Molecular and cellular mechanisms of leukemic hemopoietic cell differentiation: an analysis of the Friend system

Murine erythroleukemia (MEL or Friend) cells grown in culture and induced to differentiate into cells resembling orthochromatic normoblasts provide a suitable system for uncovering molecular and cellular mechanisms of hemopoiesis and for understanding globin gene regulation. Inducer-treated cells undergo an irreversible commitment to maturation and accumulate large amounts of hemoglobin. Clonal analysis of commitment of individual cells combined with biochemical measurements has revealed that MEL cell differentiation is a highly coordinated set of events (program) leading to the differentiated erythroid state. The developmental program of MEL cells consists of early and late processes. The early events appear to be membrane-mediated processes which operate independently of each other and lead to commitment to terminal maturation and hemoglobin synthesis. Inducer-treated cells express an ability to remember ("memory response") previous exposure to inducer and to continue their differentiation after discontinuous exposure to inducer; expression of "memory response" occurs early in differentiation and affects both the initiation of commitment and accumulation of globin mRNA in a similar manner in inducer-treated cells. Commitment to maturation appears to be the central process responsible for determining the pattern of gene expression, limitation of proliferative activity and nuclear condensation. Commitment, however, can occur independently of hemoglobin synthesis. Although initiation of commitment is associated with early membrane-mediated events (e.g., ion-transport), maintenance and completion of maturation erythroid state is a result of a number of cellular processes. These processes are discussed in relation to the molecular and cellular mechanisms of initiation and completion of MEL cell differentiation. The role of the MEL system as a model for studying mouse and human globin gene regulation is presented.     

Chromatin remodeling gene SMARCA5 is dysregulated in primitive hematopoietic cells of acute leukemia.

We identified a subset of genes involved in chromatin remodeling whose mRNA expression changes in differentiating mouse erythroleukemia (MEL) cells. We furthermore tested their mRNA expression patterns in normal and malignant CD34+ bone marrow cells. SMARCA5, imitation switch gene homologue, was rapidly silenced during in vitro erythroid differentiation of MEL cells whereas it was up-regulated in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Moreover, SMARCA5 mRNA levels decreased in AML CD34+ progenitors after the patients achieved complete hematologic remission. We detected high levels of SMARCA5 mRNA in murine bone marrow and spleen and monitored its expression in these hematopoietic tissues during accelerated hematopoiesis following hemolytic anemia induced by phenylhydrazine. SMARCA5 expression levels decreased after the onset of accelerated erythropoiesis. Our data suggest that both in vitro and in vivo induction of differentiation is followed by down-regulation of SMARCA5 expression. In CD34+ AML progenitors over-expression of SMARCA5 may thus dysregulate the genetic program required for normal differentiation.     

Characteristic patterns of hox gene expression in different types of human leukemia

Homeobox-containing genes are a network of genes encoding DNA-binding proteins highly conserved throughout evolution. They are involved in the control of normal development as well as in the regulation of gene expression in adult differentiating systems, including hematopoiesis. Aberrant expression of homeobox-containing genes has recently been related to leukemic phenotype. Human homeobox-containing genes of the HOX family are organized into 4 large clusters. We have analyzed the expression of HOX genes in different types of human leukemia to investigate whether the physical organization of HOX loci reflects a regulatory hierarchy involved in the differentiation of hematopoietic cells or whether HOX gene expression might contribute to the leukemic phenotype. Our results show that HOX genes are coordinately regulated in blocks in myeloid cells whereas they appear to function as isolated genes in lymphoid cells. Six contiguous genes of the HOX2 locus, highly expressed in acute non-lymphocytic leukemia, are switched off in chronic myelogenous leukemia, suggesting that down-regulation of HOX2 genes might be required for cell maturation of the myeloid lineages. In contrast, a few scattered genes are active in lymphoid populations. These observations suggest that hematopoietic cells express a repertoire of HOX genes characteristic of a particular cell lineage at a specific stage of differentiation. The characteristic patterns of HOX gene expression may reflect the potentially important role that these genes play in cell lineage determination during both normal and leukemic hematopoiesis.     

Survival of B lineage leukemic cells: signals from the bone marrow microenvironment

Stromal cells are an essential component of the bone marrow microenvironment that regulate development of immature hematopoietic progenitor cells. Through production of soluble cytokines, and signaling through adhesion molecule interactions, stromal cells impact survival, proliferation, and differentiation of hematopoietic progenitor cells. Similarities between normal pro-B and pre-B cells and B lineage acute lymphoblastic leukemic (ALL) progenitors have been well characterized which provide a model for investigation of the mechanisms by which ALL cells respond to bone marrow microenvironment signals. In addition to providing survival signals to B lineage ALL during initiation of disease, the bone marrow has long been recognized as a "sanctuary site" for leukemic cells during traditional chemotherapy. In the current review, mechanisms by which stromal cells contribute to leukemic cell survival, and the potential impact on treatment efficacy, are discussed. A growing appreciation of the significance of the bone marrow microenvironment in the progression of ALL, and further investigation of the signaling between leukemic progenitors and stromal cells, may contribute to novel treatment strategies aimed at enhancing sensitivity of ALL cells to currently available chemotherapeutic agents.     

Characteristics and analysis of normal and leukemic stem cells: current concepts and future directions.

Acute myeloid leukemias (AML) are considered to be clonal disorders involving early hematopoietic progenitor cells. The recent advances in characterization of early stem cells give rise to the question whether it is possible to distinguish healthy progenitors from cells of the leukemic clone in leukemia patients. Differences and similarities in phenotype, genotype and biology are described for leukemic cells and normal hematological progenitors. Recent new insights into human stem cell development offer the perspective that distinction between benign and malignant progenitors might be possible in the future at a very early stage of maturation.     

Id1 immortalizes hematopoietic progenitors in vitro and promotes a myeloproliferative disease in vivo

Id1 is frequently overexpressed in many cancer cells, but the functional significance of these findings is not known. To determine if Id1 could contribute to the development of hematopoietic malignancy, we reconstituted mice with hematopoietic cells overexpressing Id1. We showed for the first time that deregulated expression of Id1 leads to a myeloproliferative disease in mice, and immortalizes myeloid progenitors in vitro. In human cells, we demonstrate that Id genes are expressed in human acute myelogenous leukemia cells, and that knock down of Id1 expression inhibits leukemic cell line growth, suggesting that Id1 is required for leukemic cell proliferation. These findings established a causal relationship between Id1 overexpression and hematologic malignancy. Thus, deregulated expression of Id1 may contribute to the initiation of myeloid malignancy, and Id1 may represent a potential therapeutic target for early stage intervention in the treatment of hematopoietic malignancy.     

Acute leukemia

The acute leukemias continue to present a formidable challenge for which there is not yet a reliably curative 'standard approach' for the majority of adults with this family of diseases. In order to make progress in terms of curing these devastating diseases, we must understand leukemia biology on the clinical, cellular and molecular levels, with exploitation of the leukemia-associated molecular targets in designing strategies aimed at eradicating the leukemic clone. In this review, we will discuss a few key mechanisms of leukemogenesis that represent convergent pathways of malignant transformation and, as such, present pivotal molecular targets for therapy. Specifically, we focus on normal and leukemic hematopoietic cell cycle regulation, issues surrounding DNA damage and repair, programmed cell death (apoptosis) and drug responsiveness, and multidrug resistance as a marker for stem cell involvement and as a novel target for intervention. When functioning normally, such mechanisms determine a cell's ability to respond to DNA damage, traverse the cell cycle and maintain genomic integrity. And in addition to the target cell itself, there are crucial extracellular determinants of hematopoietic cell proliferation and differentiation that modulate net signalling activity and gene expression, cell-cell contact and growth-modulating factors for instance. The molecular dissection of these intersecting pathways, from the extracellular milieu to the genes themselves, in both the normal and transformed states will elucidate the means by which cells escape treatment-induced death. Such understanding should, in turn, lead to the development of targeted therapeutic strategies that exploit differences between normal and malignant cells, overcome the mechanisms by which leukemic cells acquire drug resistance, and enhance the curability of these devastating diseases.     

C-Jun modulates apoptosis but not terminal cell differentiation in murine erythroleukemia cells.

The proto-oncogene c-Jun has been implicated in the control of cell proliferation and differentiation and more recently in the regulation of apoptosis. We have previously reported the involvement of c-Jun in the erythroid differentiation block in murine erythroleukemia (MEL) cells. As reported here, we investigated the role of c-Jun in the regulation of terminal differentiation and apoptosis of MEL cells by studying different stable transfectant clones containing c-jun constructs in sense or antisense orientation. c-Jun did not prevent cell growth arrest in G0/G1 and p21 induction that are normally associated with terminal differentiation induced by DMSO treatment, suggesting that c-Jun may uncouple phenotypic differentiation and terminal cell division in the MEL cell system. Spontaneous apoptosis was accelerated in c-jun expressing MEL cells before and after DMSO treatment. Moreover, c-Jun sensitized apoptosis induced by various drugs. Drug-induced apoptosis was associated with c-Jun N-terminal kinase (JNK) activation and c-Jun N-terminal phosphorylation (JNP). In contrast, overexpression of c-jun delayed apoptosis in serum-starved cells, indicating that c-Jun may reduce or accelerate apoptosis in MEL cells depending on the nature of the apoptotic stimulus. These results suggest that the proto-oncogene c-Jun may modulate differentiation and apoptosis of leukemic cells.     

Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells.

Gradual increase of CD38 on cells expressing CD34 characterizes the early cell differentiation pathway of normal human hematopoietic progenitors. In this study the coordinated expression pattern of CD34 and CD38 was assessed on leukemic blasts from bone marrow aspirates of 95 patients with newly diagnosed acute myeloid leukemia (AML). Expression was divided into six categories analogous to the differentiation pathway of normal bone marrow. The CD38 antigen was expressed on the leukemic cells of all patients and CD34+ leukemic cells were found in 79 patients (83%). In 93 patients, the leukemic cells were found along the differentiation pathway defined by CD34 and CD38. In 33 of the 93 patients, a part of the CD34+ cells did not express the CD38 antigen (categories 1 and 2). In another 33 patients, all CD34+ cells expressed CD38 (categories 3 and 4). In the remaining 27 patients, only cells were found which dimly expressed CD34 or did not express CD34 (categories 5 and 6). Of the 93 patients, 88 were treated with intensive chemotherapy according to the protocol of the German AML Cooperative Group. Of these, 21 died early and were not evaluable for treatment response. Complete remission was achieved in 14 of 22 patients (64%) in categories 1 and 2, in 19 of 26 patients (73%) in categories 3 and 4, and in 18 of 19 patients (95%) in categories 5 and 6. The event-free survival was significantly longer in patients of categories 5 and 6 compared to patients in categories 1 and 2 (p less than 0.01) and categories 3 and 4 (p less than 0.05), respectively. We conclude that in the majority of AML patients the immunophenotype of leukemic cells follows the early cell differentiation pathways defined by coordinated expression of CD34 and CD38 similar to that of normal hematopoietic progenitors. The presence of cells in the late cell differentiation stages (CD34+/-, CD38 /+) identifies patients with a higher complete remission rate and longer complete remission duration.     

Altered natural killer cell differentiation in CD34+ progenitors from chronic myeloid leukemia patients

IL-15 and SCF fail to induce NK differentiation and proliferation of CD34+ hematopoietic progenitors from chronic myeloid leukemia patients in contrast to normal stem cells although, both normal and leukemic CD34+ cells display comparable expression of c-kit or IL-15 receptor subunits. Interestingly, confocal microscopy analysis revealed that leukemic and most normal CD34+ cells produce and secrete IL-15, as shown by its trafficking through the Golgi apparatus and early endosomes. However, only leukemic progenitors express the membrane bound IL-15. Colocalization and internalization of IL-15Rbeta/gammac and IL-15Ralpha/gammac complexes indicated that IL-15 was specifically uptaken by leukemic progenitors. We also demonstrated that in both normal and leukemic progenitors, the signaling kinase Jak3 is constitutively pre-associated with the gammac chain. Anti-IL-15 neutralizing mAb treatment resulted in down-regulation of gammac chain and disruption of gammac/Jak3 interaction in normal but had no effect in leukemic progenitors. Our results suggest the existence in both normal and leukemic CD34+ cells of a constitutive production of a bioactive IL-15 that does not lead to NK differentiation and further indicate that membrane bound IL-15 and constitutive activation of gammac are hallmarks of leukemic progenitors. Oncogene (2000).     

Cytokine control of developmental programs in normal hematopoiesis and leukemia

The establishment of a system for in vitro clonal development of hematopoietic cells made it possible to discover the cytokines that regulate hematopoiesis. These cytokines include colony stimulating factors and others, which interact in a network, and there is a cytokine cascade which couples growth and differentiation. A network allows considerable flexibility and a ready amplification of response to a particular stimulus. A network may also be necessary to stabilize the whole system. Cells called hematopoietic stem cells (HSC) can repopulate all hematopoietic lineages in lethally irradiated hosts, and under appropriate conditions give rise to neuronal, muscle, and epithelial cells. Granulocyte colony stimulating factor induces migration of both HSC and in vitro colony forming cells from the bone marrow to peripheral blood. Granulocyte colony stimulating factor is also used clinically to repair irradiation and chemotherapy associated suppression of normal hematopoiesis in cancer patients, and to stimulate normal granulocyte development in patients with infantile congenital agranulocytosis. It is suggested that there may also be appropriate conditions under which in vitro colony forming cells have a wider differentiation potential similar to that shown by HSC. An essential part of the developmental program is cytokine suppression of apoptosis by changing the balance in expression of apoptosis inducing and suppressing genes. Decreasing the level of cytokines that suppress therapeutic induction of apoptosis in malignant cells can improve cancer therapy. Cytokines and some other compounds can reprogram abnormal developmental programs in leukemia, so that the leukemic cells differentiate to mature non dividing cells, and this can also be used for therapy. There is considerable plasticity in the developmental programs of normal and malignant cells.     

Regulation of the genes for interleukin-6 and granulocyte-macrophage colony stimulating factor by different inducers of differentiation in myeloid leukemic cells

Different clones of myeloid leukemic cells can be induced to differentiate to mature macrophages and/or granulocytes by hematopoietic regulatory proteins and by other compounds. We now show that induction of differentiation in different clones of myeloid leukemic cells with the normal hematopoietic proteins granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), or interleukin 3 and by compounds such as dexamethasone or cytosine arabinoside (ara C) induces the expression of genes for the myeloid differentiation inducing protein MGI-2 that we have shown is interleukin 6 (IL-6) and for GM-CSF. We have previously shown that induction of differentiation with interleukin-1, IL-6, or bacterial lipopolysaccharide (LPS) also induces IL-6 and GM-CSF gene expression. Treatment of these leukemic clones with hematopoietic proteins that do not induce differentiation did not induce IL-6 or GM-CSF gene expression. The results indicate that induction of IL-6 and GM-CSF gene expression is part of the normal differentiation program in myeloid cells and support our previous evidence that there is transregulation of gene expression between different hematopoietic regulatory proteins.     

Hematopoietic growth and differentiation factors and the reversibility of malignancy: cell differentiation and by-passing of genetic defects in leukemia

Our development of systems for the in vitro cloning and clonal differentiation of normal hematopoietic cells made it possible to identify: the factors that regulate growth and differentiation of these normal cells; the changes in the normal development program that result in leukemia, and how to reverse malignancy in leukemic cells. I have mainly used myeloid cells as a model system. Normal hematopoietic cells require different proteins to induce growth (growth factors) and differentiation (differentiation factors). There is a multigene family for these factors. Identification of these factors and their interaction has shown how growth and differentiation can be normally coupled. The development of leukemia involves the uncoupling of growth and differentiation. This can occur by changing the requirement for growth without blocking cell response to the normal inducers of differentiation. Addition of normal differentiation factors to these malignant cells still induces their normal differentiation, and the mature cells are then no longer malignant. Genetic changes which inhibit differentiation by normal differentiation factors can occur in the progression of leukemia, but even these leukemic cells may still be induced to differentiate by other compounds, including low doses of compounds now being used in cancer therapy, that can induce differentiation by alternative pathways. The differentiation of leukemic to mature cells results in the reversion of malignancy by by-passing genetic changes that produce the malignant phenotype. We have obtained this differentiation of leukemic cells in vitro and in vivo, and by-passing genetic defects by inducing differentiation can be a useful approach to therapy.     

Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34+CD34 -phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38+, some are CD38 -. AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo . Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 -cell lines, HL60 and CB-1, as well as in normal CD34+CD34 -hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34+ leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34+-AML (M 0 and M 1 ) patients, RA caused an increase in CD38+ that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 -cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34+ cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 -cells are the targets of leukemic transformation, transition of these cells into CD38+ phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.     

Proto-oncogene expression and dissection of the myeloid growth to differentiation developmental cascade

Physiological inducers of myeloid cell growth and differentiation were used to simultaneously analyze the expression of the proto-oncogenes c-myc, c-myb, c-fos, c-fes and c-fms during normal myelopoiesis, where growth is coupled to differentiation, as compared with that in leukemia, where growth has been uncoupled from differentiation as well as upon suppression of the leukemic phenotype via induction of differentiation and growth arrest. Proto-oncogene expression was also used as a tool to dissect the growth to differentiation developmental cascade. Myeloid cell growth was correlated with high c-myc and c-myb RNA levels, decreasing to undetectable levels in terminally differentiated cells. No c-myc RNA was detected in normal myeloid progenitors induced for differentiation without growth, using media conditioned by mouse granulocytes (GCM), indicating that c-myc may play either no role or an inhibitory one in differentiation. RNA levels of the proto-oncogenes c-fos, c-fes and c-fms were undetectable in normal or M1 differentiation inducible (D+) leukemic myeloblasts, and were stably induced upon stimulation of the normal precursors for growth and differentiation, with highest levels at the time when most of the cells had undergone terminal differentiation. Only c-fes RNA was induced upon M1D+ differentiation. It was also shown to be induced upon induction of differentiation without growth in normal myeloid precursors. Using c-myc and c-myb RNA suppression as molecular markers for induction of M1D+ differentiation, the existence of myeloid differentiation factor(s), distinct from myeloid growth factors, has been demonstrated. Such differentiation inducing activity was found in media conditioned by mouse lungs or granulocytes, and was induced in normal myeloid precursors by the myelopoietic growth factors IL3, GM-CSF, G-CSF, and M-CSF. Taken together, the results of this study enhance and add to previous work to better correlate the expression of the proto-oncogenes myc, myb, fes, fos and fms with several parameters of normal and abnormal myeloid cell growth and differentiation. The results indicate that the normal myeloid growth to differentiation developmental cascade entails a mechanism whereby myeloid growth factors induce myeloid differentiation factors, subsequently suppressing c-myc and c-myb RNA expression, leading to the induction of differentiation and growth arrest, including early accumulation of c-fes RNA followed by accumulation of c-fos and c-fms RNAs. It was also indicated that this cascade is impaired in leukemia.