PubMed Health⌕ Search

Biomedical subjects

A Eaves

Publications and source records attributed to A Eaves.

At least 19 recordsLinked to original sources
PubMed2005

BCR-ABL-transduced human cord blood cells produce abnormal populations in immunodeficient mice.

In this study, we describe the successful use of a gene transfer approach to demonstrate the ability of forced BCR-ABL expression to deregulate the growth and differentiation of primitive naive human hematopoietic cells after their transplantation into immunodeficient mice. Human CD34+ cord blood cells were exposed to an MSCV retrovirus containing a BCR-ABL-IRES-GFP (P210) cassette and then injected immediately into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD/SCID) or NOD/SCID-beta2microglobulin-/- mice. P210- and control-transduced (GFP+) human hematopoietic cells were produced in the bone marrow of the mice at similar levels until termination of the experiments 5-6 months later. However, the P210-transduced cells produced a markedly different spectrum of progeny, with an increased ratio of myeloid to B-lymphoid cells and a frequently prolonged increase in erythroid and megakaryocytic cells. After 5 months, several of the mice transplanted with P210-transduced cells developed an increased WBC count and/or splenomegaly due to an expansion of the human GFP+ population. These findings demonstrate that forced expression of BCR-ABL in primitive transplantable human hematopoietic cells is sufficient to cause a rapid and persistent deregulation of their growth and differentiation in vivo with occasional evidence after several months of progression to an early stage of disease.

Animals↗
PubMed2002

Changes in the proliferative activity of human hematopoietic stem cells in NOD/SCID mice and enhancement of their transplantability after in vivo treatment with cell cycle inhibitors.

Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [(3)H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-beta was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered G(o) and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.

Animals↗
PubMed2000

Autonomous multi-lineage differentiation in vitro of primitive CD34+ cells from patients with chronic myeloid leukemia.

Neoplastic CD34+ cells from chronic myeloid leukemia (CML) patients proliferate in vitro in the absence of serum or defined growth factors due to an autocrine mechanism involving IL-3 and G-CSF (Jiang et al. Proc Natl Acad Sci USA 1999; 96: 12804). Detailed examination of the various cell types produced in such cultures has now demonstrated the rapid, factor-independent, generation of clonogenic progenitors for all lineages (granulocyte-macrophage, megakaryocyte and erythroid) with the additional appearance within 10 days of large numbers of mature granulocytes, macrophages, and megakaryocytes, as well as occasional erythroid cells. Inclusion of flt3-ligand, Steel factor, IL-3, IL-6, and G-CSF +/- erythropoietin (EPO) in the cultures enhanced only slightly the output of mature cells (except for the erythroid population which was much larger when EPO was added). Analogous subpopulations of normal CD34+ cells produced similar numbers and types of cells but, as expected, only when growth factors were added. Thus primitive CD34+ CML cells proliferating autonomously in vitro recapitulate the full spectrum of differentiation responses of normal CD34+ cells stimulated by IL-3 and G-CSF. These findings point to a role of autocrine IL-3 and G-CSF in the similar multi-lineage expansion of differentiating CD34+ CML cells that occurs in vivo.

Antigens, CD34↗
PubMed1999

Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia.

Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph(+)/BCR-ABL(+) chronic myeloid leukemia display an abnormal ability to proliferate in vitro in the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34(+) leukemic cells. When these cells differentiate into CD34(-) cells in vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growth in vitro despite their continued expression of BCR-ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewal in vitro and slow accumulation in vivo, in spite of an increased cycling activity and selective expansion of later compartments.

Base Sequence↗
PubMed1999

Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph(+)/BCR-ABL(+)) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G(0) cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA. CD34(+) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE(+)) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.

Animals↗
PubMed1999

Introduction to stem cell biology in vitro. Threshold to the future.

Transplantable hematopoietic cells with multilineage reconstituting ability can be quantitated in suspensions of human or murine cells using similar assay procedures. The incorporation into these assays of stringently defined functional endpoints ensures a high degree of specificity for the cells detected. Application of these assays to stem cell-containing suspensions after they have been stimulated for several days with defined cytokines in vitro, or by a mixture of defined and/or undefined factors in vivo, has shown that net amplifications in these populations can be obtained under both circumstances. Such studies have allowed cytokine conditions that support stem cell self-renewal divisions to be identified and have also provided evidence that stem cell regeneration can be manipulated both in vitro and in vivo by altering the molecular milieu of the responding cells. These observations pave the way to future delineation of mechanisms that control the normal behavior, pathology and future clinical exploitation of hematopoietic stem cell populations.

Animals↗
PubMed1998

BCR-ABL accelerates C2-ceramide-induced apoptosis.

In patients with chronic myeloid leukemia (CML), the neoplastic (BCR-ABL+) progenitor cells are characterized by an increased proliferative activity. Whether these cells are also resistant to apoptosis and if so, under what conditions remains controversial. We now show that highly purified populations of very primitive neoplastic progenitor cells obtained directly from CML patients survive and proliferate in vitro for several weeks in the absence of any added growth factors (except insulin). In contrast, purified primary normal progenitors maintained under the same conditions die rapidly. Nevertheless, both primary CML cells and BCR-ABL+ BAF3 cells show the same dose-dependent sensitivity to TNF-alpha or ceramide-induced apoptosis as their respective normal counterparts. In fact, time course studies demonstrated an even faster onset of apoptosis in ceramide-treated BCR-ABL+ BAF3 cells as compared to normal controls. BCR-ABL+ cells treated with ceramide also showed a rapid and sequential increase in the tyrosine phosphorylation of p210(BCR-ABL), p46-56SHC and p120Cbl. These findings suggest growth factor deprivation and treatment with TNF-alpha or ceramide trigger different initial events both of which can lead to apoptosis in factor-dependent hematopoietic cells. However, in the first case, activation of apoptosis is blocked by the basal activity of p210(BCR-ABL), whereas in the second, the presence of p210(BCR-ABL) appears to accelerate the onset of apoptosis by a mechanism that may involve an activation of its kinase function.

Apoptosis↗
PubMed1998

Defective regulation of leukemic hematopoiesis in chronic myeloid leukemia.

Over the last two decades considerable knowledge has been acquired about the distribution of cell types within the dominant leukemic (Ph+/BCR-ABL+) clone that results in human chronic myeloid leukemia (CML). Evidence is now growing to indicate that three key biological changes affecting the development of such clones are: (1) an increased probability of differentiation at the level of the most primitive leukemic stem cells; (2) an increased turnover rate of the leukemic progenitors at all stages of differentiation: and (3) their increased ability to survive under conditions of factor-deprivation. Such a model explains the long latent period for the development of CML as well as why normal stem cells may persist in large numbers but still fail to compete in contributing to the daily output of mature blood cells in patients with disease. The recent development of new genetic and transplant models of human CML may now allow the molecular basis of these biological disturbances to be delineated and more effective therapeutic strategies developed.

Cell Count↗
PubMed1997

Hematopoietic stem cells: inferences from in vivo assays.

Mice and humans both contain a population in their marrow which can permanently regenerate all of the hematopoietic lineages. This developmental potential was first demonstrated in myeloablated mice transplanted with genetically marked marrow obtained from congenic donors. More recently, this approach has been used to devise an in vivo limiting dilution assay for "competitive (lymphomyeloid) repopulating units" (CRU) that allows murine hematopoietic stem cells to be quantitated. Measurements of murine CRU have shown that this population expands concomitantly with the total hematopoietic system during ontogeny and to some extent post-transplant. During these periods of expansion, defective c-kit function can be seen to preferentially compromise CRU self-renewal more than early CRU detection (which requires differentiation and amplification of the progeny of CRU, but may not require extensive CRU self-renewal). In humans, a similar cell type with transplantable lymphomyeloid differentiation potential can be identified in cord blood on the basis of its ability to engraft sublethally irradiated immunodeficient nonobese diabetic/severe combined immunodeficient mice. Quantitation of these human CRU by limiting dilution analysis of unseparated, highly purified (CD34+CD38-) and cultured (CD34+CD38-) human cord blood cells indicates that their numbers (like the long-term culture-initiating cell [LTC-IC] population) can be slightly expanded in cytokine-supplemented serum-free media, although not as extensively as anticipated from analogous studies of human marrow LTC-IC cultured under the same conditions. Taken together, the results of our studies suggest that the self-renewal of mitotically activated hematopoietic stem cells can be enhanced by their interactions with particular cytokine combinations whose effectiveness in this regard may change during ontogeny.

Animals↗
PubMed1997

Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay.

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to > 100 Mk, detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions, the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However, optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors, of which a combination of IL-3, IL-6, GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large "pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+, approximately half of which are CD41+. Thus, CFC-Mk are more similar to primitive clonogenic erythroid progenitors than to their granulopoietic counterparts in their expression of CD34, CD45RA and CD71. Taken together, these findings support the concept that some erythroid and Mk progenitors may share a common developmental pathway. The availability of sensitive and reproducible procedures for isolating and detecting human Mk progenitors should facilitate future investigations of their biology and role in a variety of haematological conditions.

Cell Division↗
PubMed1996

Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis.

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.

Animals↗
PubMed1994

Differential manipulation of normal and chronic myeloid leukemia stem cell proliferation in vitro.

The long-term marrow culture (LTC) system allows the sustained production of primitive normal and neoplastic (chronic myeloid leukemia [CML]) hematopoietic cells in vitro for many weeks. This is achieved in the absence of exogenously added hematopoietic growth factors because of the presence in the cultures of supportive "stromal" cells of the fibroblast-endothelial-adipocyte lineages. These latter cells form a confluent adherent layer with which the most primitive hematopoietic cells become associated and which locally regulates their behavior. The LTC system has thus been considered as a model of the microenvironment of the bone marrow and used to delineate potentially physiologically relevant mechanisms that regulate the proliferation, self-renewal and differentiation of primitive normal hematopoietic cells. It has also been used to analyze the molecular basis of the altered proliferative behavior that characterizes primitive neoplastic cells from patients with CML. Most of the information obtained to date has emerged from experiments designed to shift the balance of stimulatory and inhibitory factors present in order to favor either the cycling or quiescence of primitive normal or CML cells in LTC. This has been achieved either by addition of soluble factors (or antagonists) to the LTC medium or by the use of genetically engineered factor-producing stromal cells. Such experiments have allowed the identification of a number of cytokines that promote one or the other of these responses (i.e., primitive progenitor cycling or quiescence), including some that are involved in control mechanisms endogenous to the LTC system. Recent studies suggest that the retention of primitive normal cells in a reversible G(o) state in this system is mediated by the cooperating action of limiting concentrations of at least two endogenously produced inhibitory factors (transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha)), either of which, however, if added exogenously at a sufficient concentration, can exert this action on its own. Interestingly, the heightened turnover characteristic of primitive CML cells appears to be due to a selective unresponsiveness to only one of these two inhibitors (MIP-1 alpha). These findings are consistent with a complex model of the extrinsic regulation of primitive hematopoietic cells in which a multiplicity of intracellular signaling intermediates within the target cells converge at different points ultimately to control their entry into S phase. Our findings further suggest that only some of these pathways may be affected by intracellular expression of the BCR-ABL fusion gene.

Cell Differentiation↗
PubMed1993

The biology of normal and neoplastic stem cells in CML.

Chronic myeloid leukemia (CML) has long served as a prototype malignancy for basic as well as clinical studies aimed at developing curative cancer treatment protocols. Well established features of chronic phase CML are its origin in a pluripotent stem cell, a now well defined molecular genetic basis involving the creation of a BCR-ABL fusion gene and evidence of resultant abnormalities in the mechanisms that normally control primitive hemopoietic cell proliferation. We have recently shown how the long-term marrow culture system can be adapted to quantitate and characterize a very primitive cell type in normal blood and marrow samples, as well as their normal and leukemic counterparts in patients with CML. This system has also been used to dissect mechanisms of normal progenitor regulation and to identify specific anomalies affecting leukemic (CML) progenitors. Our studies show that cells detected by their ability to initiate long-term cultures (LTC) of leukemic cells (i.e., CML LTC-initiating cells or LTC-IC) are differently distributed between marrow and blood by comparison to LTC-IC in normal individuals and, although functionally similar in terms of the number and differentiation types of clonogenic cells they produce, CML LTC-IC exhibit defective self-maintenance. Phenotypically these primitive leukemic cells are heterogeneous; the majority display features of activated/proliferating cells but a significant proportion do not. We have also documented heterogeneity in primitive CML cell responses to two factors that specifically and reversibly arrest the cycling of primitive normal hemopoietic cells; i.e., TGF-beta and MIP-1 alpha, to which CML cells are normally responsive and abnormally unresponsive, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Cells↗
PubMed1985

Long-term marrow culture of cells from patients with acute myelogenous leukemia. Selection in favor of normal phenotypes in some but not all cases.

Long-term cultures were initiated with leukemic marrow aspirate cells from each of 13 newly diagnosed acute myelogenous leukemia (AML) patients. Initial assessment of the clonogenic potential of the marrow suggested that normal hemopoietic progenitors were reduced in most cases and progenitors of abnormal colonies and clusters were present in 10 cases. Subsequent assays of both nonadherent and adherent fractions of long-term cultures revealed two patterns of progenitor cell behavior. The most common pattern (nine cases) featured the detection after 1-4 wk of near normal numbers of typical erythroid, granulopoietic, and mixed colony-forming progenitor cells. Progenitors of abnormal (blast) colonies and clusters initially demonstrable in eight of these nine cases were, in these cases, not sustained in long-term culture and could not be found after 4 wk. Conversion to cytogenetic normalcy in long-term culture was confirmed in two experiments in this group. The second pattern (four cases) was characterized by the failure of progenitors capable of normal differentiation to become detectable in long-term cultures, and the concomitant maintenance of blast progenitors in the two cases in this group where such cells were initially demonstrable. Although progenitors capable of producing abnormal (blast) colonies or clusters in methylcellulose were not detected in either of the other two experiments, the maintenance for 6 wk of a hypercellular nonadherent blast population in one of these suggested the persisting activity of an "adherent layer-dependent" leukemic progenitor cell. Taken together, these findings indicate a strong correlation between the presence of leukemic blasts and their progenitors and a decreased level of normal hemopoiesis. In addition, the failure of leukemic cells to be maintained in long-term marrow cultures from some (but not all) AML patients suggests new applications of this methodology for studies of early stages of leukemic cell development.

Adult↗