-expression of dominant negative M-Ras in MGSA/ GROa-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes.

Excessive accumulation of phospholipids results in phospholipidosis (PL), which may interfere with cellular functions, leading to acute or chronic disease or even death. Electron-microscopic detection of cytoplasmic lamellar bodies is often used as a diagnostic criterion of PL, but a faster, more convenient procedure is required for high-throughput assay of the PL-inducing potential of candidate drugs. We have developed a 96-well microplate cell-culture method for detecting PL, using a phosphatidylcholine-conjugated dye (NBD-PC) and a fluoro-microplate reader. The fluorescence intensity due to NBD-PC was normalized to that of Hoechst33342, used as an indicator of cell number, to obtain the amount of NBD-PC taken up per living cell. To select a suitable cell type, we examined the PL-detection sensitivity of five cell lines, as well as human and rat primary hepatocyte cultures, with five cationic amphiphilic drugs (CAD) as PL inducers and a negative control compound. The cell lines CHO-K1 and CHL/IU gave the best results. The NBD-PC uptake per CHO-K1 cell showed a high correlation with the pathological score of PL for 24 compounds, including PL-positive and negative compounds. This high-throughput screening assay for PL-inducing potential (HTS-PL assay) offers high sensitivity and accuracy, and it allows simultaneous determination of cytotoxicity.

Rho family small GTPases regulate organization of the actin cytoskeleton. Among them, RhoA plays essential roles in the formation of the actin stress ®bers, the associated focal adhesions, and the contractile rings necessary for cytokinesis. Recently, RhoD, a novel member of Rho family has been identi®ed. The amino acid sequences of its eector domain is distinct from those of the other Rho family proteins, suggesting its unique cellular functions. Introduction of the constitutively active form of RhoD G26V into ®broblasts by microinjection or transfection resulted in disassembly of the actin stress ®bers and the focal adhesions, whereas the dominant negative form of RhoD T31K did not aect these structures. The degree of cell migration assessed by the phagokinetic tracks on a substrate covered with gold particles was diminished by the expression of RhoD . Thus, cytoskeletal alterations including the loss of stress ®bers and focal adhesions by RhoD seems to lead to the retardation of cell migration. Transfection of RhoD G26V cDNA into cultured cells also induced multinucleation. Moreover, RhoD G26V microinjected into fertilized eggs and embryos of Xenopus laevis caused cleavage arrest only in the injected cells, and the uncleaved cells contained multiple nuclei. These results imply that RhoD does not aect nuclear division but can interfere with cytokinesis presumably by preventing the formation of the actin-based contractile ring. Enhancement of the stress ®bers by RhoA or RhoA-activating lysophosphatidic acid was reversed by the transfection of RhoD cDNA. Accordingly, the cellular functions of RhoD are likely to be antagonistic to those of RhoA.