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Blood, 1 December 2000, Vol. 96, No. 12, pp. 3985-3987
BRIEF REPORT
An expansion phase precedes terminal erythroid
differentiation of hematopoietic progenitor cells from cord blood in
vitro and is associated with up-regulation of cyclin E and
cyclin-dependent kinase 2
Mu-Shui Dai,
Charlie R. Mantel,
Zhen-Biao Xia,
Hal E. Broxmeyer, and
Li Lu
From the Departments of Microbiology/Immunology,
Medicine (Hematology/ Oncology), and The Walther
Oncology Center, Indiana University School of Medicine, Indianapolis,
and the Walther Cancer Institute, Indianapolis, IN.
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Abstract |
The dynamics of cell cycle regulation were investigated during in
vitro erythroid proliferation and differentiation of CD34+
cord blood cells. An unusual cell cycle profile with a majority of
cells in S phase (70.2%) and minority of cells in G1 phase (27.4%)
was observed in burst-forming unit-erythrocytes (BFU-E)-derived erythroblasts from a 7-day culture of CD34+ cells
stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1
phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was
moderately up-regulated during the proliferation phase, and both cyclin
E and D3 were rapidly down-regulated during terminal
differentiation. This suggests that the high proliferation potential of
erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.
(Blood. 2000;96:3985-3987)
© 2000 by The American Society of Hematology.
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Introduction |
Cell proliferation, differentiation, and fate are
controlled by the cell cycle, which depends on a highly ordered
formation and activation of cyclin-cyclin-dependent kinase (cdk)
complexes. Transition of cells from G1 to S phase depends on activation
of kinase complexes containing D-type cyclins, on the catalytic subunit cdk4 or cdk6, and formation of a complex containing cyclin E and cdk2;
S phase progress requires synthesis of cyclin A and activation of the
cdk2/cyclin A complex; transition from G2 to M phase is initiated by
the mitotic cdk complex composed of cyclin B and p34cdc2.1
G1 cyclins, such as cyclin D and E, play crucial roles as rate-limiting
activators in G1/S phase transition.2-6 Cyclin E binds to
and activates cdk2 protein kinase, and in proliferating cells the
assembly of catalytically active cyclin E-cdk2 complexes is directly
related to the abundance of cyclin E protein.2
Hematopoiesis is a highly regulated process of cell proliferation and
differentiation from a small number of quiescent stem cells, which give
rise to nonproliferative and terminally differentiated mature lineage
cells.7 Regulation of the cell cycle of stem/progenitor cells by cyclins, cdks, and cdk inhibitors may contribute to
proliferation and differentiation of various lineage
cells.8-12 Less information is known about cell cycle
regulation in normal erythropoiesis. Cdc2, cdk2, cdk4, cyclin D1, and
cyclin A are induced by multiple cytokines in human CD34+
cells and chicken erythroid progenitors.13,14 Thus, we
evaluated cell cycle regulatory proteins during erythroid proliferation and differentiation from normal CD34+ cord blood (CB) cells.
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Study design |
Recombinant human (rhu) Epo, Steel factor (SLF),
and granulocyte colony-stimulating factor (G-CSF) were purchased from
Amgen Corp and R & D Systems (Thousand Oaks, CA). Rhu
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin 3 (IL-3) were kind gifts from Immunex (Seattle,
WA). Fluorescein isothiocyanate (FITC)-conjugated antihuman
CD34 (Becton Dickinson Immunocytometry System, San Jose, CA),
FITC-conjugated anti-CD71, antiglycophorin A (GPA), phycoerythrin
(PE)-conjugated anti-CD34, and anti-CD117 (PharMingen, San Diego,
CA) were used for flow cytometry analysis. For bivariate flow
cytometry analysis, FITC-conjugated mouse or rabbit antibodies against
cyclin A, B1, D2, D3 (Santa Cruz Biotechnology, Santa Cruz,
CA), and FITC-conjugated mouse or rabbit anticyclin D1, E, cdk
2, and cdc2 (PharMingen) were used. For immunoblot analysis, monoclonal
or polyclonal mouse or rabbit antibodies against cyclin D1, D2, D3, E,
B1, A, cdk2, cdk4, and cdc2 were purchased from Santa Cruz Biotechnology.
Cells were obtained from CB scheduled for discard. Eighty percent
to 95% pure CD34+ cells were isolated with MACS beads
(Miltenyi Biotec, Auburn, CA).15
CD34+ cells were cultured either in suspension or semisolid
culture for differentiation. To prepare burst-forming unit-erythrocytes (BFU-E)-derived cells, 200 cells/mL were plated in semisolid
media.15
Cells were stained with 50 µg/mL propidium iodide (PI, Sigma,
St Louis, MO) and analyzed by FACscan flow cytometry
(Becton Dickinson) for cell cycle status, stained with PE- or
FITC-conjugated anti-CD34, anti-CD71, or anti-GPA antibodies and
analyzed for cell surface antigens. Cells were fixed and permeabilized
using Cytofix/Cytoperm reagent (PharMingen). Intracellular staining was
performed with 10 µg/mL FITC-conjugated antibodies against cyclin D1,
D2, D3, E, A, B1, cdk2, and cdc2, and cells were counterstained with PI
for bivariate flow cytometry analysis.16
Equal amounts of protein in cell lysates were separated by 10%
SDS-PAGE, and electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA). Membranes were incubated with antibodies against cell cycle regulatory proteins, followed by incubation with horseradish peroxidase-conjugated goat antirabbit or goat antimouse antibodies, and proteins were detected by Enhanced Chemi-luminescence
(ECL)-Western blotting system (Amersham Life Sciences).
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Results and discussion |
Freshly separated CD34+ cells were quiescent with
99.9% of cells in G1/G0 phase (Figure
1). On stimulation with cytokines, CD34+ cells entered S phase. The proportion of S phase
cells increased 50% to 55%, whereas G0/G1 phase cells decreased 48%
and 36%, respectively, at day 3 and 5 of suspension cultures (Figure
1A). An unusual cell cycle profile with a majority of cells in S phase
(70.2%) and a minority of cells in G1 phase (27.4%) was observed in
BFU-E-derived erythroblasts from day 7 cultures. After 7 days of
culture, the proportion of S phase cells rapidly decreased and G1 phase
cells increased. Only 15% of BFU-E-derived cells remained in S phase and 80.1% of cells were in G0/G1 phase on day 14, suggesting terminal differentiation of erythroid progenitor cells and final exit from the
cell cycle. Fresh CD34+ cells are small and contain little
cytoplasm (Figure 1B,C). After culture in the presence of cytokines,
cell size increased. Ninety-eight percent of cells resembled
proerythroblasts at day 4. Seventy-five percent of day 7 BFU-E-derived
cells were proerythroblasts and erythroblasts, small numbers were
orthochromatic erythroblasts, almost no mature red blood cells were
detected, and cell size decreased after 7 days. In BFU-E-derived
cells, the percentage of orthochromatic erythroblasts increased on day
10 to a maximal level on day 14, at which time some mature erythrocytes
were also present and increased from that noted on day 10 (Figure 1C),
indicating that these cells underwent terminal differentiation. Day 7 BFU-E-derived cells have a large proliferative capacity and can
generate many CFU-E colonies (data not shown). Expression of
c-kit and CD34 antigen decreased with cell maturation,
whereas differentiated cells expressed CD71 and GPA, indicating a more
mature nature (Figure 1D).
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| Figure 1.
Cell cycle status and morphology during erythroid differentiation of CB
CD34+ cells.
(A) Freshly separated CD34+ cells
(2 × 104/mL) (day 0), or cells from suspension culture
of CD34+ cells in the presence of IL-3 (200 units), GM-CSF
(200 units), SLF (50 ng) and Epo (1 unit) per milliliter for 3 and 5 days or from BFU-E-derived cells from semisolid cultures after 7, 10, 12, and 14 days of growth in the presence of IL-3, GM-CSF, SLF, Epo,
vitamin B12 (10 µg, Sigma), folic acid (15 µg, Sigma), and insulin
(10 µg, Sigma) per milliliter were used for cell cycle analysis.
Results are the mean ± SEM from 5 experiments. (B) Wright-Giemsa
stained day 0 CD34+ cells (i) and cells from suspension
culture after 4 days (ii), or from BFU-E-derived colonies grown in the
presence of cytokines for 7 (iii), 10 (iv), and 14 (v) days (original
magnification × 1000). (C) Morphologic classification of
BFU-E-derived cells. Cell types were determined by examining 500 cells
per time point and are expressed as a percentage. (D) Expression of
surface antigen in day 0 CD34+ cells and BFU-E-derived
cells. Results are the mean percentage of positive cells ± SEM
from 5 experiments.
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Proliferation of early erythroblasts is possibly due to a rapid
transition of cells from the G1 to S phase. Therefore, we first
observed G1 cyclins by flow cytometry (Figure
2). Fresh CD34+ cells
expressed high levels of cyclin D1, but not cyclins D2 or D3 (Figure
2A). Cyclin D3, but not cyclin D2, was moderately up-regulated in day 4 BFU-E-derived cells and expressed at maximal levels in day 7 BFU-E-derived cells, after which cyclin D3 decreased. Expression of
cyclin E correlated dramatically with S phase cells (Figure 2B,C).
Expression of cyclins E and D3 rapidly decreased during terminal
differentiation after 10 days, whereas cyclin D1 was moderately
down-regulated. This was confirmed by immunoblot analysis (Figure 2D).
Increases in cyclins D3 and E were associated with the G1/S phase
transition and remained at high levels during the S and G2/M phases
(Figure 2C). Increased expression of cyclins A and B1 was seen after
cells entered the cell cycle with maximal expression during the S and
G2/M phases. Expression of these cyclins decreased on terminal
differentiation of erythroid progenitors (Figure 2D). Increases of
cyclins D3 and E were associated with initiation of the G1/S phase
transition, and they remained at high levels during the S and G2/M
phases (Figure 2C). Cyclin binding and activation of cdk is essential
for cell cycle regulation. Expression of cdk2 was remarkably similar to
cyclin E (Figure 2B,C,D). Western blot analysis confirmed this similar
pattern, and cdk2 and cdk4 both decreased with terminal differentiation (Figure 2D). In summary, our results suggest that an expansion phase
preceding terminal erythroid differentiation is tightly associated with
the expression of high levels of cyclin E and cdk2, and that
down-regulation of cyclin E and cdk2 might be essential for initiation
of terminal erythroid differentiation.
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| Figure 2.
Expression of cyclin proteins during erythroid differentiation of CB
CD34+ cells.
(A) and (B) Cells were analyzed by bivariate cell cycle analysis.
Results are the mean ± SEM from 4 experiments. (C) Scatter
density diagrams of bivariate flow cytometric analysis of cell cycle
proteins. G0/G1 (2N) and G2/M (4N) phase populations are indicated.
Above the solid horizontal lines are cutoffs for positive staining
based on the isotype controls. One of 5 representative experiments is
shown. (D) Expression of cyclin and cdk proteins determined by
immunoblot analysis. Equal amounts of total cell protein were subjected
to SDS/PAGE and immunoblotting, with tublin used as control. Results
are 1 of 3 representative experiments.
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Acknowledgment |
We thank Patricia Mantel for her help in preparing the manuscript.
| |
Footnotes |
Submitted March 10, 2000; accepted August 1, 2000.
Supported by Public Health Service Grants R01 HL 56416, and R01 DK
53674 from the National Institutes of Health to H.E.B., and by grants
from the Phi Beta Psi Sorority to L.L.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Li Lu, The Walther Oncology Center, Indiana
University School of Medicine, 1044 W Walnut St, R4, Rm 302, Indianapolis, IN, 46202-5121.
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References |
1.
Sherr CJ.
Cancer cell cycles.
Science.
1996;274:1672-1677[Abstract/Free Full Text].
2.
Koff A, Cross F, Fisher A, et al.
Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.
Cell.
1991;66:1217-1228[Medline]
[Order article via Infotrieve].
3.
Quelle DE, Ashmun RA, Shurtleff SA, et al.
Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.
Genes Dev.
1993;7:1559-1571[Abstract/Free Full Text].
4.
Resnitzky D, Gossen M, Bujard H, Reed SI.
Acceleration of the G1/S phase transition by expression of cyclin D1 and E with an inducible system.
Mol Cell Biol.
1994;14:1669-1679[Abstract/Free Full Text].
5.
Ando K, Ajchenbaum-Cymbalista F, Griffin JD.
Regulation of G1/S transition by cyclin D2 and D3 in hematopoietic cells.
Proc Natl Acad Sci U S A.
1993;90:9571-9575[Abstract/Free Full Text].
6.
Ohtsubo M, Roberts JM.
Cyclin-dependent regulation of G1 in mammalian fibroblasts.
Science.
1993;259:1908-1912[Abstract/Free Full Text].
7.
Ogawa M.
Differentiation and proliferation of hematopoietic stem cells.
Blood.
1993;81:2844-2853[Abstract/Free Full Text].
8.
Wang Z, Zhang Y, Kamen D, Lees E, Ravid K.
Cyclin D3 is essential for megakaryocytopoiesis.
Blood.
1995;86:3783-3788[Abstract/Free Full Text].
9.
Wang Z, Zhang Y, Lu J, Sun S, Ravid K.
Mpl ligand enhances the transcription of the cyclin D3 gene: a potential role for Sp1 transcription factor.
Blood.
1999;93:4208-4221[Abstract/Free Full Text].
10.
Kato J-Y, Sherr CJ.
Inhibition of granulocyte differentiation by G1 cyclins D2 and D3 but not D1.
Proc Natl Acad Sci U S A.
1993;90:11513-11517[Abstract/Free Full Text].
11.
Kiyokawa H, Richon VM, Rifkind RA, Marks PA.
Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells.
Mol Cell Biol.
1994;14:7195-7203[Abstract/Free Full Text].
12.
Kiyokawa H, Ngo L, Kurosaki T, Rifkind RA, Marks PA.
Changes in p34cdc2 kinase activity and cyclin A during induced differentiation of murine erythroleukemia cells.
Cell Growth Differ.
1992;3:377-383[Abstract].
13.
Engelhardt M, Kumar R, Albanell J, Pettengell R, Han W, Moore MAS.
Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells.
Blood.
1997;90:182-193[Abstract/Free Full Text].
14.
Dolznig H, Bartunek P, Nasmyth K, Mullner EW, Beug H.
Terminal differentiation of normal chicken erythroid progenitors: shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control.
Cell Growth Differ.
1995;6:1341-1352[Abstract].
15.
Lu L, Heinrich MC, Wang LS, et al.
Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor- and transforming growth factor- 1.
Blood.
1999;94:2319-2332[Abstract/Free Full Text].
16.
Juan G, Traganos F, James WM, et al.
Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis.
Cytometry.
1998;32:71-77[Medline]
[Order article via Infotrieve].
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Abstract
Introduction