Yoshifumi Ogiso

Suppression of Erk activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y.

Our previous studies demonstrated that introduction of a dominant negative H‐ras mutant, N116Y, inhibits the growth of various types of cancer cells in vitro. In this study, we tested the efficacy of N116Y in blocking the growth of esophageal cancer cells using an adenoviral vector. Infection with N116Y adenovirus, (AdCMV‐N116Y), in which N116Y expression is driven by the cytomegalovirus promoter, significantly reduced the in vitro growth of all esophageal cancer cell lines studied. Esophageal cancer cells that contained wild‐type K‐ras and H‐ras (TE8, SGF3, SGF7) were more sensitive to AdCMV‐N116Y than HEC46 cells that expressed mutant K‐ras protein. Most importantly, direct injection of AdCMV‐N116Y into TE8‐ or SGF3‐induced tumors in nude mice suppressed their growth significantly. To examine the suppressive mechanism of N116Y, cell cycle profile and the activation of extracellular signal‐regulated kinase 2 (Erk2) were examined by flow cytometry and Western blot analysis, respectively. In TE8 cells, progression into S phase was clearly blocked after infection with AdCMV‐N116Y. Infection with AdCMV‐N116Y did not strongly suppress the activation of Erk2 after EGF stimulation in serum‐starved HEC46 cells, whereas it completely suppressed activation in TE8, SGF3 and SGF7 cells. Our observations suggest that N116Y reduces growth of human esophageal cancer cells and suppresses the activation of Erk2; they also indicate that N116Y is a potential candidate gene for human esophageal cancer gene therapy. Int. J. Cancer 78:366–371, 1998.© 1998 Wiley‐Liss, Inc.

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

The significance of Ras guanine nucleotide exchange factor, son of sevenless protein, in renal cell carcinoma cell lines.

PURPOSEnThe aim of the present study is to clarify the significance of the Ras guanine-nucleotide exchange reaction in the proliferation of human renal cell carcinoma cell lines.nnnMATERIALS AND METHODSnWe examined the expression of human son of sevenless-1 (hSos-1) protein and the epidermal growth factor (EGF) receptor in human renal cell carcinoma cell lines by Western blot analysis. Additionally, a dominant negative H-ras mutant, N116Y, which is known to inhibit the Ras guanine-nucleotide exchange reaction, was transfected into these cell lines by lipofection.nnnRESULTSnHuman renal cell carcinoma cell lines expressed much higher amounts of the EGF receptor and hSos-1 protein than normal kidney tissue. Moreover, the N116Y ras mutant could strongly suppress cellular proliferation in these cell lines.nnnCONCLUSIONSnAugmentation of the Ras guanine-nucleotide exchange reaction might be essential to the proliferation of human renal cell carcinoma cells.

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.

Enhanced expression of ras gene products in psoriatic epidermis

SummaryThe ras oncogene product ras p21 is structurally homologous to guanine nucleotide-binding proteins and plays an important role in transducing signals elicited by membrane receptors into intracellular metabolism. We examined psoriatic tissues for expression of ras p21 and compared them with normal skin, using the indirect immunofluorescence technique with the anti-ras p21 monoclonal antibody (MoAb), rp-35. In normal epidermis of five healthy individuals and uninvolved epidermis of three psoriatic patients, only the basal layer was positively stained by rp-35. The spinous layer was negative or faintly positive. In contrast, all psoriatic epidermis obtained from 13 psoriatic patients had strong reactivity with rp-35 throughout the epidermis. There were no differences in the staining pattern of hair follicles, sebaceous glands, eccrine glands, and eccrine ducts, which positively reacted with rp-35, between psoriatic and normal skin. The functions of ras p21 have not been clearly identified in mammalian cells; however recent reports reveal that cyclic AMP production is inhibited by the transfection of activated ras gene into normal cells. Enhanced expression of ras p21 in psoriatic epidermis may be indicative of some mechanism of defective β-adrenergic responsiveness, which is considered to be one of the important pathophysiological phenomena causing the hyperproliferative condition in psoriasis.

INVOLVEMENT OF RAS IN THE EXPRESSION OF GLYCOLIPID SULFOTRANSFERASE IN HUMAN RENAL CANCER CELLS

Glycolipid sulfotransferase activity in a human renal cell carcinoma cell line, SMKT‐R3, is enhanced by epidermal growth factor (EGF); tyrosine kinase inhibitors suppress this enhancement. To investigate the involvement of Ras in the signal transduction pathway from the EGF receptor to the expression of glycolipid sulfotransferase, we introduced v‐H‐ras into SMKT‐R3 cells. In a quiescent state, the percent GTP bound to Ras in v‐H‐ras‐expressing cells increased about 2.5‐fold compared with control cells, suggesting that v‐Ras introduced into the renal cancer cells is in an active form without EGF stimulation. Glycolipid sulfotransferase activity in v‐H‐ras‐expressing cells was higher than in control cells. The sulfotransferase activity was affected neither by EGF nor by genistein, a tyrosine kinase inhibitor, in v‐H‐ras‐expressing cells, whereas it was enhanced by EGF and reduced by genistein in control cells. Our observations suggest that Ras mediates the regulation pathway of glycolipid sulfotransferase activity in SMKT‐R3 cells. Int. J. Cancer 71:620‐623, 1997.

Dome formation induced by v-H-ras oncogene in a human choriocarcinoma cell line

To investigate the role of ras genes in trophoblastic cell lineage, we transfected the viral H- or K-ras oncogene into a human choriocarcinoma cell line, CCI, and analysed the biological properties of CCI cells expressing an activated ras oncogene. All v-H-ras-expressing clones distinctively formed the hemispherical domes, which represents an in vitro morphological expression of vectorial transport function and are characteristic of the polarized epithelial cells, but none of v-K-ras-expressing clones and control clones did. Microscopic observation demonstrated that those domes were cavities filled with fluid which accumulated between the cell layer and the surface of culture dish. Scanning electron microscopy revealed that the domes were aggregates of round cells with long numerous microvilli and were morphologically similar to a blastocyst. Furthermore, Na(+)-K(+)-ATPase activity, which is associated with the vectorial fluid transport in transporting epithelial cells, was significantly higher in the v-H-ras-expressing clones than that in the v-K-ras-expressing clones and the parental cells. Those domes flattened within 24 h after treatment with a specific inhibitor of Na(+)-K(+)-ATPase, ouabain, and the number of domes decreased in dose-dependent manner, indicating that Na(+)-K(+)-ATPase activity was required for maintainance of domes. These results suggest that up-regulated activity of H-ras but not of K-ras facilitates the vectorial fluid transport through a chorionic cell layer and leads to the dome formation. The function of II-ras in trophoblasts, may therefore, be essential for embryogenesis, especially for supplying the nutrients.

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.

Biological activity of a K-ras mutant that contains the 12R/59T/116Y mutations

The 12R/59T/116Y mutations have been shown to confer a dominant negative activity on H-ras oncogene (H-ras 116Y). To determine whether this event is unique for H-ras, we introduced the same mutations into K-ras oncogene. This mutant, K-ras 116Y, suppressed transformed phenotypes induced by overexpression of H-ras proto-oncogene. NIH3T3 cells expressing K-ras 116Y were resistant to transformation by v-fes oncogene. Analysis of chimaeras between H- and K-ras 116Y showed that the C-terminal variable region determines the level of suppressor activity. These results suggest that these mutations are applicable to other GDP/GTP binding proteins.

Reduced DNase I sensitivity of the rearranged c-myc gene in somatic cell hybrids between murine plasmacytoma cells and fibroblasts☆

In mouse plasmacytoma (MPC) S194, the rearranged c-myc gene was much more sensitive to DNase I digestion than the nonrearranged gene. The sensitivity of the rearranged c-myc was markedly reduced to the same extent as that of the nonrearranged one in hybrids between the MPC cells and the fibroblasts, but not in a hybrid between the MPC and the spleen cells. These results suggest that trans-acting factors in fibroblasts alter the DNase I-sensitive structure of the rearranged c-myc gene.