Potential role of the src gene product in inhibition of gap-junctional communication in NIH/3T3 cells.

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...

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

“…This phenomenon is consistent with observations reported by others that pp60 ~''~ overexpression reduces cell-adhesion (Warren and Nelson, 1987) and cell-ceU communication (Azarnia et al, 1988). It is worthwhile to mention that pp60 ~-s' overexpression appears to mimic here the effect of pp60 "-~ since it has been reported that pp60 .... also impairs cell-cell communication (Chang et al, 1985;Crow et al, 1990). The presence of the long cellular processes filled with pp60 ~-~r~ in the NIH (pM c-src/focus)B cells plated at low density is also indicative of a lack of adhesive strength between these cells and the substratum.…”

“…It is interesting to note that the two proteins appear to exert here the same function in reducing intercellular adhesion and communication (Warren and Nelson, 1987;Azarnia et al, 1988;Chang et al, 1985;Crow et al, 1990).…”

Induction of cell transformation by mutated 16K vacuolar H+-atpase (ductin) is accompanied by down-regulation of gap junctional intercellular communication and translocation of connexin 43 in NIH3T3 cells

“…These results suggest that transformed NIH3T3 cells do not necessarily show lower levels of GJIC, which in turn, provides evidence that lower GJIC observed in NIH3T3 cells transfected with mutant 16 K proteins may not be a consequence of cell transformation. The results also con®rm our previous ®ndings that v-src induced NIH3T3 cell transformation is not associated with reduced GJIC ability (Bignami et al, 1988), which is at variance with other reports (Azarnia and Loewenstein, 1984;Chang et al, 1985). In order to examine whether GJIC modulation of NIH3T3 cells by mutated 16 K proteins was associated with changes in C643 expression, mRNA and protein levels of C643 were determined by RT ± PCR and Western blot analysis respectively.…”

Aberrant Expression and Function of Gap Junctions during Carcinogenesis

“…Earlier studies on the relationship between oncogenes and GJIC examined whether oncogenes can modulate GJIC. Such studies have shown that certain viral oncogenes such as the v-src gene can indeed inhibit GJIC (46)(47)(48). Other oncogenes have also been shown to inhibit GJIC.…”