The Role of Cyclin C on Hematopoietic Cells.

The regulation of pluripotent hematopoietic stem cells (HSCs) is essential for the maintenance of multilineage blood production throughout life. The ability to self-renew or differentiate is a critical factor in the regulation of HSC numbers and cell fate, but so is the ability to enter and reside in a quiescent state (G0 phase of the cell cycle). The regulation of the G0 stem cell compartment is important for maintaining an inexhaustible pool of HSCs, protected from cellular stress. We have recently shown that the ETS transcription factor MEF is an important regulator of HSC quiescence (H. D. Lacorazza, Cancer Cell, 2006). The mechanism by which MEF or cell cycle regulatory proteins like p21 control entry into (and exit from) the G0 stage of the cell cycle is poorly understood. Insights into the mechanisms of HSC regulation could have important therapeutic potential.

Recently, a Cyclin C/cdk3 interaction was reported to play an important role in cell cycle re-entry from quiescent state (G0 to G1) by phosphorylating the retinoblastoma protein (Rb) in human fibroblasts (S. Ren, Cell, 2004). To elucidate if cyclin C is involved in the regulation of cell cycle in hematopoietic cells, especially human CD34+ HSCs, we have optimized a FG12 lentivirus based shRNA strategy (kindly provided by D. Baltimore, PNAS, 2002). shRNA directed against cyclin C successfully decreased both cyclin C mRNA and protein expression levels. Using this vector, we have consistently achieved a 90 % transduction efficiency and a 60–70 % reduction of cyclin C levels in human CD34+ cells as evidenced by real time PCR. Now we are analyzing the cell cycle and biological effects of cyclin C knockdown and overexpression on hematopoietic cells using both human CD34+ cells and human leukemic cell lines.