Cellular sublocalization of Cx43 and the establishment of functional coupling in IMR-32 neuroblastoma cells.
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.
Arnold
Jennifer M
JM
SIMS, PharmaGap, National Research Council of Canada, Ottawa, Ontario, Canada.
Phipps
Mikael W
MW
Chen
Jiahua
J
Phipps
Jenny
J
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D018031:Connexin 43 / Q000032:analysis* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D009447:Neuroblastoma / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects
D018031:Connexin 43; D003609:Dactinomycin; D005576:Colforsin; D020126:Brefeldin A; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003513:Cycloheximide; D017868:Cyclic AMP-Dependent Protein Kinases