Stem Cells, Vol. 19, No. 6, 483-491,
November 2001
© 2001 AlphaMed Press
Steel Factor Regulates Cell Cycle Asymmetry
Charlie Mantel,
Paul Hendrie,a,
Hal E. Broxmeyer
Departments of Microbiology/Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, and the Walther Cancer Institute, Indianapolis, Indiana, USA;
a Current address: Department of Medicine, Division of Hematology, University of Washington, Seattle, Washington, USA
Key Words. Stem cell asymmetry • Stem cell factor • P27kip-1 • Cell cycle heterogeneity
Charlie R. Mantel, Ph.D., Walther Oncology Center, 1044 West Walnut Street, Indianapolis, Indiana 46202-5121, USA. Telephone: 317-274-7550; Fax: 317-274-7592; e-mail: cmantel{at}iupui.edu
 |
ABSTRACT
|
|---|
Asymmetric segregation of cell-fate determinants during mitosis (spatial asymmetry) is an essential mechanism by which stem cells are maintained while simultaneously giving rise to differentiated progenitors that ultimately produce all the specialized cells in the hematopoietic system. Temporal cell cycle asymmetry and heterogeneity are attributes of cell proliferation that are also essential for maintaining tissue organization. Hematopoietic stem cells (HSCs) are regulated by a complex network of cytokines, some of which have very specific effects, while others have very broad ranging effects on HSCs. Some cytokines, like steel factor (SLF), are known to synergize with other cytokines to produce rapid expansion of progenitor cells. Using the human growth factor-dependent MO7e cell line as a model for synergistic proliferation, we present evidence that links proliferation asymmetry to SLF synergy with GM-CSF, and suggests that temporal asymmetry and cell cycle heterogeneity can be regulated by SLF in vitro. We also show that CDK-inhibitor and cell cycle regulator, p27kip-1, may be involved in this temporal asymmetry regulation. We propose that SLF/GM-CSF synergy is, in part, due to a shift in proliferation pattern from a heterogeneous and asymmetric one to a more synchronous and symmetric pattern, thus contributing dramatically to the rapid expansion that accompanies SLF synergy observed in MO7e cells. This kinetic model of asymmetry is consistent with recent evidence showing that even though SLF synergy results in a strong proliferative signal, it does not increase primary HSC self-renewal, which is believed to be highly dependent on asymmetric divisions. The factor-dependent MO7e/SCF- synergy/asymmetry model described here may therefore be useful for studies of the effects of various cytokines on cell cycle asymmetry.
|
INTRODUCTION
|
|---|
The fundamental principle underlying development of all multicellular organisms with diverse cell types is the ability of primitive pluripotent cells to multiply and segregate cell-fate determinants to give rise to the vast array of specialized somatic cells found in these organisms [1–3]. This process follows certain cues that are both extrinsic (i.e., cytokines and accessory cells) and intrinsic (i.e., telomere length, stochastic factors) [2,4]. These processes continue in fully developed adult organisms because of the need for replacement cells in certain tissues that are frequently damaged or otherwise die because of the nature of their specialization. This is perhaps more evident in blood than in any other tissue. Hematopoietic stem cells (HSC) must maintain a steady supply of highly differentiated and specialized cells while also maintaining a renewing pool of pluripotent stem cells. Recent evidence demonstrates that HSCs, as well as many other primitive stem cells, accomplish this in large part by the ability to asymmetrically segregate certain cell-fate determinants during cell division, thus giving rise to two cells with very different proliferative and differentiative fates; i.e., one daughter cell remains a stem cell (self-renewal) while the other becomes a differentiated progenitor destined to give rise to a pool of specialized cells [3,5]. This "spatial stem cell asymmetry" during mitosis is therefore crucial to both development and maintenance of blood cells [6,7].
There is also another type of asymmetry during cell division that is equally crucial for development and maintenance, but which is frequently overlooked; this is temporal asymmetry or cell cycle heterogeneity [8–11]. It has been known for decades that proliferating populations of cells in vitro and in vivo have considerable heterogeneity in cell cycle kinetics [8,10]. Most of this variation arises during the G1 phase [8,10,12]. This is the reason that an artificially synchronized population of cells will very rapidly (usually within the first or second generations) once again become asynchronous. This natural tendency toward asynchrony is essential for stable tissue formation in multicellular organisms because synchronous proliferation would cause tremendous disruption in tissue organization. The molecular mechanisms involved in this temporal asymmetry are unknown.
The proliferation and differentiation of HSCs are under very tight control of numerous hematopoietic cytokines. An important cytokine is steel factor (SLF), which is believed to have an essential role in the regulation of primitive HSCs and progenitor cells [13-15]. SLF is the prototype cytokine of a small group of hematopoietic growth factors that can synergize with other selected growth factors to induce a replicative burst of mitosis much greater than the sum of the effects of either cytokine alone. Its use clinically, for example, ex vivo HSC expansion, and other uses are under investigation [16]. However, the fundamental molecular mechanisms leading to its synergistic properties remain largely unknown.
During our investigations of the intracellular mechanisms of SLF proliferative synergy with GM-CSF using an MO7e cell line as a model of stem cell factor (SCF) synergy [17–20], we discovered growth patterns, described herein, that may link SLF-induced synergy to regulation of temporal asymmetry via the cell cycle threshold regulator, p27kip-1. These data suggest that an important relationship between spatial and temporal asymmetry exists in this cell line and that these attributes might be regulated by certain combinations of cytokines. Therefore, while the MO7e cell line is not a proven model of HSC proliferation, it remains a useful model of cytokine-induced proliferation and now also as a model of cytokine effects on cell cycle symmetry.
|
METHODS AND MATERIALS
|
|---|
Cells
MO7e cells were obtained and maintained as previously described [21]. To synchronize cells in the G0/G1 phase of the cell cycle, they were incubated in growth factor-free and serum-free medium for 18 hours as described [20].
Cell Cycle Analysis and Flow Cytometry
Cell cycle phases were assessed by DNA content [22,23] by suspending cells in 200 µl of propidium iodide solution (0.3% saponin, 20 µg/ml propidium iodide, 0.2 mg/ml RNAse-A, 1% bovine serum albumin, and 0.5 mM EDTA, ph 7.2; all from Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com) for 15 minutes at room temperature. Fluorescence intensity was measured using a FACScan flow cytometer (Becton Dickinson; San Jose, CA; http://www.bd.com) and analyzed using Cell Quest software (Becton Dickenson) and ModFit software (Verity Software House; Topsham, ME; http://www.vsh.com). PKH26 (Sigma) labeling was done according to the manufacturer's instructions, fluorescence intensity was measured by flow cytometry, and the generational analysis was done using a kinetic model program (Sigma) designed for the ModFit program. Cell sorting of various PKH26-labeled populations was done with a FACStar Cell Sorter (Becton Dickinson).
Western Blotting
SDS-PAGE, immunoblotting, and chemiluminescent labeling were performed as previously described [20].
|
RESULTS
|
|---|
SLF Synergy Cannot Be Explained Solely by a Decreased Cell Cycle Time
We have previously used the MO7e cell line as a model of synergistic cell proliferation and to investigate the molecular mechanism of SLF synergy [17–20]. During these studies, we performed cell cycle analysis of MO7e cultures stimulated with GM-CSF or SLF alone or with the synergistic combination of the two (SC). Table 1
shows the cell cycle/DNA distribution of MO7e cells grown under these various conditions. There was an obvious increase in the percentage of cells in S and G2/M phases at the expense of G0/G1 phase, when cells were stimulated with the SC. This result suggested that the time required to progress from quiescence into active cycling was less for the SC than for the single cytokines. However, an alternative possibility is that the proportion of cells "recruited" into cycle from quiescence was greater for the SC, while the kinetics remain essentially the same. We therefore investigated the average interdivision time (IDT) of these cultures using the established technique of stathmokinetic analysis [24]. These data (Fig. 1) clearly indicated that MO7e cells responding to the SC had an average IDT that was shorter than those with the single cytokine. However, this technique makes the assumption that the "growth fraction" is 100%, an assumption that could be very misleading. We reasoned that if the average IDT was indeed shorter, then we should be able to see this difference between SC and single cytokine stimulation when factor-starved cells emerge from quiescence, and transit G1 phase into S phase, as measured by 3H-thymidine incorporation. Figure 2 shows that the earliest 3H-thymidine incorporation after stimulation of factor-starved MO7e cells occurred between 10 and 12 hours, which was the same irrespective of the cytokine used. The convergence of these indices (i.e., no difference in lag time between SC and GM-CSF alone) suggests that the average G0/G1 transit time was similar for SC and the single cytokine and that the proportion of cells responding was different. Therefore, SLF-induced synergistic proliferation cannot be explained solely by a shortened average IDT, but rather that the proportion of quiescent cells recruited into cycle is also involved.
View this table:
[in this window]
[in a new window]
|
Table 1. Cell cycle distribution of MO7e cells proliferating in response to GM-CSF, SLF, and the synergistic combination of GM-CSF plus SLF (SC)
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. Time to reach S-phase for MO7e cells stimulated with GM-CSF, SLF, or the SC. Factor-starved MO7e cells were stimulated as in Table 1 in 96-well plates. At time zero, 1uCi 3H-thymidine was added to each well and incubated for the indicated times, then harvested and the amount of incorporated 3H-thymidine was measured as previously described [21]. Results are representative of six separate experiments.
|
|
SLF also Regulates Population Cell Cycle Synchrony
To gain further insight into the nature of these growth patterns, and to measure the degree of cell cycle variation and heterogeneity, we considered following individual MO7e cells by microcinematography. However, this approach is considerably complicated by the fact that MO7e cells are not adherent. We therefore resorted to a unique alternative experimental design to determine cell cycle heterogeneity, which is explained below.
There is a body of literature that describes the kinetics of cell proliferation variation, which details the mathematical concepts used to quantitate this variation [8–10]. By coupling these older principles with the newer techniques of fluorescence membrane labeling and flow cytometry, we developed methods that allow measurement of IDT heterogeneity and generational diversity in in vitro cultures of MO7e cells. These mathematical concepts are illustrated in Figure 3. In panel A, a plot of the percent of a population of cells in culture that have not yet divided (
) versus time is shown. The solid line illustrates the results for a perfectly synchronized culture, i.e., all cells divide simultaneously. The dashed line describes an asynchronously dividing population. It was discovered that by plotting the natural log of versus time, the asynchronous relationship to time becomes linear, the slope of which is proportional to what is referred to as a "probability of transition" (Tp) [8,10]. This term, in effect, describes the population variation or heterogeneity of IDTs, which we refer to as Vc. The greater the slope, the less variation in population IDT; i.e., more synchronous proliferation.
View larger version (10K):
[in this window]
[in a new window]
|
Figure 3. Mathematical analysis of IDT variation and heterogeneity. A) A plot of the percent of cells in a population that has not yet divided versus time for a synchronized and asynchronous growth pattern. B) Linear transformation of the data illustrated in panel A and the relationship of slope of this line to IDT variation. Tp = transition probability; Vc = variation of cell cycle time (see text for details).
|
|
We labeled factor-starved MO7e cells with the nondegradable membrane fluorescent label, PKH26 [25,26], and stimulated them with GM-CSF or the SC of GM-CSF plus SLF and measured the amount of fluorescence heterogeneity by flow cytometry and calculated the generational diversity using a proliferative model program (Sigma) designed for the ModFit analysis program. Since PKH26 permanently labels the cell membrane, its fluorescence intensity will decrease (in theory by one-half) each time the cell divides, and the fluorescence intensity will be proportional to the number of times the cell has divided since being labeled (generation number). Thus, flow-cytometric analysis of PKH26 allows determination, at a single-cell level, whether or not a cell has divided since being labeled (allowing determination of ""), and if so, how many times it has divided since being labeled (allowing determination of generational heterogeneity). No consideration was given to differentiation when interpreting PHK26 levels since MO7e cells do not differentiate in vitro. Figure 4A shows the generational profile of MO7e cells stimulated for 3 days with GM-CSF alone, or with the SC of GM-CSF plus SLF. As would be expected for a population with a shorter IDT, and as suggested by the data in Table 1 and Figure 1, there are more total cell divisions and more generations for cells stimulated with SC compared to GM-CSF alone. This is consistent with the stathmokinetic analysis in Figure 1A. However, generation number one represents the fraction of cells not yet divided (), and results from three experiments (Fig. 3B) show that was approximately fourfold lower for the SC compared to GM-CSF alone. This is consistent with the findings of Figure 3B that there are more cells recruited into cycle from quiescence over time by the SC.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 4. Cell cycle heterogeneity in MO7e cultures proliferating in response to GM-CSF, SLF, or the SC. A) Factor-starved MO7e cells were labeled with PKH26 according to the manufacturer's instructions (Sigma), then stimulated with GM-CSF, SLF, or the SC as in Table 1 for 3 days, then cells were harvested and generational analysis was performed as outlined in the Materials and Methods section. The percent of the total population for each generation (relative frequency histogram) is shown. B) Statistical analysis of "" measurements from the average of three separate experiments, mean ± SD is shown, * = p < 0.05. C) The natural log (ln) of(generation 1 population, i.e., cells that have not yet divided) was plotted versus time for the indicated time points after factor starvation and stimulation as in Table 1. A linear regression line was constructed, and the mean slope ± SD from three separate experiments is shown.
|
|
Importantly, when the -versus-time plot is constructed from the data in Figure 4A at different time points, it was found that Vc was nearly twice as large for the SC compared to GM-CSF alone (Fig. 4C,D). Thus, there was a significant decrease in the population IDT variation. In other words, SLF-induced synergistic proliferation is characterized by a shortened average IDT plus a more synchronized recruitment from quiescence and a more synchronous proliferation pattern (i.e., less temporal asymmetry).
Cellular p27kip-1 is Higher in Cells with Delayed Recruitment from Quiescence
Even though the natural tendency of cell populations and tissues to become and remain asynchronous has been long known [8], any aggressive attempt to understand the molecular mechanism of this behavior has been thwarted by a lack of techniques to easily separate and enrich large numbers of cells from specific parts of the IDT spectrum to analyze their molecular contents. The technique of fluorescent membrane labeling coupled to cell sorting has now allowed us to enrich for a population of cells with a very long IDT (compared to the population average) from an asynchronous cell culture and to subject them to conventional biochemical analysis. We have previously associated the cell cycle regulator, p27kip-1, with SLF synergy [20], by showing that p27kip-1 is lower in cultures stimulated with SCF plus GM-CSF compared to levels in cultures stimulated with GM-CSF alone. Because p27kip-1 has profound effects on cell cycle timing and tissue organization [27–29], we first investigated the content of p27kip-1 in enriched long IDT populations by immunoblotting. We used MO7e cultures stimulated with GM-CSF alone for these sorting experiments because the proportion of cells that have not yet divided is greater under these conditions (Fig. 4), and therefore it is easier to obtain the large numbers of cells needed for biochemical analysis. Figure 5 shows that p27kip-1 co-enriches in the long IDT cells, and is significantly higher in cells that take a long time to divide compared to the average of unsorted starting population cells. In other words, if you increase the proportion of cells in the culture that are taking a long time to divide (as indicated by no change in PKH26 fluorescence) and analyze p27kip-1 in this sorted population, then compare these to p27kip-1 levels analyzed in the starting, unsorted population, you will find p27kip-1 levels are higher in the sorted population. Therefore, p27kip-1 levels must be higher in the population of cells that take longer to divide compared to the population average. This suggests an important link between asymmetric segregation of cell cycle regulators and temporal asymmetry. This is consistent with our previous findings that SLF synergy was associated with decreased levels of p27kip-1 [20], and is also consistent with the gross organomegally and tissue disruption seen in p27kip-1 knock-out mice (i.e., disruption of temporal asymmetry by p27kip-1 loss causes severe tissue disruption) [27–29].
View larger version (25K):
[in this window]
[in a new window]
|
Figure 5. Intracellular p27kip-1 content is increased in cells with a long IDT. Long IDT cells were sorted and enriched from MO7e cultures stimulated with GM-CSF alone as described in the text (the upper 5% of the cell population with the greatest PKH26 content was considered to be long IDT cells), and Western blots of p27kip-1 were done as previously described [20] and compared to unsorted cells. Equal cellular protein was loaded onto each lane. P27kip-1 specific band intensity was measured as described [20] and the mean ± SD from three separate experiments is shown. * = p < 0.05.
|
|
Together, these data indicate that SLF synergy in the model cell line, MO7e, results in a highly symmetrical proliferation pattern in addition to shortened cell cycle duration, and this is associated with lowered intracellular levels of the cell cycle regulator, p27kip-1.
|
DISCUSSION
|
|---|
Previous investigations by us have been able to exclude some potential intracellular mechanisms for SLF synergy such as receptor number and affinity modulation [21], and have also been able to implicate a few signal transduction pathways that lead to synergistic proliferation [17–20]. SLF synergy has also been attributed in part to survival properties of this cytokine [30]. However, despite the efforts of numerous groups, the principal mechanisms have remained elusive. Nonetheless, it is important to understand these mechanisms because of the great potential clinical benefit of controlling primary HSC proliferation and differentiation in vitro with this cytokine. In this study, we present evidence suggesting a new and potentially important and useful clue into the basic nature of SLF synergy. At the same time, these studies help to reinvigorate a nearly forgotten, but important, area of cell biology by linking the comparatively recent studies of SLF proliferative synergy to the more antiquated concepts of cell cycle heterogeneity. Furthermore, we have introduced the new, but simple, technique of sorting and enriching "long-IDT" cells so that the molecular contents and properties of this unique cell population can, for the first time, be studied and compared to the unseparated population. Thus, the molecular mechanisms of temporal asymmetry and cell cycle heterogeneity can be addressed. Using these methods, we have, through association, suggested a potential role for the cell cycle regulator, p27kip-1, in regulating temporal asymmetry. These data suggest that this CDK inhibitor is asymmetrically segregated or expressed in cell populations such that it is greater in cells that will ultimately take a long time to enter and transit the cell cycle. This is consistent with the established role of p27kip-1 as a cell cycle "threshold regulator" and its role in maintaining cells in a quiescent state [31].
In order to visualize how such a seemingly simple conclusion as is reached from the analysis in Figure 4C and D (i.e., synergistic proliferation patterns are more symmetrical) can potentially explain such a large "burst" in proliferation as is observed during cytokine synergy, it might be useful to consider the simplified illustration in Figure 6. If the IDT of a proliferating group of MO7e cells is shortened in a symmetrical way (panel B) by a certain particular cytokine combination, then many more cells will arise in the culture in the same time than would arise with a cytokine combination that acts to shorten the IDT in an asymmetrical way (panel A). The proliferation pattern illustrated in Figure 6A is similar to that described by Smith and Martin [10], and typifies that which is found throughout nature. It is believed to be the underlying cause of the so-called "right-hand skew" of IDT histograms found in proliferating populations of cell types ranging from prokaryotes to complex multicellular organisms. This large variation in IDT is the kinetic source of cell cycle heterogeneity. This is illustrated in Figure 7 (solid line). We envision the histogram produced by a synergistic and symmetrical proliferation pattern to be more like that indicated by the dashed line in Figure 7, where the average IDT is shortened, but there is the added attribute that, because the IDT distribution is more symmetrical, the proliferating fraction (i.e., the growth fraction) at any one time is much larger. This could further compound the effects described in Figure 6 and therefore lead to an even greater and more rapid increase in cell number (i.e., a "burst" of proliferation).
View larger version (15K):
[in this window]
[in a new window]
|
Figure 6. Illustration of synchronous (A) and asynchronous (B) cell proliferation patterns. IDT is represented by arrow length. See text for details.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
Figure 7. Illustration of a relative frequency histogram of cell population IDTs. The solid line represents an asynchronous and asymmetrical proliferation pattern, and the dashed line represents a shorter IDT, plus a more symmetrical and synchronous proliferation pattern.
|
|
It is perhaps not surprising to find that an important cell cycle regulator such as p27kip-1 is involved in temporal asymmetry; especially since cell cycle checkpoints have already been implicated in spatial asymmetry [32,33]. It now appears likely that these same checkpoint regulators may also be involved in regulating temporal asymmetry and cell cycle heterogeneity. Nonetheless, we believe that this is the first description of the "molecular nature" of cell cycle temporal heterogeneity. Obviously, many more questions now arise, such as the significance of temporal asymmetry in tissue and organ development, whether it has any influence on spatial asymmetry and also if these effects are relevant to primary HSCs. Also, are there any potential benefits to manipulating these types of proliferation patterns in primary cells? Does a reduced temporal asymmetry imply that there is also reduced spatial asymmetry, and therefore less cell fate determinant segregation? Could this lead to more differentiation and less self-renewal? One very interesting connection between the data presented here and other recent investigations of primary human HSCs is that SLF has been shown to provide a strong proliferation stimulus but does not simultaneously increase the number of self-renewing divisions [34]. This fits well with the notion that SLF-induced reduction of temporal/spatial asymmetry could result in less self-renewal. It has been documented that as many as 40% of isolated single human HSCs will divide in vitro to produce one daughter cell that undergoes rapid proliferation, while the other remains quiescent for various periods up to at least 8 days, even in the presence of numerous cytokines [26,35,36]. Blast colony formation and self-renewal were highly associated with the fraction of cells undergoing these asymmetric divisions [26]. Therefore, there appears to be a causal relationship between asymmetric cell division and HSC self-renewal. However, these same studies indicated that asymmetry was regulated by cell-autonomous, intrinsic factors, and its prevalence was the same regardless of the cytokine combination that was used to induce proliferation. Our findings suggest that the temporal form of asymmetry may indeed be modifiable by cytokines in our model cell line. Model cell lines are useful for molecular studies where large numbers of cells are required. By combining single-cell techniques, with molecular analysis of enriched long IDT populations, we are now beginning to ask what molecules are involved in regulating asymmetric growth patterns. The methodologies outlined here should be extendable to investigations of primary cells.
Finally, it has been suggested that there is a cell-cycle-associated mechanism that silences HSC transplantability such that engraftment is thought to be restricted to the G1 phase population of cycling HSCs when transplanted [37]. It was therefore suggested that the optimal engraftment "window" might be very sensitive to the cell cycle and proliferative status of HSCs at the time of transplantation. Hence, understanding every detail of ex vivo HSC division dynamics, including temporal cell cycle asymmetry and how cytokines affect it, could be critical for maximizing the use of proliferating HSC populations for transplantation. The asymmetry model described here may thus be useful to investigate the effects of various cytokines on cell cycle heterogeneity within the context of these cell division dynamics.
|
ACKNOWLEDGMENT
|
|---|
This study was supported by Public Health Service Grants RO1 HL 56416, RO1 HL 67384, and RO1 DK 53674 from the National Institutes of Health and a grant from the Phi Beta Psi Sorority to H.E.B.
|
REFERENCES
|
|---|
-
Jan Y-N, Jan LY. Asymmetric cell division. Nature 1998;392:775–778.[CrossRef][Medline]
-
Hawkins N, Garriga G. Asymmetric cell division: from A to Z. Genes Dev 1998;12:3625–3638.[Free Full Text]
-
Rhyu MS, Knoblich JA. Spindle orientation and asymmetric cell fate. Cell 1995;82:523–526.[CrossRef][Medline]
-
Jan Y-N, Jan LY. Asymmetry across species. Nat Cell Biol 1999;1:E42–E44.[CrossRef][Medline]
-
Keller G. Clonal analysis of hematopoietic stem cell development in vivo. Curr Top Microbiol Immunol 1992;177:41–57.[Medline]
-
Leary AG, Ogawa M, Strauss LC et al. Single cell origin of multilineage colonies in culture: evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes. J Clin Invest 1984;74:2193–2197.
-
Leary AG, Strauss LC, Civin CI et al. Disparate differentiation in hematopoietic colonies derived from human paired progenitors. Blood 1985;66:327–332.[Abstract/Free Full Text]
-
Shields R. Transition probability and the origin of variation in the cell cycle. Nature 1977;267:704–707.[CrossRef][Medline]
-
Murphy JS, Landsberger FR, Kikuchi T et al. Occurence of cell division is not exponentially distributed: differences in the generation times of sister cells can be derived from the theory of survival of populations. Proc Natl Acad Sci USA 1984;81:2379–2383.[Abstract/Free Full Text]
-
Smith JA, Martin L. Do cells cycle? Proc Natl Acad Sci USA 1973;70:1263–1267.[Abstract/Free Full Text]
-
Brummendorf TH, Dragowska W, Lansdorp PM. Asymmetric cell divisions in hematopoietic stem cells. Ann N Y Acad Sci1999;872:265–272; discussion 272-273.[Abstract/Free Full Text]
-
De Maertelaer V, Galand P. Some properties of a "G0"-model of the cell cycle. II. Natural constraints on the theoretical model in exponential growth conditions. Cell Tissue Kinet 1977;10:35–42.[Medline]
-
Broxmeyer HE, Maze R, Miyazawa K et al. The kit receptor and its ligand, steel factor, as regulators of hemopoiesis. Cancer Cells 1991;3:480–487.[Medline]
-
Ashman LK. The biology of stem cell factor and its receptor C-kit. Int J Biochem Cell Biol 1999;31:1037–1051.[CrossRef][Medline]
-
Sawyer ST, Jacobs-Helber SM. Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells. J Hematother Stem Cell Res 2000;9:21–29.[CrossRef][Medline]
-
Glaspy J. Clinical applications of stem cell factor. Curr Opin Hematol 1996;3:223–229.[Medline]
-
Horie M, Broxmeyer HE. Involvement of immediate-early gene expression in the synergistic effects of steel factor in combination with granulocyte-macrophage colony-stimulating factor or interleukin-3 on proliferation of a human factor-dependent cell line. J Biol Chem 1993;268:968–973.[Abstract/Free Full Text]
-
Miyazawa K, Hendrie PC, Mantel C et al. Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase. Exp Hematol 1991;19:1110–1123.[Medline]
-
Miyazawa K, Hendrie PC, Kim YJ et al. Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, M07e. Blood 1992;80:1685–1692.[Abstract/Free Full Text]
-
Mantel C, Luo Z, Canfield J et al. Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo. Blood 1996;88:3710–3719.[Abstract/Free Full Text]
-
Hendrie PC, Miyazawa K, Yang YC et al. Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of the human factor-dependent cell line, M07e. Exp Hematol 1991;19:1031–1037.[Medline]
-
Mantel C, Braun SE, Reid S et al. p21(cip-1/waf-1) deficiency causes deformed nuclear architecture, centriole overduplication, polyploidy, and relaxed microtubule damage checkpoints in human hematopoietic cells. Blood 1999;93:1390–1398.[Abstract/Free Full Text]
-
Mantel CR, Braun SE, Lee Y et al. The interphase microtubule damage checkpoint defines an S-phase commitment point and does not require p21(waf-1). Blood 2001;97:1505–1507.[Abstract/Free Full Text]
-
Kimmel M, Darzynkiewicz Z, Staiano-Coico L. Stathmokinetic analysis of human epidermal cells in vitro. Cell Tissue Kinet 1986;19:289–304.[Medline]
-
Berardi AC, Wang A, Levine JD et al. Functional isolation and characturization of human hematopoietic stem cells. Science 1995;267:104–108.[Abstract/Free Full Text]
-
Huang S, Law P, Francis K et al. Symmetry of initial cell divisions among primitive hematopoietic progenitors is independent of ontogenic age and regulatory molecules. Blood 1999;94:2595–2604.[Abstract/Free Full Text]
-
Fero ML, Rivkin M, Tasch M et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 1996;85:733–744.[CrossRef][Medline]
-
Kiyokawa H, Kineman RD, Manova-Todorova KO et al. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 1996;85:721–732.[CrossRef][Medline]
-
Nakayama K, Ishida N, Shirane M et al. Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 1996;85:707–720.[CrossRef][Medline]
-
Katayama N, Clark SC, Ogawa M. Growth factor requirement for survival in cell-cycle dormancy of primitive murine lymphohematopoietic progenitors. Blood 1993;81:610–616.[Abstract/Free Full Text]
-
Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149–1163.[Free Full Text]
-
Ohshiro T, Yagami T, Zhang C et al. Role of cortical tumor-suppressor proteins in asymmetric division of drosophila neuroblast. Nature 2000;408:593–596.[CrossRef][Medline]
-
Peng C-Y, Manning L, Albertson R et al. The tumour-suppressor genes lgl and dlg regulate basal protein targeting in drosophila neuroblasts. Nature 2000;408:596–600.[CrossRef][Medline]
-
Domen J, Weissman IL. Hematopoietic stem cells need two signals to prevent apoptosis; bcl-2 can provide one of these, kitl/c-kit signaling the other. J Exp Med 2000;192:1707–1718.[Abstract/Free Full Text]
-
Brummendorf TH, Dragowska W, Zijlmans J et al. Asymmetric cell divisions sustain long-term hematopoiesis from single-sorted human fetal liver cells. J Exp Med 1998;188:1117–1124.[Abstract/Free Full Text]
-
Mayani H, Dragowska W, Lansdorp PM. Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines. J Cell Physiol 1993;157:579–586.[CrossRef][Medline]
-
Glimm H, Oh I-L, Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0. Blood 2000;96:4185–4193.[Abstract/Free Full Text]
Received May 18, 2001;
accepted for publication July 24, 2001.
This article has been cited by other articles:
|
|
|
|
 
S. Ito, C. R. Mantel, M.-K. Han, S. Basu, S. Fukuda, S. Cooper, and H. E. Broxmeyer
Mad2 is required for optimal hematopoiesis: Mad2 associates with c-Kit in MO7e cells
Blood,
March 1, 2007;
109(5):
1923 - 1930.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Verhoeyen, M. Wiznerowicz, D. Olivier, B. Izac, D. Trono, A. Dubart-Kupperschmitt, and F.-L. Cosset
Novel lentiviral vectors displaying "early-acting cytokines" selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells
Blood,
November 15, 2005;
106(10):
3386 - 3395.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Shayakhmetov, Z.-Y. Li, A. Gaggar, H. Gharwan, V. Ternovoi, V. Sandig, and A. Lieber
Genome Size and Structure Determine Efficiency of Postinternalization Steps and Gene Transfer of Capsid-Modified Adenovirus Vectors in a Cell-Type-Specific Manner
J. Virol.,
September 15, 2004;
78(18):
10009 - 10022.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
, where