Catalytic Activities of G1 Cyclin-Dependent Kinases and Phosphorylation of Retinoblastoma Protein in Mobilized Peripheral Blood CD34+ Hemato
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《干细胞学杂志》
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@insel.ch
ABSTRACT
For autologous transplantations, peripheral blood progenitor cells (PBPCs) have completely replaced the use of bone marrow . Moreover, for allogeneic transplantations, there is an increasing experience of use of PBPCs . To assess the stem and progenitor cell content in a transplant, the number of CD34+ cells is a reliable surrogate marker .
Mobilized CD34+ cells have been demonstrated to be either in G0 or G1 phase of the cell cycle . 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) , indicating a cell cycle status beyond the G0 phase. Alternatively, only 1%–3% of mobilized CD34+ cells are in S/G2+M phase . 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 , 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 . The activity of pRB is controlled by post-translational modifications that include phosphorylation . 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 . 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 . 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 , 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
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 , was detected with anti-PCNA antibody, thereby revealing small or nonexistent levels in quiescent NIH3T3 reference fibroblasts and resting mononuclear cells, respectively (Fig. 1, lanes 1, 3, 4). Substantial upregulation of PCNA expression was observed in NALM-6 extracts (Fig. 1, lane 2) and after in vitro stimulation of mononuclear (Fig. 1, lanes 5, 6) and CD34+ cells (Fig. 1, lanes 9, 10), whereas unstimulated mobilized CD34+ extracts accumulated moderate amounts of PCNA protein (Fig. 1, 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. 1, 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. 1, 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 , membranes were immunoblotted with anti-MARCKS antibody. Quiescent NIH3T3 lysates and resting mononuclear cell extracts had elevated levels of MARCKS protein (Fig. 1, lanes 1, 3, 4). Expression was attenuated in extracts of in vitro–stimulated mononuclear cells (Fig. 1, lanes 5, 6), whereas small or nonexisting levels of MARCKS were detected in proliferating NALM-6 cells and in cytokine-stimulated CD34+ extracts (Fig. 1, lanes 2, 9, 10). Very low levels of MARCKS expression were observed in unstimulated CD34+ extracts (Fig. 1, lanes 7, 8) and was similar to that of in vitro–stimulated mononuclear cells (Fig. 1, lanes 5, 6). Together the results indicated that the number of resting G0 cells in unstimulated mobilized CD34+ samples was small or nonexistent.
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 1, bottom line). The results indicated that the percentage of S-phase cells in mobilized CD34+ cell samples was small.
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, 2B). 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. 2A). 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. 2B). 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, 2C). The results indicated that CDK2 catalytic activity was low in unstimulated mobilized CD34+ extracts and changed dramatically with the growth rate of cells.
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. 2C). CDK4 activity was twofold enhanced in extracts of PHA-stimulated mononuclear cells compared with untreated cells (Fig. 2C). 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. 2C). 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. 2C). Exposure of mononuclear cells to PHA generated a twofold increase in CDK6 activity (Fig. 2C). 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. 3A). 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. 3A). Because vehicle alone did not interfere with CDK2 activity, inhibition of CDK2 was due to roscovitine (Fig. 3A). 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. 3A). 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. 3B). 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. 3B).
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. 4B, lane 2), mononuclear cells stimulated in vitro with PHA (Fig. 4B, lanes 5, 6), or CD34+ cells after in vitro stimulation with cytokines (Fig. 4B, 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 to be unphosphorylated at Ser-608 (hypo-p110RB) (Fig. 4B, 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. 4B, lanes 1–10). Different to using phosphorylation site–specific antibodies, immunoblotting with antibody against total pRB revealed the protein in every cell lysate (Fig. 4B, 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. 4B, 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 , complying with an early G1-phase cell cycle status.
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 1. 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 , 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 1, Fig. 5).
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
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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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@insel.ch
ABSTRACT
For autologous transplantations, peripheral blood progenitor cells (PBPCs) have completely replaced the use of bone marrow . Moreover, for allogeneic transplantations, there is an increasing experience of use of PBPCs . To assess the stem and progenitor cell content in a transplant, the number of CD34+ cells is a reliable surrogate marker .
Mobilized CD34+ cells have been demonstrated to be either in G0 or G1 phase of the cell cycle . 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) , indicating a cell cycle status beyond the G0 phase. Alternatively, only 1%–3% of mobilized CD34+ cells are in S/G2+M phase . 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 , 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 . The activity of pRB is controlled by post-translational modifications that include phosphorylation . 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 . 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 . 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 , 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
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 , was detected with anti-PCNA antibody, thereby revealing small or nonexistent levels in quiescent NIH3T3 reference fibroblasts and resting mononuclear cells, respectively (Fig. 1, lanes 1, 3, 4). Substantial upregulation of PCNA expression was observed in NALM-6 extracts (Fig. 1, lane 2) and after in vitro stimulation of mononuclear (Fig. 1, lanes 5, 6) and CD34+ cells (Fig. 1, lanes 9, 10), whereas unstimulated mobilized CD34+ extracts accumulated moderate amounts of PCNA protein (Fig. 1, 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. 1, 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. 1, 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 , membranes were immunoblotted with anti-MARCKS antibody. Quiescent NIH3T3 lysates and resting mononuclear cell extracts had elevated levels of MARCKS protein (Fig. 1, lanes 1, 3, 4). Expression was attenuated in extracts of in vitro–stimulated mononuclear cells (Fig. 1, lanes 5, 6), whereas small or nonexisting levels of MARCKS were detected in proliferating NALM-6 cells and in cytokine-stimulated CD34+ extracts (Fig. 1, lanes 2, 9, 10). Very low levels of MARCKS expression were observed in unstimulated CD34+ extracts (Fig. 1, lanes 7, 8) and was similar to that of in vitro–stimulated mononuclear cells (Fig. 1, lanes 5, 6). Together the results indicated that the number of resting G0 cells in unstimulated mobilized CD34+ samples was small or nonexistent.
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 1, bottom line). The results indicated that the percentage of S-phase cells in mobilized CD34+ cell samples was small.
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, 2B). 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. 2A). 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. 2B). 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, 2C). The results indicated that CDK2 catalytic activity was low in unstimulated mobilized CD34+ extracts and changed dramatically with the growth rate of cells.
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. 2C). CDK4 activity was twofold enhanced in extracts of PHA-stimulated mononuclear cells compared with untreated cells (Fig. 2C). 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. 2C). 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. 2C). Exposure of mononuclear cells to PHA generated a twofold increase in CDK6 activity (Fig. 2C). 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. 3A). 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. 3A). Because vehicle alone did not interfere with CDK2 activity, inhibition of CDK2 was due to roscovitine (Fig. 3A). 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. 3A). 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. 3B). 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. 3B).
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. 4B, lane 2), mononuclear cells stimulated in vitro with PHA (Fig. 4B, lanes 5, 6), or CD34+ cells after in vitro stimulation with cytokines (Fig. 4B, 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 to be unphosphorylated at Ser-608 (hypo-p110RB) (Fig. 4B, 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. 4B, lanes 1–10). Different to using phosphorylation site–specific antibodies, immunoblotting with antibody against total pRB revealed the protein in every cell lysate (Fig. 4B, 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. 4B, 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 , complying with an early G1-phase cell cycle status.
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 1. 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 , 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 1, Fig. 5).
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
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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