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Biomedical subjects

B V Madhukar

Publications and source records attributed to B V Madhukar.

At least 19 recordsLinked to original sources
PubMed1999

Potential involvement of calcium, CaM kinase II, and MAP kinases in PCB-stimulated insulin release from RINm5F cells.

Polychlorinated biphenyls (PCBs) are environmental contaminants that induce release of insulin in rat insulinoma cells, RINm5F (Fischer et al., Life Sci. (1996) 59, 2041-2049). In the present study the mechanisms of this effect were investigated using noncytotoxic concentrations (10 microg/ml) of a PCB mixture, Aroclor-1254, and the pure PCB congeners 2,2',4,4'-tetrachlorobiphenyl and 2,2',4,4',5, 5'-hexachlorobiphenyl. Treatment of RINm5F cells with each of these agents resulted in a rapid increase in intracellular free calcium. The presence of extracellular calcium was required for PCB-induced insulin release because removal of calcium from the medium attenuated the effect. In addition, pretreatment of RINm5F cells with the calcium channel blocker verapamil also blocked PCB-induced insulin release. To determine whether PCB-related insulin release could be associated with the enzyme, calcium/calmodulin-dependent kinase II (CaM kinase II), RINm5F cells were pretreated with the CaM kinase II inhibitor KN-93. PCB-induced insulin release was completely blocked by KN-93. Under similar treatment conditions, PCBs also induced the activity of mitogen-activated protein kinases (MAPK) 1 and 2. However, inhibition of MAPK activation by a specific inhibitor, PD-98059 (10.0 microM) did not prevent insulin release induced by PCBs. The results of the present investigation suggest a role for calcium and CaM kinase II in PCB-induced insulin release. Furthermore, the results suggest that insulin release by PCBs is independent of the activation of MAPKs.

Animals↗
PubMed1998

Altered biologic activities of commercial polychlorinated biphenyl mixtures after microbial reductive dechlorination.

The reductive dechlorination of polychlorinated biphenyls (PCBs) by anaerobic bacteria has recently been established as an important environmental fate of these compounds. This process removes chlorines directly from the biphenyl ring with replacement by hydrogen, resulting in a product mixture in which the average number of chlorines per biphenyl is reduced. In this study, dechlorination of commercial PCB mixtures (Aroclors 1242 and 1254) by microorganisms eluted from PCB-contaminated sediments of the River Raisin (Michigan) and Silver Lake (Massachusetts) caused a depletion in the proportion of highly chlorinated PCB congeners and an accumulation of lesser-chlorinated congeners. Dechlorination occurred primarily at the meta and, to a much lesser extent, para positions of biphenyl. The concentrations of the coplanar congeners including 3,3',4,4',5-pentachlorobiphenyl, the most potent dioxinlike congener, were significantly lowered by reductive dechlorination. Microbial reductive dechlorination of commercial PCB mixtures caused a substantial reduction in biologic activities in several instances. It significantly lowered or eliminated the inhibitory effects of Aroclors on fertilization of mouse gametes in vitro. Similarly, the dechlorinated product mixtures had substantially lower ethoxyresorufin-O-deethylase induction potencies and showed less ability to induce activating protein 1 transcription factor activity as compared to the unaltered Aroclors. In other assays the same dechlorinated product mixtures demonstrated biologic activities similar to the nondechlorinated Aroclors, including the ability of PCB mixtures to stimulate insulin secretion and cause neutrophil activation. The data presented here establish that the biologic activities of commercial PCB mixtures are altered by microbial reductive dechlorination and that an assessment of their toxic potential requires an array of tests that include the different mechanisms associated with PCBs.

Animals↗
PubMed1997

Xenobiotics released from fat during fasting produce estrogenic effects in ovariectomized mice.

The pesticide residues 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p'-DDT) and beta-hexachlorocyclohexane (beta-HCH) act as weak estrogens, producing uterotrophic responses in ovariectomized rodents and stimulating human breast cancer cells in culture. Such activity suggests that these compounds may act as tumor promoters in estrogen-responsive tissues. Organochlorine compounds such as o,p'-DDT and beta-HCH are concentrated in body fat. The present report tests whether sufficient compound can be released from fat depots to produce estrogenic effects in uteri of ovariectomized mice. Adult animals were "loaded" with test compound by three daily injections of vehicle (DMSO), 17beta-estradiol (E2), beta-HCH, or o,p'-DDT. Uterotrophic effects were assessed at 24 h after the last loading dose of test compound and at 2 weeks after the loading regimen, with or without a prior 2-day period of fasting. The initial 3-day treatment with either beta-HCH or o,p'-DDT doubled the relative dry weight of the uterus: 102 +/- 8.6 mg/kg body weight (BW) and 104 +/- 4.4 mg/kg BW for beta-HCH and o,p'-DDT, respectively, compared to 49 +/- 1.9 mg/kg BW for vehicle-treated animals. E2-treated animals had uterine dry weights of 228 +/- 11 mg/kg BW. After 2 weeks without further treatment, a 2-day fast produced a decrease in body mass of 4.1 g/animal (fasted, 25.9 +/- 1.89 g versus fed, 30.0 +/- 2.82 g). Animals that had been loaded with beta-HCH and fasted had uterine weights (88 +/- 12 mg/kg BW) significantly greater (P < 0.05) than those of vehicle-loaded, fasted animals (51 +/- 2.9 mg/kg BW) or of beta-HCH-loaded, fed animals (59 +/- 4.6 mg/kg BW). The uterine weights of the fasted and fed o,p'-DDT-loaded or E2-loaded animals were not different from those of control weights. The difference between wet and dry weights showed that fasting of beta-HCH-loaded animals also increased water imbibition in the uterus; there was no effect from fasting in the other groups. Generally, epithelial cell height reflected the same responses as uterine weight with the exception that cell heights of beta-HCH-loaded, fed animals were slightly higher (P < 0.05) than corresponding controls, indicating that there may have been some active compound available to the tissues even without fasting. The effects of fasting show that during periods of lipolysis beta-HCH can be released in quantities sufficient to stimulate estrogen target tissues, suggesting a novel mechanism linking obesity and the progression of estrogen-responsive tumors. The lack of effect from fasting in o,p'-DDT-loaded animals indicates that these compounds are differentially mobilized from fat depots.

Adipose Tissue↗
PubMed1996

Novel estrogenic action of the pesticide residue beta-hexachlorocyclohexane in human breast cancer cells.

The estrogenic action of some persistent organochlorine pesticide residues may play a role in the progression of hormonally responsive tumors of the breast and uterus. The prototypical xenoestrogen o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) acts by binding and activating the estrogen receptor (ER). The present study focuses attention on the mechanisms through which another organochlorine compound, beta-hexachlorocyclohexane (beta-HCH), exerts estrogen-like effects in human breast cancer cells. Both o,p'DDT and beta-HCH stimulated proliferation in a dose-dependent manner in the ER-positive cell lines MCF-7 and T47D but not in the ER-negative lines MDA-MB231, MDA-MB468, and HS578T. Both compounds produced an increase in the steady state level of pS2 mRNA in MCF-7 cells. These responses were equal in magnitude to the maximal effect of estradiol, and they were inhibited by inclusion of the antiestrogen ICI164384. On the other hand, when tested in a competitive binding assay, beta-HCH did not displace 17beta-[3H]estradiol from the ER even at a concentration that was 40,000-fold higher than the tracer steroid. Furthermore, nuclear retention of the ER during homogenization procedures was induced by a 2- or 24-h treatment of MCF-7 cells with o,p'-DDT and 17beta-estradiol but not by treatment with beta-HCH; this indicates that beta-HCH nether activates the ER, nor is it converted intracellularly to an ER ligand. Transcriptional activation by beta-HCH occurs in estrogen-responsive GH3 rat pituitary tumor cells transfected with a luciferase reporter construct driven by a complex 2500-bp portion of the PRL gene promoter; this trans-activation response is inhibited by inclusion of ICI164384. However, beta-HCH is ineffective in stimulating a reporter construct driven only by a consensus estrogen response element and a minimal promoter derived from the herpes simplex virus thymidine kinase gene. Thus, beta-HCH cannot act on a simple, single estrogen response element; rather, it requires the combinatorial regulation found in a complex promoter. These data are consistent with the notion that beta-HCH stimulation of cell proliferation and gene expression is ER dependent, but its action is not through the classic pathway of binding and activating the ER. beta-HCH may represent a new class of xenobiotic that produces estrogen-like effects through nonclassic mechanisms and, therefore, may be of concern with regard to breast and uterine cancer risk.

Adenocarcinoma↗
PubMed1996

Pulse treatment with the tumor promoter TPA delays the onset of desensitization response and prolongs the inhibitory effect on gap junctional intercellular communication of a rat liver epithelial cell line WB F-344.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is an inhibitor of gap junctional intercellular communication (GJIC) of the rat liver epithelial cell line, WB F-344. We have previously reported that prolonged treatment of the WB cells with TPA (10 ng/ml) caused a reversal of the inhibition of GJIC that was initially induced (Oh, S.Y., et al. (1988) Carcinogenesis, 9, 135-139). Under this condition, addition of fresh TPA did not inhibit GJIC of these cells. In the present investigation we examined whether pulse exposure to TPA delays the onset of this desensitization response. Cultures were treated for 5 or 15 min with TPA and shifted to normal medium. Intercellular communication was measured at 15 min, 1 h and 6 h after the 5 or 15 min pulse treatments. Under these pulse treatment conditions, GJIC of the cells was markedly inhibited for up to 4 h and gradually reverted to near control levels by 6-8 h. At every sixth hour of pulse treatment the cells were given an additional pulse treatment (5 or 15 min) and the inhibitory effect of TPA on the GJIC of the cells was assayed 15 min after each such treatment. The results clearly showed that, when the cells were treated with 10 ng/ml TPA for 5 or 15 min every 6 h they maintained their sensitivity to the inhibitory effect of TPA on GJIC. This response to TPA was sustained for a considerably longer time when the duration of the pulse treatment was 5 min. Our data suggested that pulse exposure to TPA delays the desensitization response normally observed in prolonged treatment regimens and that this delay is possibly due to maintenance of the TPA activatable pool of protein kinase C under these conditions.

Animals↗
PubMed1995

Inhibition of gap junctional intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat hepatocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent rodent hepatic tumor promoter. Unlike observations with the majority of tumor promoting chemicals studied to date, most investigations have failed to demonstrate down-regulation of gap junctional intercellular communication (GJIC) in cultured cells by TCDD. The present study examined the effect of TCDD on GJIC in rat hepatocytes in primary culture. At non-cytolethal doses TCDD inhibited GJIC in a time- (1, 4, 24 and 48 h) and concentration (1 x 10(-8) - 1 x 10(-14) M)-dependent manner. This inhibition occurred within 4 h of treatment at doses of 1 x 10(-8) - 1 x 10(-12) M TCDD and persisted for up to 48 h, despite removal of TCDD. Treatment of rat hepatocytes with TCDD resulted in a decrease in hepatocyte connexin 32 mRNA, but had no apparent effect on connexin 26 mRNA. Co-incubation of rat hepatocytes with TCDD and alpha-napthoflavone abolished down-regulation of GJIC by TCDD. Similarly, co-treatment with a cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) prevented down-regulation of GJIC by TCDD. The results of this investigation demonstrated, for the first time, that TCDD inhibits GJIC in the in vivo target of its tumor promoting effect and that this effect may, in part, be mediated through the Ah receptor. In addition, this study showed that inhibition of GJIC by TCDD may be due to transcriptional down-regulation or stability of the connexin 32 gap junction mRNA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

The role of modulated gap junctional intercellular communication in epigenetic toxicology.

The normal development and health of all multicellular organisms, including the human being, depend on the adaptive maintenance of the integrity of the genetic information (e.g., DNA protective and repair mechanisms), as well as of the homeostatic and cybernetic regulatory systems within and between tissues. The primary focus of the past and current toxicological studies and risk assessment practices has been to ascertain and predict the "genotoxicity" of various physical and chemical agents. The paradigm of "carcinogen as mutagen," while valuable for stimulating studies of the detection of mutagens and of their potential role in "causing" somatic and germ line diseases, has tended to blunt research on the role of nongenotoxic mechanisms in disease causation. This brief analysis will emphasize the need to consider the role of modulated gap junctional intercellular communication (GJIC) in any biological risk assessment model. It is based on the following assumptions and facts. Because gap junctions exist in all metazoans, they have been associated with the regulation of cell proliferation, development, differentiation, and the adaptive function of both excitable and nonexcitable coupled cells. A highly evolutionarily conserved family of genes codes for proteins (connexins), which, as hexameric units (connexons), form membrane-associated channels of gap junctions. Cells coupled by gap junctions will have their ions and small regulatory molecules equilibrated. Regulation of GJIC can be at the transcriptional, translational, or posttranslational levels. Transient down or up regulation of GJIC can be induced by endogenous or exogenous chemicals via many mechanisms at any of these three levels. Stable abnormal regulation has been associated with activated oncogenes, and normal regulation has been associated with several tumor suppressor genes. The dysfunction of these gap junctions might play a role in the actions of various toxic chemicals that have cell type/tissue/organ specificity. This could bring about distinct clinical consequences, such as embryo lethality or teratogenesis, reproductive dysfunction in the gonads, neurotoxicity of the central nervous system, hyperplasia of the skin, and tumor promotion of initiated tissue. Modulation of GJIC should be viewed as a scientific basis of "epigenetic toxicology" because the alteration of intercellular communication would alter the internal physiological state of the cell. The inhibition of GJIC is a necessary component of mitogenesis (a necessary component of the multistage carcinogenic process). The modulation of GJIC can have both toxicological, as well as therapeutic potential.

Animals↗
PubMed1994

Activated neutrophils from rat injured isolated hepatocytes.

BACKGROUND: Activated neutrophils (PMNs) release cytotoxic agents that can damage surrounding tissue. These studies were performed to determine whether activated PMNs from rat could injure isolated, rat hepatic parenchymal cells (HCs) in vitro. EXPERIMENTAL DESIGN: HCs were cocultured with unstimulated rat PMNs or with PMNs activated with either f-met-leu-phe (FMLP) or phorbol myristate acetate (PMA), that stimulate predominantly degranulation or superoxide production, respectively. Toxicity to HCs was evaluated from release of alanine aminotransferase into the medium. RESULTS: Alanine aminotransferase release was greater in HCs cocultured with FMLP- or PMA-stimulated PMNs compared with unstimulated PMNs. Toxicity was observed by 16 hours after stimulation of PMNs. To test the possible involvement of a soluble mediator released by activated PMNs, HCs were incubated with conditioned medium from PMNs. Compared with unstimulated PMNs, toxicity to HCs was greater in the presence of conditioned medium from FMLP-stimulated PMNs, but not conditioned medium from PMA-activated PMNs. Reactive oxygen species do not appear to be involved in the mechanism by which activated PMNs damage HCs since superoxide dismutase, catalase, superoxide dismutase+catalase, or desferrioxamine failed to prevent the injury. Furthermore, less superoxide anion was detected in PMA-stimulated PMNs when either HCs or HC-conditioned medium was present. Proteolytic enzymes released by stimulated PMNs may play a role in HC damage since an inhibitor of proteases diminished injury due to PMNs activated by either FMLP or PMA. CONCLUSIONS: These results indicate that activated, rat PMNs damage HCs in culture. The data suggest that reactive oxygen species are not involved in the mechanism, but that release of proteolytic enzymes may play a role in the toxic response.

Alanine Transaminase↗
PubMed1993

Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin.

The plasma-membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farnesyl synthesis and p21 ras farnesylation are inhibited by hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v-Ha-ras-transformed rat liver epithelial cell line (WB-ras cells) and if changes were associated with the enhancement of gap-junctional intercellular communication (GJIC). WB-ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1-0.5 microM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB-ras tumors were induced in male rats by intraportal-vein injection of WB-ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB-ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin-treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.

Animals↗
PubMed1993

Restoration of gap-junctional intercellular communication in a communication-deficient rat liver cell mutant by transfection with connexin 43 cDNA.

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.

Animals↗
PubMed1993

Protein kinase C is not involved in Ah receptor transformation and DNA binding.

Induction of cytochrome P4501A1 by 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) is mediated by the Ah receptor (AhR) complex, a ligand-dependent DNA-binding transactivator. Recently a role for protein kinase C (PKC) in the induction response has been reported in which PKC or a related kinase positively modulates AhR activity. We have examined the role of PKC by determining the effect of two nonspecific PKC inhibitors, H7 and staurosporine, and one specific PKC inhibitor, calphostin c, on AhR functionality. Although no kinase activity was detectable in cytosol, under the conditions used for our assays, AhR transformation and DNA binding still occurred. Addition of relatively high concentrations of the kinase inhibitors also had no significant effect on TCDD:AhR:DRE complex formation. Thus, our results indicate that protein kinase activity does not appear to be necessary for TCDD-dependent AhR transformation and DNA binding and they imply that protein kinases must play a role in another step(s) in the AhR-dependent mechanism of P4501A1 induction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Endogenous and exogenous modulation of gap junctional intercellular communication: toxicological and pharmacological implications.

During the evolution of single-celled organisms to multicellular metazoans, a family of highly conserved genes coding for proteins (connexins), which as hexameric units (connexins), has evolved to form intercellular channels (gap junctions). These gap junctions allow ions and small molecular weight molecules to flow between coupled cells, thereby facilitating synchronization of electrotonic or metabolic cooperation. Control of cell proliferation, cell differentiation and adaptive responses of differentiated cells have been speculated to be biological roles of gap junctions. The regulation of these gap junctions can occur at the transcriptional, translational and posttranslational levels. Transient downregulation by endogenous or exogenous chemicals can bring about adaptive or maladaptive consequences depending on circumstances. Stable abnormal regulation of gap junction function has been associated with the activation of several oncogenes. Several tumor suppressor genes have also been associated with the up-regulation of gap junction function. Since gap junctions exist in all organs of the multi-cellular organisms, the dysfunction of these gap junctions by various toxic chemicals which have cell type/tissue/organ specificity could bring about very distinct clinical consequences, such as embryo lethality or teratogenesis, reproductive dysfunction in the gonads, neurotoxicity of the CNS system, hyperplasia of the skin, and tumor promotion of initiated tissue. Understanding how many non-mutagenic chemicals might alter normal gap junction function should form the basis of "epigenetic" toxicology. On the other hand, restoring normal gap junction function to cells which have dysfunctional intercellular communication could be the basis for a new approach for therapeutic pharmaceuticals.

Animals↗
PubMed1993

Comparison of alpha-tubulin mRNA and heat shock protein-70 mRNA in gerbil brain following 10 min of ischemia.

In situ hybridization and Northern blot analysis were used to characterize the mRNA expression of alpha-tubulin, a neuroprotein crucial for neuronal structural and functional restoration, in comparison to that of the stress inducible heat shock protein-70 (HSP-70), in the same gerbil brain following 10 min of forebrain ischemia. The HSP-70 expression was noted in the dentate granule layer 1 h postischemia (PI) and became prominent in all pyramidal cell fields of the hippocampus in addition to the dentate layer at 6 h PI. The induction of HSP-70 persisted in CA1 and CA2 regions and partly in dentate gyrus for up to the 1 day PI period examined. There was no significant HSP-70 expression in any of the regions of the nonischemic or 15 min PI brain. alpha-Tubulin, on the other hand, was expressed in all pyramidal fields of the hippocampus as well as dentate gyrus in nonischemic controls. A decline was noted in the CA1 region 1 h PI onward and was maximal at 6 h PI. Its expression, however, increased at 24 h PI (significant only in comparison to 15 min and 6 h PI but not to control) when it became rather strong in the dentate gyrus. Thus, the temporal pattern of expression of alpha-tubulin sharply contrasted with that of HSP-70 in the PI brain as it declined in the vulnerable CA1 region during the 1st 24 h PI, i.e., the period when HSP-70 was induced and its expression was lowest in the 6 h group when HSP-70 peaked. It was maximum in the dentate gyrus at 24 h PI when HSP-70 was marginally detectable in that region. These studies indicate that in early recirculation period following prolonged ischemia, HSP-70 mRNA is expressed in both vulnerable regions as well as in regions of the brain that are destined to survive while alpha-tubulin is diminished in vulnerable regions. These data suggest a positive correlation between the loss of alpha-tubulin mRNA and delayed neuronal necrosis that follows in the vulnerable CA1 region.

Animals↗
PubMed1993

Immortalized hypothalamic luteinizing hormone-releasing hormone neurons express a connexin 26-like protein and display functional gap junction coupling assayed by fluorescence recovery after photobleaching.

Expression of gap junction proteins was studied in the LHRH neuronal cell line, GT1-7, as a first step in defining the signalling mechanisms responsible for the pulsatile secretion of LHRH. GT1-7 cells were found to express a connexin 26-like protein that comigrated with mouse liver connexin 26 and that reacted with connexin 26-specific antibodies on Western blots. Immunofluorescent staining revealed punctate staining in a fraction of the cells, often present at points of apparent contact with neighboring cell bodies or processes. Fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein loaded GT1-7 cells showed dye coupling among 20-30% of cells that made contact with other cells, suggesting the presence of functional gap junctions in this cell line.

Alkaline Phosphatase↗
PubMed1993

Characterization of gap junctional communication-deficient mutants of a rat liver epithelial cell line.

To understand the mechanism(s) regulating gap junctional communication, we isolated gap junctional intercellular communication-deficient (GJIC-) mutant clones of a rat liver epithelial cell line, WB F-344, which is hypoxanthine-guanine phosphoribosyl transferase deficient (HGPRT-). The cells were exposed to a mutagenesis regimen and cocultured with the wild type HGPRT+ cells. Four GJIC- and one positive clones were characterized in the present study. Northern analysis of RNA isolated from both mutant and parental cells showed a single RNA species of about 3.0 kb which hybridized to connexin43 (Cx43) cDNA. Western blot analysis confirmed the expression of this junctional protein in all these clones. However, in the GJIC- clones the slowest migrating band corresponding to a hyperphosphorylated form, P2, of Cx43 protein (approximately 46 kDa) was absent suggesting that loss of this phosphorylated form of Cx43 may be involved in the failure of the mutants to establish cell-cell communication. Immunofluorescence analysis of the mutants did not reveal any differences in the distribution and localization of Cx43 between GJIC+ and GJIC- clones suggesting that the loss of phosphorylation did not affect the membrane association of this protein. Taken together, these data suggest that one mechanism for the loss of communication in these GJIC- mutants may be the consequence of a change in the intrinsic phosphorylation state of Cx43 protein.

Animals↗
PubMed1990

Chemical, oncogene and growth factor inhibition gap junctional intercellular communication: an integrative hypothesis of carcinogenesis.

Most, if not all, cancer cells have some dysfunction in gap-junction-mediated intercellular communication, either because of defects in cell adhesion or inability to have functional gap junctional communication. In addition, most, if not all, tumor-promoting chemicals and conditions down-regulate gap junction function, while some antitumor-promoting chemicals can up-regulate gap junctional communication. Several oncogenes are associated with down-regulation of gap junction function and several hormone and growth regulators, known to be tumor promoters, are also able to down-regulate gap junction function. On the other hand, some tumor suppressor genes have been linked to the up-regulation of gap junctions. Based on these observations, it is hypothesized that, if a progenitor cell is unable to perform gap junctional intercellular communication, normal growth control and cell differentiation would not be possible, thereby favoring the development of malignant neoplasia.

Animals↗
PubMed1990

Modulation of intercellular communication during radiation and chemical carcinogenesis.

Carcinogenesis is a multistep process, involving the irreversible conversion of a stem cell to a terminal-differentiation-resistant cell ("initiation"), followed by the clonal expansion of this cell ("promotion") and by the acquisition of other genetic alterations leading to malignancy ("progression"). The initiation and progression steps seem to be facilitated by mutagenesis. Promotion has been associated with agents and conditions that cause mitogenesis. Gap junctional intercellular communication, a fundamental biological process regulating cell growth and differentiation, has been postulated to play a major role in carcinogenesis. The hypothesis is supported by the fact that many cancer cells have some dysfunction in gap junctional intercellular communication, many tumor-promoting chemicals and several oncogenes (i.e., ras, src, mos, neu, but not myc) reduce gap junctional intercellular communication, and several growth factors (i.e., EGF, TGF-beta, bovine pituitary extract) inhibit gap junction function. This integrative concept postulates that chemical promoters, oncogenes coding for growth factors, receptors, or transmembrane signaling elements, and growth factors can isolate an initiated cell from the suppressing influence of surrounding normal cells by down-regulating the transfer of ions and small molecules through gap junctions.

Animals↗