Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells.

Differentiation of murine erythroleukemia cells induced by hexamethylene bisacetamide (HMBA) is associated with accumulation of underphosphorylated retinoblastoma protein (pRB) and an increase in retinoblastoma (RB) gene expression. Here we show that HMBA causes a rapid decrease in the level of cyclin-dependent kinase 4 (cdk4) protein. This decrease results from decreased stability of the protein, while the rate of synthesis of the protein is not affected by HMBA. The decrease in the level of cdk4 protein is followed by suppression of the pRB kinase activity associated with cdk4. Cyclin D3, which can bind and activated cdk4, is increased in HMBA-induced cells and is found in complex with pRB and the transcription factor E2F. In uninduced cells cyclin D3 complexes with pRB and E2F are barely detected. At the later stages of differentiation, MEL cells become arrested in G1 and cdk2 kinase activity is suppressed; this is accompanied by a decrease in the level of cyclin A and cdk2 proteins. Cells transfected with cdk4, which continue to overexpress cdk4 protein during culture with HMBA, are resistant to HMBA-induced differentiation. In contrast, overexpression of cdk2 protein does not inhibit induced differentiation. These findings suggest that suppression of cdk4 is a critical event in the pathway leading to terminal differentiation of erythroleukemia cells.