Erythropoietin induces the expansion of c-kit+ progenitors for myeloid and erythroid cells, but not for lymphoid cells, in the bone marrow and liver

In humans, the numbers of erythrocytes and granulocytes, but not that of lymphocytes, tend to increase in parallel. To determine the mechanism, we investigated how the administration of erythropoietin induces the expansion of erythroid cells and other lineage cells in the bone marrow, liver, and other organs of mice. When mice were injected twice (days 1 and 2) with erythropoietin at a dose of 20 or 200 IU/day/ mouse, a prominent expansion of TER 19+ (erythroid cells) and Gr-1high cells (granulocytes) occurred in the liver, spleen, and bone marrow day 3 after the initial injection. On the other hand, lymphoid cells, including NK cells, extrathymic T cells, and conventional T cells, did not expand. In parallel with the expansion of erythroid cells and granulocytes, the levels of c-kit(+)Lin- cells increased in the liver and bone marrow. Despite the increase in the proportion of c-kit(+) Lin(-) cells, the generation of lymphocytes (e.g., T cells) decreased when such bone marrow cells were injected to scid mice. These results suggest that erythropoietin has the ability to induce the expansion of not only erythroid cells but also granulocytes in the liver, spleen, and bone marrow. Furthermore, c-kit+ progenitors which may commit themselves to erythroid and myeloid cells, but not to lymphoid cells, were also activated in the liver and bone marrow of mice treated with erythropoietin.     

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen

Background

The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated.

Design and Methods

Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry.

Results

Erythropoiesis in Rac1^−/−;Rac2^−/− mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen.

Conclusions

These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.     

Interferon-alpha overrides the deficient adhesion of chronic myeloid leukemia primitive progenitor cells to bone marrow stromal cells

Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN-alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI-CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG-dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.     

Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte- CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.     

Antigen-selected, immunoglobulin-secreting cells persist in human spleen and bone marrow

Plasma cells (PCs) represent the final stage of B-cell differentiation and are devoted to the production of immunoglobulin (Ig). Perturbations to their development can result in human disorders characterized by PC expansion and hypergammaglobulinemia. Ig-secreting cells (ISCs) have been identified in secondary lymphoid tissues and bone marrow (BM). Most ISCs in lymphoid tissue are short-lived; in contrast, ISCs that migrate to the BM become long-lived PCs and continue to secrete immunoglobulin for extended periods. However, a small population of long-lived PCs has been identified in rodent spleen, suggesting that PCs may persist in secondary lymphoid tissue and that the spleen, as well as the BM, plays an important role in maintaining long-term humoral immunity. For these reasons, we examined ISCs in human spleen and identified a population that appears analogous to long-lived rodent splenic PCs. Human splenic ISCs shared morphologic, cellular, molecular, and functional characteristics with long-lived PCs in BM, demonstrating their commitment to the PC lineage. Furthermore, the detection of highly mutated immunoglobulin V region genes in splenic ISCs suggested they are likely to be antigen-selected and to secrete high-affinity immunoglobulin. Thus, our results suggest that splenic ISCs have an important role in humoral immunity and may represent the affected cell type in some B-cell dyscrasias.     

A case of agnogenic myeloid metaplasia evolving into acute myelogenous leukemia associated with the development of trisomy 11 in bone marrow cells

An 83-year-old woman who had a 5-year history of agnogenic myeloid metaplasia and a previous normal marrow cell karyotype presented with acute myelogenous leukemia (AML) associated with the development of trisomy 11 in 100% of cultured marrow cells. Isolated trisomy 11 in marrow cells has been reported previously in five cases of AML, suggesting that trisomy 11 is a nonrandom chromosomal aberration associated with AML.     

Interleukin-11 stimulates multilineage progenitors, but not stem cells, in murine and human long-term marrow cultures

Interleukin-11 (IL-11) is a bone marrow microenvironment-derived growth factor with pleiotropic effects on a variety of hematopoietic cells. To more accurately assess the effects of IL-11 on stem and progenitor compartments within the hematopoietic microenvironment (HM), we added recombinant human (rh) IL-11 to human and murine long-term bone marrow cultures (LTMC) and analyzed primitive (high proliferative potential- colony forming cells [HPP-CFC], long-term culture-initiating cells [LTC- IC], and long-term reconstituting stem cells) and progenitor (day 12 colony forming unit-spleen [CFU-S12], colony forming unit-megakaryocyte [CFU-Mk] and colony forming unit-granulocyte/macrophage [CFU-GM]) compartments throughout the duration of the cultures. rhIL-11 (100 ng/mL) added twice weekly resulted in significantly increased nonadherent (NA) cellularity, CFU-GM, and CFU-Mk production in human LTMC. Addition of rhIL-11 to murine LTMC was associated with a 5- to 40- fold increase in CFU-GM and a four- to 20-fold increase in day 12 CFU-S in NA cells. However, IL-11 had no significant effect on total HPP-CFC concentration and decreased the size of the more primitive stem/progenitor compartment as evidenced by both decreased LTC-IC frequency in human LTMC and decreased frequency of long-term reconstituting stem cells in murine LTMC. These data suggest that IL-11 may increase commitment of stem cells into a multipotential progenitor compartment.     

Roles of Gi and Gq/11 in mediating desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes

Although desensitization of most guanine nucleotide-binding (G) protein receptors is triggered by phosphorylation of the receptor, desensitization of the LH/CG receptor (-R) in porcine follicular ovarian membranes appears to be independent of LH/CG-R phosphorylation. We therefore evaluated whether desensitization of the LH/CG-R reflected a direct inhibition of adenylyl cyclase (AC) activity by either the alpha-subunit of Gi or betagamma-subunits derived from any of the membrane G proteins activated in response to LH/CG-R activation or whether desensitization reflected a competition between Gs and a G protein that activated phospholipase C for binding sites on the LH/CG-R. The results showed that follicular membrane AC activity was not inhibited upon activation of the LH/CG-R despite evidence that the ACs in follicular membranes, when maximally activated by forskolin, could be inhibited when membrane G proteins were activated by guanyl-5'-yl imidodiphosphate, and that pertussis toxin pretreatment of membranes raised forskolin-stimulated AC activity, consistent with a tonic inhibition of follicular membrane AC activity. Similarly, agonist-stimulated desensitization of LH/CG-R-stimulated AC activity was not inhibited by pertussis toxin. Therefore, desensitization is not the result of inhibition of AC mediated by an inhibitory Gi subunit. Follicular membrane AC was also not inhibited by Gbetagamma subunits freed with activation of Gs Gq/11, or G13, based on the inabilities of exogenous Gbetagamma to promote desensitization and of a protein that sequesters Gbetagamma to inhibit desensitization. Desensitization was also not inhibited by a Gq/11 C-terminal peptide or antiserum directed toward the C-terminus of Gq/11, nor was it reversed with the addition of Gbetagamma to membranes exhibiting desensitized LH/CG-R, suggesting that desensitization is independent of coupling of the LH/CG-R to Gq/11. These results indicate that agonist-dependent desensitization of LH/CG-R-stimulated AC activity is mediated by a unique mechanism.     

Flavonoid quercetin, but not apigenin or luteolin, induced apoptosis in human myeloid leukemia cells and their resistant variants

Flavonoids and their in vivo metabolites are neuroprotective, cardioprotective and chemopreventive agents acting as hydrogen-donating antioxidants or modulators functioning at protein kinase and lipid signaling pathways. In presented study treatments of human leukemia cells HL60 and their MDR-1 resistant subline HL60/VCR by flavonoids apigenin (API), luteolin (LUT), quercetin (QU) and anticancer drug doxorubicin (DOX) are reported. Of all flavonoids used only QU treatments led in both cell lines to DNA fragmentation, cleavage of poly (ADP- ribose) polymerase (PARP), up-regulation of proapoptotic Bax and posttranslational modification (phosphorylation) of antiapoptotic Bcl-2. Cytochrome c and p21WAF1/CIP1 levels remained unchanged in these cells. Furthermore, treatments of both cell lines by QU and in its combined application with DOX increased phosphorylation of ERK, while Akt-1 and phosphorylated Akt-1 levels were not changed. All these events resulted in effective induction of apoptosis associated with down-regulation of P-glycoprotein in resistant cells. Presented results suggest that in human leukemia cells QU is a potent regulator of the cell apoptotic program associated with the modulation of several signaling molecules.     

Roles of the bone marrow niche in hematopoiesis,leukemogenesis, and chemotherapy resistance in acute myeloid leukemia

ABSTRACT

Objectives: To summarize the effects of the bone marrow niche on hematopoiesis and leukemogenesis and discuss the chemotherapy resistance that can arise from interactions between the niche and leukemia stem cells.

Methods: We review the major roles of the bone marrow niche in cell proliferation, adhesion and drug resistance. The signaling pathways and major molecular participants in the niche are discussed. We also address potential niche-targeting strategies for the treatment of acute myeloid leukemia (AML).

Results: The bone marrow niche supports normal hematopoiesis and affects acute myeloid leukemia (AML) initiation, progression and chemotherapy resistance.

Discussion: AML is a group of heterogeneous malignant diseases characterized by the excessive proliferation of hematopoietic stem and/or progenitor cells. Even with intensive chemotherapy regimens and stem cell transplantation, the overall survival rate for AML is poor. The bone marrow niches of malignant cells are remodeled into a leukemia-permissive environment, and these reformed niches protect AML cells from chemotherapy-induced cell death. Inhibiting the cellular and molecular interactions between the niche and leukemia cells is a promising direction for targeted therapies for AML treatment.

Conclusions: Interactions between leukemia cells and the bone marrow niche influence hematopoiesis, leukemogenesis, and chemotherapy resistance in AML and require ongoing study. Understanding the mechanisms that underlie these interactions will help identify rational niche-targeting therapies to improve treatment outcomes in AML patients.     

Human bone marrow granulocyte-macrophageal colonies (GM-Cs) in vitro: myeloid cells in health and in cases of myeloid leukemia

The morphological characteristics of granulocyte/macrophageal (GM-) colonies and clusters, obtained in vitro (in semi-solid agar cultures) from bone marrow hematopoietic myeloid progenitors pertain to leukocyte hemorheology of healthy persons and patients with myeloid leukemias. The morphological features of in vitro growing myeloid progenitors, granulocytes and macrophages of healthy persons differ in their cell size, shape and degree of differentiation from the cultivated marrow cells in cases of acute and chronic myeloid leukaemia. In this malignant disease, the rheological properties of leukocytes (granulocytes/macrophages) were found to provide diagnostic information. Further studies should be undertaken to examine whether the method could be useful in defining survival, prognosis and therapeutical approach in cases of myeloid leukemia.     

Different composition of the erythropoietic tissue in bone marrow, spleen and liver in myelofibrosis.

Aspirates from bone marrow, spleen and liver were analysed in 10 untreated patients with idiopathic myelofibrosis (MF). The proportion of erythroblasts was higher in the spleen and the liver than in the bone marrow. The mitotic indices of the erythropoietic precursor cells were subnormal in the extramedullary sites and significantly lower in the liver compared with the spleen. There was a "shift to the left" within the liver erythropoiesis and a significant megaloblastosis in the spleen. The same tendencies have formerly been found in patients with chronic myeloid leukemia and it is suggested that the discrepancies may be due to differences in the microenvironment of the erythropoietic cells.     

A comparison of allogeneic bone marrow transplantation, autologous bone marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia in remission

Intensive, myelosuppressive therapy is necessary to maximizeoutcomes for patients with acute myeloid leukemia (AML). A comparison was made of 3 aggressive postremission approaches for children andadolescents with AML in a randomized trial, CCG-2891. A total of 652 children and adolescents with AML who achieved remission on 2 inductionregimens using identical drugs and doses (standard and intensivetiming) were eligible for allocation to allogeneic bone marrowtransplantation (BMT) based on matched related donor status (n = 181)or randomization to autologous BMT (n = 177) or to aggressivehigh-dose cytarabine-based chemotherapy (n = 179). Only 115 patients (18%) refused to participate in the postremission phase ofthis study. Overall compliance with the 3 allocated regimens was 90%.At 8 years actuarial, 54% ± 4% (95% confidence interval) of allremission patients remain alive. Survival by assigned regimen ("intent to treat") is as follows: allogeneic BMT, 60% ± 9%;autologous BMT, 48% ± 8%; and chemotherapy, 53% ± 8%.Survival in the allogeneic BMT group is significantly superior toautologous BMT (P = .002) and chemotherapy(P = .05); differences between chemotherapy and autologous BMT are not significant (P = .21). Nopotential confounding factors affected results. Patients receivingintensive-timing induction therapy had superior long-term survivalirrespective of postremission regimen received (allogeneic BMT,70% ± 9%; autologous BMT, 54% ± 9%; chemotherapy,57% ± 10%). Allogeneic BMT remains the treatment of choice forchildren and adolescents with AML in remission, when a matched relateddonor is available. For all others, there is no advantage to autologousBMT; hence, aggressive nonablative chemotherapy should be used.     

Naive recirculating B cells mature simultaneously in the spleen and bone marrow

We have recently demonstrated that IgD(hi) B cells can occupy an extravascular perisinusoidal niche in the bone marrow in addition to the well-established follicular niche in conventional secondary lymphoid organs. The spleen has long been considered to be the site at which newly formed B lymphocytes mature into IgD(hi) naive recirculating B cells, but the existence of mutant mice that have selectively lost mature B cells in the bone marrow prompted an examination of B-cell maturation at this latter site. Following a single pulse of BrdU in intact mice, sequential labeling of more mature B-cell populations in the bone marrow suggested ongoing maturation at this site. Further evidence for B-cell maturation in the bone marrow was obtained from analyses of transitional B cells in splenectomized lymphotoxin alpha-deficient mice that lack all secondary lymphoid organs. In these mice, antibody-secreting cells recognizing multivalent antigens were also observed in the bone marrow following an intravenous microbial challenge. These data suggest that newly formed B cells mature into IgD(hi) B cells simultaneously in the spleen and the bone marrow and establish in a stringent manner that humoral immune responses can be initiated in situ in the bone marrow.