Tumor promoting properties of a cigarette smoke prevalent polycyclic aromatic hydrocarbon as indicated by the inhibition of gap junctional intercellular communication via phosphatidylcholine‐specific phospholipase C

Abstract

Inhibition of gap junctional intercellular communication (GJIC) and the activation of intracellular mitogenic pathways are common hallmarks of epithelial derived cancer cells. We previously determined that the 1-methyl and not the 2-methyl isomer of anthracene, which are prominent cigarette smoke components, activated extracellular receptor kinase, and inhibited GJIC in WB-F344 rat liver epithelial cells. Using these same cells, we show that an immediate upstream response to 1-methylanthracene was a rapid (<1 min) release of arachidonic acid. Inhibition of phosphatidylcholine-specific phospholipase C prevented the inhibition of GJIC by 1-methylanthracene. In contrast, inhibition of phosphatidylinositol specific phospholipase C, phospholipase A 2 , diacylglycerol lipase, phospholipase D, protein kinase C, and tyrosine protein kinases had no effect on 1-methylanthracene-induced inhibition of GJIC. Inhibition of protein kinase A also prevented inhibition of GJIC by 1-methylanthracene. Direct measurement of phosphatidylcholine-specific phospholipase C and sphingomyelinase indicated that only phosphatidylcholinespecific phospholipase C was activated in response to 1-methylanthracene, while 2-methylanthracene had no effect. 1-methylanthracene also activated p38-mitogen activated protein kinase; however, like extracellular kinase, its activation was not involved in 1-methylanthracene-induced regulation of GJIC, and this activation was independent of phosphatidylcholine-specific phospholipase C. Although mitogen activated protein kinases were activated, Western blot analyzes indicated no change in connexin43 phosphorylation status. Our results indicate that phosphatidylcholinespecific phospholipase C is an important enzyme in the induction of a tumorigenic phenotype, namely the inhibition of GJIC; whereas mitogen activated protein kinases triggered in response to 1-methylanthracene, were not involved in the deregulation of GJIC. (Cancer Sci 2008; 99: 696-705)

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells

“…The dysregulation of GJIC by VIN was attenuated by an inhibitor of p38, which is mechanistically similar to effects induced by 1-MeA in lung but not liver epithelial cells (Osgood et al, 2013;Upham et al, 2008), or by anisomycin in WB-F344 cells (Ogawa et al, 2004). Interestingly, the effects of MXC and VIN on GJIC were prevented by an inhibitor of PC-PLC.…”

“…The extent of GJIC was evaluated using ImageJ as a function of the area of the cells stained with lucifer yellow dye. The fluorescence area from the positive control treated with 1-MeA (70 mM, 10 min) inducing complete inhibition of GJIC (Upham et al, 2008) was subtracted from each treatment. Each experiment was repeated at least 3 times independently.…”

“…As optimized and used previously (Upham et al, 2009), the cells were placed in a serum-free cell culture medium for 4 h prior the addition of the tested chemicals to minimize basal activity of MAPKs (Pirkmajer and Chibalin, 2011). TPA (50 nM, 30 min) was used as a positive control to induce MAPK ERK1/2 and Cx43 phosphorylation, 1-MeA (70 mM, 30 min) was used as a positive control for MAPK ERK1/2 and p38 activation (Upham et al, 2008). After exposure, the cells were rinsed with PBS and the proteins extracted with 20% SDS (v/v) containing protease and phosphatase inhibitors .…”

“…After wash with PBST and nuclear counterstaining (5 min with 0.1 mg/ml DAPI, Sigma-Aldrich), the cover slips were mounted using ProLong Antifade reagent (Life Technologies). TPA (50 nM) was used as a positive control inducing Cx43 translocation and internalization (Upham et al, 2008). Negative controls for nonspecific antibody binding were prepared using non-treated cells subjected to the same staining procedure without either the primary or the secondary antibody incubation.…”

“…These effects, which are relevant for transcriptional and epigenetic control of gene expression and normal cell behavior (Trosko, 2011), were studied in a rat liver epithelial cell line WB-F344. This cell line is a well-established in vitro model for assessment of GJIC and mechanistic studies in responses to environmental contaminants (Sovadinova et al, 2015;Upham et al, 2007Upham et al, , 2008, which has been validated by in vivo studies (Sai et al, 2000;Upham et al, 2009) and matches the most extensively used rodent model system, F344 rats, in the National Toxicology Program. Both of these EDCs exhibited dose, time and mechanistic differences on GJIC, MAPKs, and phospholipase C, which were elicited by the parental compounds and occurred independently of ER-or AR-mediated genomic mechanisms.…”