The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.

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Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (-0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.A small set of cellular genes, termed proto-oncogenes, appear to be frequent targets for genetic alterations leading to cancer. One family of such oncogenes, the ras gene family, has been directly implicated in the malignant process in both animals and humans. The normal ras gene product, a 21,000-molecular-weight protein, exhibits GTP and GDP binding as well as an associated GTPase activity (14,30,34). Mutations that commonly activate the transforming properties of ras genes involve point mutations around codon 12 or 61 (4,13,21,31,35,39). It has been shown that microinjection of activated ras p21 into quiescent fibroblasts results in transient morphological transformation and cell proliferation (6,11,33). Microinjection of the unaltered proto-oncogene p21 failed to produce a response. Thus, microinjection of activated p21 into quiescent cells provides an excellent system for examining the rapid biological effects of this transforming protein on normal cells (1).One of the earliest effects of a variety of growth factors on quiescent cells is a rapid influx of Na+ into the cell. Stimulation of Na+ influx results from activation of a membranebound Na+/H+ antiporter and leads to cytoplasmic alkalinization (16,20,25,32). There is recent evidence that increased intracellular pH (pHj) may be an important determinant for the initiation of DNA synthesis (19). In this study we have developed a novel system for monitoring pHi in single cells following the microinjection of ras p21 to assess the role of pHi changes in mediating the mitogenic effect of p21.Our assay of pHi utilized the pH-sensitive indicator 2',7'bis-(carboxyethyl)-carboxy fluorescein (BCECF) (22). Rodent fibroblasts were labeled by exposure to the permeant ester derivative BCECF-tetraacetoxymethyl ester at 10-5 M for 1 h, and fluorescence was monitored by microspectrofluorometry with a Farrand microscope spectrum analyzer. Emissions were collected from a single cell with a pinhole spotting device. The fluorescence signal was recorded at excitation and emission maxima of 490 and 550 nm, respectively. In these studies fluorescence was translated into pH with a calibration curve generated by the nigericin equilibration method (23, 36). Cells were incubated in buffers containing the K+/H+ ionophore nigericin at various pH values. It was found that BCECF-labeled fibroblasts exhibited a nearly linear relationship between fluorescence and pH over a pH range of 6.4 to 7.4 (Fig. 1A). To test the sensitivity of * Corresponding author. Following an NH4-induced acid load, pH, recovery was rapid and complete in different cells in 3 to 8 min (-70% of cells responded, n = 50) (Fig. 1B). Since phorbol esters have been ...

The major neoplastic transformation-inducing genes of human solid tumors are members of the ras oncogene family. We used an immunohistochemical assay to assess expression of both the unaltered and the mutated ras oncogene protein (p21) in normal and neoplastic prostatic cells. With the concentration of monoclonal antibody used in this study, epithelial and stromal cells from subjects with normal prostates and from 19 patients with benign prostatic hyperplasia were negative for p21 antigen. This antigen was detected in 2 of 6 prostates with Grade I carcinoma, 4 of 6 with Grade II, and all of 17 with higher grades. A semiquantitative immunohistochemical method demonstrated that expression of the p21 antigen in a carcinoma strongly correlated with nuclear anaplasia and was inversely related to the degree of glandular differentiation. However, markedly anaplastic tumors were often more heterogeneous in expression of p21 and contained areas of low staining for the antigen. Comparison of p21 antigen with tumor carcinoembryonic antigen and prostate-specific antigen demonstrated that ras p21 was the only phenotypic marker that correlated with histologic tumor grade. Thus, ras oncogene p21 may represent a new class of biologically relevant tumor markers and may be a useful adjunct to histopathologic examination in determining the prognosis of patients with prostate cancer.