Antiproliferative effect of nitric oxide on epidermal growth factor-responsive human neuroblastoma cells.
Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009589:Nitrogen Oxides; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured
D003470:Culture Media; D006834:Hydrazines; D007527:Isoenzymes; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D009569:Nitric Oxide; D004815:Epidermal Growth Factor; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D019001:Nitric Oxide Synthase; D066246:ErbB Receptors; D006152:Cyclic GMP