PHOSPHOPRYLATION OF CYCLIN-DEPENDENT KINASE AND RETINOBLASTOMA PROTEIN IN RAT SPLEEN FOLLOWING ANILINE EXPOSURE

Jianling Wang, M.D., M.S. and M Firoze Khan, Ph. D.

Department of Pathology, UTMB

Background: Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron-overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. Objective: This study was focused on evaluating the expression of cell cycle proteins (cyclins) and cyclin-dependent kinases (CDKs) associated with phosphorylation of the retinoblastoma protein (Rb) in the spleen, in an experimental condition preceding a tumorigenic response. Methods: Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, expression of cyclins, and phosphorylation of cell cycle-related proteins were measured. Results: Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclin D2, cyclin D3, cyclin E, and cyclin A, as compared to the controls. The overexpression of these cyclins was associated with significant increases in the expression of CDK2, CDK4, as well as phosphorylation of CDK2 and Rb proteins. Aniline treatment also resulted in significant increases in splenocyte proliferation, based on cell counts and MTT assay. Conclusions: Our data suggest that changes in the expression of cyclins, and phosphorylation of CDK2 and Rb proteins could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in the spleen. Supported by grant ES006476 from NIEHS (NIH).