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First published online June 7, 2005
Stem Cells Vol. 23 No. 7 August 2005, pp. 1002 -1011
doi:10.1634/stemcells.2004-0088; www.StemCells.com
© 2005 AlphaMed Press

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Catalytic Activities of G1 Cyclin-Dependent Kinases and Phosphorylation of Retinoblastoma Protein in Mobilized Peripheral Blood CD34+ Hematopoietic Progenitor Cells

Kurt Leibundguta,b, Nicole M.R. Schmitzb, Andreas Hirta,b

a Department of Pediatrics and
b Department of Clinical Research, University of Bern, Bern, Switzerland

Key Words. Cyclin-dependent kinases • Retinoblastoma protein • Cell cycle • CD34+ cells

Correspondence: Kurt Leibundgut, M.D., Department of Pediatrics and Department of Clinical Research, University of Bern Inselspital, CH-3010, Bern, Switzerland. Telephone: 41-31-632-9495; Fax: 41-31-632-9507; e-mail: kurt.leibundgut{at}insel.ch


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Depending on the source of cells, the cell cycle status of hematopoietic stem and progenitor cells capable of repopulating the marrow of transplant recipients is controversial. In this study, using biochemical methods, the cell cycle status of mobilized CD34+ cells was analyzed. It was demonstrated in CD34+ cell extracts that there was high catalytic activity of G1 cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) but low activity of CDK2. This was in contrast to the resting reference cells that showed only minimal or no activity of these CDKs. Since at the G0->G1->S transition CDK4/6 and CDK2 sequentially phosphorylate the retinoblastoma protein (pRB), its phosphorylation status was analyzed. Previously, we showed that p110RB was unphosphorylated at serine (Ser)-608 in CD34+ cells, consistent with the ability to suppress cell growth. Here, it was established that this form of pRB was phosphorylated at Ser-780, Ser-795, and Ser-807/811 in CD34+ but not in resting reference cells. This result was therefore consistent with the presence of high CDK4/6 activities in CD34+ cells. Conversely, CDK2 activity was low and the pRB residues Ser-612 and threonine (Thr)-821, which are exclusively phosphorylated by CDK2 in conjunction with either cyclin E or A, were unphosphorylated in >90% of CD34+ cells. We therefore show for the first time the exact position of mobilized CD34+ cells within the cell cycle; that is, they do not reside in G0 but in early G1 phase and did not cross the restriction point into late G1 phase.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For autologous transplantations, peripheral blood progenitor cells (PBPCs) have completely replaced the use of bone marrow [1]. Moreover, for allogeneic transplantations, there is an increasing experience of use of PBPCs [1, 2]. To assess the stem and progenitor cell content in a transplant, the number of CD34+ cells is a reliable surrogate marker [3–5].

Mobilized CD34+ cells have been demonstrated to be either in G0 or G1 phase of the cell cycle [6–8]. However, most mobilized CD34+ cells express proliferation markers such as the p120 nucleolar antigen, the Ki-67 antigen, and the proliferating-cell nuclear antigen (PCNA) [7, 9–12], indicating a cell cycle status beyond the G0 phase. Alternatively, only 1%–3% of mobilized CD34+ cells are in S/G2+M phase [7, 9–13]. Therefore, this would support the suggestion that most mobilized CD34+ cells reside in G1 phase.

Despite the major components of the cell cycle machinery, which include cyclin-dependent kinases (CDKs), cyclins, and cyclin-dependent kinase inhibitors, being identified, little is known about their respective activities in hematopoietic stem and progenitor cells. Whereas in primary human fibroblasts and T lymphocytes G0 -> early G1 phase progression may require the activation of CDK 4,6-cyclin D2 complexes [14, 15], the activity levels of G1 CDKs have not yet been investigated in mobilized CD34+ cells.

Cyclin D-CDK4,6 and cyclin E-CDK2 sequentially phosphorylate the retinoblastoma protein (pRB) at the restriction point, a window of diminishing options covering the transition from G1a to G1b. This is triggered following mitogen stimulation by cyclin D-CDK4,6 protein kinases and accelerated by the cyclin E-CDK2 complex [16–20]. The activity of pRB is controlled by post-translational modifications that include phosphorylation [21, 22]. pRB contains 16 consensus sequences for CDK phosphorylation, including threonine (Thr)-373, serine (Ser)-608, Ser-612, Ser-780, Ser-795, Ser-807/811, and Thr-821 [23–25]. Although the central role of pRB in control of the cell cycle is well known, there is little available data on its functional status in hematopoietic stem and progenitor cells.

Because mobilized CD34+ cells entered G1 phase, we hypothesized that their CDK activities differ significantly from those of resting cells such as growth-arrested NIH3T3 or peripheral blood mononuclear cells that spontaneously arrest in G0 phase [14, 15, 26, 27]. As a consequence, we expected different phosphorylation patterns between the pRB in mobilized CD34+ and in resting reference cells. We show here a low catalytic activity of CDK2 and a high activity of CDK4 and CDK6 in CD34+ cell extracts. This was reflected by the detection of phosphorylated pRB at Ser-780, Ser-795, or Ser-807/811 in CD34+ cells, whereas in mononuclear or NIH3T3 cells these pRB consensus sequences for CDK phosphorylation were unphosphorylated. The specificity of CDK catalytic activities was confirmed by inhibition experiments during in vitro kinase assays. In addition, we showed at a single-cell level that the pRB residues Ser-612 and Thr-821, which are exclusively phosphorylated by CDK2 in conjunction with either cyclin E or A [24, 28], were unphosphorylated in more than 90% of CD34+ cells, reflecting the low CDK2 catalytic activity.

We conclude that mobilized CD34+ cells have attained the early G1 phase but did not cross the restriction point in late G1 and, therefore, are still susceptible to external stimuli, inducing cell cycle arrest, apoptosis, differentiation, or proliferation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Sources and Preparation
Hematopoietic progenitor cells were collected by leukapheresis from patients with solid tumors or hematological malignancies after combined mobilization treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). CD34+ cells were enriched by the Isolex 300i stem cell concentration system (Baxter AG, Volketswil, Switzerland, http://www.baxter.com) The mean purity (± 1 SD) of enriched CD34+ cells was 94.6% ± 1.5%, as assessed by both flow cytometry and staining of cyto-centrifuge smears with a monoclonal anti-CD34 antibody. After the selection procedure, the cells were frozen in a controlled-rate freezer and stored in liquid nitrogen until use. Peripheral blood mononuclear cells from healthy volunteers were isolated by Lymphoprep (Nycomed, Oslo, Norway, http://www.nycomed.com) gradient centrifugation. NALM-6 cells (a commercially available acute lymphoblastic pre-B-cell line) were maintained in Roswell Park Memorial Institute-1640 medium (Sigma, St. Louis, http://www.sigmaalrich.com) supplemented with 10% fetal bovine serum (FBS) (Sigma) in a 5% CO2 incubator at 37°C. NIH3T3 cells (a commercially available mouse fibroblast cell line) were maintained in minimal essential medium (Sigma) supplemented with 10% FBS in a 5% CO2 incubator at 37°C. NIH3T3 cells were contact inhibited and serum starved in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco BRL, Basel, Switzer-land, http://www.invitrogen.com) supplemented with 2% FBS and incubated for 1 week at 37°C in 5% CO2. This study has been reviewed and was approved by the Institutional Review Board of the Department of Pediatrics, University Hospitals in Bern, and by the Institutional Review Board of the Medical Faculty of the University of Bern.

In Vitro Cultures
CD34+ cells were thawed at 37°C, slowly reconstituted with IMDM, and divided into aliquots [12]. One aliquot was analyzed immediately, and the others were used for in vitro cultures. CD34+ cells were incubated for 48 hours in 5 ml IMDM culture medium supplemented with 20% FBS, 5 x 10–5 mol/L 2-mercaptoethanol (Fluka Chemie AG, Buchs, Switerland, http://www.sigmaaldrich.com) and recombinant human cytokines that were added at the following final concentrations: stem cell factor, 20 ng/ml; inter-leukin 3 (IL-3), 50 ng/ml; IL-6, 20 ng/ml; erythropoietin, 6 U/ml (all from R & D Systems, Minneapolis, http://www.rndsystems.com), and G-CSF, 100 ng/ml (Amgen [Europe] GmbH, Luzern, Switerland, http://www.amgen.com) After 48 hours of incubation at 37°C in a fully humidified 4.5% CO2 atmosphere, the cells were washed and thereafter used for the experiments. As determined by Trypan blue exclusion, the viability was always >95%. Mononuclear cells were either analyzed immediately or cultured at a density of 106/ml in IMDM supplemented with 10% FBS. After adding phytohemagglutinin (PHA) (Becton Dickinson, AG, Allschwil, Switzerland, http://www.difco.com) at a concentration of 10 µg/ml, the cells were incubated at 37°C in a fully humidified 4.5% CO2 atmosphere for 48–72 hours.

Antibodies
The following phospho-specific antibodies to pRB were purchased from New England Biolabs (Beverly, MA, http://www.neb.com): phosphorylated (pp) Thr-373RB:9306S, ppSer-780RB:9307S, ppSer-795RB:9301S, and ppSer-807/811RB:9308S. Antibodies ppThr-821RB:44-582G and ppSer-612RB:44-572 were from Biosource International (Camarillo, CA, http://www.biosource.com). The following antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, http://www.scbt.com): HDAC1:sc-7872, MARCKS:sc-6454, sc-7905 (amino acids 769–921 of mouse pRB, human reactive), CDK2:sc-163, CDK4:sc-260, CDK6: sc-177, cyclin D1 (sc-246), cyclin D2:sc-181, cyclin D3:sc-182, cyclin E:sc-198. Antibody to cyclin A (Ab-3):cc17 was purchased from Oncogene Research Products (Boston, http://www.oncresprod.com). The anti-p120 antibody (Na061) was purchased from Calbiochem (Darmstadt, Germany, http://www.emdbiosciences.com) the Anti-EF1a antibody, clone CBP-KK1, from Upstate Biotechnology (Lake Placid, NY, http://www.upstatebiotech.com), and the anti-PCNA from DakoCytomation (Glostrup, Denmark, http://www.dakocytomation.com). Anti-bromodeoxyuridine (anti-BrdU) (BMC 9318) was obtained from Roche Dianostics AG (Rotkreuz, Switzerland, http://www.roche-applied-science.com).

Immunocytochemistry
The pRB was detected in individual cells by immunocytochemistry on cytocentrifuge preparations with phosphosite–specific antibodies to pRB in duplicates or triplicates according to the alkaline phosphatase:anti-alkaline phosphatase method [29]. The percentage of cells in S phase was determined by BrdU incorporation (BrdU Labeling and Detection Kit, Boehringer Mannheim Biochemica) and expressed as BrdU-labeling index (BrdU-LI). The smears were evaluated by light microscopy, and at least 1,000 cells per smear were counted.

In Vitro Kinase Assays
Cells were lysed into lysis buffer (1% NP-40, 20 mM Na2HPO4, pH 7.4, 250 mM NaCl, 5 mM EDTA, 5 mM DTT, 25 mM ß-glycerophosphate, 0.1 mM Na3VO4, 1 mM phenylmethyl sulfonyl fluoride (PMSF), 2 µg/ml leupeptin, and 1.5 µg/ml–1 aprotinin). To measure in vitro kinase activity toward human biologically active recombinant pRB (amino acids 1–928, No. 3108; QED Biosciences, San Diego, http://www.qedbio.com), endogenous CDKs were immunoprecipitated for 1 hour at 4°C using 6 µganti-CDK2, anti-CDK4, or anti-CDK6 antibodies. Extracts of 10 million cells were used for each immunoprecipitation. Immunocomplexes were coupled to protein A-agarose beads for 1 hour at 4°C using a rotating wheel and washed four times with lysis buffer, once with 100 mM Tris, pH 7.5, and 0.5 M LiCl, and once with kinase buffer (20 mM Tris, pH 7.5, 7.5 mM MgCl2, 1 mM DTT, 0.5 mM EGTA, 25 mM ß-glycerophosphate, 0.5 mM Na3VO4, 1 mM PMSF, 2 µg/ml leupeptin, and 1.5 µg/ml–1 aprotinin). Kinase reactions were carried out in the presence of 2 µCi {gamma}[33P]ATP (AH9968, Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) for 30 minutes at 37°C using 5 µg pRB as a substrate (No. 3108; QED Biosciences). For specific competition with unlabeled ATP, immunocomplexes coupled to protein A-agarose beads were split in two aliquots and the substrate was added in the presence or absence of 7 mM ATP. Reactions were terminated by boiling for 3 minutes in SDS-PAGE sample buffer.

Inhibition of CDK Catalytic Activities During In Vitro Kinase Assays
In vitro kinase assays were carried out according to Matsushime et al. [30]. For inhibition of CDK2 catalytic activity, 1 µl roscovitine 14 mM (Sigma, No. R-135) in DMSO (vehicle) was added to immune complexes resuspended in 30 µl kinase buffer. Inhibition of CDK4 or CDK6 activity occurred after addition of 1 µg recombinant p16INK4a (No. 07-316; AmProx, Carlsbad, CA, http://www.amprox.com) to the lysate used for immunoprecipitation.

Densitometric Analysis
Densitometric quantification of the data obtained by in vitro kinase assays (x-ray films) was done using the Lumi-imager F1 Workstation (Roche).

Protein Extracts
Total or nuclear extracts were prepared for Western analysis. pRB was not detectable in the cytoplasmatic extracts prepared from primary cells. For total cell extracts, washed mononuclear cells or CD34+ cells were lysed in buffer containing 420 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% NP-40. All buffers were supplemented before use with ß-glycerophosphat 10 mM, NaF 10 mM, sodium orthovanadate 2 mM, protease inhibitor cocktail (Roche), PMSF 1 mM. After incubation for 30 minutes on ice, cell lysates were cleared by centrifugation at 13 krpm for 30 minutes at 4°C. For whole nuclear extracts, washed cells were resuspended in HEPES 20 mM, MgCl2 3 mM, KCl 20 mM, and the cytoplasmatic membrane broken by homogenizing for 10 seconds at 20 krpm using a Polytron PT 3100 (Kinematica, Inc., Cincinnati, http://www.kinematica.ch). After pelleting the nuclei at 4.7 krpm for 5 minutes at 4°C, they were resuspended in HEPES 40 mM, glycerol 50%, NaCl 420 mM, and MgCl2 3 mM. This high-salt buffer solubilized nuclear matrix proteins, including tethered pRB. Extracts were cleared by centrifugation at 13 krpm for 30 minutes at 4°C. The protein concentration was determined with the Bradford protein assay (Bio-Rad, Hercules, IL, http://www.bio-rad.com), and aliquots of 40 µg were stored at –80°C until use.

Immunoprecipitation
Cell lysates were precleared by incubation with 20 µl of protein A-agarose (Roche) at 4°C for 30 minutes using a rotating wheel, followed by centrifugation for 5 minutes at 3 krpm at 4°C, and then subjected to specific immunoprecipitation for 4 hours on ice. Immunocomplexes were recovered by incubation with 40 µl of protein A-agarose at 4°C for 1 hour using a rotating wheel. Tubes were centrifuged at 3 krpm for 5 minutes at 4°C, and then agarose was washed several times with 600 µl phosphate-buffered saline. Proteins were eluted in sample buffer for 3 minutes at 90°C.

Western Analysis
Equal amounts of protein (40 µg) were used for immunoblotting, and different membranes were probed with phospho-specific anti-bodies. Detection of pRB by immunoblotting with phospho-specific antibodies to Thr-373, Ser-780, Ser-795, or Ser-807/811 was controlled by using nuclear extracts of contact-inhibited NIH3T3 cells or actively dividing NALM-6 cells. Equal loading was controlled by the Bio-Rad protein assay followed by Coomassie gels. Protein extract was heated for 3 minutes at 80°C in sample buffer and then loaded on a 7% SDS-gel. Transfer of proteins to PVDF membrane (Roche) was for 45 minutes at 60 V, 220 mA in a Mini Trans Blot Cell (Bio-Rad). Blots were saturated with Tris-buffered saline containing 0.25% gelatin for 1 hour at room temperature and then washed two times for 5 minutes in Tris-buffered saline, 0.02% gelatin, and 0.25% Triton X-100. The blots were further incubated with diluted antibody at 0.5 µg/ml of Tris-buffered saline containing 0.02% gelatin, 0.25% Triton X-100 for 1 hour at room temperature. After washing in 0.02% gelatin and 0.25% Triton X-100 for two times for 5 minutes at room temperature, the blots were incubated with recombinant protein G conjugated with HPO (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) at 0.15 µg/ml for 1 hour at room temperature. After washing four times for 15 minutes at room temperature in Tris-buffered saline containing 0.02% gelatin and 0.25% Triton X-100, the proteins were detected by chemiluminescence using Lumi-Light Plus and the F1 Lumi-imager Workstation (Roche).

Statistical Analysis
Statistical evaluation was performed on a personal computer applying the STATGRAPHICS Plus (5.0) software (Manugistics, Inc., Rockville, MD, http://www.manugistics.com). Means and standard deviations (SDs) were calculated. When indicated, immunocytochemical results were compared by paired and unpaired t-tests. A p value < .05 was considered statistically significant.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression Profile of Proliferation Markers
Patterns of G0- or G1-specific gene expression were determined in equal amounts of cell lysates from mobilized CD34+ cells or reference mononuclear cells, highly proliferating NALM-6 and quiescent NIH3T3 cells subjected to Western analysis. The PCNA protein, which is synthesized in G1- and S-phase cells [31], was detected with anti-PCNA antibody, thereby revealing small or nonexistent levels in quiescent NIH3T3 reference fibroblasts and resting mononuclear cells, respectively (Fig. 1Go, lanes 1, 3, 4). Substantial upregulation of PCNA expression was observed in NALM-6 extracts (Fig. 1Go, lane 2) and after in vitro stimulation of mononuclear (Fig. 1Go, lanes 5, 6) and CD34+ cells (Fig. 1Go, lanes 9, 10), whereas unstimulated mobilized CD34+ extracts accumulated moderate amounts of PCNA protein (Fig. 1Go, lanes 7, 8). Expression of HDAC1 and the cell proliferation–associated nucleolar protein p120 resembled that of PCNA, because quiescent NIH3T3 and resting mononuclear cells expressed little if any HDAC1 or p120 protein (Fig. 1Go, lanes 1, 3, 4). Compared with NALM-6 or in vitro–stimulated cells, the abundance of PCNA, HDAC1, and p120 proteins was lower in mobilized CD34+ cells (Fig. 1Go, lanes 7, 8). Because quiescent cells in G0 express high levels of myristoylated alanine-rich C kinase substrate (MARCKS) mRNA and protein, and cell division causes a striking downregulation of MARCKS expression [32], membranes were immunoblotted with anti-MARCKS antibody. Quiescent NIH3T3 lysates and resting mononuclear cell extracts had elevated levels of MARCKS protein (Fig. 1Go, lanes 1, 3, 4). Expression was attenuated in extracts of in vitro–stimulated mononuclear cells (Fig. 1Go, lanes 5, 6), whereas small or nonexisting levels of MARCKS were detected in proliferating NALM-6 cells and in cytokine-stimulated CD34+ extracts (Fig. 1Go, lanes 2, 9, 10). Very low levels of MARCKS expression were observed in unstimulated CD34+ extracts (Fig. 1Go, lanes 7, 8) and was similar to that of in vitro–stimulated mononuclear cells (Fig. 1Go, lanes 5, 6). Together the results indicated that the number of resting G0 cells in unstimulated mobilized CD34+ samples was small or nonexistent.



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  		Figure 1. Proliferation markers. Western blotting of extracts derived of quiescent NIH3T3 cells, proliferating NALM-6 cells, MNCs before (–) and after (+) in vitro stimulation with PHA, and mobilized CD34+ cells before (–) and after (+) in vitro stimulation with cytokines (CYTO). Mononuclear cells (–PHA) were cells spontaneously arrested in G0 phase. Equal loading was controlled by immunoblotting with anti-EF1a antibody.

 
BrdU-Labeling Index
To analyze the proliferative capacity of CD34+ cells, S-phase cells were detected using BrdU labeling. Whereas no BrdU was incorporated in resting mononuclear cells, slowly proliferating CD34+ cells showed significantly lower BrdU-labeling indexes than cells with proliferative activity, such as NALM-6 cells or in vitro–stimulated cells (Table 1Go, bottom line). The results indicated that the percentage of S-phase cells in mobilized CD34+ cell samples was small.


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  		Table 1. Phosphorylation of the pRB- and BrdU-labeling index
 
Comparison of CDK Activities in Mobilized CD34+ and Resting Reference Cells
CDK catalytic activities derived of CD34+ and reference cell extracts were subjected to in vitro kinase assays toward recombinant pRB followed by Western blotting and autoradiography (Figs. 2A, 2BGo). First, specific pRB labeling was determined in NALM-6 extracts. When immune complexes obtained with anti-CDK2, anti-CDK4, or anti-CDK6 antibody were split and tested for kinase activity, pRB phosphorylation occurred only in the absence of unlabeled ATP (Fig. 2AGo). The intensity of autoradiographic signals was quantitated by densitometry relative to CDK activities of NALM-6 extracts. It was found that CDK2 catalytic activity was detectable at low level in CD34+ extracts whereas the activity was nonexistent in resting mononuclear or quiescent NIH3T3 cells (Fig. 2BGo). Induction of high CDK2 activity was dependent on in vitro stimulation of CD34+ with cytokines (fivefold increase) or of mononuclear cells with PHA (sixfold increase) (Figs. 2B, 2CGo). The results indicated that CDK2 catalytic activity was low in unstimulated mobilized CD34+ extracts and changed dramatically with the growth rate of cells.



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  		Figure 2. Autoradiographies of CDK activities. IPs were obtained using anti-CDK2, -CDK4, or -CDK6 antibody. (A): Specific labeling of recombinant pRB due to CDKs. Immunocomplexes from NALM-6 cells were split after immobilization to protein A agarose beads and tested for kinase activity toward recombinant human pRB in the absence (–) or presence (+) of unlabeled ATP. (B): Phosphorylation of recombinant human pRB by CDK2, CDK 4, and CDK6 precipitated from cell lines (contact-inhibited NIH3T3 and proliferating NALM-6), from resting (–PHA), and from invitro–stimulated MNCs (+PHA) as well as from untreated (–CYTO) and stimulated (+CYTO) mobilized CD34+ cells. (C): Densitometric analysis of autoradiographies. The intensity of radioactive signals on the x-ray films was quantitated and expressed relative to the corresponding CDK activity in NALM-6 cells that arbitrarily has been set to 1. Abbreviations: IP, immunoprecipitates; MNC, mononuclear cell; PHA, phytohemagglutinin; pRB, phosphorylated retinoblastoma protein.

 
When CDK4 activity was determined, there was a threefold to fourfold higher level in extracts of CD34+ than in resting mono-nuclear cells or in quiescent NIH3T3 lysates (Fig. 2CGo). CDK4 activity was twofold enhanced in extracts of PHA-stimulated mononuclear cells compared with untreated cells (Fig. 2CGo). Conversely, no significant difference in CDK4 activity was revealed in response to either no treatment or in vitro stimulation of CD34+ cells with cytokines (Fig. 2CGo). When CDK6 activity was tested, it was found that the level was twofold to threefold enhanced in CD34+ extracts compared with resting mononuclear cells lysates and fivefold compared with quiescent NIH3T3 extracts (Fig. 2CGo). Exposure of mononuclear cells to PHA generated a twofold increase in CDK6 activity (Fig. 2CGo). Conversely, CDK6 activity decreased in CD34+ extracts after stimulation with cytokines. The reasons for this decrease remain to be evaluated. Measuring catalytically active CDK4,6 in unstimulated mobilized CD34+ extracts indicated that these cells had entered the mitotic division cycle and therefore were not in G0 phase.

To further determine whether the activities in unstimulated CD34+ extracts toward pRB were specifically due to CDKs, roscovitine or recombinant p16INK4a was added to immunoprecipitates followed by in vitro kinase assays, SDS-PAGE, and autoradiography. Compared with nonspecific labeling in the absence of antibody, CDK activity toward pRB was present in immunoprecipitates of CDK2, CDK4, or CDK6 (Fig. 3AGo). The autoradiographic signal of CDK2 immunoprecipitates treated with roscovitine resembled that of nonspecific labeling, whereas immunoblotting indicated that CDK2 was present in anti-CDK2 immunoprecipitates. This indicated that CDK2 was not active in the presence of roscovitine (Fig. 3AGo). Because vehicle alone did not interfere with CDK2 activity, inhibition of CDK2 was due to roscovitine (Fig. 3AGo). The data indicated that CDK2 activity was present in unstimulated mobilized CD34+ lysates and was specifically inhibited by roscovitine. While CDK4 and CDK6 were detected by immunoblotting, addition of recombinant p16INK4a to immunoprecipitates resulted in a signal similar to that of nonspecific labeling (Fig. 3AGo). The results indicated that CDK4 and CDK6 were present in CD34+ lysates and were specifically inhibited by p16INK4a. To quantitate inhibition of CDKs, autoradiographies from in vitro kinase assays derived of three different extracts of unstimulated CD34+ cells were subjected to densitometric analysis. The signal obtained for CDK2, CDK4, or CDK6 was set to 1. Inhibition of CDK2 by roscovitine was threefold, and no residual activity was detected compared with the signal in the absence of antibody, whereas vehicle alone was without effect (Fig. 3BGo). Inhibition of CDK4 or CDK6 by p16INK4a was twofold and 2.5-fold, respectively, and only minor residual activity was observed compared with the signal in the absence of antibody (Fig. 3BGo).



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  		Figure 3. Specific inhibition of CDKs in vitro. (A): Autoradiographies of in vitro kinase assays toward 33P-pRb subjected to SDS-PAGE. Cell lysates of different unstimulated CD34+ samples were used for immunoprecipitation of CDKs. Immunoblotting with antibody to CDK2, CDK4, and CDK6 indicated that the catalytic subunits were present during in vitro kinase assays. Together Western blotting and autoradiographies indicated that CDK2 was inactive in the presence of roscovitine while addition of the vehicle alone was without effect. Furthermore, specific inhibition of CDK4 or CDK6 occurred in the presence of p16INK4a. (B): The inhibition of CDK activities derived from three different unstimulated CD34+ lysates (n = 3) was quantitated by densitometric analysis of autoradiographies, and mean values were expressed relative to the corresponding CDK activity that arbitrarily has been set to 1. Abbreviations: CDK, cyclin-dependent kinase; IgG, immunoglobulin G; 33P-pRb, recombinant retinoblastoma protein.

 
Effects of CDK Activities on Site-Specific Phosphorylation of pRb
Because CDKs were active in extracts of unstimulated mobilized CD34+ cells, we examined the phosphorylation status of pRB. Equal amounts of CD34+ lysates were subjected to SDS-PAGE for Western analysis with pp site–specific antibodies against pRB at Thr-373, Ser-780, Ser-795, or Ser-807/811. Specific staining of the pRB protein was validated using quiescent NIH3T3 cells that were serum-starved and contact-inhibited for 1 week and by dividing NALM-6 cells. In contrast to unphosphorylated pRB in extracts of resting NIH3T3 cells, immunoblotting revealed different forms in extracts of actively dividing cells. These included NALM-6 cells (Fig. 4BGo, lane 2), mononuclear cells stimulated in vitro with PHA (Fig. 4BGo, lanes 5, 6), or CD34+ cells after in vitro stimulation with cytokines (Fig. 4BGo, lanes 9, 10). In unstimulated CD34+ cell extracts, the antibodies detected phosphorylated residues of pRB at Thr-373, Ser-780, Ser-795, and Ser-807/811. However, in contrast to NALM-6 or to in vitro–stimulated mono-nuclear and CD34+ cells, Western blot revealed only one form of pRb at 110 kDa, which has previously been shown [12] to be unphosphorylated at Ser-608 (hypo-p110RB) (Fig. 4BGo, lanes 7, 8). Immunoblotting with the phosphorylation site–specific antibody to Thr-373 detected pRB in extracts of all cell populations, including resting and proliferating cells; even in extracts derived of quiescent NIH3T3 cells, a weak signal was observed (Fig. 4BGo, lanes 1–10). Different to using phosphorylation site–specific antibodies, immunoblotting with antibody against total pRB revealed the protein in every cell lysate (Fig. 4BGo, bottom panel, lanes 1–10). The appearance of different forms of pRB migrating with retarded mobility in extracts of dividing cells was consistent with the presence of hypophosphorylated and hyperphosphorylated forms of pRB (Fig. 4BGo, bottom panel, lanes 2, 5, 6, 9, 10). These data indicated that phosphorylation of pRB in extracts of mobilized CD34+ cells has occurred at Thr-373, Ser-780, Ser-795, and Ser-807/811 but not at Ser-608 [12], complying with an early G1-phase cell cycle status.



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  		Figure 4. Phosphorylation of pRB. (A): Schematic representation of the human retinoblastoma protein (pRB) and consensus CDK phosphorylation sites. Numbers at the bottom delineate amino acids comprising the pocket domain A and B. Approximate locations for serine (Ser) and threonine (Thr) CDK phosphorylation sites, as well as amino acid position, are indicated on the upper side. Indicated phosphoacceptor sites were analyzed in this study. (B): Purified mobilized peripheral blood CD34+ cells or mononuclear cells were induced to proliferate by the addition of cytokines (+CYTO) for 48 hours or phytohemagglutinin (+PHA) for 72 hours, respectively. Negative control: Contact-inhibited NIH3T3 cells. Positive control: Exponentially growing NALM-6 cells. Equal amounts of total or nuclear cell lysates were subjected to SDS-PAGE and Western blotting. Blot probes: Anti-phospho (pp) ppThr-373RB, anti–ppSer-780RB, anti–ppSer-795RB or anti–ppSer-807/811RB antibody. The anti–total pRB antibody (sc-7905) detected all forms of pRB (total pRB).

 
Alternatively, the expression of pRB was examined by immunocytochemical analysis. Specific staining was validated by using exponentially dividing NALM-6 cells and quiescent peripheral blood mononuclear cells that had spontaneously arrested in G0 phase. Results are summarized in Table 1Go. Whereas in >99% of NALM-6 and >90% of CD34+ cells, respectively, pRB phosphorylated at Thr-373, Ser-780, Ser-795, or Ser-807/811 was detected, this was the case in <1% for mono-nuclear cells. Interestingly and consistent with results obtained by immunoblotting, a subset of resting mononuclear cells (15%) was reactive for antibody to ppThr-373. After in vitro stimulation of mononuclear cells with PHA, the percentage of labeled cells almost approximated that of proliferating NALM-6 cells. Conversely, for CD34+ cells, the percentage of immunoreactive cells did not change after in vitro stimulation.

Because pRB phosphorylation sites Ser-612 and Thr-821 are exclusively phosphorylated by CDK2, in conjunction with either cyclin A or E, but not by CDK4/6 [24, 28], we obtained indirect evidence for low CDK2 activity in CD34+ cells by staining with anti–ppSer-612 and anti–ppThr-821. Whereas before in vitro stimulation only a small percentage of CD34+ cells was stained, the fraction of positive cells increased dramatically thereafter (Table 1Go, Fig. 5Go).



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  		Figure 5. Phosphorylation of retinoblastoma protein (pRB) at Ser-612 and Thr-821. Mobilized CD34+ cells before (left side) and after (right side) in vitro stimulation with cytokines. The cells were stained with anti–ppSer-612 (upper panel) or anti–ppThr-821 (bottom panel), respectively. Note that after in vitro stimulation, the CD34+ cells also have changed their morphological appearance; they have become larger, and in the red-stained chromatin, conspicuous nucleoli are visible.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Today, instead of bone marrow, most autologous transplantations and an increasing number of allogeneic transplantations are performed using hematopoietic stem and progenitor cells mobilized to the peripheral blood [1, 2]. In the clinical transplantation setting, the number of CD34+ cells has been shown to be a reliable surrogate marker to predict long-term engraftment [3–5].

Depending on the source of cells (adult bone marrow, mobilized peripheral blood, umbilical cord blood, or human fetal hematopoietic cells), the cell cycle status of hematopoietic stem cells capable of repopulating the marrow of transplant recipients is controversial [33–37, reviewed in 38]. Analyzing a set of proliferation markers, we could confirm results obtained by others [7, 9, 10] indicating that mobilized CD34+ cells do not reside in G0 phase. To supplement these data, immunoblotting with an anti-MARKS antibody was performed. Quiescent cells in G0 express high levels of MARCKS mRNA and protein, but cell division causes a striking downregulation [32]. Whereas quiescent NIH3T3 and resting mononuclear cells had elevated levels of MARCKS protein, only small or nondetectable levels were traced in proliferating cells. MARCKS expression in unstimulated CD34+ extracts was similar to that of in vitro–stimulated mono-nuclear cells, indicating that the former have entered the cell cycle. Because BrdU-LI was less than 3% and there was strong expression of proliferation markers as well as low levels of MARCKS expression in unstimulated CD34+ cells, it can be concluded that they reside in G1 phase.

Most studies reporting on cell cycle analysis in hematopoietic stem and progenitor cells rely on flow-cytometric methods [6, 7, 10, 13, 33–35, 39, 40]. In this study, it was decided to adopt an alternative approach based on comparative analysis between G1 CDK activities in resting reference cells and in mobilized CD34+ hematopoietic stem and progenitor cells. Because pRB is both a substrate for phosphorylation by CDKs and a critical regulator of mid/late G1-phase progression at the restriction point [16, 19], we also compared the pRB phosphorylation status in cycling and resting cell populations.

After stimulation of quiescent G0 cells by signals from the environment, the concerted activities of multiple CDKs that phosphorylate substrates in a cell cycle–specific fashion is required to promote entry of the early G1 phase and transition across the restriction point into late G1 phase [16, 18, 38, 41, 42]. Beyond the restriction point, cells no longer need mitogenic stimulation to enter S phase and proliferate [43]. In contrast to the restriction point in late G1 phase, the point that controls the G0->G1 transition has been only partially defined. In human T-lymphocytes and human diploid fibroblasts, there is evidence that cyclin D-CDK4/6 complexes are the early G1-pRB-hypophosphorylating kinases [14, 15]. Consistent with previous work [14], virtually no activity of CDK4, 6, and 2 was detected in extracts of quiescent NIH3T3 and mononuclear cells that spontaneously arrested in G0 phase, and resting NIH3T3 and mononuclear cells were found to express only unphosphorylated pRB, in keeping with a previous report [27]. However, CDK4 and CDK6 activities were clearly detected in extracts of mobilized CD34+ cells, and accordingly, extracts contained pRB that was phosphorylated at Thr-373, Ser-780, Ser-795, and Ser-807/811. These residues have been identified to be phosphorylated initially by cyclin D-CDK4/6 protein kinase [23–25], whereas Ser-612 and Thr-821 are exclusively phosphorylated by CDK2 in conjunction with either cyclin E or A [24, 28]. We showed at a single-cell level that the latter pRB residues were unphosphorylated in more than 90% of CD34+ cells. While initially at the G0->G1 transition pRB is phosphorylated by CDK4/6-cyclin D, CDK2-cyclin E protein kinase is required in late G1 phase to completely inactivate pRB by successive phosphorylation, allowing progression through the cell cycle [20, 44, 45]. Previously we have shown that mobilized CD34+ cell extracts contain pRB unphosphorylated at Ser-608 [12]. This fast-migrating form of pRB (p110RB) exhibits a growth-suppressive function [46]. Thus, in mobilized CD34+ cells, the process of sequential phosphorylation of pRB has been initiated by cyclin D-CDK4/6 protein kinases, indicating exit from G0 and entry into G1 phase. However, due to the low levels of CDK2 catalytic activity, no transition beyond the restriction point into late G1 phase is possible. The present results were consistent with previous immunocytochemical analysis using anti-statin, a monoclonal antibody against a nonproliferation-specific protein [47], demonstrating that unstimulated CD34+ cells contained only 2%–3% resting G0-phase cells [12]. These results are contrary to some reports [6] but consistent with others [7, 8]. Williams et al. [6] reported that 94.53% ± 3.95% of mobilized CD34+ cells were in G0, whereas according to Fruehauf et al. [7] and Horwitz et al. [8], most resided in G1 phase. This discrepancy might be due to different methods applied for cell cycle analysis. While Williams et al. [6] relied on simultaneous measurement of cellular DNA and protein content in CD34+ cells, Fruehauf et al. [7] and Horwitz et al. [8] used triple staining for CD34, DNA, and Ki-67.

Relative quiescence is a defining characteristic of hematopoietic stem cells that is critical to prevent exhaustion under conditions of stress. There is some evidence that CD34+ cells have a decreased engraftment capacity after progression from G0 to G1 phase or even lose their engraftment potential during S/G2/M transit [33–35, 48, 49]. Although the present study does not help to explore the role of the cell cycle status of transplanted cells for long- or short-term engraftment, biochemical analyses demonstrated for the first time that most mobilized CD34+ cells reside in early G1 phase. Since there are virtually no engraftment failures if the number of CD34+ cells in the graft is 2.5 to 5.0 x 106 per kg body weight [50], we propose two alternative hypotheses to fit the present results in the context of a decreased engraftment capacity after progression from G0 to G1 phase. First, a minority of 2%–3% of mobilized CD34+ cells in G0 phase may be sufficient to provide long-term hematopoietic reconstitution after autologous or allogeneic transplantation, whereas CD34+ cells in G1 phase may be responsible for the fast short-term recovery. Second, after homing of CD34+ cells to the bone marrow, their cell cycle status will be retained in a low-cycling state by communication with the hematopoietic stroma [51–53], and it could be imagined that they may return to G0 phase.

In summary, based on biochemical analyses, we gave clear evidence that most mobilized CD34+ cells in the peripheral blood have attained early G1 but did not cross the restriction point in late G1 phase.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors gratefully acknowledge the skilled technical assistance of Regula Bürgi, Friedgard Julmy, Annemarie Schmid, and Christine Zala. This work was supported by the Bernese Cancer League (Bern, Switzerland); the Swiss National Science Foundation (Bern, Switzerland; grant 32-59005.99); the Foundation for Clinical and Experimental Cancer Research, Bern, Switzerland; and the Stammbach Foundation, Basel, Switzerland.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on April 15, 2004; accepted for publication on March 14, 2005.




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