6-Aminonicotinamide Induces G_1 Arrest by Elevating p27~(kip1) as well as Inhibiting cdk2, Cyclin E and p-Rb in IMR 32 Neuroblastoma Cell Line

摘要

The effects of 6-aminonicotinamide (6-AN) on viability of IMR32 neuroblastoma cells in the presence of ATP or NAD~+ have been investigated. 6-AN caused marked reduction in cell viability and similar observations were also made with cells treated with6-AN+ATP. However, cells treated with 6-AN+NAD~+ showed cell viability similar to untreated cells. Morphologically, 6-AN and 6-AN+ATP treated cells showed loss of neurites, polyhedric shapes, shrinkage of cell bodies and formation of lysed cells, while 6-AN+NAD~+ cells did not show any such changes. The flow cytometry analysis demonstrated that 6-AN increased cell population in GO/G! phase and decreased cell population in S and G_2/M phase following a 72 h exposure. Western blot analysis showed that 6-AN stimulated a substantial increase in the level of the cdk inhibitor p27~(klp1), but lowered the levels of cdk2, cyclin E and p-Rb. However, cdc25A and p53R2 were not significantly affected. Immunofluorscence staining of p27~(klp1), cdk2, cyclin E and p-Rb revealed close correlation between the signal observed in the Western blot analysis. 6-AN+ATP treated cells showed similar results obtained with 6-AN treated cells in expression of cdk2, cyclin E, p-Rb proteins and p27~(klp1). 6-AN+NAD~+cells showedgreater expression of cdk2, cyclin E and p-Rb than those in 6-AN and 6-AN+ATP treated cells. The results suggest that 6-AN induced the G_0IG_1 phase arrest in IMR32 neuroblastoma cell lines through the increase of p27~(klp1) and the decrease of cdk2, cyclin E and p-Rb.