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Delineation of Cell Cycle State and Correlation to Adhesion Molecule Expression of Human CD34⁺ Cells from Steady-State Bone Marrow and Peripheral Blood Mobilized Following G-CSF-Supported Chemotherapy

^a Department of Internal Medicine V, University of Heidelberg;
^b German Cancer Research Center, Division D 0200, Heidelberg;
^c III. Medizinische Klinik, Klinikum Mannheim, University of Heidelberg, Heidelberg, Germany

Key Words. Adhesion antigens • CD34 • CD49d • Cell cycle • Cyclins • Hematopoietic stem cells • Human • Ki-67

Dr. Stefan Fruehauf, Department of Internal Medicine V, University of Heidelberg, Hospitalstrasse 3, D-69115 Heidelberg, Germany.

	Abstract

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Treatment with a combination of chemotherapy and G-CSF leads to the release of hematopoietic stem cells from the bone marrow (BM) to the peripheral blood (PB), where they can be harvested for transplantation. Premobilization BM CD34⁺ cells were reported to proliferate actively, while virtually none of the mobilized PB CD34⁺ cells were in the S/G₂M phase. We were interested in elucidating the cell cycle state further and in investigating the role of adhesion molecule expression on marrow-adherent and circulating CD34⁺ cells during different phases of the cell cycle. Consecutive premobilization BM and leukapheresis product (LP) samples were obtained from 14 patients following G-CSF-supported chemotherapy. Steady-state BM and LP CD34⁺ selected cells were triple-stained for CD34, for DNA using the intercalating dye 7-aminoactinomycin D, and for Ki-67, cyclins, or adhesion antigens. Ki-67 is expressed in all phases of the cell cycle except G₀ and was found in 69.14% ± 3.46% (mean ± standard error [SE]) of BM CD34⁺ cells and 62.78% ± 3.37% of LP CD34⁺ cells, while in BM significantly more CD34⁺/Ki-67⁺ cells were in the S/G₂M phase of the cell cycle than in LP (8.6% ± 0.9% versus 1.8% ± 0.3%, respectively, p = 0.0001). Therefore, most circulating mobilized CD34⁺ cells are in the G₁ phase, similar to their steady-state BM counterparts. Cyclin A became detectable in the 2n DNA peak. As expected, a higher proportion of CD34⁺/cyclin A⁺/S/G₂M cells was found in BM than in LP (p < 0.05). Antigen density of the cyclins D₃ and D₂ tended to be higher on LP than on BM CD34⁺ cells, while D₁ was found at low levels in similar density. The adhesion antigens CD18, CD49b, CD49d, CD49e, CD58, and CD62L were expressed in a significantly higher proportion of S/G₂M-phase than in G₀/G₁-phase CD34⁺ cells. The strongest association to the proliferative status was observed for CD49d, which was coexpressed by 85.9% ± 2.6% (BM) or 90.8% ± 2.5% (LP) of CD34⁺/S/G₂M cells, whereas a distinct CD34⁺/CD49d^–/S/G₂M population could not be detected. The average coexpression of the other antigens was 57% (CD49e, CD18) or lower. Our results demonstrate that the majority of PB CD34⁺ cells mobilized following G-CSF-supported chemotherapy and steady-state BM CD34⁺ cells are in the late G₁ phase of the cell cycle and show a correlation between the expression of adhesion receptors and cell cycle status of CD34⁺ cells in both BM and LP.

	Introduction

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Mobilized peripheral blood progenitor cells (PBPC) have replaced steady-state bone marrow (BM) as hematopoietic support following high-dose conditioning in patients with malignancies because mobilized PBPC mediate a more rapid hematological reconstitution than steady-state BM [1]. In recent reports, differences in the cell cycle distribution of BM progenitor cells and mobilized PBPC using cytokine administration alone [2-4] or following cytokine-supported chemotherapy [5] were addressed, and a significantly greater fraction of PB progenitor cells was found in the G₀/G₁ phase than in steady-state BM. Analysis of the G₀/G₁ phase and the G₁/S transition may allow further assessment of the "quiescent" state of PBPC [5] suggested by these results. New techniques allow a simultaneous characterization of cell surface and intracellular antigens as well as measurement of the DNA content [6]. Ki-67 is an intranuclear antigen associated with cellular activation [7]. In murine and human cells, Ki-67 is expressed from the late G₁ to the G₂/M phase of the cell cycle and disappears from postmitotic cells [8, 9]. Differences in cyclin expression allow further discrimination of cells having the same DNA content but residing at different phases of the cell cycle [10]. The D-type cyclins, D₃ and D₂ but not D₁, were upregulated during the G₁ phase of proliferating hematopoietic cells following cytokine-activation [11] or on isolated BM CD34⁺ cells [12], whereas cyclin A mRNA levels increased when DNA synthesis was initiated in hematopoietic cells from the G₁/S transition on [13].

In normal mesenchymal cells, loss of adhesion leads to cell cycle arrest at the G₁/S boundary [14]. We therefore asked whether the expression of adhesion molecules on steady-state BM and mobilized PBPC CD34⁺ cells is correlated to the cell cycle state, as suggested by some investigators [15, 16]. In previous experiments, we and others found a significantly greater fluorescence intensity of the adhesion molecule CD49d and the leukocyte function associated molecule-1 (LFA-1) complex which consists of the L/ß2 (CD11a/CD18) chains on BM CD34⁺ cells than with mobilized PBPC [17, 18]. In this analysis also, CD49b, CD49e, CD58 (LFA-3), and CD62L (L-Selectin), which were all reported on CD34⁺ cells, were included [19-21]. Leukapheresis product samples were obtained from 14 patients with hematological malignancies and solid tumors following G-CSF-supported cytotoxic chemotherapy and were compared with BM cells obtained prior to mobilization from these patients with regard to cell biological and immunological progenitor cell subsets as well as cell cycle state.

The results presented here indicate that the majority of mobilized CD34⁺ PBPC are in the late G₁ phase. Further cell cycle progression occurred primarily in the BM and was associated with an increased expression of adhesion molecules in the S/G₂M phase. The strongest association to the proliferative status was observed for CD49d.

	Materials and Methods

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Patients
Patients with breast cancer, non-Hodgkin's lymphoma, multiple myeloma, germ cell tumor, and ovarian tumor received G-CSF-supported (RmetHuG-CSF; filgrastim; Neupogen^TM; Amgen, Thousand Oaks, CA) cytotoxic chemotherapy, which depended on diagnosis. Patient characteristics are given in Table 1. Samples were obtained from BM before the start of mobilization therapy and from the first leukapheresis product (LP). PBPC were collected as soon as a distinct population of CD34⁺ cells became detectable in the peripheral blood. A COBE Spectra blood cell separator was used for harvesting (COBE BCT, Inc.; Lakewood, Colorado). The study took place under the guidelines of the ethical committee of the University of Heidelberg. Informed consent was obtained from each patient.

Colony-Forming Cell (CFC) Assay
The concentration of hematopoietic progenitor cells in each BM sample and LP product was assessed using a semisolid clonogenic culture assay (MethoCult H4433, Stem Cell Technologies, Inc.; Vancouver, Canada). The culture medium consisted of 30% fetal calf serum (FCS), 10% medium conditioned by phytohemagglutinin-stimulated leukocytes, 1 IU recombinant human erythropoietin, 5 x 10^–5 M 2-mercaptoethanol, and 0.9% methylcellulose. BM cells (1.5 x 10⁵) or 5 x 10⁴ LP cells were plated in duplicate and incubated at 37°C and 5% CO₂ in humidified atmosphere. After 14 days, colonies were scored using an inverted microscope.

Cobblestone-Area-Forming Cell (CAFC) Assay
Confluent stromal layers of the preadipocyte FBMD-1 cell line in 96-well plates were overlaid with steady-state BM or LP mononuclear cells from 6 of the 14 patients in a limiting dilution set-up as described [22]. Some patients presented to the transplant unit for a BM tap "day 0" on short notice so that confluent stromal layers were not available, and it was not possible to set up long-term cultures of BM "day 0" and LP samples of these patients. All samples inoculated in stromal cultures were growing and could be evaluated. In the first dilution, 2 x 10⁵ cells were overlaid per well. Twelve dilutions twofold apart were used for each sample, with 15 replicate wells per dilution. The cells were cultured at 33°C in -modified Dulbecco's minimal essential medium (DMEM) supplemented with HEPES (3.5 mM), glutamine (2 mM), sodium selenite (10^–7 M), 2-mercaptoethanol (10^–4 M), 20% horse serum, and hydrocortisone 21-hemisuccinate (10^–5 M final concentration). Half of the medium was changed weekly. IL-3 (10 ng/ml) and G-CSF (20 ng/ml) were added weekly to the cultures. The percentage of wells with at least one phase-dark hematopoietic clone of at least five cells (cobblestone area) beneath the stromal layer was determined at weeks 2 and 6 after overlay, and CAFC frequencies were calculated using Poisson statistics as described previously [22, 23].

Long-Term Culture Colony-Forming Cell (LTC-CFC) Assay
Confluent stromal layers of FBMD-1 cells in 25 cm² flasks were overlaid with 2-3 x 10⁴ CD34 selected PBPC. The cells were cultured in the same medium and under the same conditions as the CAFC assays. After culture periods of two weeks and six weeks, the number of colony-forming cells of the pooled nonadherent and adherent fractions was determined. To this purpose, the medium was removed from the flasks and replaced by 3 ml of 0.1% trypsin (GIBCO; GIBCO Life Technologies; Eggenstein, Germany) for 5 min. Proteolysis was stopped by adding 1 ml of ice-cold FCS. A single-cell suspension was obtained by sieving the cell suspension through a 100 mm nylon filter. Cells were resuspended in -modified DMEM and plated in duplicate in a semisolid clonogenic cell assay as described above.

Fluorescence-Activated Cell Sorting Analysis
For surface staining of antigens, BM or LP cells were incubated for 30 min at 4°C with fluorescein-isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated monoclonal antibodies directed against the following epitopes: CD18 (FITC; Dianova; Hamburg, Germany); CD7 (FITC), CD19 (PE), CD33 (PE), CD34 (PE), CD38 (PE), CD45 (FITC), CD45RA (FITC), CD62L (FITC), CD71 (FITC; Becton-Dickinson; Heidelberg, Germany); CD49b, CD49d, CD49e, CD117 (all FITC; Immunotech; Krefeld, Germany); CD58 (FITC; Cymbus Biotechnology; Southhampton, Hampshire, UK) and CDw90 (PE; Pharmingen; San Diego, CA). Isotype-specific FITC and PE monoclonal antibodies (IgG₁-FITC, IgG_2a-FITC, IgG₁-PE and IgG_2a-PE, all from Becton-Dickinson) served as controls.

For intracellular antigen staining and DNA analysis, a fixation and permeabilization protocol reported previously was followed [22]. In brief, following surface antigen staining, CD34⁺ selected cells were washed twice with ice-cold PBS and resuspended in 500 µl PBS. While slowly vortexed, 500 µl ice-cold PBS containing 2% paraformaldehyde (Merck; Darmstadt, Germany) and L--lysolecithin (160 µg/ml) (Sigma; Deisenhofen, Germany) were added dropwise and incubated for 5 min at 4°C. Lysolecithin activity was blocked by adding 2 ml PBS containing 1% bovine serum albumin ([PBS/1% BSA]; Sigma). Cells were washed twice with PBS/1% BSA and resuspended in 100 µl PBS/1% BSA. For intracellular staining of the nuclear protein Ki-67, cells were incubated for 30 min with the MIB-1 FITC (Dianova) antibody, which recognizes the FKELF amino acid sequence in the Ki-67 protein [24]. Alternatively, FITC-labeled antibodies directed against the human cyclins D₁ (clone G124-326) [25], D₂ (clone G132-43) [25], D₃ (clone G107-565) [26], and cyclin A (clone BF683) [27] were employed (all PharMingen). Isotype-specific FITC-labeled control antibodies were used. After antigen staining, cells were washed twice in PBS/1% BSA and resuspended in 100 µl PBS/1% BSA. For staining of cellular DNA, cells were incubated for 30 min in a final concentration of 25 µg/ml 7-aminoactinomycin D ([7-AAD] Molecular Probes; Leiden, The Netherlands) in PBS/1% BSA at room temperature. Cells were analyzed within one h after staining. Acquisition and analysis were performed on a FACScan flow cytometer (Becton-Dickinson) mounted with an air-cooled 488 nm Argon laser using Lysis II software. Emission from FITC, PE, and 7-AAD was measured simultaneously using short-band pass 530 nm and 585 nm filters and a 650 nm long-pass filter, respectively. Ten thousand CD34⁺ selected cells stained simultaneously for surface antigens, intracellular antigens, and DNA were acquired at a flow rate of 10-50 events per second. DNA histograms were displayed from primitive hematopoietic cells fitting into a lymphoblastoid region in a forward versus sideward scatter dot plot, in a CD34⁺ gate, and in a singlet gate defined by a fluorescence-area against fluorescence-width display ( Fig. 1). The coefficient of variation (CV) values for the G₀/G₁ peak of the CD34⁺ cell DNA-histograms following 7-AAD staining were 7.8% ± 0.5% in BM and 7.4% ± 0.4% in LP. The FL-1 channel representing the antigen stainings and the FL-3 channel representing the DNA 7-AAD staining were displayed in a two-dimensional dot plot to evaluate coexpression on CD34⁺ cells (FL-2 channel). All coexpression data were corrected for unspecific binding of isotype-matched control antibodies.

View larger version (25K): [in this window] [in a new window]   Figure 1. DNA histogram of CD34+ selected hematopoietic progenitor cells falling into the lymphoblastoid region (R1), fulfilling the criteria for singlets (R2) in an area-versus-width display of 7-aminoactinomycin D fluorescence (7-AAD) and expressing the CD34 antigen (R3). Matched samples from steady-state bone marrow (BM) and leukapheresis products (LP) of 14 patients were analyzed for cell cycle distribution. A significantly higher proportion of BM than LP CD34+ cells was in the proliferative phase (S/G2M) (p < 0.0001). Mean values ± 1 SE.

	Results

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Progenitor Cell Characteristics in Steady-State BM and Mobilized Peripheral Blood
The BM and LP samples (n = 14, each) contained 1.88% ± 0.26% and 1.72% ± 0.44% CD34⁺ cells, respectively. After immunomagnetic CD34⁺ cell selection, the percentage of CD34⁺ cells in BM was 89.18% ± 3.17% and 91.05% ± 2.26% in LP samples. When we analyzed the coexpression on CD34⁺ cells of the lineage-associated and maturation-associated antigens CD7, CD19, CD33, CD38, CD45RA, CDw90, CD117, and HLA-DR as well as the activation molecule CD71, we found that greater than 95% of hematopoietic cells in both compartments coexpressed the HLA-DR and CD38 antigens, whereas in LP compared to BM, the proportion of phenotypically primitive CD34⁺/CDw90⁺ cells and of myeloid precursors (i.e., CD34⁺/CD33⁺) was significantly greater at the expense of B-lymphocyte precursors (CD34⁺/CD19⁺), as already reported [28, 29] (data not shown).

The proliferative activity of the BM- and PB-derived CD34⁺ cells was assessed in CFC assays and in a group of 6 of the 14 patients in stroma-dependent long-term cultures plated after two and six weeks of cultivation as well as in a limiting dilution set-up with the direct visual endpoint cobblestone area formation two and six weeks after overlay. The CAFC frequencies determined here were in the previously reported ranges for BM and LP samples [22, 30] (data not shown). The mean concentration of lineage-committed and primitive progenitor cell subsets contained in the LP CD34⁺ cells tended to be greater than that contained in the BM CD34⁺ cells as has been found by other investigators that compared normal BM and LP of tumor patients [30] ( Table 2). Significance was reached in the week-2 replate (p < 0.05).

Expression of Cell-Cycle-Associated Proteins in CD34⁺ Cells
DNA-staining with 7-AAD does not discriminate G₀ from G₁ cells, which is due to a similar DNA content. Ki-67 is expressed from the late G₁ phase on through to mitosis. The majority of CD34⁺ cells in steady-state BM and PB mobilized following G-CSF -supported chemotherapy expressed the Ki-67 antigen (69.2% ± 3.5% and 62.8% ± 3.4% of CD34⁺ cells, respectively, Fig. 2). In conjunction with the DNA analysis, this suggests an accumulation in the late G₁ phase from which primarily BM CD34⁺ cells and only a few PB CD34⁺ cells can progress to the S phase. Cell cycle progression could be dissected further by analysis of cyclin A expression, which is active during S phase and degrades during anaphase. Expression of cyclin A started in the 2n DNA peak. Expression levels increased with cell cycle progression, and as expected, a significantly greater proportion (p < 0.05) of CD34⁺/cyclin A⁺/S/G₂M cells were found in BM than in LP ( Fig. 2). The antigen density of the D-type cyclins that are expressed in a tissue-specific fashion and that promote G₁ transition was similar between BM and LP for D₁. Cyclin D₂ was expressed at higher proportions in LP (mean 6.6%) than steady-state BM CD34⁺ cells (mean 2.1%) in three of four patients ( Fig. 2). Cyclin D₃ was observed at higher proportions in LP (mean 38.9%) than BM CD34⁺ cells (mean 13.6%) in all patients (p = 0.08; Fig. 2).

View larger version (37K): [in this window] [in a new window]   Figure 2. Proportions of CD34+ cells expressing cell-cycle-related proteins in the G0/G1 phase (I) or the S/G2M phase (II). Matched samples from steady-state BM and LP of 11 patients (Ki-67), eight patients (cyclin A), or four patients (D-type cyclins) were analyzed. Mean values ± 1 SE.

View larger version (15K): [in this window] [in a new window]   Figure 3. Adhesion molecule expression on BM and LP CD34+ cells during the cell cycle. For the G0/G1 phase and the S/G2M phase, ratios of coexpressing CD34+ cells relative to all CD34+ cells in the specific cell cycle compartment were calculated. Adhesion molecule expression on G0/G1 phase and S/G2M phase CD34+ cells was compared in each sample using the paired Student's t-test. Significance levels of p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) were chosen. Mean values ± 1 SE (n = 10: BM/LP CD18, CD49b, CD49d, CD49e, CD58; n = 8, BM CD62L; n = 9, LP CD62L).

View larger version (16K): [in this window] [in a new window]   Figure 4. Representative triple-color analysis of the adhesion molecule CD49d and cell cycle status of CD34+ cells in steady-state bone marrow (BM) and mobilized peripheral blood (LP). CD49d expression was associated with the progression of CD34+ cells through the DNA-synthesis-and-mitosis phase (upper right quadrant). A population of CD34+/CD49d cells could not be identified in the S/G2M phase (lower right quadrant).

	Discussion

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For separation of the G₀/G₁ phase population, the Ki-67 antigen was used which is expressed in all stages of the cell cycle except for the G₀ and early G₁ phase [8, 9]. In steady-state PB, Ki-67 was not detected on smears of CD34⁺ selected cells [32]. Using a similar staining protocol as in our study, Ki-67 was detected in 15%-25% of CD34⁺ cells from peripheral blood of normal donors mobilized with G-CSF alone [6], while following G-CSF-supported chemotherapy, we found a proportion of 62.78% ± 3.37% of CD34⁺ PBPC expressing Ki-67. This may result from an increased proliferation of hematopoietic progenitor and stem cells following myelotoxic therapy and G-CSF stimulation [33]. In comparison to CD34⁺ cells from BM, circulating CD34⁺ cells transited to the S phase in very low numbers [2-5, 31, 34], while their proliferative activity was comparable to or even higher than that of steady-state BM CD34⁺ cells [30] as observed in this study ( Table 2) and in a G-CSF mobilization study in allogeneic donors [4]. Upon cytokine stimulation of plastic adherent (P-delta) cells, a rapid cell cycle induction occurred which resulted in similar kinetics of granulocyte maturation between PBPC and BM [35].

Analysis of cyclins was used to further dissect the cell cycle state of CD34⁺ cells. D-type cyclins are induced upon cell stimulation with growth factors or mitogens and are required for progression through the G₁ phase [10]. Cyclin D₃ and cyclin D₂ tended to be expressed on a higher proportion of mobilized PBPC than steady-state BM CD34⁺ cells, while no difference was observed for cyclin D₁ ( Fig. 2). Interestingly, in transfection experiments, cell lines overexpressing D₂ and D₃ showed a shortening of the G₁ phase [11]. Pulse chase experiments are required to test the hypothesis of a shorter G₁ transit time in mobilized PBPC.

Cyclin A is active during S phase when bound to the cyclin-dependent kinase 2 (cdk2). We observed initiation of cyclin A expression in the 2n DNA peak of CD34⁺ BM cells and PBPC ( Fig. 2) which may correspond to late G₁ phase cells [14]. As expected, significantly fewer CD34⁺/cyclin A⁺ cells were found in the S/G₂M phase in mobilized PB than in steady-state BM (p < 0.05), which points to a lack of stimulatory signals or an inhibition of G₁/S phase transition. One line of further investigation is suggested by the observation that cell cycle arrest of mesenchymal cells in suspension occurs at the G₁/S boundary; this was found to be due to the stabilization of the cyclin-dependent kinase inhibitor, p27^kip-1, which leads to an inhibition of the retinoblastoma gene product, Rb, with continuing sequestration of transcription factors required for cyclin A expression by Rb or an Rb family member [14, 36].

Since cell cycle activation and adhesion are tightly connected in hematopoietic cells cultured in vitro [16], we studied whether cell cycle progression of bone-marrow-adherent and circulating hematopoietic cells is correlated to the expression of adhesion molecules. We determined 8.48% ± 0.42% of steady-state BM CD34⁺ cells and 1.73% ± 0.21% of LP CD34⁺ cells to be in the S/G₂M phase. Coexpression analysis was based on 10⁴ CD34⁺ cells per sample. The low variation in the small proportion of adhesion receptors expressing CD34⁺/S/G₂M cells in 8 to 10 patients demonstrates the reproducibility and validity of our data ( Fig. 3). Cell cycle progression was associated with the co-expression of the ₄ß₁ integrin subunit CD49d in the majority of steady-state BM (85.9% ± 2.6%) and mobilized PB (90.8% ± 2.5%) CD34⁺ cells in the S/G₂M phase, while the other adhesion molecules of the integrin, selectin, or immunoglobulin superfamily showed a significant yet lower S/G₂M-phase-related coexpression of on average 57% (CD49e, CD18) or less. CD49d is involved in the homing and mobilization of primitive hematopoietic progenitor cells within the BM, since administration of a function-blocking antibody to CD49d to nonhuman primates resulted in a mobilization of hematopoietic progenitor cells [37]. Previous investigations of our group and others showed a significantly greater fluorescence intensity of the adhesion molecules CD18 and CD49d on BM CD34⁺ cells compared to mobilized PBPC [17-19]. Levesque and colleagues [38] proposed a concept of an initial activation of low-affinity CD49d and CD49e receptors generated by cytokine receptors for, for example, IL-3, that is followed by a secondary proliferation-inducing signal resulting from fibronectin binding to CD49d or CD49e [16]. One molecule which was shown to integrate fibronectin-mediated adhesion and cell cycle signals in fibroblasts is the product of the c-abl proto-oncogene, which functions as a tyrosine kinase and can transmit integrin signals to the nucleus [39]. Binding of fibronectin to its receptor, CD49d, and CD49e expressed on hematopoietic progenitors and leukemic cell lines [40] results in a proliferation induction [16]. Our DNA analysis, Ki-67, cyclin, and adhesion molecule coexpression data support the hypothesis that mobilized PBPC are ready to enter the S/G₂M phase but lack stimulatory signals by bone marrow stroma components.

	Acknowledgments

 
We are grateful for the technical assistance of Mrs. Sigrid Heil and for the support of the stem cell transplantation laboratory team. IL-3 was provided by Dr. Färber, Novartis, Nürnberg. This work was supported in part by grant 10-1018-Ze-I (to S.F. and W.J.Z.) of the Deutsche Krebshilfe/Dr. Mildred-Scheel-Stiftung.

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