Cell Division Tracking Research Articles

BMS-214662 Targets Quiescent Chronic Myeloid Leukaemia Stem Cells and Enhances the Activity of Both Imatinib and Dasatinib (BMS-354825).

Chronic myeloid leukaemia (CML) is a clonal disease of stem cell origin associated with expression of the Philadelphia chromosome and its oncogenic fusion protein product Bcr-Abl. Despite an impressive rate of complete cytogenetic response in chronic phase CML, the majority of patients treated with imatinib mesylate (IM) show persistent molecular disease. Recent work by our group shows that this molecular persistence results from a population of quiescent CML stem cells which are not effectively targeted by IM, the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825; which targets Bcr-Abl and Src kinases), or several rationally designed drug combinations1. Further in vitro studies by our group have demonstrated that the only combination to have an improved response in the quiescent stem cell sub-population was IM with the farnesyl transferase inhibitor (FTI) lonafarnib. BMS-214662 is an atypical non-peptidomimetic cytotoxic FTI, which has been shown to preferentially kill non-dividing cells2 and has anti-leukaemic activity in acute myeloid leukaemia. We assessed the efficacy of this compound alone and in combination with IM and dasatinib in primary CD34+ CML cells in vitro using a CFSE-based flow cytometry method to track cell division, caspase-3 activity to measure apoptosis and dephosphorylation of Crkl to determine Bcr-Abl kinase activity. Primary CD34+ CML cells were cultured for 6 days in serum free medium supplemented with 5 growth factors (IL-3, IL-6, Flt-3 ligand, G-CSF and SCF). Conditions studied were: (1) no drug control, (2) IM (5μM; ~IC90 dose), (3) dasatinib (150nM; ~IC90 dose) (4) BMS-214662 (250nM; ~IC50 dose), (5) IM plus BMS-214662, (6) dasatinib plus BMS-214662. After 6 days culture, there was a significant reduction in total viable cells in all treatment arms relative to the no drug control (P=0.001). The combinations of IM plus BMS-214662 and dasatinib plus BMS-214662 showed increased cytotoxic effect over either IM or dasatinib alone (P=0.024 and P=0.034, respectively). While the IM and dasatinib arms showed significant accumulation of undivided CFSEmax CD34+ CML cells over the no drug control (P=0.04 and P=0.023, respectively), the arms containing BMS-214662 either alone or in combination showed a reduction in these primitive cells to <50% of the no drug control. This reduction was highly statistically significant when either IM or dasatinib alone was compared to the combination with BMS-214662 (P=0.01 and P=0.043, respectively). There were no significant differences in undivided CFSEmax CD34+ CML cells between the BMS-214662 containing arms. At 72 hours, caspase-3 activity was increased in the BMS-214662-containing arms with increased apoptosis in the undivided CFSEmax CD34+ CML cells. BMS-214662 induced dephosphorylation of Crkl in remaining viable cells at 72 hours and 6 days, suggesting inhibition of Bcr-Abl kinase activity. In conclusion, BMS-214662 is highly effective against CML cells, including, for the first time, the primitive quiescent stem cell fraction, overcoming the accumulation of this population seen with IM or dasatinib in vitro.

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow‐FISH‐DDD) to determine telomere length dynamics in proliferating cells

Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.

High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro:correlation with apoptosis and Kit expression

AbstractT-helper 1 (TH1) (interferon-γ [IFN-γ]) and TH2 (interleukin-4 [IL-4] and IL-5) cytokines have been variably reported to alter human mast cell numbers in complex culture systems. The effects of these cytokines on the kinetics of cell division and cell death are unknown, and their effect on mast cell behavior is relevant to anticipate the consequences of in vivo strategies that alter cytokine levels. To determine the effect of these cytokines on stem cell factor (SCF)–dependent human mast cell production, we used highresolution tracking of cell division and correlated the results with cell apoptosis, expression of Kit, and mast cell degranulation. When IFN-γ, IL-5, or IL-4 was administered over 8 weeks, we found each cytokine decreased the mast number through a different mechanism. IFN-γ inhibited early progenitor cell division, IL-4 down-regulated early Kit expression, and IL-5 blocked later cell division. Further, IL-4 and IFN-γ had the greatest suppressive effect on degranulation and FcϵRI expression. When these cytokines were administered to mature mast cells, IFN-γ and IL-5 had no effect on degranulation and cell division, but IL-4 induced division and potentiated FcϵRI-mediated degranulation. Thus, exposure of human mast cells to IL-4, IL-5, and IFN-γ during growth and differentiation generally down-regulated mast cell number and function, whereas IL-4 increased mature mast cell division and degranulation.

Effect of Growth Factor Stimulation on Imatinib-Mediated Proliferation Inhibition and Apoptosis of CML CD34+ Cells.

Imatinib mesylate (Gleevec) is a highly effective treatment for CML, but the long-term durability of response is unclear. We have identified residual BCR/ABL+ progenitors in patients achieving CCR on imatinib (Blood 2003, 101:4701). We have shown that imatinib inhibits CML progenitors through suppression of abnormally enhanced proliferation (Blood 2002, 99:3792) and that apoptosis is restricted to dividing cells with quiescent progenitors being resistant to apoptosis (Blood 2003, 102:652a). Here we have investigated whether growth factor (GF) stimulation of imatinib-treated CML progenitors could enhance proliferation and sensitivity to apoptosis and reduce the number of viable quiescent cells. CML and normal CD34+ cells were analyzed for apoptosis following culture in the presence or absence of imatinib (1μM) for 72 hours with either low (1μg/ml SCF and FL3, 0.2μg/ml IL6, G-CSF and IL3) or high (100μg/ml SCF and FL3, 20μg/ml IL6, G-CSF and IL3) GF concentrations. Cells were labeled with CFSE prior to culture to track cell division and allow separate examination of apoptosis in quiescent and proliferating cells. High GF conditions increased proliferation of both normal and CML CD34+ cells. Suppression of CML progenitor proliferation by imatinib was reduced in high GF conditions, compared to low GFs, when IL-3 was present (p=0.009), but not when IL3 was absent (p=0.065). Interestingly, normal CD34+ cells stimulated with high GFs demonstrated increased sensitivity to imatinib-mediated inhibition of proliferation, both with (p=0.005) and without IL3 (p=0.013). High GF conditions also resulted in reduced apoptosis of CML CD34+ cells, both with (p=0.003) and without IL-3 (p=0.001). In contrast, normal CD34+ cells demonstrated increased sensitivity to imatinib-induced apoptosis in high GFs. Imatinib did not induce apoptosis in undivided cells under either low or high GF conditions. For proliferating CML CD34+ cells we observed a reduction in apoptosis induced by imatinib in high GF conditions (+IL3: p=0.012; −IL3: p=0.019). The percent of cells used to initiate culture that remained as viable, undivided cells following exposure to imatinib was calculated. There was a trend towards a reduction in the number of quiescent, viable CML CD34+ cells after imatinib treatment in high GF conditions (p=0.071). However, high GF conditions also led to a reduction in the number of quiescent, viable normal cells. In conclusion, we show that high GF conditions counter imatinib-mediated proliferation suppression in CML CD34+ cells but also protect cells from apoptosis. GF stimulation also enhances the sensitivity of normal CD34+ cells to imatinib. A trend towards a reduction in viable, quiescent CML progenitors was seen, even with short-term GF exposure, supporting further evaluation of the use of GF stimulation to reduce residual CML progenitors during imatinib treatment.Effect of Imatinib on CD34+ CellsLow Growth FactorsHigh Growth Factors+IL3−IL3+IL3−IL3Proliferation Suppression¹CML54 ± 256 ± 245 ± 252 ± 1Normal25 ± 430 ± 451 ± 153 ± 2Apoptosis Increase¹CML23 ± 229 ± 317 ± 320 ± 3Normal3.4 ± 3.64.4 ± 3.913 ± 413 ± 2CML-Divided²27 ± 341 ± 89.9 ± 1.88.6 ± 2.5CML-Undivided²−4.1 ± 3.7−2.8 ± 4.1−1.4 ± 5.50.6 ± 5.0% remaining undivided and viable³CML25 ± 722 ± 520 ± 822 ± 8Normal46 ± 1146 ± 1225 ± 237 ± 7¹relative to cells not exposed to imatinib. ²CFSE compared to starting population: less=divided; same=undivided. ³% of input cells that remain undivided & non-apoptotic with imatinib.

Gene Expression Profiles, Differentiation and Lineage Commitment of Human Myeloid Progenitors.

With the objective to further elucidate the mechanism behind commitment to erythroid and neutrophil lineages, we isolated by cell sorting common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), based on the surface expression of CD123 (IL3R-α) and CD45RA on human CD34+ bone marrow cells (first described by Manz et al. PNAS , 2002; 99:11872). Methylcellulose cultures supporting the growth of myeloid and erythroid progenitors, and real-time RT-PCR mapping the gene expression of Flt3, c-kit, TpoR, GATA-2, GATA-1, SCL, NF-E2, EpoR, ABO, β-globin, GPA, PU.1, C/EBPα, C/EBPε, G-CSFR, proteinase 3 (PR3) and lactoferrin, were used to validate and characterize the progenitors and their progeny. Cell sorted progenitors were labeled with CFDA, SE to track cell division and cultured in suspension to induce neutrophil or erythroid differentiation (SCF+G-CSF for neutrophil and Epo+IL–3+GM–CSF for erythroid culture). After 3–5 days, cells that had gone through 1–8 divisions were sorted and changes in clonogenicity and gene expression were studied. The CMP-population retained some clonogenicity after as many divisions as were tested (at the most six divisions in erythroid and eight in neutrophil culture) and the CMPs differentiated along the lineage defined by the culture system, as evidenced both by the methylcellulose cultures and an increasing expression of GATA-1 and EpoR in erythroid and PU.1, G-CSFR and PR3 in neutrophil cultures, respectively. On the other hand, the GMP-population displayed granulocyte/monocyte (G/M)-differentiation irrespective of the culture system used, although it divided fewer times and lost its clonogenic capacity faster in erythroid culture. Little or no clonogenicity remained after 4–5 divisions in erythroid culture, while some colony-forming capacity remained even after seven divisions in neutrophil culture (maximum number tested). The increased expression of the granulopoiesis-associated genes was also less pronounced in the erythroid culture. The MEP-population dominated by erythroid differentiation capacity retained colony-forming capacity for at least 6–7 divisions in both erythroid and neutrophil cultures, although with a higher overall clonogenicity in erythroid culture. Unexpectedly, however, MEPs were restricted to G/M-differentiation when cultured in neutrophil culture. In cells from erythroid culture the expression of GATA-1, EpoR and β-globin increased, while a corresponding pattern was seen for PU.1, G-CSFR and PR3 in neutrophil culture. Overall, our data support the progenitor classification, based on the surface expression of CD123 and CD45RA, with regard to CMP and GMP populations but question it with regard to the MEP-population. The change in differentiation course for the MEPs in neutrophil culture could be a result of an initially present G/M-potential or a less strict commitment susceptible to cytokine-induced redifferentiation.

Expression of DLK/PREF1 Results in Inhibition of Human Myeloid Cell Differentiation and Proliferation through Distinct Molecular Mechanisms.

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. Microarray based analyses have shown that the Delta-Like/Preadipocyte-Factor 1 (DLK/PREF1) gene is overexpressed in CD34+ cells from patients with MDS. DLK encodes an EGF-like transmembrane protein subject to proteolytic cleavage and release. Stromal expression of DLK appears to play a role in maintenance of murine hematopoietic stem cells and in supporting B- and T-lymphopoiesis. However, the functional consequences of DLK overexpression in human hematopoietic cells are not known. We have studied the effects of ectopic expression of DLK on the differentiation and growth of HL-60 promyelocytic cells, and found that ectopic expression of DLK inhibits induced differentiation in response to ATRA, PMA and DMSO exposure. DLK mutants were generated to explore mechanisms underlying its effects on hematopoietic cells. These included DLK-EC, expressing only the extracellular portion of the molecule; DLK-dIC, a transmembrane form lacking the intracellular domain; and DLK-ALT, an alternatively spliced form lacking the protease sensitive site. We observed that proteolytic cleavage and release of the extracellular domain was not required for inhibition of hematopoietic cell differentiation, whereas intracellular domain interactions were critical to this DLK function. We have also tested the effect of DLK expression on myeloid cell proliferation. DLK expressing cells demonstrated significantly reduced proliferation compared with controls (91-fold expansion of control cells after 7 days culture vs. 66-fold expansion for DLK expressing cells, p<0.0001). Studies using SNARF-1 labeling to track cell division confirmed the inhibitory effect of DLK expression on proliferation (proliferation index of 6.39±1.0 for control vs. 4.99±0.3 for DLK expressing cells, p=0.04, n=3). Cell cycle analysis revealed that DLK expressing cells exhibited slower progress through G0/G1 phase into S-phase than controls following release from G2/M phase synchronization with Nocodazole treatment (45±5% of DLK expressing cells vs. 66±10% of control cells were in S-phase after 14 hours, p=0.027, n=3). We also evaluated the effects of mutations in the DLK molecule on its anti-proliferative effect. DLK-dIC inhibited cell proliferation to a similar extent as wild type DLK (p=0.42), whereas the DLK-ALT resulted in further reduction in proliferation (p=0.0014). In contrast, the DLK-EC resulted in significantly enhanced cell proliferation compared with wild type DLK (p<0.0001). These results indicate that the inhibitory effects of DLK on proliferation require its expression as a transmembrane molecule but do not require intracellular domain mediated interactions. On the other hand proteolytic cleavage and release of the extracellular domain appears to have a stimulatory effect on cell proliferation. In summary, DLK overexpression in myeloid cells has important functional consequences including inhibition of proliferation and differentiation. The inhibitory effects of DLK are independent of proteolytic cleavage of the molecule and require its expression as a transmembrane protein. The intracellular portion of the molecule is critical to inhibition of differentiation but not to proliferation inhibition. In contrast, proteolytic release of the extracellular portion of the molecule may stimulate cell proliferation but does not affect differentiation. Further investigation of the role of DLK overexpression in abnormal hematopoiesis in MDS is warranted.

Functional ABCG2 Is Expressed on CML Stem Cells and Its Inhibition Selectively Depletes CML CD34+ Cells.

We have previously described a population of deeply, but reversibly, quiescent stem cells (qSC) found in patients with chronic phase (CP) CML at diagnosis. In vitro studies have proven this population to be highly insensitive to imatinib mesylate (IM; Gleevec, STI571) induced killing, and more worryingly shown that qSC are accumulated after CML CD34+ cells are treated with IM. As it is likely that CML qSC closely resemble normal HSC, we hypothesise that they too may express the stem cell-associated ABCG2 and have therefore examined the expression and function of this drug efflux pump on CML cells. In agreement with other studies we show the interaction between ABCG2 and IM. Using ABCG2 over-expressing cells (AML6.2 and HL60-BCRP) we found that ≥0.5μM IM reduced efflux of the ABCG2 substrate BODIPY-Prazosin by a similar degree as the inhibitor fumitremorgin C (FTC; 10μM). We have now examined expression and function of ABCG2 on primary CML cells taken from patients in chronic phase (CP) and prior to any treatment. Quantitative Taqman analysis of 8 CD34+ enriched (≥90%+) CML samples revealed that the level of expression is 2.46 fold higher than that in normal mobilised CD34+ cells (n=8 CML, n=4 normal). In addition, we undertook microarray analysis of normal or CML CP CD34+ cells fractionated according to cell cycle using Hoechst-Pyronin (G0, G1 and G2/S/M). These analyses (n=3 normal, n=5 CML) show that at all stages of the cycle CML cells express more ABCG2 than normal cells and that G0 CML cells express 2.48 fold more than those in G1 , confirming both the over-expression in CML and relationship to the most primitive subset of cells. Using the antibody BXP21 we found that 8 of 9 samples contain ABCG2+ve cells (5 of 9 ≥60% of cells ABCG2+). We also examined the function of ABCG2 on CML CD34+ cells by performing efflux assays, 4 of 6 showed efflux that was inhibited by 10μM FTC or ≥0.5μM IM, and this efflux capacity correlated with BXP21 staining. We therefore considered whether the combination of IM therapy and ABCG2 inhibition would overcome the accumulation of CML qSCs we have previously reported after treatment with IM. Using CFSE to track cell division we treated CD34+ enriched CML samples with 5μM IM +/− FTC or with 10μM FTC alone for 3 days. In comparison to untreated controls 5μM IM reduced the total number of cells to 31.9±9.2 % and the number of CD34+ cells to 43.2±17.6%. However, the non-cycling qSC significantly increased to 318±75.8% of control. In contrast, the ABCG2 inhibitor FTC did not effect a reduction in total cells (99.5±11.9%) but gave a significant reduction of CD34+ cells (58.6±8.4%; p=0.02) and no accumulation of qSC (104.6±33.8%) when used alone. We saw no cumulative effect when IM and FTC were given concurrently. These data suggest strongly that FTC may be used to deplete CD34+ ‘stem cells' from CML, as the total cell number is unchanged it is likely that this depletion is by the induction of differentiation. We propose that the expression of ABCG2 may be clinically significant in CP CML and that inhibition of this pump may result in a ‘stem cell targeted therapy' that could be followed by IM treatment to reduce the tumor load. Such reduction of CML stem cells would result in elimination of minimal residual disease and effect a lasting remission.

'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations.

The extension of airway smooth muscle cell (ASMC) functions, from just contractile, to synthetic and/or proliferative states, is an important component of airway remodelling and inflammation in asthma. Whereas all these functions have been demonstrated in ASM, currently, it is not known whether ASMC can be differentiated on the basis of their proliferative and synthetic functions. We used flow-cytometric techniques to determine, first, whether human ASMC are phenotypically heterogenous with regard to their secretory function, and second, the proliferative status of secretory cells. ASMC were induced to synthesize GM-CSF by stimulation with IL-1beta and TNF-alpha followed by 10% human serum. Flow-cytometric detection of intracellular GM-CSF revealed that only a proportion of cells in culture (approximately 20-60%) synthesize GM-CSF. To determine the proliferative status of GM-CSF producing cells, ASMC were pretreated with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), a fluorescein based dye used to track cell division, prior to cytokine/serum stimulation. Simultaneous analysis of intracellular GM-CSF and CFSE revealed that GM-CSF producing cells were present in both the divided and undivided ASMC populations. Thus, cytokine production and proliferation occurred in overlapping ASMC populations and prior progression through the cell cycle was not essential for ASMC cytokine production.

Integration of Cell Cycle Signals by Swe1 Protein Kinase

Cell division in budding yeast depends on the formation of a filamentous collar containing septin proteins. This collar forms at the base of the bud, marking the place in the membrane between the mother cell and the emerging bud that constricts during cytokinesis, ultimately forming two cells when mitosis is complete. New evidence now indicates that protein complexes formed with septins are critical in coordinating morphogenesis with cell division and proper tracking of temporal progression through the cell cycle. Sakchaisri et al. show that yeast cells use the status of multiple phosphorylation sites on the protein kinase Swe1 to keep track of cell cycle progression and assembly of the necessary complexes organized at the bud neck. Genetic analysis and use of a designed form of protein kinase Cla4 sensitive to a specific inhibitor showed that the Cla4 protein contributes to phosphorylation of Swe1 during S phase of the cell cycle. Mutants with weakly functioning alleles of another protein kinase, Cdc5, lacked further phosphorylation of Swe1 that normally occurs right before entry into mitosis. Thus, accumulation of highly phosphorylated Swe1 provides the cell with an indicator that the cycle is progressing properly and that the necessary components are assembled in septin complexes. These phosphorylation events tag Swe1 for ubiquitin-dependent degradation, which in turn relieves Swe1's inhibitory phosphorylation of the critical cyclin-dependent kinase (Cdc28) that controls initiation of mitosis. K. Sakchaisri, S. Asano, L.-R. Yu, M. J. Shulewitz, C. J. Park, J.-E. Park, Y.-W. Cho, T. D. Veenstra, J. Thorner, K. S. Lee, Coupling morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. U.S.A . 101 , 4124-4129 (2004). [Abstract] [Full Text]

CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis).

Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell-stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28.7 +/- 12%vs 18.1 +/- 6.1% of input cells within 24 h, mean +/- SD, n = 8), whereas 9.4 +/- 12.6% (mean +/- SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks alpha4beta1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that alpha4beta1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment.

Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34(+) hematopoietic progenitor cells.

Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.

CHARACTERIZATION AND EFFICACY OF PKH26 AS A PROBE TO STUDY THE REPLICATION HISTORY OF THE HUMAN HEMATOPOIETIC KG1a PROGENITOR CELL LINE

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).

Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro

In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34(+) progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin(-)CD34(+)) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured for 3 days with and without growth factors +/- STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34(+) cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy.

Immune reconstitution after autologous peripheral blood progenitor cell transplantation: effect of interleukin-15 on T-cell survival and effector functions.

The aim of this study was to evaluate the occurrence of T-cell spontaneous apoptosis (A(spont)) and its modulation in vitro by the interleukin-2 receptor (IL-2R) gamma-chain (gammac)-signaling cytokine IL-15 in patients transplanted with autologous peripheral blood progenitor cells (PBPC) for hematologic malignancies. Patients were examined on days 30-60, 60-90, and 90-120 after PBPC infusion. Dissipation of mitochondrial transmembrane potential, a hallmark of T-cell apoptosis, has been detected using the fluorescent probe 3,3'-dihexyloxacarbocyanine iodide, after short-term T-cell culture in the absence or presence of exogenous cytokines. Expression of Bcl-2 family members has been studied by flow cytometry and reverse transcriptase polymerase chain reaction. T-cell proliferative responses to recall antigens have been estimated in autologous mixed leukocyte cultures. A(spont) was seen in 45% +/- 6% of CD4(+) and 55% +/- 6% of CD8(+) T cells cultured in the absence of cytokines. Of interest, IL-15 and, to a lesser extent, its structural cousin IL-2 counteracted T-cell A(spont) by inhibiting the processing of caspase-3 and up-regulating Bcl-2 mRNA and protein levels. Cell division tracking confirmed that IL-15 did not rescue T cells from A(spont) by promoting proliferation but rather acted as a genuine survival factor. Addition of a gammac-blocking antibody to cytokine-conditioned cultures abrogated both apoptosis inhibition and Bcl-2 induction by IL-15, suggesting involvement of the IL-2Rgammac signal transduction pathway. Whereas cytokine-unprimed posttransplant T cells mounted inadequate responses to recall antigens, T cells conditioned with IL-15 expanded vigorously, indicating restoration of antigen-specific proliferation. T cells recovering after autologous PBPC transplantation are highly susceptible to spontaneous apoptosis in vitro. This phenomenon can be counteracted by the gammac-signaling cytokine IL-15. These findings suggest that IL-15 might be a promising immunomodulating agent to improve postgrafting T-cell function.

Mycobacterial di-O-acyl-trehalose inhibits mitogen- and antigen-induced proliferation of murine T cells in vitro.

2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.

Flow cytometric analysis of human hemopoietic progenitor differentiation by assessing cell division rate and phenotypic profile

The fate of a single hemopoietic progenitor (HP) is determined by both an intrinsic hemopoietic potential typical of each HP and the activity of various cytokines that regulate HP survival, proliferation, and differentiation. Maturation of HP into functional hemopoietic cells in vitro is monitored with success at the population level by the use of flow cytometry. This approach exploits the availability of monoclonal antibodies (MAb) to specific surface markers defining the maturative stage and the specific lineage a given precursor is committed to and the capability of certain fluorescent probes to track cell division history. In these systems, cells are loaded with a probe that equally redistributes into daughter cells at each round of cell division, generating progenies with progressively halved fluorescence signal. This chapter focuses on the problems the flow cytometrist faces when attempting to analyze variations in human HP phenotype and correlates them with carboxyfluorescein diacetate–succinimidyl ester (CFDA–SE) halving during in vitro cytokine-driven differentiation toward the major hemopoietic lineages, namely, granulocytic/monocytic, erythroid, and megakaryocytic.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

AbstractAlthough long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper-IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE(+) cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE(+) cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE(+) "start" population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE(+) cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

Analysis of growth kinetics by division tracking.

Cell division tracking using fluorescent dyes, such as carboxyfluorescein diacetate succinimidyl ester, provides a unique opportunity for analysis of cell growth kinetics. The present review article presents new methods for enhancing resolution of division tracking data as well as derivation of quantities that characterize growth from time-series data. These include the average time between successive divisions, the proportion of cells that survive and the proliferation per division. The physical significance of these measured quantities is interpreted by formulation of a two-compartment model of cell cycle transit characterized by stochastic and deterministic cell residence times, respectively. The model confirmed that survival is directly related to the proportion of cells that enter the next cell generation. The proportion of time that cells reside in the stochastic compartment is directly related to the proliferation per generation. This form of analysis provides a starting point for more sophisticated physical and biochemical models of cell cycle regulation.