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T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors

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T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors
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   Experimental Hematology 29 (2001) 401–415 0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.PIIS0301-472X(01)00617-8  T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors  Sergio Rutella   a  , Luca Pierelli   a  , Carlo Rumi   a  , Giuseppina Bonanno   b  , Maria Marone   b  ,Simona Sica   a  , Ettore Capoluongo   c  , Franco Ameglio   c  , Giovanni Scambia   b  , and Giuseppe Leone   a   a   Department of Hematology and b   Department of Gynecology, Catholic University Medical School, Rome, Italy; and c   Department of Clinical Pathology, IRCCS San Gallicano, Rome, Italy  (Received 28 September 2000; revised 16 November 2000; accepted 29 November 2000)  Objective.  Peripheral blood progenitor cells (PBPC) mobilized by granulocyte colony-stimu-lating factor (G-CSF) promptly engraft allogeneic recipients after myeloablative chemother-apy for hematologic malignancies. Surprisingly, no exacerbation of acute graft-vs-host diseasehas been observed despite a 10-fold higher T-cell content in PBPC compared with bone mar-row allografts. Because G-CSF can suppress T-cell proliferation in response to mitogens andenhance their activation-induced apoptosis, we examined the molecular mechanisms underly-ing G-CSF–induced immune dysfunction.   Materials and Methods.  Normal allogeneic lymphocytes were challenged with phytohemagglu-tinin in the presence of serum collected after G-CSF administration (postG) to healthy PBPCdonors, and the expression of key components of the cell cycle and apoptotic machineries wasinvestigated by flow cytometry and Western blotting.   Results.  Lymphocyte stimulation was associated with collapse of mitochondrial transmem-brane potential, hypergeneration of reactive oxygen intermediates, and activation of caspase-3 and DNA fragmentation. Lymphocytes were arrested in a G   1  -like phase of the cell cycle, asmeasured by G   1  -phase cyclin expression and bromodeoxyuridine (BrdUrd) incorporation.Cell tracking experiments confirmed the occurrence of a lower number of population dou-blings in postG compared with preG cultures. Unexpectedly, the phosphorylation state of theprotein encoded by the retinoblastoma susceptibility gene (pRB) was unaltered in postG cul-tures, and the inhibition of cell cycle progression occurred without the recruitment of the cy-clin-dependent kinase inhibitors p15   INK4B  , p16   INK4A  , and p27   Kip1  . We eventually evaluated theability of antioxidant/cytoprotectant agents to prevent the G-CSF–induced mitochondrial dys-function and inhibition of cell cycle progression. Of interest, both  N   -acetylcysteine and amifos-tine reduced apoptotic cell death by 45% on average, inhibited the activation/processing of caspase-3, and increased BrdUrd incorporation in postG cultures.  Conclusions.  Based on these experimental findings, a model is proposed in which T-cell acti-vation in the presence of serum immunoregulatory factor(s) induced by G-CSF is associatedwith a molecular phenotype mimicking the G   1  –S transition and consisting of pRB phosphory-lation, lack of CDKI recruitment, and reduced cyclin-E expression. The putative relationshipbetween lymphocyte mitogenic unresponsiveness and apoptosis induction would occur at thelevel of key molecules shared by the cell cycle and apoptotic machineries. Whether theG-CSF–mediated modulation of lymphocyte functions in vitro is beneficial in transplantationmedicine remains to be determined.© 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.  Offprint requests to: Sergio Rutella, M.D., Department of Hematology, Catholic University Medical School, Largo A. Gemelli 8-00168 Rome, Italy; E-mail:sergiorutella@tin.it   402  S. Rutella et al./Experimental Hematology 29 (2001) 401–415  Introduction  Recombinant human granulocyte colony-stimulating factor(G-CSF) can be administered to human leukocyte antigen(HLA)-identical as well as to HLA-A, HLA-B, and HLA-DRcompatible unrelated donors to mobilize peripheral blood pro-genitor cells (PBPC) for subsequent transplantation into allo-geneic recipients [1,2]. Contrary to expectations, incidenceand severity of acute graft-vs-host disease (aGVHD) were notincreased after PBPC compared with bone marrow allogeneictransplantation, despite a 10-fold higher T-cell content inG-CSF–mobilized grafts [3–5]; however, the incidence of clinical extensive chronic GVHD might be increased [6].The regulatory effects of G-CSF on T lymphocytes werereviewed recently [7,8]. Several investigations suggest a hu-moral modulation of T-cell function both in animal modelsand in humans, which consists of 1) downregulation of Thelper-1 (Th1)-derived cytokines and polarization toward aTh2 functional profile [9]; 2) increase of serum immunoreg-ulatory mediators, i.e., lactoferrin, interleukin-1 receptor an-tagonist (IL-1ra), and IL-10 [10,11]; 3) lymphocyte unre-sponsiveness; and 4) induction of lymphocyte partialactivation after mitogenic challenge [12,13]. However, ad-ditional mechanisms of immune suppression by G-CSFhave been claimed, i.e., monocyte-mediated inhibition of T-cell proliferation through CD28 responsive complex, aninducible T-cell transcription factor binding in the promoterof the interleukin-2 (IL-2) gene [14–16].We recently described an enhanced tendency to activa-tion-induced apoptosis of allogeneic CD4     and CD8     Tcells stimulated with phytohemagglutinin (PHA) in the pres-ence of serum collected after G-CSF administration [17].Conceivably, those phenomena were mediated by Bax pro-tein overexpression with subsequent collapse of mitochon-drial transmembrane potential, hypergeneration of reactiveoxygen intermediates (ROIs), and fragmentation of genomicDNA. The neutralization of surface CD95 abrogated the per-turbation of mitochondrial function, strongly suggesting theinvolvement of the CD95–CD95L signaling pathway [17].The mitogen-dependent progression through the G   1  phaseof the cell cycle and the initiation of DNA synthesis are co-operatively regulated by the cyclin-dependent kinases(CDKs), whose activity is controlled by their associationwith cyclins and CDK inhibitors (CDKI) [18]. Upon stimu-lation with mitogens, the protein encoded by the retinoblas-toma susceptibility gene (pRB) is inactivated by phosphory-lation mediated by the sequential intervention of D-typecyclins and cyclin E. Hyperphosphorylation renders pRBincapable of binding E2F-type transcription factors, leadingto the activation of genes necessary for DNA synthesis andfor the advancement through the cell cycle [19–21].Experimental evidence supports an interconnection be-tween cell cycle and apoptosis, a ubiquitous physiologic pro-cess through which multicellular organisms eliminate dam-aged or unwanted cells. In particular, pRB, D-type cyclins,and CDKI have been proposed as candidate molecules af-fecting both cell cycle progression and apoptotic cell death[22]. Recently, a novel and intriguing function of mitochon-dria has emerged in apoptosis research, in addition to theirestablished role as energy-producing organelles. In particu-lar, mitochondria might be central executioners of apoptoticprocesses, and both collapse of mitochondrial transmem-brane potential and hypergeneration of ROIs might representgeneral features of programmed cell death [23].Because the molecular mechanisms underlying lympho-cyte unresponsiveness to mitogenic challenge after exposureto G-CSF–induced soluble immunoregulatory mediators re-main largely unknown [24], we investigated the expressionof key components of the cell cycle and apoptotic machiner-ies in normal allogeneic lymphocytes activated with PHA inthe presence of serum collected after G-CSF administration(postG) to healthy PBPC donors. To this purpose, lympho-cyte mitochondrial function, phosphorylation state of cell cy-cle related proteins (pRB, p107), and expression of G   1  -phasecyclins and representative CDKI (p15   INK4B  , p16   INK4A  ,p21   Cip1  , p27   Kip1  ) were measured. This study clearly demon-strates that lymphocyte stimulation in the presence of postGserum is associated with molecular events mimicking thetransition through the G   1  –S phase of the cell cycle but ulti-mately leading to the execution of an apoptotic program.  Materials and methods  Characteristics of PBPC donors and collection of donor serum  Six Caucasian HLA-identical healthy donors (three men and threewomen; median age 36 years) received G-CSF (16   g/kg/day;Granocyte Rhone-Poulenc Rorer, Milan, Italy) subcutaneously for6 days to mobilize PBPC for allogeneic transplantation. Donor se-rum was collected before (preG) and on day 4 of G-CSF adminis-tration (postG), aliquoted, and stored at   80    C until use, as de-tailed previously [10,25]. Informed consent was obtained fromPBPC donors, and the investigations were approved by the Institu-tional Human Research Committee.  Preparation of peripheral blood lymphocytes and culture conditions  Heparin-anticoagulated peripheral blood samples from normalG-CSF–untreated subjects were diluted 1:1 in RPMI 1640 culturemedium, layered on a Ficoll-Hypaque gradient (density 1,077 g/L;Uppsala, Sweden), and centrifuged at 1,700 rpm for 30 minutes at20    C. Peripheral blood mononuclear cells (PBMC) were harvestedat the interface, washed in RPMI 1640 at 1,500 rpm for 6 minutes,and seeded at a concentration of 1   10   6   /mL in RPMI 1640 me-dium either under serum-free (SF) conditions (10% BIT HCC-9500serum substitute; StemCell Technologies, Vancouver, BC, Canada)or with preG or postG serum (20% final concentration, v/v). Cellswere stimulated with PHA (0.5   g/mL) for 72 hours at 37    C in hu-midified atmosphere (5% CO   2  and 95% air), harvested, counted,and used as described in detail later.   Evaluation of lymphocyte mitochondrial function  The simultaneous assessment of surface markers, mitochondrialinner transmembrane potential (     m  ), and generation of ROIs was   S. Rutella et al./Experimental Hematology 29 (2001) 401–415  403  performed as follows [17,23]. Cells were stained with phycoeryth-rin (PE)-conjugated anti-CD4 (S3.5 clone, IgG   2a  ) or anti-CD8(3B5 clone, IgG   2a  ) monoclonal antibodies (mAb) or with PE-con- jugated isotype-matched irrelevant mAb (Caltag Laboratories,Burlingame, CA, USA) for 30 minutes at 4    C and then exposed for15 minutes to 3,3    -dihexyloxacarbocyanine iodide [DiOC   6  (3), 40nM] and to hydroethidine (HE; 2 mM; Molecular Probes, Eugene,OR, USA) prior to flow cytometric analysis. In control experi-ments, cells were incubated with carbonyl cyanide m-chlorophe-nyl-hydrazone (mClCCP, 50   M; Sigma Chemical Corp., St.Louis, MO, USA), an uncoupler of oxidative phosphorylation, at37    C for 30 minutes before DiOC   6  (3) labeling. MClCCP treatmentcompletely abolishes    m  , demonstrating that DiOC   6  (3) uptake isdriven by    m  and does not involve significant binding to othercellular components. In selected experiments aimed at protectinglymphocytes from activation-induced apoptosis, PBMCs were pre-incubated with  N   -acetylcysteine (NAC; 10 mM/L) or amifostine(60   g/mL) for 30 minutes prior to PHA challenge in the presenceof preG or postG serum [26,27].   Detection of processed caspase-3  After PHA activation, cells were treated with fixation medium (so-lution A; Fix & Perm, Caltag Laboratories) and incubated for 15minutes at room temperature. After washings in phosphate-buff-ered saline (PBS) supplemented with 1% bovine serum albumin(BSA), cells were resuspended in permeabilizing medium (solu-tion B) containing saturating amounts of rabbit polyclonal anti-activecaspase-3 antibody (PharMingen, San Diego, CA, USA), followedby incubation with fluorescein isothyocyanate (FITC)-conjugatedgoat anti-rabbit immunoglobulins (Caltag Laboratories) for 30minutes at 4    C. The anti-active caspase-3 antibody reacts with aconformational epitope exposed on the processed forms of caspase-3 and recognizes cells undergoing apoptosis [28].   Bromodeoxyuridine incorporation assay  Lymphocyte proliferation was analyzed after 24, 48, and 72 hoursof PHA stimulation by evaluating the incorporation of bromodeox-yuridine (BrdUrd), a thymidine analogue, in newly synthesizedDNA strands. Briefly, cells were exposed to BrdUrd (25   M finalconcentration; Sigma Chemical Corp.) during mitogenic chal-lenge. Aliquots of cells were harvested at indicated time points andwere fixed with ice-cold 70% ethanol. Because anti-BrdUrd mAbsreact with single-stranded DNA, partial denaturation was achievedby incubation with 3N HCl containing 0.5% Tween-20 for 20 min-utes at room temperature. After washings with PBS, cells were re-suspended in 0.1 M Na   2  B   4  O   7  to neutralize residual HCl and subse-quently incubated with pretitrated dilutions of FITC-conjugatedanti-BrdUrd mAb (BR-3 clone, IgG   1  ; Caltag Laboratories) for 30minutes at 4    C. Background fluorescence was established with flu-orochrome-conjugated isotype-matched irrelevant immunoglobu-lins. Cells not exposed to BrdUrd during PHA activation wereused to evaluate the specificity of the anti-BrdUrd staining. Cellu-lar DNA content was measured by resuspending the BrdUrd-labeled cells in DNA staining buffer (5   g/mL propidium iodide,2 mg/mL RNAse) for 30 minutes prior to flow cytometric analysis.   Detection of G   1  -phase cyclins  PHA-challenged PBMCs were sequentially fixed with 0.1%paraformaldehyde and 70% ethanol, as previously reported [29]. Af-ter washings with PBS containing 0.05% Tween-20, cells were incu-bated overnight with FITC-conjugated anti-D-type cyclin mAb(G107-565 clone, IgG   1  ) or unconjugated anti-cyclin E mAb (HE12clone, IgG   1  ; both from PharMingen). Samples labeled with the pri-mary unconjugated anti-cyclin E mAb were further incubated withFITC-conjugated F(ab    )   2  goat anti-mouse immunoglobulins (CaltagLaboratories) for 30 minutes at 4    C [30]. Controls were preparedidentically as described, except that an isotype-matched mAb wasadded instead of the anti–D-type cyclin and anti-cyclin E mAb. Forthe evaluation of cyclin vs DNA content, cells were resuspended inDNA staining buffer (5   g/mL propidium iodide, 2 mg/mL RNAse)and kept on ice for 30 minutes prior to flow cytometric analysis [31].   Detection of cell cycle-related proteins and CDKI   PHA-activated PBMCs were sequentially fixed and permeabilizedas described earlier. After washings in PBS supplemented with 1%BSA, cells were incubated for 30 minutes at 4    C with saturatingamounts of antibodies directed against pRB (G3-245 clone, mouseIgG   1  ), p107 (SD9 clone, mouse IgG   1  ), p16   INK4A  (G175-405 clone,mouse IgG   1  ), p21   Cip1  (SX118 clone, mouse IgG   1  ), p27   Kip1  (G173-524clone, mouse IgG   1  ; all from PharMingen), and p15   INK4B  (sc-611clone, rabbit IgG; Santa Cruz Biotechnology, Santa Cruz, CA,USA). Cells were then washed with PBS supplemented with 1%BSA and labeled with FITC-conjugated F(ab    )   2  goat anti-mouse orwith FITC-conjugated goat anti-rabbit immunoglobulins (CaltagLaboratories) for 30 minutes at 4    C. Isotypic controls were pre-pared with the appropriate mouse or rabbit irrelevant antibodies.  Western blot analysis  Cell lysate and Western blotting were performed as follows. Thepellet obtained from 1   10   6  cells was washed in PBS and dis-solved in lysis buffer (20 nM TRIS HCl at pH 7.4, 0.1 M NaCl, 5 mMMgCl   2  , 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 U/mLkallikrein inhibitor aprotinin, 50 mM NaF, 2 mM Na   3  VO   4  , 1 mMPMSF, 2   g/mL leupeptin). Protein concentration was determinedwith the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,CA, USA). Thirty micrograms of each protein sample was sepa-rated on 6% polyacrylamide SDS gel and electroblotted on polyvi-nylidene fluoride membranes (Millipore Co., Bedford, MA, USA).Membranes were incubated with 6% nonfat dry milk in 1    TBSTfor blocking (0.1 M Trizma base, 0.15 M NaCl, 0.05% Tween-20,pH 7.4) and then with the primary rabbit antibodies in 3% nonfatdry milk in 1    TBST (anti-pRB and anti-p107; both from SantaCruz Biotechnology). Following incubation with a goat anti-rabbitsecondary antibody conjugated with horseradish peroxidase (Bio-Rad), detection was performed with the ECL Plus system (Amer-sham International, Buckingamshire, United Kingdom), and theblots were exposed to X-AR-5 OMAT Kodak films. The blotswere reused by stripping at 50    C for 30 minutes in 100 mM 2-mer-captoethanol, 2% SDS, and 62.5 mM TRIS HCL, pH 6.7, followedby extensive blocking and reprobing with a different antibody. TheBenchMark prestained protein ladder (Life Technologies Inc.,Rockville, MD, USA) was used as a reference for relative mobil-ity. Images of x-ray films were acquired with a Cohu CCD camera,and quantification of the bands was performed with Photoretix 1D(Photoretix International Ltd., Newcastle Upon Tyne, UnitedKingdom). Band intensity was expressed as relative absorbanceunits.   Measurement of lymphocyte divisions by carboxyfluorescein-diacetate succinimidyl-ester   PBMC were resuspended in PBS containing 2.5   M carboxyfluo-rescein-diacetate succinimidyl-ester (CFDA-SE; Molecular Probes)   404  S. Rutella et al./Experimental Hematology 29 (2001) 401–415  for 10 minutes at room temperature. To quench the labeling pro-cess, an equal volume of fetal calf serum (FCS) was added. Afterwashings in PBS supplemented with 3% FCS, cells were chal-lenged with PHA (0.5   g/mL) for 72 hours at 37    C under SF condi-tions or in the presence of either preG or postG-serum, as describedearlier. CFDA-SE labeled cells then were incubated with PE-conju-gated anti-CD4 or anti-CD8 mAb or with PE-conjugated isotype-matched mAb as negative control (Caltag Laboratories) for 30 min-utes at 4    C. After washings with ice-cold PBS supplemented with1% BSA, cells were kept on ice until flow cytometric analysis. Cellkinetic parameters were calculated as detailed in the Appendix.  Flow cytometry  Samples were run through a FACScan flow cytometer (Becton-Dickinson [BD], Mountain View, CA, USA) equipped with an ar-gon laser emitting at 488 nm. The fluorescence of FITC andDiOC   6  (3) was recorded in FL1 (525 nm), the fluorescence of PEwas recorded in FL2 (575 nm) and the fluorescence of ethidium(Eth) was recorded in FL3 (670 nm), after suitable electronic com-pensation. A minimum of 10,000 events was acquired in list modeusing CellQuest software. Forward scatter (FSC) and side scater(SSC) were collected as linear signals, and fluorescent emissionswere collected on four-decade logarithmic scales. Optical align-ment and gain setting stability were verified daily with fluorescentmicrospheres (Calibrite; BD).For DNA content analysis, a minimum of 30,000 events wasacquired after instrument calibration with chicken erythrocyte nu- clei (DNA QC; BD), setting PE emission channel to a linear scalefor propidium iodide fluorescence. The calculation of cell cyclecompartments was performed by deconvoluting DNA content his-tograms on a 1,024-channel scale with ModFit LT 2.0 software(Verity Software House Inc., Topsham, ME, USA).  Statistical methods  The approximation of population distribution to normality wastested using y(g) statistics for kurtosis and symmetry. Results arepresented as mean (    ) and standard deviation (SD). Correlationswere examined by Spearman rank analysis, and all comparisons byanalysis of variance (ANOVA) or the Student’s t   -test for pairedand unpaired data, as appropriate. The criterion for statistical sig-nificance was defined as  p     0.05.  Results   Lymphocyte stimulation in the presence of postG serum is associated with collapse of     m   , hypergeneration of ROI, and caspase-3 activation  Following our observation that immunoregulatory solublefactor(s) induced by G-CSF render lymphocytes unrespon-sive to mitogenic challenge and enhance their activation-induced apoptosis [10,17], we evaluated mitochondrial in-ner transmembrane potential (     m  ) and generation of ROI Figure 1. Measurement of lymphocyte  m  and generation of ROI after mitogenic challenge in the presence of postG serum. Normal PBMCs were chal-lenged with PHA (0.5  g/mL) for 72 hours in the presence of preG or postG serum (20% final concentration, v/v). Control cultures were performed underserum-free (SF) conditions. (A)  Lymphocyte  m  and production of superoxide anion, as measured by DiOC 6 (3) retention and oxidation of HE to Eth, respec-tively, were evaluated both in cultures performed with unselected lymphocyte subsets and in those performed with FACS-purified CD4   and CD8   T cells.*  p     0.01 and **  p     0.001 compared with preG cultures. Bars represent the mean percentage of apoptotic DiOC 6 (3) low Eth high  T cells from six independentexperiments performed in duplicate. (B)  Representative flow cytometric profile. The percentage of apoptotic DiOC 6 (3) low Eth high  CD4(8)   T cells is indicated.The purity of FACS-selected CD4   and CD8   T-cell fractions always exceeded 99.3%.  S. Rutella et al./Experimental Hematology 29 (2001) 401–415 405 after PHA stimulation in the presence of preG or postG se-rum. To this purpose, the lipophilic cation DiOC 6 (3) wasused, which accumulates in the mitochondrial matrixdriven by the electrochemical gradient following theNernst equation [32]. Cells were simultaneously labeledwith hydroethidine (HE), which is oxidized by superoxideanion to Eth, emitting red fluorescence [23]. Interestingly,26%   7% of CD4   and 39%   11% of CD8   T cells inpostG cultures were apoptotic, as measured by reducedDiOC 6 (3) retention and hypergeneration of ROI. Con-versely, 8%   5% DiOC 6 (3) low Eth high CD4   T cells (  p    0.01) and 6   3% DiOC 6 (3) low Eth high CD8   T cells (  p    0.001) were found in cultures stimulated in the presence of preG serum (Fig. 1). When FACS-purified CD4   andCD8   T cells were challenged separately with the mitogen,the proportion of apoptotic T cells was still higher in postGcompared with preG cultures (Fig. 1), confirming that mi-tochondrial activation occurs both in unfractionated and inFACS-purified CD4   and CD8   T cells [17]. To addresswhether the perturbation of lymphocyte mitochondrialfunction is associated with caspase activation, cells werelabeled with an antibody directed against a conformationalepitope of caspase-3, an “effector” caspase responsible foramplification of the apoptotic signal and likely to be re-quired for cells to adopt a typical apoptotic phenotype [33].Of interest, active caspase-3 was detected in 45%   9% of T cells from postG cultures vs 8%   3% of T cells in preGcultures (  p     0.001). As expected, less than 2% of T cellsreacted with the anti-active caspase-3 antibody in culturesperformed under SF conditions. The enhanced tendency toactivation-induced T-cell apoptosis in postG cultures wasconfirmed by a highly sensitive LM-PCR ladder assay(data not shown).  Lymphocyte stimulation in the  presence of postG serum is associated with low expression of cyclin E and low incorporation of BrdUrd  We reasoned that the perturbation of lymphocyte  m  andthe hypergeneration of ROI might directly impact on the ca-pacity to progress through the cell cycle; thus, the expres-sion of G 1 -phase cyclins was investigated and correlatedwith the cell cycle position through the bivariate analysis of cyclin vs DNA content. Following the observation that themaximal level of D-type cyclins can be detected early aftermitogenic challenge [31], cells were harvested after 24, 48,and 72 hours of stimulation in the presence of preG orpostG serum. No difference in the overall level of D-typecyclin-associated fluorescence was found in postG vs preGcultures after 24 hours of mitogenic stimulation (Fig. 2). At48 hours of culture, the percentage of D-type cyclin   cellswith a G 0  /G 1 -like DNA content (G 0 -like cells) was higher inpostG compared with preG cultures. At 72 hours of culture,the percentage of D-type cyclin   cells with a G 0  /G 1 -likeDNA content (G 1 -like cells) was higher in postG comparedwith preG cultures, indicating a hindered exit from quies-cence (Fig. 2). Because the expression of D-type cyclinsprecedes that of cyclin E, the fraction of cyclin E   lympho-cytes was measured after 72 hours of PHA stimulation. Of interest, an average 72% reduction of the percentage of cy-clin E   cells was found in postG compared with preG cul-tures (Fig. 3A). A representative flow cytometric profile of cyclin E vs DNA content is shown in Figure 3B.When DNA synthesis was evaluated through BrdUrd in-corporation, the majority of cells stimulated in the presenceof postG serum resided in the BrdUrd   fraction at any timeof culture. Conversely, T cells actively synthesized DNAand incorporated BrdUrd in preG cultures after 24, 48, and Figure 2. Cell cycle position and expression of D-type cyclins after mitogenic challenge in the presence of postG serum. Normal PBMCs were activated withPHA (0.5  g/mL) in the presence of preG or postG serum (20% final concentration, v/v). Control cultures were performed under serum-free (SF) conditions.T cells were gated based on CD3 specific fluorescence. D-type cyclin   T cells with a G 0  /G 1  DNA content were considered to be in the G 0  phase of the cellcycle. D-type cyclin   T cells with a G 0  /G 1  DNA content were considered to be in the G 1  phase of the cell cycle. Cells were considered in the G 2 -M phase of the cell cycle if they expressed a G 2 -M DNA content and they were positive for D-type cyclins. *  p     0.01 and **  p     0.001 compared with preG cultures.Datapoints represent the mean and standard deviation obtained in six independent experiments performed in duplicate.
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