Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43

Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of con nexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomati tis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-exp ress the green fluorescent protein (GFP) and Cx43 in the communication-defi cient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx 43 GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form u nits (CFU)/ml and was free of replication-competent retroviruses. After a 7 -day treatment with retinoic acid (20 muM), N2a transformants (N2a-Cx43 and N2a-Cx43 GFP) maintained the expression of Cx43 and Cx43 GFP. Expression o f both constructs resulted in functional coupling, as evidenced by electrop hysiological and dye-injection analysis. Suppression of cell growth correla ted with expression of both Cx43 or Cx43 GFP and retinoic acid treatment. B ased on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expre ssing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2 a cells does not alter retinoic acid-induced neuronal differentiation but d oes enhance growth inhibition.