The action of v-src on gap junctional permeability is modulated by pH

The product of the viral src gene (v-src) is the protein tyrosine kinase pp60v-src. Among the known consequences of pp60v-src activity is the reduction in permeability of gap junctions, an effect that is counteracted by the calcium antagonist TMB-8 (8-N,N-[diethylamino]octyl-3,4,5-trimethoxybenzoate). We show here that a decrease in intracellular pH (pHi) also counteracts the v-src effect: junctional permeability of cells containing active v-src kinase rose with decreasing pHi in the range 7.15 to 6.75, whereas junctional permeability of cells containing inactive v-src kinase or no v-src at all was insensitive to pH in that range. Low pH also counteracted the known action of diacylglycerol on junction, but only when pp60v-src kinase was inactive. Immunoblots of whole-cell lysates using an antibody against phosphotyrosine show that phosphorylation on tyrosine of at least one cellular protein, specific for pp60v-src kinase activity, was reduced by low pH but not by TMB-8. These results suggest that TMB-8 does not inhibit v-src action on junctional permeability by interfering with tyrosine phosphorylation of a protein crucial for closure of gap junction channels, but that the inhibition by low pH may be via this mechanism.     

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes

Transformation of a rat thyroid epithelial cell line (FRTL5-C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid-soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC-C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v-mos oncogene) and when transformed by transfection with the c-myc proto-oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c-myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI-like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.     

Phosphorylation of connexin43 induced by Src: regulation of gap junctional communication between transformed cells

Cx43 is a widely expressed gap junction protein that mediates communication between many cell types. In general, tumor cells display less intercellular communication than their nontransformed precursors. The Src tyrosine kinase has been implicated in progression of a wide variety of cancers. Src can phosphorylate Cx43, and this event is associated with the suppression of gap junction communication. However, Src activates multiple signaling pathways that can also affect intercellular communication. For example, serine kinases including PKC and MAPK are downstream effectors of Src that can also phosphorylate Cx43 and disrupt gap junctional communication. In addition, Src can affect the expression of other proteins that may affect intercellular communication. Indeed, disruption of gap junctions by Src appears to be complex. It has become clear that Src can affect Cx43 activity by multiple mechanisms. Here, we review how Src may orchestrate events that regulate intercellular communication mediated by Cx43.     

Altered expression and distribution of the cytoskeletal-associated p35 protein in NIH 3T3 cells transformed with the Harvey sarcoma virus v-ras oncogene

1. Cytoskeletal events associated with retroviral oncogene (v-ras)-mediated transformation were studied in NIH 3T3 fibroblasts and their v-ras-transfected counterparts (3T3/H-1 cells). 2. Abnormal microfilament networks seen in 3T3/H-1 cells reflected significant decreases (approximately 90%) in two cytoskeletal-associated proteins (tropomyosin-1, p35). Neither actin content nor actin mRNA levels were altered, however, v-ras transfectants. 3. p35 mRNA activity in both NIH 3T3 and 3T3/H-1 cells was similar although differential compartmentalization of p35 to the detergent-resistant cytoskeletal fraction was evident only in normal fibroblasts. 4. Proper cytoskeletal organization may be a factor in the regulation of p35 mRNA translation in situ or influence the stability of p35 independent of translational rate.     

Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60^v-src, pp60^v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60^v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl₂, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60^v-src activity by a vanadium-sensitive protein phosphatase activity.     

Nontransformed cells can normalize gap junctional communication with transformed cells

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Different regulation of vascular endothelial growth factor expression by the ERK and p38 kinase pathways in v-ras, v-raf, and v-myc transformed cells

Here we show that vascular endothelial growth factor (VEGF) mRNA expression is up-regulated in oncogene transformed rat liver epithelial (RLE) cell lines and that the extracellular signal-regulated kinase (ERK) and p38 kinase differentially regulate the oncogene-mediated stimulation of VEGF. The highest level of VEGF mRNA expression was observed in the v-H-ras transformed RLE cell line, followed by the v-raf and v-myc transformed lines. The PD98059 MEK inhibitor was used to block the ERK pathway and SB203580 inhibitor to block the p38 pathway. The parent and the v-H-ras transformed RLE cell lines showed up-regulation of VEGF RNA expression through the ERK pathway and down-regulation of VEGF through the p38 pathway. VEGF was regulated in a comparable manner in a human breast carcinoma cell line. In the v-raf and v-myc transformed RLE lines, positive regulation of VEGF was transduced through the p38 pathway. These findings suggest that (1) oncogenic ras differs from raf and myc in the recruitment of the MAPK signaling pathways for VEGF regulation; (2) that VEGF is regulated in ras transformed and human cancer cell lines in a positive and negative manner by the ERK and p38 signaling pathways.     

Growth inhibition of transformed cells correlates with their junctional communication with normal cells

The growth of various chemically and virally transformed cell types in culture is inhibited when they are in contact with normal cell types. We show that this growth inhibition is contingent on the presence of junctional communication between the normal and transformed cells (heterologous communication), as probed with a 443 dalton microinjected fluorescent tracer. In cell combinations where heterologous communication is weak or absent there is no detectable growth inhibition; the inhibition appears when communication is induced by cyclic AMP-dependent phosphorylation, and only then. In cell combinations where heterologous communication is spontaneously strong, the growth inhibition is present, but it is abolished when the communication is blocked by retinol or retinoic acid. The cell-to-cell membrane channels of gap junctions are the likely conduits of the signals for this growth control.     

Immortalization of murine primary spleen cells by v-myc, v-ras, and v-raf.

Growth factor-independent cell lines, including four lines characterized as macrophages, were isolated by infection of BALB/c mouse primary spleen cells with combinations of three retroviruses encoding v-myc, v-ras, and v-myc/v-raf. Proliferating cell lines were isolated only rarely, and after long crisis periods, following the introduction of myc and raf by infection with J2 virus, or of myc and ras by coinfection with myc309 and raszip6 viruses. However, sequential infections with all three viruses--myc plus ras cells reinfected with J2, or J2 followed by myc plus ras coinfection--resulted in rapid outgrowth of cell lines which grew at high growth rates to high densities. When cells were treated with anti-IgG F(ab')2/IL-4/IL-5 to specifically stimulate B cells, cell lines were isolated readily by infection with myc plus ras alone, J2 alone, or all three viruses. These cell lines arose after shorter crisis times and all grew at high growth rates and to high densities. Analysis of cell surface markers and immunoglobulin gene arrangement revealed no lymphoid characteristics in any of the lines. Four cell lines express all three macrophage markers analyzed (F4/80, Mac1, FcR), and many others are Mac1+ and/or FcR+. Out of 20 immortalized cell lines tested, 13 show clonal growth in soft agar, and 3/6 of these produced tumors in BALB/c mice, indicating that fully transformed cells may be isolated by these procedures. In at least one of the cell lines, integration of all three infecting viruses has occurred.     

Increased activity of c-Src and Csk in fibroblasts transformed by v-src oncogene.

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.     

Altered cytoskeletal structures in transformed cells exhibiting obviously metastatic capabilities

Cytoskeletal changes in transformed cells (LM-51) exhibiting obviously metastatic capabilities were investigated by utilization of double-fluorescent labelling through combinations of: (1) tubulin indirect immuno(?)uorescence plus Rhodamine-phalloidin staining of F-actins; (2) indirect immunofiuorescent staining with actinin polyclonal-and vinculin monoclonal antibodies. The LM-51 cells which showed metastatic index of >50％ were derived from lung metastasis in nude mice after suboutaneous inoculation of human highly metastatic tumor DNA transfected NIH3T3 cell transformants. The parent NIH3T3 cells exhibibed well-organized microtubules, prominent stress fibers and adhesion plaques while their transformants showed remarkable cytoskeletal alterations: (1) reduced microtubules but increased MTOC fluorescence; (2) disrupted stress fibers and fewer adhesion plaques with their protein components redistributed in the cytoplasm; (3) Factin- and actinin/vinculin aggregates appeared in the cytoplasm. These aggregates were doe-like, varied in size (0.1--0.4m) and number, located near the ventral surface of the cells. TPA-indueed actin/vinculin bodies were studied too, Indications that actin and actinin/vinculin redistribution might be important alterations involved in the expression of metastatic capabilities of LM-51 transformed cells were discussed.     

Altered protein kinase activities of lymphoid cells transformed by Abelson and Moloney leukemia viruses

Five different types of protein kinase activities have been evaluated in cell lines from murine lymphomas induced by Abelson leukemia virus (A-MuLV), whose oncogene codes for a tyrosine protein kinase. Such activities were compared with those of normal cells and of cells transformed by Moloney leukemia virus (M-MuLV), lacking oncogene sequences in its genome. While cAMP-dependent protein kinase and casein kinase-1 do not undergo significant changes, casein kinase-2 rises in both A-MuLV and M-MuLV infected lymphocytes, becoming largely associated with the particulate fraction of transformed cells. Protein kinase-C on the other hand is unchanged in M-MuLV transformed cells but it undergoes a 2-3-fold increment in both soluble and particulate fractions of A-MuLV transformed lymphocytes, which also display high tyrosine protein kinase activity.     

Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB,src,ras and raf, but not sis and dbltransformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src- and ras-transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme γ-glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down-regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.     

Expression of pp60v-src alters the ionic permeability of the plasma membrane in rat cells

The transmembrane potential of Rous sarcoma virus (RSV)-infected Rat-1 cells, expressing the pp60v-src protein kinase, is markedly less negative (by approximately 30 mV) than that of their normal counterparts. By contrast, the membrane potential of Rat-1 cells infected with Kirsten sarcoma virus is virtually unaltered. The RSV-induced membrane depolarization is shown to be due to a severalfold increase in the cation permeability ratio (PNa/PK) of the plasma membrane. When cells infected with a temperature-sensitive mutant of RSV (ts LA29), encoding a src protein with heat-labile kinase activity, are shifted from the nonpermissive to the permissive temperature, a rapid and sustained membrane depolarization is observed. Conversely, thermal inactivation of the ts LA29 pp60v-src kinase activity rapidly restores the membrane potential to near normal levels. Addition of epidermal growth factor, platelet-derived growth factor, or insulin to uninfected cells fails to cause a detectable change in membrane potential. We conclude that, unlike growth factor receptor tyrosine kinases, pp60v-src can induce, either directly or indirectly, a major change in the membrane permeability to monovalent cations.     

Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor

Summary Mammalian cells in culture were exposed to cyclic AMP, dibutyrul cyclic AMP, the phosphodiesterase inhibitor caffeine, or a combination of the last two, while junctional molecular transfer was probed with the series of microinjected, fluorescentlabelled linear molecules Glu, Glu-Glu, Glu-Glu-Glu, and Leu-Leu-Leu-Glu-Glu. The junctional permeability for these molecules increased with each of the agents, most markedly with the dibutyryl cyclic AMP-caffeine combination, as the intracellular cyclic nucleotide concentration rose. The junctional permeability effect developed over several hours. When probed with molecules close to the limit of cell-to-cell channel permeation (the most sensitive setting), the effect was detectable both, as an increase in the (relative) junctional transit rate and as an increase in the number of transferring cell interfaces in the test populations. The number of transferring cell interfaces reached a maximum by 4 hr, when the junctional transit rate, hence the junctional permeability, was still rising. Nonjunctional membrane permeability for the probe molecules, as determined by intracellular fluorescence loss, was not significantly changed (nor was there significant nonjunctional cell-to-cell transfer of molecules before or after the treatments). The rise in junctional permeability was associated with an increase in the number of gap junctional membrane particles, as determined by freeze-fracture electron microscopy: the average size of the particle clusters increased, and the frequency of the clusters increased, particularly that of the smaller (and presumably newer) clusters. This effect was blocked by treatments with the protein synthesis inhibitors cycloheximide or puromycin. These agents caused particle diminution (diminution of cluster frequency but not of average cluster size), with or without cyclic nucleotide. The junctional effects may represent a cyclic AMP-promoted proliferation of cell-to-cell channels. Some physiological implications, in particular, implications for hormone-regulated tissues, are discussed.     

Glutamic acid decarboxylase activity is stimulated in quail retina neuronal cells transformed by Rous sarcoma virus and is regulated by pp60v-src.

Rous sarcoma virus (RSV) stimulates in quail embryo neuro-retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY-68, a thermodependent transformation-defective mutant of RSV, stimulation of GAD activity is regulated by pp60v-src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY-68-transformed established NR cell lines, have a high GAD activity which is regulated by pp60v-src, while other clones have a low GAD activity apparently not regulated by pp60v-src. These data indicate that pp60v-src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v-mil.     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

Abstract. The tumour promotor 4 β -phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

The tumour promotor 4 beta-phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.