Quiescent Fraction Research Articles

Inhibition of eIF2α Dephosphorylation Maximizes Bortezomib Efficiency and Eliminates Quiescent Multiple Myeloma Cells Surviving Proteasome Inhibitor Therapy

The proteasome inhibitor bortezomib (Velcade) effectively eradicates multiple myeloma (MM) cells, partly by activating endoplasmic reticulum (ER) stress apoptotic signaling. However, MM recurrences in bortezomib-treated patients are invariable. We have shown that ER stress signaling can also induce growth arrest and survival in cancer cells. Thus, we hypothesized that bortezomib therapy could induce quiescence and survival of residual MM cells, contributing to disease recurrence. Here, we report that in MM cells, proteasome inhibition with MG-132 or bortezomib results in a surviving cell fraction that enters a prolonged quiescent state (G(0)-G(1) arrest). Mechanism analysis revealed that bortezomib-surviving quiescent cells attenuate eIF2alpha phosphorylation and induction of the ER stress proapoptotic gene GADD153. This occurs independently of the eIF2alpha upstream kinases PERK, GCN2, and PKR. In contrast, the prosurvival ER-chaperone BiP/Grp78 was persistently induced. The bortezomib-surviving quiescent fraction could be eradicated by a simultaneous or sequential combination therapy with salubrinal, an inhibitor of GADD34-PP1C phosphatase complex, and, in consequence, eIF2alpha dephosphorylation. This effect was mimicked by expression of a phosphorylated mimetic eIF2alpha-S51D mutant. Our data indicate that bortezomib can induce growth arrest in therapy-surviving MM cells and that attenuation of eIF2alpha phosphorylation contributes to this survival. Most importantly, this survival mechanism can be blocked by inhibiting eIF2alpha dephosphorylation. Thus, strategies that maintain eIF2alpha in a hyperphosphorylated state may be a novel therapeutic approach to maximize bortezomib-induced apoptosis and reduce residual disease and recurrences in this type of cancer.

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice

Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.

Inhibition of eIF2α Dephosphorylation Maximizes Bortezomib Efficiency and Eliminates Quiescent Multiple Myeloma Cells Surviving Therapy

The proteasome inhibitor Bortezomib (Velcade®) effectively eradicates multiple myeloma (MM) cells, partly by activating endoplasmic reticulum (ER) stress apoptotic signaling. However, MM recurrences in Bortezomib-treated patients are invariable. We have shown that ER stress signaling can also induce growth arrest and survival in cancer cells. Thus, we hypothesized that Bortezomib therapy could induce quiescence and survival of residual MM cells, contributing to disease recurrence. Here we report that in MM cells, proteasome inhibition with MG-132 or Bortezomib results in a surviving cell fraction that enters a prolonged quiescent state (G0-G1 arrest). Mechanism analysis revealed that in Bortezomib-surviving quiescent cells eIF2α phosphorylation and induction of the ER stress pro-apoptotic gene, GADD153, were attenuated. In contrast, p38 and the ER-chaperone BiP/Grp78 were persistently activated and induced, respectively. The Bortezomib-surviving quiescent fraction could be eradicated by a simultaneous or sequential combination therapy with Salubrinal, an inhibitor of GADD34-PP1C phosphatase complex, and in consequence, eIF2α dephosphorylation. This effect was mimicked by expression of a phospho-mimetic eIF2α-S51D mutant. Our data indicate that Bortezomib can induce growth arrest in therapy-surviving MM cells and that attenuation of eIF2α phosphorylation and GADD153 induction contribute to this survival. Most importantly, this ability of MM cells to evade Bortezomib toxicity can be prevented by inhibiting eIF2a dephosphorylation. Thus, strategies that maintain eIF2α in a hyper-phosphorylated state may be a novel therapeutic approach to maximize Bortezomib-induced apoptosis and reduce recurrences in this type of cancer.

Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2

In addition to its role in megakaryocyte production, signaling initiated by thrombopoietin (TPO) activation of its receptor, myeloproliferative leukemia virus protooncogene (c-Mpl, or Mpl), controls HSC homeostasis and self-renewal. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. We found that HSCs from Lnk-/- mice have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeat treatments with cytoablative 5-fluorouracil in vivo compared with WT HSCs. We further provide genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal, predominantly through Mpl. Consistent with this observation, Lnk-/- HSCs displayed potentiated activation of JAK2 specifically in response to TPO. Biochemical experiments revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Of note, the JAK2 V617F mutant, found at high frequencies in myeloproliferative diseases, retains the ability to bind Lnk. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells and TPO/Mpl/JAK2/Lnk as a major regulatory pathway in controlling stem cell self-renewal and quiescence.

Characterization of Differentiated Quiescent and Nonquiescent Cells in Yeast Stationary-Phase Cultures

Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental changes. Our results indicate that Q and NQ cells differentiate significantly, with Q cells providing genomic stability and NQ cells providing nutrients to Q cells and a regular source of genetic diversity through mutation and transposition. These studies are relevant to chronological aging, cell cycle, and genome evolution, and they provide insight into complex responses that even simple organisms have to starvation.

The Tyrosine Kinase Inhibitor TK258 Differentially Targets Proliferating Human Hematopoietic Progenitor Cells but Does Not Eliminate the Quiescent Fraction.

NOD-SCID mouse models have shown that, as with normal hematopoietic stem cells, leukemic stem cells (LSC) exist in a quiescent state and are thus capable of sustaining disease in vivo. Targeted therapy of LSC may therefore have a significant impact on disease eradication, relapse rates and toxicity reduction. Inhibition of FLT3 and c-kit is a current therapeutic strategy for AML but the efficacy of this approach on quiescent cells is unknown. Furthermore, although both FLT3 and c-kit play crucial roles in the development of normal human hematopoietic progenitor (HPC) and stem cells, the hematopoietic consequences of FLT3 and c-kit inhibition remain undefined. Here we show that the FLT3, c-kit and PDGF directed tyrosine kinase inhibitor TK258 specifically targets slowly dividing human HPCs, but not rapidly proliferating HPCs or quiescent cells displaying an immature phenotype. For normal CD34+ cells, treatment of short-term cultures with TK258 resulted in a dose dependent inhibition of cell expansion. Moreover, treatment of normal CD34+ cells with TK258 within the clinical dose range [0.2μM to 1.5μM] inhibited myeloid colony growth but had no effect on erythroid colony output, suggesting a differential effect on lineage-committed progenitors. To further elucidate the effect of TK258 on dividing and also quiescent CD34+ cells, normal CD34+ cells were labelled with a non-toxic fluorescent cell membrane dye, PKH26, prior to TK258 treatment in vitro. Coupled with flow cytometry, this dye enabled the discrimination of progenitor cells with different proliferative capacities. Thus, using this technique, we defined a CD34+/CD133+ quiescent population of cells that retained the same PKH fluorescence intensity as from day 0 of culture. TK258 treatment in culture generated a significant increase in the CD34+/CD133+ quiescent fraction (15.8% treated versus 5.9% untreated of total viable cells). In contrast with untreated cells this fraction maintained c-kit expression, suggesting maintenance of an immature population by TK258. Total viable cell recoveries were higher in the TK258 treated quiescent fraction (24% treated versus 13.5% untreated, p=0.037). Rapidly dividing progenitors were unaffected by TK258, while progenitors with a slower division rate showed significant sensitivity to the compound. The anti-proliferative effect of TK258 was mediated via G0/G1 cell cycle arrest, but not by apoptosis of CD34+ cells. We assessed the functional read-out of TK258 pre-treated progenitors in CFU assays, by sorting cells on the basis of their PKH26 fluorescence. These assays demonstrated a higher total colony output and diverse colony type for TK258 pre-treated ‘quiescent' cells compared to untreated controls, in keeping with the preservation of a more immature population by TK258. Rapidly proliferating populations displayed a lower CFU potential, attributable to a reduction in myeloid output, which was not significantly altered by TK258 pre-treatment. CAFC assays and gene expression profiling of PKH26-sorted subsets are in progress. Taken together, these data demonstrate that TK258 has significant inhibitory effects on normal human hematopoietic progenitor cells in vitro and that the sensitivity of progenitor cells to the compound can be predicted from their proliferative index. Importantly, immature hematopoietic quiescent cells are preserved, which may have implications for the activity of tyrosine kinase inhibitors on non-cycling human leukemia cells.

Bortezomib Has Anti-Proliferative and Apoptotic Effects Against CML Stem Cells, Including the Quiescent Population.

CML is a disease of the haemopoietic stem cell (HSC) caused by the constitutively active tyrosine kinase Bcr-Abl. The current first-line therapy for CML is imatinib (IM), a tyrosine kinase inhibitor (TKI) effective in establishing durable disease control in the majority of patients in chronic phase. However, the quiescent leukaemic HSC (Ph+HSC) population is insensitive to the apoptotic effects of this and more potent TKIs - such as dasatinib or nilotinib. These TKIs exert potent anti-proliferative effects against CML stem and progenitor cells, resulting in accumulation of quiescent cells in vitro. This Ph+HSC reservoir of disease in treated patients is now recognised to be a critical target if we wish to achieve complete eradication of residual disease. Bcr-Abl+ cells have abnormally high levels of proteasome activity and expression and in common with other cancer cells have been shown to be relatively more sensitive to the effects of proteasome inhibitors (PI) than their non-malignant counterparts. We have assessed the potential role for PI in CML using Bcr-Abl+ cell lines (K562 and Ba/F3 with mutant/wild type Bcr-Abl) and Ph+HSC obtained from leukapheresis products of patients, taken with consent, at time of diagnosis with CML. We have demonstrated apoptotic and anti-proliferative effects of PI (MG132 and bortezomib) in wild type and mutated Bcr-Abl+ cell lines (including T315I) and in Ph+HSC (n=7) by viable cell counts, thymidine incorporation and flow cytometry for annexin V and 7AAD. The apoptotic effects also occurred in the quiescent Ph+HSC fraction with an IC50 of 5–7.5nM (bortezomib)- similar to that seen in the total CD34+ cell population and lower than that previously reported for cells from multiple myeloma patients. These results were compared to parallel FACS-sorted cell populations from non-CML Ph- leukapheresis products and a therapeutic window was seen. CFSE tracking of cell division in CML samples in vitro indicated depletion of the quiescent fraction, in contrast to the accumulation of these cells demonstrated with IM, nilotinib or dasatinib, either alone or in combination. The intracellular effects of exposure of these cell populations to bortezomib were assessed by quantitatively measuring the intracellular accumulation of polyubiquitinated proteins seen following PI treatment. Interestingly no clear effect on Bcr-Abl activity was demonstrated with PI treatment of Ph+HSC (measured by immunoblotting with antibody to phosphorylated Crkl), though in Bcr-Abl+ cell lines detectable activity was reduced. Drug combination experiments (Calcusyn) were performed with bortezomib and IM or dasatinib to assess the effect of simultaneous inhibition of Bcr-Abl and the proteasome - the combination of a TKI with a PI resulted in additive effects, however no clear synergistic effects of significance were seen. Our results indicate that PI are capable of inducing CML stem cell specific apoptosis, suggesting the proteasome may be a relevant target for eradication of residual disease in CML following TKI therapy.

Mll Has a Critical Role in Fetal and Adult Hematopoietic Stem Cell Self-Renewal

The Mixed Lineage Leukemia (Mll) gene is a homolog of Drosophila Trithorax commonly rearranged in infant leukemia. Comprehensive analysis of the role of Mll in hematopoiesis in fetal and adult knockout mice has been prevented by the lethality of Mll(-/-) mice. We have established a conditional deletion model that allows us to study adult hematopoiesis in the absence of Mll. In this study, Mll(-/-) embryos survive to E16.5 and have reduced numbers of HSCs. The quiescent fraction of these HSCs is greatly reduced, and they are unable to compete with wild-type cells in transplantation assays. Mice with Mll expression conditionally deleted in the hematopoietic system have grossly normal hematopoiesis in bone marrow, thymus, and spleen. However, transplanted Mll-deficient bone marrow cells are highly compromised in their ability to competitively reconstitute irradiated recipients. These results suggest a critical role for Mll in regulating stem cell self-renewal.

The hierarchical build-up of bulges in CDM

AbstractWe investigate the hierarchical build-up of stars in bulges within the standard Λ-cold dark matter scenario. By separating the population into stars born during starbursts that accompany the formation of spheroids in major mergers (starburst component), and stars that are previously formed in discs of progenitor galaxies (quiescent component) and added to the spheroid by dynamical interaction. Our results are summarised as follows: bulges that form early have larger starburst fraction and hence should be smaller than their counter parts that form later. The quiescent fraction in bulges is an increasing function of bulge mass, becoming constant at Mq/Mbul ~ 0.8, mainly due to the infall of satellite galaxies that contribute disc stars to the bulge. Minor mergers are an order of magnitude more frequent than major mergers and must play a significant role in the evolution of bulges. Above the critical mass Mc ~ 3 × 1010 M⊙ most of the stars in the universe are in spheroids, which at high redshift are exclusively elliptical galaxies and at low redshifts partly bulges. Due to the enhanced evolution of galaxies ending up in high density environments, the starburst fraction and the surface mass densities of bulges below Mc should be enhanced with respect to field galaxies. Dissipation during the formation of massive bulges in present day early-type spirals is less important than for the formation of present day elliptical galaxies of the same mass thereby explaining the possible difference in phase-space densities between spiral galaxies and elliptical galaxies.

Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction

AbstractDasatinib (BMS-354825), a novel dual SRC/BCR-ABL kinase inhibitor, exhibits greater potency than imatinib mesylate (IM) and inhibits the majority of kinase mutations in IM-resistant chronic myeloid leukemia (CML). We have previously demonstrated that IM reversibly blocks proliferation but does not induce apoptosis of primitive CML cells. Here, we have attempted to overcome this resistance with dasatinib. Primitive IM-resistant CML cells showed only single-copy BCR-ABL but expressed significantly higher BCR-ABL transcript levels and BCR-ABL protein compared with more mature CML cells (P = .031). In addition, CrKL phosphorylation was higher in the primitive CD34+CD38– than in the total CD34+ population (P = .002). In total CD34+ CML cells, IM inhibited phosphorylation of CrKL at 16 but not 72 hours, consistent with enrichment of an IM-resistant primitive population. CD34+CD38– CML cells proved resistant to IM-induced inhibition of CrKL phosphorylation and apoptosis, whereas dasatinib led to significant inhibition of CrKL phosphorylation. Kinase domain mutations were not detectable in either IM or dasatinib-resistant primitive CML cells. These data confirm that dasatinib is more effective than IM within the CML stem cell compartment; however, the most primitive quiescent CML cells appear to be inherently resistant to both drugs.

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.

TCL1 Expression in B-Cell Tumors Is Dynamically Regulated by Maturation Stage, Proliferation, and the Follicular Microenvironment.

The kinase comodulator TCL1 is the primary initiating oncogene in T-cell prolymphocytic leukemia and can produce B-cell or T-cell chronic lymphocytic leukemia (CLL) following transgenic expression in mice. Given its strong expression in some non-neoplastic B-cell subsets, the role of TCL1 as an oncogene in human B-cell tumors is less clear. Using a recently developed TCL1 monoclonal antibody (clone 1–21), we examined the relationship between TCL1 expression and B-cell maturation stage in tumor tissue arrays, lymphoma cell lines, primary tumor samples and in vitro stimulation assays. Results were compared with immunohistochemical expression of a variety of maturation, activation and cell proliferation markers and with the somatic hypermutation status determined by VH transcript analysis (<2% sequence divergence in VH regarded as “pre-germinal center”). In non-neoplastic B-cells, TCL1 was strongly and uniformly expressed in naive B-cell subsets, but was variably expressed in subsets of germinal center B-cells, with complete absence of expression in immunoblasts and plasma cells. In germinal center B-cells, TCL1 was expressed more strongly in the quiescent centrocyte fraction than in the proliferating centroblasts. This pattern was replicated in human B-cell lymphoma lines and tumors with complete absence of TCL1 in myeloma cases (n = 35) and monocytoid B-cells in marginal zone lymphoma (n = 8) and dim or absent expression in the majority of the mutated/post-germinal center subset of B-CLL (10/15, 67%). In contrast, TCL1 showed strong but modulated expression in the majority of unmutated/pre-germinal center type of B-CLL (14/19, 74%), and mantle cell lymphoma (MCL, 44/57, 84%), and less frequently in follicular lymphoma (FL, 21/47, 45%). In B-CLL, TCL1 was overexpressed in non-proliferating cells within the pseudo-follicular proliferation centers but was markedly downregulated in the CD23+bright PCNA+ proliferating tumor cell component. Similarly in MCL, TCL1 was downregulated in the proliferative component but upregulated in tumor cells in follicular and mantle zone locations versus the diffuse areas. In FL, the highest levels of TCL1 expression were found in those cases that most strongly expressed the germinal center markers CD10 and bcl-6, but TCL1 staining was inversely correlated with expression of proliferation and activation markers, such as CD23. In 3 FL cases with multiple biopsies at different tissue sites, TCL1 was downregulated in tumor cells at extranodal sites as compared to those within lymph node follicles. Thus, the dynamic regulation of TCL1 in B-cells and derived tumors is likely due to changes in the balance between stimulatory microenvironmental influences within the lymphoid follicle and inhibitory signals during cell cycle progression/proliferation. In post-germinal center tumors, TCL1 expression is effectively silenced, and no longer exhibits this dynamic regulation pattern. These studies strongly suggest that TCL1 exerts its effects in promoting cell survival in quiescent B-cell subsets prior to and in the absence of an (antigenic) proliferative signal.

Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18(INK4c), p21(waf1/cip1), and p27(Kip1) all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18(INK4c) and p27(Kip1) expression positively correlate with growth arrest, whereas p21(waf1/cip1) is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18(INK4c) is found in increasing association with CDK6, whereas p27(Kip1) associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

The presence of 19-kDa Bcl-2 in dividing cells.

The 26-kDa bcl-2 gene product inhibits apoptosis and cell proliferation. Cleavage of Bcl-2 into a 22-kDa fragment inactivates its anti-apoptotic activity and is a key event in apoptosis. Here, and in recent work, we describe massive 19-kDa Bcl-2 immunoreactivity in non-apoptotic cells, suggesting a link with viability rather than cell death. Loss of 19 kDa Bcl-2 in adriamycin-induced apoptotic cells underlines this. G2/M-phase accumulation of cells by nocodazole-treatment also results in loss of 19 kDa Bcl-2. Next to its well-documented cytoplasmic localization, a substantial pool of Bcl-2 resides in nuclei. Hampered nuclear localization of Bcl-2 leads to a loss of cell cycle repression. This has led us to point at a pivotal role for nuclear Bcl-2 in cellular proliferation. In this report, cellular fractionation of bcl-2 transfected cells in various phases of the cell cycle reveals a constitutive cytoplasmic pool of 19 kDa Bcl-2. Nuclear 19-kDa Bcl-2 immunoreactivity is far more pronounced in rapidly dividing nuclei compared with more quiescent nuclear fractions. This implicates that ongoing cell proliferation involves cleavage of nuclear Bcl-2 with a 19-kDa fragment.

Modelling Complex Populations Formed by Proliferating, Quiescent and Quasi-quiescent Cells: Application to Plant Root Meristems

A proliferating population of cells may be considered complex when its proliferative or growth fractionP is lower than 1 and/or when it is formed by subpopulations with different mean cycle times. The present paper shows that in such complex populations exponential growth is consistent with a steady-state distribution of cells. Obviously, whenP =1 then cell distribution is only a function of cell age. An analytical model has been developed to study complex populations including both quiescent fractions formed by cells with unreplicated genome (G0 cells) and cells with fully duplicated chromosomes (Q2 cells). The model also considers those quasi-quiescent cells in their last transit through G1 and S (Q1 and Qs cells) before becoming quiescent. In order to solve the difficulties of a direct analysis of the whole population, its kinetic parameters have been obtained by studying the negative exponential distribution of two subpopulations: one formed by the proliferating cells and another formed by the quasi-quiescent cells. Additionally, the model could be applied when quiescence is initiated at any other cycle phase different from G1 and G2, for instance, cells in the process of replicating their DNA or being at any other mitotic phases. The utility of the method was illustrated in populations which constitute the root meristems of both Allium cepa L. and Pisum sativum L. Three facts should be stressed: (1) the method seems to be rather powerful because it can be carried out from different sets of experimentally measured parameters; (2) the rate of division and, therefore, the population doubling time can be easily estimated by this method; and (3) it also allows the determination of the amount of cells that had become quiescent either before they had replicated their DNA (G0) or after having completed their replication (Q2), as well as those quasi-quiescent cells which are progressing throughout their last pre-replicative and replicative periods (thus Q1 and Qs, respectively).

Asynchronous exponential growth in an age structured population of proliferating and quiescent cells

A model of a proliferating cell population is analyzed. The model distinguishes individual cells by cell age, which corresponds to phase of the cell cycle. The model also distinguishes individual cells by proliferating or quiescent status. The model allows cells to transit between these two states at any age, that is, any phase of the cell cycle. The model also allows newly divided cells to enter quiescence at cell birth, that is, cell age 0. Sufficient conditions are established to assure that the cell population has asynchronous exponential growth. As a consequence of this asynchronous exponential growth the population stabilizes in the sense that the proportion of the population in any age range, or the fraction in proliferating or quiescent state, converges to a limiting value as time evolves, independently of the age distribution and proliferating or quiescent fractions of the initial cell population. The asynchronous exponential growth is proved by demonstrating that the strongly continuous linear semigroup associated with the partial differential equations of the model is positive, irreducible, and eventually compact.

Homing and engraftment potential of Sca-1+lin− cells fractionated on the basis of adhesion molecule expression and position in cell cycle

AbstractEngraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1+lin− cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e+ or CD43+ Sca-1+lin− cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1+lin− cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential–colony forming cells, low proliferative potential–colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1+lin− AM− cells contained a higher percentage of cells in G0/G1 than their AM+ counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G0/G1 following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1+lin− AM+ or AM−cells were further fractionated by Hoechst 33342 into G0/G1 or S/G2+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1+lin− engrafting cell as one that is mitotically quiescent, CD43+, CD49e+, CD11a−, CD49ddim, and CD62L−. Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs.

Homing and engraftment potential of Sca-1+lin− cells fractionated on the basis of adhesion molecule expression and position in cell cycle

Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1+lin− cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e+ or CD43+ Sca-1+lin− cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1+lin− cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential–colony forming cells, low proliferative potential–colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1+lin− AM− cells contained a higher percentage of cells in G0/G1 than their AM+ counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G0/G1 following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1+lin− AM+ or AM−cells were further fractionated by Hoechst 33342 into G0/G1 or S/G2+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1+lin− engrafting cell as one that is mitotically quiescent, CD43+, CD49e+, CD11a−, CD49ddim, and CD62L−. Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs.

Flt3LP3nduces the ex-vivo amplification of umbilical cord blood committed progenitors and early stem cells in short-term cultures.

Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.