No abstract

The effects of the src gene on the activity of protein kinase C and intercellular communication have been studied in transformed NIH/3T3 clones isolated from soft agar following transfection with the plasmid carrying the v-src gene (psrc-l1). Six transformed clones that were studied contained newly incorporated v-src genes in the genome, had an increased amount of pp6src, and showed enhanced activities of protein kinase C. Intercellular communication, studied by observing with autoradiography the transfer of [3H]uridine nucleotide from prelabeled donor cells to recipient cells in contact, was found to be reduced in transformed clones as compared to parental NIH/3T3 cells. Treatment with phorbol 12-myristate 13-acetate was also found to increase protein kinase C activity and to reduce intercellular communication in normal NIH/3T3 cells. These results suggest that the v-src gene product, in a manner similar to some of the powerful tumor promoters, may directly or indirectly affect cell-cell communication.The identification of the role of many genes involved in the complex multistep carcinogenic process has begun with the development of techniques to detect oncogenes in vitro (1). The recent demonstrations that some oncogenes code for a growth factor (2-4), growth factor receptor (5), and several tyrosine-directed protein kinases (6) suggest that many of these oncogenes have growth-promoting effects. More recently, the discovery that some oncogene products can phosphorylate phosphatidylinositol (7,8) MATERIALS AND METHODSIsolation of src Gene-Transformed NIH/3T3 Clones. NIH/3T3 cells, used as DNA recipients, were grown in Dulbecco's modified minimal essential medium supplemented with 10% fetal calf serum at 37°C in water-jacketed incubators that provide 5% CO2 in air and humidity. The donor DNA carrying the v-src gene is a plasmid (psrc"l) provided to us by G. Cooper (Harvard Medical School). This plasmid contains an intact Rous sarcoma virus v-src gene with two long terminal repeat promoters positioned both upstream and downstream from the gene, in such a way that the v-src messages can be effectively expressed (see Fig. 10. The transfection experiments followed the procedure described by Wigler et al. (24). The total DNA per plate was 20 ,ug which includes plasmid DNA (0-10 ,ug per plate) and salmon DNA (10-20 ,tg per plate). After 5 hr of incubation with calcium phosphate/DNA precipitate, the medium was removed and the attached cells were treated with 20% glycerol in phosphate-buffered saline (P1/NaCl) for 1 min.After rinsing with Pi/NaCl, fresh medium was added to each plate. The cells were cultured for 2 weeks with a change of medium every 3 days before the plates were overlayed with 0.28% agarose. After an additional week, colonies that developed into the soft agar were isolated for further studies.Iletermination of the Presence of Transfected src Gene in NIH/3T3 Clones. DNA of NIH/3T3 clones was isolated by the procedure of Maniatis et al. (25). Forty micrograms of DNA from each cell line was dig...

ABSTRACT2,3,7,8,-Tetrachlorodibenzo-p-dioxin (TCDD) administered in vivo causes drastic reduction in the weight of the mouse thymus at low doses (e.g., 30 pg/kg single i.p. i jection), the reduction becoming statistically significant after 2 days. To understand the cause for such thymic involution TCDD-evoked changes in various biochemical parameters in this tissue were examined. The most noticeable change was observed in the increased activity of specific protein-tyrosine kinases and protein kinase C and an increased level of p21-associated binding of [3H]GTP. Since no significant change was observed with cAMP-stimulated protein kinases and cAMP levels, the above changes appear to be a selective effect on these special classes of proteins. As a result of a time sequence study it has become apparent that the rise in proteintyrosine kinase activities becomes significant within 24 hr, whereas the rise in protein kinase C does not become sicant until 48 hr. Among protein-tyrosine kinases, pp60c-and probably pps6lskT were found to be significantly elevated by TCDD treatment. In view of similarities between TCDD and thyroid hormones in causing thymic involution, the levels of c-erb-A expression were assessed in the liver by using avian 32P-labeled v-erb-A probe and RNA transfer blot hybridization technique. The results clearly indicate that TCDD has the property to elevate levels of mRNA bearing homology to v-erb-A. Such changes in c-erb-A expression and proteintyrosine kinase occurred only in TCDD-susceptible (responsive) strains but not in tolerant (nonresponsive) strains of mice at the dose tested. Based on such observations a hypothesis has been proposed that TCDD owes its potency to its ability to stimulate the expression of one of a family of DNAs bearing homology to v-erb-A and that one of the major consequences of such an action is stimulation of various tyrosine kinases.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a very toxic, teratogenic, and carcinogenic chemical (1-4). In addition to these deleterious effects, TCDD is known to cause atrophy of thymus (5, 6) in most experimental animals. Since this lesion occurs at sublethal doses of TCDD at an early stage of poisoning, it has been used by several scientists (e.g., refs. 2, 7, 8) as a sensitive indicator of TCDD poisoning.As for the possible cause for such a thymic lesion, Neal et al. (9) have shown that an increase in serum glucocorticoid levels is unlikely. Poland and Glover (7) have found that involution of thymus occurs at low doses of TCDD (30 ,ug/ kg) in sensitive (responsive) strains of mice but only at high doses in tolerant (nonresponsive) strains of mice. These workers used such differential responses among mouse strains as a biochemical indicator for the involvement of specific cytosolic TCDD binding proteins (i.e., TCDD receptor) in the final expression of thymic involution.Our research group has been studying the effects of TCDD on the hepatic plasma membrane receptors. To date, the plasma membrane-bound epidermal growth factor (EGF) r...

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r -0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1, ammonia, NO,, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.S 1997 Society of Toxicology.

No abstract