Cyclin D1 downregulation is important for permanent cell cycle exit and initiation of differentiation induced by anchorage-deprivation in human keratinocytes.

To understand the relationship between permanent cell cycle exit and differentiation the immortalized keratinocyte cell line, SIK and the squamous cell carcinoma, SCC9 were compared during differentiation induced by anchorage-deprivation. The SIK cells when placed in suspension culture promptly lost almost all ability to reinitiate growth by 2 days concomitantly expressing the differentiation specific proteins, transglutaminase (TGK) and involucrin. These cells rapidly underwent G1 cell cycle arrest with complete disappearance of phosphorylated RB. In contrast SCC9 cells neither showed TGK expression nor increase in involucrin. They decreased their colony-forming ability much more slowly, which coordinated well with a gradual decrease in phosphorylated RB, demonstrating the significant resistance to loss of colony-forming ability and cell cycle exit. In accordance, cyclin D1, a positive regulator of cyclin-dependent kinase (CDK) 4/6 which phosphorylates RB decreased drastically in anchorage deprived SIK but not in SCC9 cells. Endogenous cyclin D1 knockdown in SCC9 cells by siRNA enhanced loss of the colony-forming ability during anchorage-deprivation. Conversely enforced expression of cyclin D1 in SIK cells and in another immortalized keratinocyte cell line, HaCaT, partly prevented loss of their colony-forming abilities. Cyclin D1 overexpression antagonized Keratin 10 expression in suspended HaCaT cells. The result demonstrates the importance of cyclin D1 down regulation for proper initiation of keratinocyte differentiation.