Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma.

Expression of c-fms proto-oncogene product by ovarian cancer cell lines with effects of macrophage colony-stimulating factor on proliferation.

We studied the production of macrophage colony-stimulating factor (M-CSF) and the expression of c-fms mRNA, an M-CSF receptor, in four human ovarian cancer cell lines. All four cell lines expressed c-fms mRNA while three secreted M-CSF into the culture medium. The exogenous administration of M-CSF caused no significant enhancement of cellular proliferation in any cell line. Interestingly, the proliferation of KK cells was not affected by anti-M-CSF antibody. These results, taken together with the fact that ovarian cancer cells simultaneously produce M-CSF and c-fms, suggest that an autocrine mechanism may modulate cellular proliferation.

Tyrosine 807 of the v-Fms oncogene product controls cell morphology and association with p120RasGAP.

Expression of the v-fms oncogene of feline sarcoma virus in fibroblasts causes surface exposure of an activated receptor tyrosine kinase, v-Fms, that is autophosphorylated at multiple sites within its cytoplasmic domain. Cellular proteins interacting with this part of v-Fms modulate the mitogenic activity and morphology of the cells. We show here that the tyrosine residue in position 807 (Y-807) of the v-Fms molecule constitutes a major autophosphorylation site. The replacement of this residue by phenylalanine (Y807F mutation) allowed us to functionally dissect v-Fms-specific mitogenic and morphogenic cascades. Cells expressing the mutant v-Fms molecule resembled wild-type (wt) v-Fms-transformed (wt-v-Fms) cells in terms of [3H]thymidine uptake rates and activation of the Ras/Raf-1 mitogenic cascade. Such cells showed, however, a flat morphology and contained intact actin cables and fibronectin network. Our studies indicate that the v-Fms molecule controls cell morphology by a cascade that involves a direct interaction with p120RasGAP and p190RhoGAP: (i) in contrast to wt v-Fms molecules, the Y807F v-Fms protein failed to associate with and phosphorylate p120RasGAP; (ii) tight complexes between p120RasGAP and p190RhoGAP as well as detectable RhoGAP activity were present exclusively in wt-v-Fms cells; and (iii) p190RhoGAP was dispersed throughout the cytoplasm of wt-v-Fms cells, whereas its distribution was restricted to perinuclear regions of cells expressing the mutant v-Fms gene.

ras p21 expression in relation to DNA ploidy, S-phase fraction and prognosis in colorectal adenocarcinoma.

ras p21 expression, as indicated by the monoclonal antibody ras 11, was estimated using immunohistochemistry on 69 primary colorectal adenocarcinomas. Also, DNA ploidy and S-phase fraction (SPF) were analysed with flow cytometry. Positive staining for ras 11 tended to be more common in DNA non-diploid tumours (P = 0.11), but was significantly correlated with high SPF (P = 0.038). Positive ras 11 staining, Dukes' stage, DNA ploidy and SPF were related to the recurrence-free interval of patients with Dukes' A-C tumours (P = 0.0014, P = 0.023, P = 0.035 and P = 0.040, respectively). ras 11 staining was a prognostic factor independent of both Dukes' stage and DNA ploidy (P = 0.011). The results indicate that pan ras p21 expression is associated with proliferative activity and has an independent prognostic value in colorectal adenocarcinoma.