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Experimental Cell Research 291 (2003) 282–288
Cell differentiation and proliferation—simultaneous but independent? Geoffrey Brown,a,* Philip J. Hughes,a and Robert H. Michellb a
Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, UK b School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK Received 18 June 2003
Abstract Despite studies over many years, it is still not clear to what extent cellular controls on proliferation and on differentiation are interrelated. For example, the idea that exit from cell cycle into G1/G0 is a necessary— or at least frequent—prelude to differentiation developed partly from studies of haemopoietic cell maturation, often using cell lines as models. The responses of cells to treatment with differentiating agents suggested that exit from cell cycle into G1/G0 occurs quite quickly, with functional differentiated characteristics acquired later, and so promoted the notion that cyclin-dependent kinase inhibitors (CDKIs) might be important initiators of normal differentiation. However, recent work has made it clear that differentiation and cell proliferation are regulated simultaneously but independently, that cells often start differentiating long before they stop dividing, and that the launching of differentiation is not restricted to any particular segment of the cell cycle. This combination of attributes allows expansion of cell numbers and acquisition of differentiated function to occur in parallel, generating abundant effector cells. Given this dichotomy, future studies to develop a more exact picture of the events that initiate and drive differentiation might be simplified by studying differentiation under experimental conditions that divorce it from concerns about cell cycle control. © 2003 Elsevier Inc. All rights reserved.
Increase in cell size, proliferation, differentiation, and apoptosis are behaviours available to all cells that have not terminally differentiated. It is often assumed that in any cell there is substantial coupling between the regulation of cell growth, proliferation, and differentiation, even though each process is to some degree independent of the others. A persistent notion has been that as many cells start to differentiate and acquire specialised functions they stop dividing—and they may move into G0 and lose the capacity for subsequent cell division [1–3]. This view assumes that there is some fairly tight coupling between differentiation, cessation of proliferation, and entry into G0 — even though it has long been known that Xenopus blastula cells can differentiate to muscle even while blocked in mitosis by colchicine or cytochalasin B . Similarly, it is commonly assumed that proliferating animal cells tend to maintain a fairly constant average size by mechanisms—like those in * Corresponding author. Division of Immunity and Infection, Medical School, University of Birmingham, Birmingham B15 2TT, UK. Fax: ⫹44121-414-3599. E-mail address: [email protected]
(G. Brown). 0014-4827/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4827(03)00393-8
yeast—in which multiple “checkpoints” sanction stepwise progression through the phases of the cell cycle only once appropriate threshold sizes are achieved. Recent work has contradicted this view [5,6]. To what extent, therefore, might there also be an illusory element to the purported links between regulation of the cell cycle and of differentiation? Below we summarise a body of information that argues against models of cell behaviour that posit some clear linkage between differentiation and progression through— or exit from— cycle. In general, there seems to be no obligatory coupling between cellular decisions to proliferate and to differentiate, even though the two behaviours are often controlled simultaneously.
The possible involvement of cyclin-dependent kinase inhibitors in initiating differentiation Cyclin-dependent protein kinases (CDKs), their regulatory cyclins, and CDK inhibitors (CDKIs) are primary regulators of the cell cycle [7,8]. Given the purported link between cell cycle exit and differentiation, the identification
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of CDKIs seemed to offer a useful starting point for identifying initiators of differentiation . CDKIs are of two types: INK4 proteins (p16INK4A, p15INK4B, and p19INK4C) interfere with cyclin D binding to CDK4 and CDK6 and so inhibit CDK activity [10,11]; and KIP family members (p21CIP1, p27KIP1, and p57KIP2) primarily inhibit CDK2 in vivo [12,13]. As an illustration of their effects, overexpression of several CDKIs in erythroid cells inhibits CDK2 or CDK4, which is followed by withdrawal of cells from cycle and differentiation . Overexpression of transfected p21CIP1 in the myelomonocytic cell line U937 provokes cell cycle arrest and the appearance of monocyte/macrophagespecific cell-surface markers . Results such as these gave credence to the idea that CDKI-driven exit from the cell cycle might be generally involved in initiating differentiation, but other data do not support this view. For example, U937 sublines that overexpress p16INK4A accumulate in G0/G1, but rather than differentiate they have an impaired capacity for D3-induced maturation . The myeloid cell lines U937 and HL60 differentiate to monocytes in response to 1␣,25-dihydroxyvitamin D3 (D3). The D3-treated cells also express p27KIP1, as do human CD34⫹ve haemopoietic progenitor cells undergoing spontaneous myeloid differentiation [17,18], and D3 transcriptionally induces p21CIP1 [15,19]. However, most of the CDK1 expression does not occur until days after the cells have started to acquire differentiated character [19,20]. In the light of such divergent observations, it is unlikely that CDKIs play any general role in controlling the onset of differentiation. These doubts are reinforced by the fact that mice in which genes encoding p16INK4A , p21CIP1 [22,23], or p27KIP1 [24 –26] are knocked out show no dramatic defects in tissue differentiation. It seems that no individual CDKI has any generally essential role in differentiation.
“Maturation divisions” accompany differentiation but are controlled independently During terminal differentiation, committed erythroid progenitor cells (CFU-E: colony forming unit— erythroid) and model cell lines such as murine erythroleukaemia (MEL) undergo ⬃5– 6 divisions before permanently leaving the cell cycle [27,28]. These maturation divisions are different from the cell cycles involved in “normal” cell proliferation. In chicken erythroid progenitors, for instance, the period needed to transit the cell cycle progressively shortens as the synthesis of haemoglobin and other erythrocytespecific proteins increases . Promyeloid cells undergoing monocytic differentiation in response to D3 traverse a similar series of rapid maturation divisions. D3-treated HL60 cells divide untypically fast for 2–3 days, probably as a result of shortening of the G1 phase of the cycle , and D3-treated U937 cells also display a transient burst of rapid proliferation . This
rapid proliferation of D3-treated myeloid cells is accompanied by functional activation of numerous genes and proteins linked to proliferation—including cyclins A, D1, and E, Ki67 antigen, ribosomal proteins L21 and S4, elevated CDK2/CDK5 activity, protein kinase C activation, upregulation of JNK and ERK2 MAP kinases (and downregulation of p38), and histone H3 phosphorylation [15,28,32–36]. Rather than causing growth arrest while it initiates differentiation, it seems that D3 provokes acceleration of HL60 cell transit through ⬃3 cell cycles — during which the cells differentiate to a substantial degree [30,37]. HL60 cells induced to differentiate to neutrophils in response to alltrans retinoic acid (ATRA) also traverse ⬃3 cycles before halting division, though in this case there is no shortening of the cycle period [30,37]. Recognition that cells induced to differentiate often also proliferate rapidly raises two immediate questions. What are the relative time courses of the two processes, and do the same or different controls regulate the concurrent proliferation and differentiation? In differentiating cells, what are the relative expression kinetics of early markers of differentiation and of correlates of proliferation? Early occurrence of the latter is documented above, with HL60 cells treated with D3 or ATRA multiplying ⬃8-fold in ⬃3 days [30,37]. mRNA array profiles of gene expression during myeloid differentiation induced by ATRA or D3 have made it clear that early steps in maturation are initiated within a few hours [38,39]. Differentiation-related genes that are expressed early include IL-8, ICAM1, granulocyte colony-stimulating factor receptor, macrophage colony-stimulating factor receptor, CD11a, and macrophage-inhibitory protein-1␣ [38,39]. Within the first 10 – 20 h, the cells both incorporate the thymidine analogue bromodeoxyuridine (BrDU) into DNA and coexpress the maturation marker CD11b and the proliferation marker PCNA [36,40]. This means that differentiation is well underway while cells continue to proliferate at an unrestrained rate: numerous gene products that contribute to mature myeloid function are expressed many hours, or days, before differentiating cells drop out of the cycle. To examine more closely the temporal relationships between control of the cell cycle and controls over myeloid differentiation, we used centrifugal elutriation to harvest cell populations at various stages of the cycle. The cells were treated with D3 or ATRA, and cell cycle progress and differentiation were monitored in parallel. When cells harvested in early G1 were treated with the differentiating agents, most were expressing CD11b before they reached the mitotic phase of their first maturation division [37,40]. HL60 cells can initiate maturation divisions only when they encounter D3 or ATRA during the first few hours of G1, indicating that the “switch” that redirects cells from unrestrained proliferation to a set program of terminal maturation divisions operates only during this brief cell cycle “window” [30,37] (summarised in Fig. 1B). By contrast,
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two types of results indicate that the same cells can launch differentiation to neutrophils or monocytes from widely separated points through much of the cell cycle [37,40,41]. First, the early kinetics of the induction of CD11b expression are similar in populations of HL60 cells that are stimulated with D3 or ATRA during G1, at the G1/S boundary, or during S phase : the cells do not have to reach some specific point in the cell cycle before expression can be induced. Secondly, cells remain capable of launching differentiation normally even when they have been halted in early G1, at the G1/S boundary or in S phase  (Fig. 1B). Given the very different regulatory behaviours of the launching of maturation divisions and of the initiation of differentiation, the controls on these two processes must be largely separate. Similar conclusions have emerged from analyses of the effects of insulin-related growth factor-1 receptor (IGF1R) signals on myeloid and adipocyte differentiation, in which IGF1 is a stimulant to both proliferation and differentiation. IGF1 promotes both proliferation and D3-primed monocytic differentiation in HL60 cells , with Erk1 and Erk2 kinase activities involved in maintaining cell proliferation during the initial proliferative phase of D3-induced monocyte differentiation of HL60 cells . Different IGF1generated signals seem to drive 34D murine myeloid cells into a burst of proliferation (signaled through IRS-1 phosphorylation, Type I phosphoinositide 3-kinase and p70S6K) and to neutrophil differentiate: overactivity of the mitogenic signals tends to extinguish the differentiation response [44 – 46]. Again, different IGF1 signals drive 3T3-L1 clonal expansion and adipocyte differentiation: Tyr-phosphorylation of c-Crk drives differentiation , and this continues normally even if clonal expansion, mediated through the Erk1 and Erk2 MAP kinase pathway(s), is delayed or prevented; and vice versa [47– 49].
“Uncoupling” differentiation from proliferation The information summarised so far establishes that a few rounds of proliferation often accompanies the commitment of progenitor cells to terminal differentiation. Are these maturation divisions essential for the cells to differentiate successfully, or might cells be able to differentiate without undergoing any cell cycle progression? It has been suggested that progressive remodeling of chromatin during these postcommitment cell cycles facilitates the sequential emergence of differentiated characteristics [50,51], which
would suggest that maturation divisions might be essential for successful differentiation. Also, that decoupling of these controls might prevent cells from differentiating properly, and could contribute to malignancy [1,52]. Although proliferation and differentiation of 3T3-L1 preadipocytes usually go hand in hand, successful adipocyte differentiation requires neither cell division nor DNA synthesis . Recent studies of HL60 cell differentiation yielded a similar conclusion. Fig. 1A shows that HL60 cells parked in early G1 by the K⫹ channel-blocking agent quinidine initiate differentiation to neutrophils (in response to ATRA) or to monocytes (in response to D3) normally . As noted earlier, and summarised in Fig. 1B, differentiation can also be initiated normally in HL60 cells that are halted at the G1/S boundary by thymidine or whose progression through S phase is greatly slowed by the DNA polymerase inhibitor aphidicolin [40,41]. Hence, differentiation can be initiated quickly in HL60 cells irrespective of whether they are permitted to proliferate normally or are arrested at various points in the cell cycle. This is in striking contrast to the control of maturation divisions in the same cells, which occurs only when the cells encounter the differentiating agent early in G1 [30,37] (Fig. 1B).
Similar patterns in immune cells Most peripheral blood T lymphocytes and B lymphocytes are out of cycle in G0, and the expansion and maturation of antigen-simulated clones are among classical situations that involve concurrent cell proliferation and differentiation . This probably serves to maximise the mature effector cell yield in vivo. Circulating T lymphocytes typically exist in a prolonged G0 state, and their interaction with antigen-presenting cells leads them to initiate several rounds of division. This selectively expands the number(s) of T lymphocytes responsive to the presented antigen(s), and the cells acquire effector functions [53–55]. Some workers have argued that these divisions are needed for the progeny to acquire effector functions (such as interferon-␥, interleukin-4, and interleukin-10 production) [56 –58], whereas others have offered evidence that IFN␥ synthesis can be initiated without cells dividing— or even leaving G1 [59,60]. One compelling study used transfection of human peripheral blood T lymphocytes with TAT-p16INK4A to prevent the initially quiescent cells from leaving G0, a process that would normally start within ⬃4 h of the addition of a polyclonal stimulus
Fig. 1. In HL60 cells, relationships between the cell cycle and the launching of differentiation and between the cell cycle and the launching of maturation divisions are quite different. (A) HL60 cells “parked” in G1 differentiate efficiently. Early G1 cells, isolated by centrifugal elutriation, were held in G1 by treatment with 0.2 mM quinidine and treated with differentiating agents: 500 nM ATRA (to neutrophils; CD11b positive/CD14 negative, at left) or 500 nM D3 (to monocytes; CD11b positive/CD14 positive, at right). The results shown are from cells treated with ATRA or D3 for 4 days: and most cells (50 –70% with ATRA; ⬃80% for D3) were CD11b positive within 1 day (from Brown et al. ). (B) A summary of those parts of the cell cycle from which differentiation and/or maturation divisions can be initiated. Launching of maturation divisions is prohibited after mid-G1 [30,37]. No prohibitions on differentiation have yet been defined [37,40].
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(anti-CD3/anti-CD28 or PMA/ionomycin). Even though the TAT-p16INK4A-transfected cells were held in G0 and prevented from increasing in size, they still responded to PMA/ ionomycin with a normal pattern of induction of several effector/marker molecules . Activated B lymphocytes also undergo substantial clonal expansion as they mature . Once again, it has been argued that the sequential acquisition of more mature B cell characteristics (in terms, for example, of immunoglobulin isotype switching and secretion, and adoption of memory cell character) depends on the number of divisions the cells complete [63–70]. However, a recent study suggests that B lymphocyte proliferation and differentiation are two sets of concurrent events that are independently controlled. Specifically, the B lymphocyte antigen receptor (surface immunoglobulin in complex with signalling elements) appears to use independent signals to regulate cell division and immunoglobulin isotype switching . Concluding comments The succession of activities that occurs through the various phases of the cell cycle maps out the “timetable” followed by proliferating cells. So to what extent might the cycling of cells — or at least their admission to particular cell cycle phases— be necessary for the correct control of other complex cell processes such as increase in size, differentiation, or apoptosis? The main conclusion from the studies discussed above is that differentiation is generally not coupled in any tight manner either to any aspect of the proliferative cycling of cells or to cycle exit. Rather, differentiation and cell cycle seem to be separate, but often concurrent, processes that are regulated independently and simultaneously—though always with a possibility of crosstalk between their controls in specific situations. Cell models are now available, principally for myeloid cells, in which differentiation proceeds even when division is prohibited and cells are held in one or other phase of cycle. Detailed examination of models of this type, ideally employing a variety of cell-types, should help future studies to pinpoint the particular cellular controls that are most central to the regulation of differentiation and also, for multipotent progenitor cells, the controls that govern lineage diversification. References  L. Sachs, Constitutive uncoupling of pathways of gene expression that control growth and differentiation in myeloid leukemia: a model for the origin and progression of malignancy, Proc. Natl. Acad. Sci. USA 77 (1980) 6152– 6156.  R. Maione, P. Amati, Interdependence between muscle differentiation and cell-cycle control, Biochim, Biophys. Acta 1332 (1997) M19 – M30.  K. Walsh, H. Perlman, Cell cycle exit upon myogenic differentiation, Curr. Opin. Genet. Dev. 7 (1997) 597– 602.
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