6‐Aminonicotinamide induces G₁ arrest by elevating p27^kip1 as well as inhibiting cdk2, cyclin E and p‐Rb in IMR32 neuroblastoma cell line

Abstract

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 with 6‐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 G0/G1 phase and decreased cell population in S and G₂/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^kip1, but lowered the levels of cdk2, cyclin E and p‐Rb. However, cdc25A and p53R2 were not significantly affected. Immunofluorscence staining of p27^kip1, 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^kip1. 6‐AN+NAD⁺cells showed greater 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 G₀/G1 phase arrest in IMR32 neuroblastoma cell lines through the increase of p27^kip1 and the decrease of cdk2, cyclin E and p‐Rb.

Key words:

• 6‐Aminonicotinamide
• IMR32 cells
• ATP
• NAD⁺
• G₁ arrest

Notes

To whom correspondence should be addressed. Tel: [phone]; Fax: [phone] E‐mail: [email protected]