Oncogene (2002) 21, 3403 ± 3413 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Cell cycle regulators and hematopoiesis Richard A Steinman*,1 1Department of Medicine and Pharmacology, E1052 BST, 200 Lothrop Street, Pittsburgh, Pennsylvania, PA 15213, USA The cell cycle behavior of hematopoietic cells varies from controls the cell cycle intersect with dierentiation extended quiescence to spectacular proliferation. Cell machinery? cycle regulators choreograph these transitions through variation in the makeup of cyclin-dependent kinase Cell cycle machinery (cdk)-containing complexes and through alteration in protein expression levels and subcellular localization. The The G1-S cell cycle stage represents a critical period mechanisms through which cell cycle regulators couple for cells to commit to growth arrest or proliferation. proliferation, dierentiation and survival is coming into It is during this stage that cells are responsive to sharper focus. Cdk-inhibitors, once thought of solely in cytokines. Once cells are committed to enter S phase, terms of a checkpoint function on cycling, are now additional stimulation by growth factors is super¯uous known to interact directly with proteins and pathways (Sherr, 1994). The following discussion therefore central to dierentiation and apoptosis. By shuttling concentrates on molecular regulators of the G0/G1-S between binding partners committed to discrete func- phase transition, with a particular emphasis on the tional pathways, cell cycle regulators may directly regulatory proteins p21/WAF1/CIP1 (p21) and coordinate proliferation with dierentiation, migration p27Kip1 (p27) that have been widely studied in and apoptosis. hematopoietic models. Oncogene (2002) 21, 3403 ± 3413. DOI: 10.1038/sj/ Both positive and negative regulators guide the onc/1205325 progression of cells into and through G1 phase (Figure 1). Distinct Rb-family-E2F repressor com- Keywords: hematopoiesis; quiescence; cyclin-dependent plexes prevent progression at dierent cell cycle p21 kinases; dierentiation stages ± E2F/p130 sustains cells in G0 phase (Grana et al., 1998; Vairo et al., 1995), whereas a Rb-E2F1 complex suppresses the transcription of genes required for progression through G1 phase phase (reviewed in Mayol and Grana, 1997). As Introduction cells proceed from G0 to S phase, cyclin/cyclin- dependent kinase (cdk) complexes are sequentially Hematopoiesis must be durable and ¯exible throughout activated (Dimri et al., 1996). Cell cycling is the lifespan of an animal. Durability requires a promoted by inactivation of the Rb-E2F1 complex population of stem cells, which continually renew the early in G1 by D-cyclin/cdk-4 or -6 complexes or, diverse pool of progeny in proportions appropriate to later in G1, by cyclin E-cdk2 complexes. Cyclin D their eector functions. Flexibility requires a pool of activity is mitogen-dependent, whereas cyclin E multipotent progenitor cells capable of spectacular activity is mitogen-independent. In hematopoietic proliferation under the spell of siren cytokines. Mature cells, the predominant D cyclins are D2 and D3. cells may be deaf to the proliferative signals of these Cyclin-cdk activity leads to Rb phosphorylation and cytokines yet respond to them with increased eector dissociation of Rb and E2F-1, which can lead to activity. It is likely that the diverse replicative status of activation of genes coding for proteins required for dierent cellular subsets in the marrow is sustained in S phase. The activity of cyclin-cdk's is opposed by part by intrinsic levels of cell cycle regulators, and in cdk-inhibitors (cdki's) of the INK4a class (p15, 16, part by modulation of certain of those regulators by 18, 19 ± inhibitors of cdk4 and 6) or the KIP/CIP environmental cues including cytokines and integrins. family (p21, p27 and p57) which are able to inhibit Drawing from this thesis, this review focuses on cyclins and cdks of multiple types but have greatest current research that illuminates the following ques- inhibitory activity against cdk2 complexes. The anti- tions: What cell cycle regulators primarily control stem proliferative activity of INK4 inhibitors is due in cell quiescence, progenitor replication and cell cycle part to their selective binding to cdk4 and cdk6 exit upon dierentiation? How does the circuitry that complexes and prevention of p21 and p27 seques- tration by those complexes. This results in greater p21 and p27 binding to cdk2, with resultant growth *Correspondence: R Steinman; E-mail: [email protected] arrest. Cell cycle regulator and hematopiesis RA Steinman 3404 Figure 1 Cell cycle control mechanisms. Selected pathways pertinent to cell cycle control in hematopoiesis are highlighted. Inhibition by cdki's prevents cyclin-cdk phosphorylation and dissociation of repressive Rb-E2F complexes The cell cycle status of hematopoietic stem cells IC) (Berardi et al., 1995). Moreover, it has been demonstrated that the LTC-IC activity of primitive An appreciation of the role of cell cycle regulators in cells is highest in G0 phase, and that cells which have hematopoiesis must consider the cell cycle status of cycled and re-entered G0 phase progressively lose their various hematopoietic cell subsets. Regulated expansion pluripotentiality, with little residual activity after three of a small stem cell subset is necessary both to sustain a cyclings (Gothot et al., 1997, 1998a). steady state level of mature blood cells and to upregulate The cell cycle phenotype of primitive cells capable of production in response to stresses such as infection or reconstituting hematopoiesis in mice is controversial, blood loss. These requirements demand either a stem cell however (Srour, 2000). It has been reported that pool capable of constant cycling without a loss of hematopoiesis can optimally be reconstituted by cells pluripotency or a larger population of quiescent stem in G0 phase (Gothot et al., 1998b), in either G0 or G1 cells with limited self-renewal capability that cycle phase (Wilpshaar et al., 2000), or by cells which have sporadically and unpredictably (the clonal succession undergone replication and subsequently entered G1 hypothesis). (but not G0) phase (Glimm and Eaves, 1999). Cells in Most data support a hierarchical organization of S/G2/ or M phase have minimal engraftment potential primitive hematopoietic stem cells (HSC) on the basis (Glimm et al., 2000). Conceivably, there may be a cell of quiescence. According to these studies, primitive cycle-dependence for homing to the bone marrow. hematopoietic cells in G0 phase of the cell cycle exhibit Hematopoietic stem cells (HSC) are functionally the highest stem cell activity (Morrison and Weissman, de®ned by the ability to generate long-term hematopoi- 1994). Two lines of in vitro evidence support this. Bone esis upon transplantation. Alternatively, HSC-enriched marrow cells resistant to cycling in response to c-kit blood subsets often are de®ned on an immunohisto- and IL-3 and surviving 5-¯uorouracil (5FU)-challenge chemical basis in terms of positivity for CD34+ are highly enriched for pluripotent cells capable of (human) or Sca-1 (murine) antigen expression. More generating hematopoietic colonies for prolonged peri- primitive subsets selected for the absence of lineage ods of time (long-term culture-initiating cells, or LTC- markers (CD387 or lin7) are further enriched for Oncogene Cell cycle regulator and hematopiesis RA Steinman 3405 reconstitution potential and reside to a higher degree in progression in HSC). If the cyclin D proteins are the G0 phase of the cell cycle (Leemhuis et al., 1996). produced, a natural question is why the cells are G0 cells are de®ned by 2N DNA content on Hoechst primarily in G0 phase and enter the cycle so slowly. staining and by low metabolic rates (pyronin, or Py-lo, One can envision that stochastic progression into the or Rhodamine, Rh-lo). Functional selection of candi- cell cycle depends on a matrix of expression levels of date stem cells exploits the quiescence of uncommitted, diverse cell cycle regulators. In this view, progression primitive cells by isolating cytokine nonresponsive cells, into the cell cycle depends on permissible combinations often through the use of suicide selection techniques of cell cycle activators and inhibitors aorded by the such as 5-¯uorouracil (5-FU) or 4-hydroperoxycyclo- (random or induced) ¯uctuation of individual protein phosphamide (4-HC) exposure. levels and activities (Gonze and Goldbeter, 2001; The appreciation that signi®cant proportions of Romond et al., 1999). immunophenotypically-isolated stem cell candidates are in G0 phase lent support to the view that the p21WAF1 and HSC's most primitive and pure stem cell populations remained out of the cell cycle, with successive, The cdki's are natural candidates for proteins that stochastic contribution of a portion of these cells to oppose the cell cycle progression of HSC's. The cdki hematopoiesis. Retroviral marking experiments (Capel p21WAF1 has been implicated as playing a predomi- et al., 1990) also lent support to clonal succession of nant role in maintaining hematopoietic stem cell stem cell activity, but have been disputed as an quiescence both in mouse and humans. In subsets of accurate model of steady-state hematopoiesis (Cheshier umbilical cord-derived blood cells enriched for HSC, et al., 1999). the expression of p21 increases concurrently with On the other hand, long-term in vivo BrDU labeling cellular quiescence and HSC phenotype. For instance, experiments indicate that in mice (Bradford et al., although human CD34+ cells are in G0/G1 phase, 1997; Pietrzyk et al., 1985) and to a lesser degree in almost all are negative for p21WAF1 protein expres- primates (Mahmud et al., 2001) hematopoietic stem sion. In contrast, p21WAF1 is expressed in a minority cells are constantly and slowly cycling. In mice of CD34+lin7 cells and is uniformly expressed in the continual BrDU administration demonstrated that 1% of lin7 cells that are cytokine-nonresponsive and Ho-lo/Rho-lo primitive HSC cycle with an average 5FU-resistant (Steinman et al., 1997). Cheng et al. turnover time of 30 days.