Retinoic acid–induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27Kip1

Abstract

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...

Regulation of the cyclin A1 protein is associated with its differential subcellular localization in hematopoietic and leukemic cells

“…In ATRA-treated cells, CDK1 appeared to be predominantly nuclear, although the total level of CDK1 declined in the treated cells ( Figure 2D). The decrease in the levels of cyclin A2 and CDK1 agreed with previous observations in which ATRA treatment resulted in cell cycle arrest in G1/S phase, leading to the downregulation of several cell cycle proteins including cyclin E and cyclin B1 in U-937 cells (Dimberg et al, 2002). To further confirm the nuclear versus cytoplasmic localization of cyclin A1, we performed immunostaining of untreated ( Figure 2E, panel a) and ATRA-treated U-937 cells ( Figure 2E, panel b).…”

“…For example, all-trans retinoic acid (ATRA) can induce clinical remission in patients with acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML) (He et al, 1999;Zhu et al, 1999). Certain leukemic cell lines respond to ATRA by switching from a state of uncontrolled proliferation, typical for malignant cells, to a state in which the cells undergo terminal differentiation like normal hematopoietic cells (Dimberg et al, 2002). It has been suggested that the role of ATRA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subcellular localization of several leukemiaassociated proteins such as PML and other nuclear domain-associated proteins (Koken et al, 1994;Weis et al, 1994, Faretta et al, 2001.…”

Signal transduction pathways that contribute to myeloid differentiation

“…STAT1 may contribute to ATRA-induced cell cycle arrest via regulation of cyclins, p27 Kip1 and c-Myc. 61,62 …”

Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2 and Cks1 proteins

“…Moreover, treatment of human embryonal carcinoma cells with all-trans-RA induced G 1 arrest through the accelerated ubiquitination of cyclin D1 (Spinella et al, 1999). Besides regulating the turnover of cyclins, retinoids were also shown to exert antiproliferative effects by modulating the levels of cyclin-dependent kinase (CDK) inhibitors, particularly of p21 WAF1/CIP1 (Liu et al, 1996;Li et al, 1998;Suzui et al, 2002) and p27 Kip1 (Pomponi et al, 1996;Zancai et al, 1998;Baldassarre et al, 2000;Hsu et al, 2000;Dow et al, 2001;Zhang et al, 2001;Dimberg et al, 2002). Nevertheless, the molecular mechanisms underlying these effects are still poorly defined.…”