Dibutyryl cAMP stimulates the proliferation of SH-SY5Y human neuroblastoma cells by up-regulating Skp2 protein

Abstract

PURPOSE: We previously found that the proliferation of SH-SY5Y neuroblastoma cells is stimulated when cAMP is up-regulated by stable expression of stimulatory G protein. Therefore, this study was performed to investigate the mechanism whereby cAMP stimulates the proliferation of SH-SY5Y cells. METHODS: To investigate the effect of cAMP on cellular proliferation, SH-SY5Y neuroblastoma cells were treated with dibutyryl cAMP (dbcAMP), and then cell growth, thymidine incorporation and cell cycle phase distribution were analyzed. The expression and the activity of the molecules that regulate cell cycle progression were monitored by Western blot, RT-PCR, and kinase activity assay. RESULTS: Treatment with dbcAMP produced a biphasic effect on cellular proliferation; especially treatment with low concentration of dbcAMP (0.5 mM) showed a higher cellular proliferation rate and promoted G1/S transition in cell cycle. The dbcAMP (0.5 mM) treatment increased CDK2 activity, and it significantly decreased p27Kip1 expression with a decreased half-life of p27Kip1 protein. Moreover, dbcAMP (0.5 mM) increased the protein level and the stability of Skp2 with a concomitant decrease in its ubiquitination. CONCLUSIONS: cAMP up-regulates Skp2 protein by reducing its degradation probably through decreasing the ubiquitination of Skp2, which might result in accelerated degradation of p27Kip1, increase in CDK2 activity, and stimulation of SH-SY5Y cell proliferation in sequence.