IntroductionHematopoietic tumors often arise as a consequence of uncontrolled proliferation of immature blasts, failing to terminally differentiate into mature blood cells. 1 A hallmark of terminal differentiation is an irreversible arrest in the G 0 /G 1 phase of the cell cycle. This arrest involves the coordinate regulation of signals that negatively control the cell cycle machinery and inhibit the G 1 /S transition. At the heart of the cell cycle lies the regulation of cyclin-dependent kinases (CDKs), which control the phosphorylation of several substrates, including retinoblastoma protein (pRb) family members, and thus the transcriptional activation or repression of E2F target genes important for cell cycle progression. [2][3][4] The G 1 /S transition is believed to be sequentially controlled by D-type cyclins, which activate CDK4 and CDK6, and subsequently by cyclin E, which associates with CDK2. 5 CDK activity is dependent on association with cyclins and subsequent phosphorylation-dephosphorylation events, and may be inhibited by 2 different groups of CDK inhibitors (CKIs), namely the Ink4 family and the Cip/Kip family. 6 Thus, growth arrest associated with differentiation could be achieved by several mechanisms including the down-regulation of cyclins or up-regulation of CKIs or both. The critical events leading to a cell cycle arrest are, however, likely to be specific for each mode of induction of differentiation and may differ for each particular cell type.Proliferation and differentiation of hematopoietic cells can be regulated by a number of physiologic agents, including all-trans retinoic acid (ATRA). ATRA treatment triggers terminal differentiation and growth arrest of several established human myeloid cell lines in vitro and has also proven to be effective in the clinical treatment of acute promyelocytic leukemia (APL) by inducing differentiation and apoptosis of the immature blasts. 7,8 Although the biologic effects of ATRA are well characterized, the molecular mechanisms regulating these processes are largely unknown. The cell cycle arrest associated with ATRA-induced differentiation may involve regulation of the expression of both cyclins and CKIs, affect phosphorylation status of CDKs, and ultimately trigger dephosphorylation of pRb pocket proteins. However, although previous studies on ATRA-induced growth arrest of myeloid cells have implicated individual proteins, for example, p21 WAF1/CIP1 , 9-11 a more complete investigation of the regulation of the cell cycle machinery is required to understand the relative importance of different events during the differentiation process.To gain further insight into the ATRA-induced events leading to G 0 /G 1 arrest during terminal differentiation of human myeloid cells, we have performed a comprehensive analysis of the regulation of CKIs, cyclins, CDKs, and pRb pocket proteins during terminal differentiation. The well-characterized human U-937 cell line 12 was chosen as a model system. This established model of monocytic differentiation can be induced t...
