Regulation of the ubiquitin proteasome pathway in human lens epithelial cells during the cell cycle.

Most proliferating cells follow a series of orderly transitions from one phase to another. These transitions are usually controlled by timed degradation of cell cycle regulators by the ubiquitin-proteasome pathway (UPP). There are no published reports regarding the timing of phases of the human lens cell cycle or regarding cell cycle-related changes in UPP components. Objectives of this study were to characterize the timing of the phases of the human lens epithelial cell cycle and to explore potential functions of critical components of the UPP in controlling lens cell cycle. Human lens epithelial cells were synchronized at G0/G1 phase by contact inhibition. Cell cycle progression upon subculturing was monitored by FACS analysis. It took approximately 40 hr for HLEC to complete one cell cycle, approximately 20 hr for G1 phase, approximately 8-10 hr for S phase and approximately 10 hr for the combination of G2 and M phases. Proteasome-dependent degradation of p21WAF and p27Kip, the dominant Cdk inhibitors, was associated with the G1/S phase transition in these cells. Proteasome inhibition experiments indicate that proteolysis is the predominant process which is responsible for the variations in these regulators during the cell cycle. Levels of specific ubiquitin conjugating enzymes, Ubc7 and Ubc10, increased 6 and 2-fold at the G2/M phase and S/G2/M phases, respectively. Levels of these E2s decreased precipitously upon completion of the M phase. In contrast, levels of ubiquitin activating enzyme (E1) and Ubc3 remained constant during the cell cycle. Cul1, a component of the SCF (an E3), remained relatively constant during cell cycle. The up-regulation of Ubc7 and Ubc10 during the G2/M and S/G2/M phases suggests that these enzymes may be involved in controlling the cell cycle progression at this phase. Taken together, the data indicate that expression of key components of the UPP in the human lens epithelial cells is regulated in a cell cycle-dependent manner. Some of the variations in levels of ubiquitin conjugating enzymes are suggestive of previously undescribed functions.