Cellular sublocalization of Cx43 and the establishment of functional coupling in IMR-32 neuroblastoma cells

Abstract

Neuroblastoma (NB) is the most common solid pediatric tumor. IMR-32 cells are a highly malignant human NB cell line with uncontrolled proliferation but with the potential to be differentiated under specific conditions. Preliminary research indicated that connexin 43 (Cx43), the most widespread of the Cx family, is aberrantly located in IMR-32 cells, which renders these cells incapable of gap junction (GJ) intercellular communication. Functioning GJ intercellular communication has been strongly associated with growth control and a decrease in tumorigenicity. 8-br-cAMP, known to initiate the differentiation process in cancer cells, was used to examine changes in Cx43 localization and expression via immunocytochemistry, Western blot analysis, and flow cytometry. Exposure of IMR-32 cells to 8-br-cAMP decreased cell proliferation, restored the abnormally localized Cx43 from around the nucleus to the cell membrane, increased de novo Cx43 protein expression, and appeared to phosphorylate Cx43 on serine (Ser) 255 and Ser262. Forskolin, an activator of cAMP dependent protein kinase (PKA), produced identical results to 8-br-cAMP demonstrating the effect that was not unique to a cAMP analog. The use of a PKA inhibitor further confirmed the specificity of 8-br-cAMP and forskolin's effect on Cx43. The cellular relocation of Cx43, combined with the increased protein expression, established first ever GJ intercellular communication between IMR-32 cells as revealed by scrape loading. These results suggest that the GJ-mediated return of growth control, as a prerequisite for further differentiation, offers a new therapeutic avenue in the treatment of NB.

NT-polyplex: a new tool for therapeutic gene delivery to neuroblastoma tumors

“…The relatively weak bystander efficiency might be explained by the decrease of functional gap junctions in the allografted N1E-115 cells as has been reported for other neuroblastoma cell lines. 40,41 Another condition that possibly limits the bystander effect may be the defective immune response mediated by the T-cell linage in the athymic mice used in our tumoral model. 42 The use of the HSVTK-GCV system in human trials to cause the death of tumoral cells has not been efficient.…”

Gap junctional intercellular communication capacity by gap‐FRAP technique: A comparative study

“…Cx43 phosphorylated forms are usually concentrated in gap junctional plaques [7], and GJIC functionality is related to the expression of phosphorylated Cx43 expression and their localization in the cell-cell contact areas, corresponding to gap junctions between contacting cells [38]. Nonphosphorylated Cx43 expression is often associated with the cytoplasmic pool of available Cx43 proteins that would normally be trafficked to the membrane [39]. Phosphorylation of Cx43 may be coupled to the assembly of gap junction plaques and to the formation of functional channels.…”

Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication

“…Primary antibodies used in this study include rabbit anti-Cx43 (Chemicon), mouse anti-Cx43 (Zymed), mouse anti-Cx40 (Zymed), mouse anti-α-tubulin (Sigma), rabbit anti-acetylated-α-tubulin (Enzo), mouse anti-α-actin antibody (Sigma), rabbit anti-acetylated lysine antibody (Abcam), and Ncad (Sigma). pSer-specific Cx43 antibodies were produced as previously described including rabbit anti-pS255 (Santa Cruz; Sirnes et al, 2009), rabbit anti-pS262 (Santa Cruz; Srisakuldee et al, 2006), rabbit anti-pS279/282 (Santa Cruz; Arnold et al, 2005; Solan and Lampe, 2008), rabbit anti-pS325/328/330 (Lampe et al, 2006), rabbit anti-pS365 (Solan et al, 2007), rabbit anti-pS368 (R&D; Solan et al, 2007), and rabbit anti-pS373 (P. D. Lampe, personal communication, manuscript in preparation).…”