Abstract
Cadmium (Cd) is a ubiquitous environmental heavy metal that has been classified as a human carcinogen. Deregulation of cell cycle progression is an important issue to cause cancer. Previous studies indicate that Cd causes mitotic arrest and decreases replicative DNA synthesis. However, the effect of Cd on molecules regulating G1 cell cycle progression remains unclear. In this thesis, we explore whether Cd affects G1 cells entering S phase at molecular levels using synchronized human non-small lung carcinoma cells, CL3. CdCl2 (40 mM, 2 h) markedly delayed S phase progression of synchronous cells at the G1/S border derived from aphidicolin procedure. Cd also delayed G1 progression of cells synchronized at G1 phase derived from either the nocodazole release procedure or the counterflow centrifugal cell elutriation system. Using western blotting and immunocomplex kinase activity analysis techniques, we have observed that Cd significantly delayed RB phosphorylation, down-regulated the protein levels of Cdk4, and decreased cyclin D1- and cyclin E-associated kinase activity in elutriated G1 cells; however, the protein levels of cyclin D1 did not altered. Furthermore, exposure these elutriated G1 cells to Cd also markedly delayed the induction of cyclin A expression, and cyclin A-associated kinase activity; however, the protein levels of Cdk2 were unchanged. On the other hand, Cd induced p38 MAPK phosphorylation and p21WAP1 protein levels, but not phospho-p53 (Ser15) in elutriated-G1 cells. SB202190, an inhibitor of p38 MAPK, delayed G1 progression of untreated or Cd-treated elutriated-G1 cells, elevated phospho-p53 (Ser15) while decreased p21WAP1 levels. Taken together, these results suggest that Cd delays RB phosphorylation by decrease Cdk4 protein levels, and cyclin D1- and cyclin E- associated kinase activities, thereby further slow-down the expression of cyclin A and cyclin A associated kinase kinase activity to delay G1 to S progression. Additionally, the activated-p38 MAPK may result in decreased phospho-p53 (Ser15), which may allow damaged cells bypass G1 checkpoint.