Mechanisms involved in the induced differentiation of leukemia cells

Pharmacology & Therapeutics 100 (2003) 257 – 290 www.elsevier.com/locate/pharmthera

Mechanisms involved in the induced differentiation of leukemia cells Asterios S. Tsiftsoglou*, Ioannis S. Pappas1, Ioannis S. Vizirianakis Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece

Abstract Despite the remarkable progress achieved in the treatment of leukemias over the last several years, many problems (multidrug resistance [MDR], cellular heterogeneity, heterogeneous molecular abnormalities, karyotypic instability, and lack of selective action of antineoplastic agents) still remain. The recent progress in tumor molecular biology has revealed that leukemias are likely to arise from disruption of differentiation of early hematopoietic progenitors that fail to give birth to cell lineage restricted phenotypes. Evidence supporting such mechanisms has been derived from studying bone marrow leukemiogenesis and analyzing differentiation of leukemic cell lines in culture that serve as models of erythroleukemic (murine erythroleukemia [MEL] and human leukemia [K562] cells) and myeloid (human promyelocytic leukemia [HL-60] cells) cell maturation. This paper reviews the current concepts of differentiation, the chemical/pharmacological inducing agents developed thus far, and the mechanisms involved in initiation of leukemic cell differentiation. Emphasis was given on commitment and the cell lineage transcriptional factors as key regulators of terminal differentiation as well as on membrane-mediated events and signaling pathways involved in hematopoietic cell differentiation. The developmental program of MEL cells was presented in considerable depth. It is quite remarkable that the erythrocytic maturation of these cells is orchestrated into specific subprograms and gene expression patterns, suggesting that leukemic cell differentiation represents a highly coordinated set of events that lead to irreversible growth arrest and expression of cell lineage restricted phenotypes. In MEL and other leukemic cells, differentiation appears to be accompanied by differentiationdependent apoptosis (DDA), an event that can be exploited chemotherapeutically. The mechanisms by which the chemical inducers promote differentiation of leukemic cells have been discussed. D 2003 Elsevier Inc. All rights reserved. Keywords: Leukemia; Differentiation; Apoptosis; Hematopoietic cells; Mechanisms; Chemical inducers Abbreviations: MEL, murine erythroleukemia cells; HL-60, human promyelocytic leukemia cells; K562, human leukemia cells.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional cancer chemotherapy: successes, failures, and obstacles . . . . . . . . . . . . Leukemias as disorders of hematopoietic cell differentiation and apoptosis . . . . . . . . . 3.1. The study of hematopoiesis under normal and leukemic states . . . . . . . . . . . . 3.1.1. Murine erythroleukemia (or Friend leukemia) cells. . . . . . . . . . . . . . 3.1.2. Human promyelocytic leukemia cells. . . . . . . . . . . . . . . . . . . . . 3.1.3. Human leukemic K562 cells . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The developmental programs and subprograms in differentiating leukemic cells . . . 3.2.1. Commitment as a central event in leukemic cell differentiation leads to irreversible growth arrest and discrete patterns of gene expression. . . . . . 3.2.2. Expression of memory proceeds commitment . . . . . . . . . . . . . . . . 3.3. Coordination of cell proliferation and differentiation in murine erythroleukemia cells Inducers of leukemic cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel.: +30-2310-997-631; fax: +30-2310-997-618. E-mail address: [email protected] (A.S. Tsiftsoglou). 1 Present address: Laboratory of Pharmacology, Department of Veterinary Medicine, School of Health Sciences, University of Thessaly, Terma Trikalon, GR-43100 Karditsa, Thessaly, Greece. 0163-7258/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2003.09.002

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4.1. 4.2. 4.3.

Retinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vitamin D3 and its analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agents that remodel chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Hypomethylating agents . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Histone deacetylase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Inhibitors of topoisomerases . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Purine and pyrimidine analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Purines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Pyrimidines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Inosine 5V-monophosphate dehydrogenase inhibitors . . . . . . . . . . . . . . . . . 4.6. Activators and inhibitors of signal transduction pathways . . . . . . . . . . . . . . . 4.7. Polar agents and ureido derivatives of pyridine . . . . . . . . . . . . . . . . . . . . 4.7.1. Dimethylsulfoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.2. Bisacetamides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.3. Benzodiazepines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.4. Cyclic ureas and thioureas . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.5. Substituted amides, lactams, and 2-pyridones . . . . . . . . . . . . . . . . 4.7.6. Pyridine derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8. Hemin and other agents as inducers of human K562 leukemia cell differentiation . . 4.9. Cytokines as inducers of leukemic cell differentiation. . . . . . . . . . . . . . . . . 5. Mechanisms of induced differentiation of leukemic cells . . . . . . . . . . . . . . . . . . . 5.1. Membrane-mediated events and signal transduction pathways . . . . . . . . . . . . 5.2. Receptor-mediated processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Remodeling of the superfine structure of chromatin . . . . . . . . . . . . . . . . . . 5.4. The role of DNA and RNA transmethylation in murine erythroleukemia cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Lineage-specific transcriptional factors as regulators of hematopoietic cell fate under normal and leukemic states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. The role of c-myc and other protoncogenes in murine erythroleukemia cell differentiation and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Induction of differentiation and apoptosis in leukemic cells . . . . . . . . . . . . . . . . . 6.1. Is differentiation-dependent apoptosis part of the developmental program of leukemic cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Coactivation of differentiation and apoptosis programs as potential target for combination chemotherapy of leukemias . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The discovery that Friend leukemia cells (virus-transformed spleen hematopoietic cells) can be induced to differentiate into nondiving mature erythroid cells resembling normal orthochromatophilic normoblasts early in 1970s (Friend et al., 1971) sparked extensive investigation worldwide and led to the so-called differentiation therapy of cancer (Lotan et al., 1990; Leszczyniecka et al., 2001), which is treatment with agents inducing terminal differentiation of neoplastic cells. Moreover, this triggered the development of suitable model systems to study differentiation of hematopoietic progenitors and intensified research on novel antineoplastic agents. Several reviews have been already published over the years on this theme (Harrison, 1976; Tsiftsoglou & Wong, 1985; Rifkind et al., 1996), and it is not our intention here to simply review the literature. The central objective of this paper is to comprehensively present and discuss the mechanisms of induced leukemic cell differentiation on light of recent

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findings in cell cycle control, programmed cell death (apoptosis), and reprogramming of cell fate via lineagespecific transcriptional factors. Emphasis was also given to the development of potent inducers of differentiation and their ability to initiate commitment to maturation via either receptor-mediated processes or signal transduction pathways.

2. Conventional cancer chemotherapy: successes, failures, and obstacles Cancer is a malignant mosaic disease of uncontrolled cellular growth, which includes a large number (>100) of neoplasms (neoplasm is an abnormal mass of tissue comprising new growth in a given tissue) (Holland et al., 1993). As clonal disease, cancer is initiated at the level of individual early progenitor cells, which exist almost in every tissue and have the potential to self-renew, proliferate, and yield differentiated progeny (Fialkow, 1984). At

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first, cancers grow locally then infiltrate the surrounding tissues and at the end metastasize into distant organs via the microvascular endothelial capillaries, blood vessels, and lymphatics, where they form secondary malignancies. Metastatic cancer cells grow unrestrictedly and finally kill the host (Holland et al., 1993). Neoplasms differ from each other in morphology, cell type involved, growth kinetics, degree of differentiation (Pierce, 1974), histology, potential for metastasis, mutations and chromosomal aberrations (Rowley, 2001), cell surface antigens, epigenetic gene regulation (Jones & Baylin, 2002), susceptibility and multidrug resistance (MDR) to chemotherapeutic agents (Biedler, 1994). Although a lot of information is known about cancer etiology and carcinogenesis, our understanding of cancer cell behavior at cellular and molecular levels is still limited despite the remarkable progress achieved in molecular biology of malignancy (Hahn & Weinberg, 2002). Conventional antineoplastic agents are currently used to treat malignancies in clinic when surgery or radiotherapy is proven ineffective in controlling metastatic carcinomas. These include alkylating agents, antimetabolites, antitumor antibiotics, folate analogues as enzyme inhibitors, blockers of mitosis, cell cycle cytotoxic inhibitors, nitrosourea derivatives, cis-platin and derivatives, taxanes, estrogen antagonists, immunosuppressants, natural and semisynthetic alkaloids like camptothecins, cytokines, and growth factors recently developed via recombinant DNA technology. Unequivocally, cancer chemotherapy has been successful in curing effectively a few malignant neoplasms (Burkitt’s lymphoma, choriocarcinoma, Hodgkin’s disease, and testicular carcinomas) and useful in treating symptoms and prolonging life for several years in some other cases like childhood leukemias. However, despite this remarkable progress, conventional chemotherapy has not been able to eradicate the large majority of solid tumors (lung carcinomas, pancreatic malignancies, osteosarcomas, and brain tumors) and hematological malignancies (Canellos, 1982; Wiernik, 1982). The major problems encountered in conventional cancer chemotherapy include (a) enormous diversity in morphology and physiology of neoplasms, (b) extensive cellular heterogeneity and heterogeneity in molecular abnormalities in malignant tumors, (c) inadequate delivery of anticancer agents to the targeted tumor sites, (d) lack of selective antitumor activity and broad-spectrum toxicity to normal tissues, (e) genome instability, and (f) development of MDR (Biedler, 1994; Rowley, 2001). The large majority of conventional cancer chemotherapeutics either kills the cancer cells directly or stumbles on their growth potential at specific phase of the cell cycle (S, M, or S/G2 interphase). Over the past several years, several novel exploitable targets for drug development have been revealed. These include enzymes like tyrosine kinases involved in signal transduction pathways, genes regulating apoptosis and cell differentiation in malignant cells, cell

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lineage transcriptional factors, angiogenesis factors, and unique proteins driving the cell cycle machinery. The development and use of Gleevec or Glivec (imatinib mesylate), a selective inhibitor of Bcr-Abl fusion tyrosine kinase involved in signaling, as an effective agent for treatment of chronic myelogenous leukemia (CML) patients represents a bright example of molecularly targeted oncotherapy agents (Capdeville et al., 2002).

3. Leukemias as disorders of hematopoietic cell differentiation and apoptosis Leukemias represent a large proportion of hematological malignancies that occur as acute or chronic diseases at relatively low frequency (5– 8/105) (Canellos, 1982; Wiernik, 1982). Evidence exists to indicate that leukemias result from disruption of differentiation of hematopoietic cells (Bonnet & Dick, 1997; Enver & Greaves, 1998; Tenen, 2003). Based on clonal analysis experiments performed in vitro, human bone marrow contains hematopoietic stem cells (HSCs) that self-renew themselves and give birth to different blood cell types (Wu et al., 1967; Metcalf, 1998; Orkin, 2000). Normally, HSCs are programmed to differentiate into cell lineage restricted pathways of blood cell development by external growth factors that keep balancing cell growth, differentiation, and programmed cell death in the bone marrow stochastically, instructively, or otherwise (Socolovsky et al., 1998; Cantor & Orkin, 2001; Fisher, 2002). Commitment of HSC into differentiation pathways is accompanied by irreversible maturation, cell cycle growth arrest, or apoptosis (Enver et al., 1998; Metcalf, 1998). Reprogramming of HSCs to cell lineage restricted phenotypes during hematopoiesis has implicated the existence of lineage-specific transcriptional factors (Cantor & Orkin, 2001; Graf, 2002). In leukemias, early hematopoietic progenitors fail to respond to external stimuli within the bone marrow microenvironment and differentiate into discrete types of blood cells (Fig. 1) (Fialkow et al., 1981). Leukemic cells acquire immortality, self-perpetuate, grow in high numbers in bone marrow at the expense of other cells, and then spill into the peripheral blood. That leukemia cells do not undergo programmed cell death (apoptosis) and fail to differentiate (Domen, 2001) makes the leukemias disorders of both hematopoietic cell differentiation and apoptosis (Fig. 1). Leukemic cells usually exhibit characteristic karyotypic abnormalities (e.g., t(9;22) chromosomal translocation), express unique cell surface antigens, and form transformed foci in soft agar (Champlin & Gale, 1987). The degree of differentiation is inversely related to their malignancy. This relationship suggests that differentiation is uncoupled from cell growth control in leukemia (Sachs, 1980). Therefore, induction of differentiation should then reduce or abrogate the malignant potential of leukemic cells.

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Fig. 1. Diagrammatic presentation of normal hematopoiesis and leukemiogenesis. Normally, long-term HSCs (LT-HSCs) that self-renew also produce shortterm HSCs (ST-HSCs) that subsequently differentiate into late progenitors (committed hematopoietic progenitors) and mature blood cells. In leukemic state, ST-HSCs regenerate early progenitors that are transformed and unable to respond to differentiation stimuli. Such cells perpetuate, grow in high numbers, and spill into the peripheral blood. Due to the blockade of differentiation, the peripheral blood is populated by blastic leukemic cells that resemble undifferentiated early hematopoietic progenitors. Leukemic hematopoietic progenitors acquire unlimited capacity to grow and immortality presumably to additional blockade of programmed cell death.

3.1. The study of hematopoiesis under normal and leukemic states Detailed investigation of cellular and molecular processes involved in normal and abnormal hematopoiesis could be valuable in understanding leukemias. This requires of course studying events occurring in the bone marrow microenvironment in vivo. Unfortunately, early studies designed to explore bone marrow hematopoiesis in vivo have not been very successful. Bone marrow microenvironment is a highly heterogeneous and complex system, where many cell types (HSCs, mesenchymal stem cells [MSCs], and early and late hematopoietic progenitors) coexist in different developmental stages. This complexity, however, did not permit an in-depth analysis of the molecular events of differentiation along cell lineage restricted pathways. Moreover, removal of normal bone marrow cells form their microenvironment and transfer into an artificial system such as methylcellulose or plasma clot cultures is accompanied by extensive cell death, reduced renewal capacity of HSCs, and other cell types. In addition, early hematopoietic pro-

genitors growing in culture away from stroma cells and growth factors like cytokines (or other as yet unidentified regulators of hematopoiesis) may not behave authentically as in their natural microenvironment. Long-term bone marrow cultures (LTBMCs) employed several years ago to investigate hematopoiesis in vitro were notorious for extensive death of early hematopoietic progenitors and outgrowth of stroma-mesenchymal cells in layers (Dexter, 1996). Experiments, however, with knockout transgenic animals lacking genes that encode growth factors, growth factors receptors, and/or transcriptional regulators provided new insights into the regulation of normal hematopoiesis (Orkin, 2000; Sanchez et al., 2001). Moreover, the use of retroviral vectors at first (Mulligan, 1993) and most recently of lentivirus-based gene transfer vectors (Evans et al., 1999; Miyoshi et al., 1999; Cui et al., 2002) permitted targeted transgene expression in human HSC/progenitor cells (pluripotent CD34 + cells) capable of engrafting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Isolation, maintenance, and expansion of HSCs and their early progenitors in recent years under well-defined

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culture conditions brought valuable information concerning their differentiation into cell-restricted lineages (Metcalf, 1998; Orkin, 2000; Domen, 2001). The development of culture conditions, which allowed maintenance and erythroid maturation of mouse hematopoietic progenitors up to the level of erythrocytes complemented with cDNA microarray technology, revealed discrete profiling of gene expression during erythroid maturation (Dolznig et al., 2001). A similar approach can be useful to identify specific pattern of gene expression in other cell lineage differentiation systems (e.g., myeloid and lymphoid). Several leukemic cells have been established over the years and used as suitable model systems (human promyelocytic leukemia [HL-60], human leukemia [K562], KG-1, murine erythroleukemia [MEL], and lymphocytic leukemia [U937]) to study the mechanisms of cell differentiation of unipotent and multipotent hematopoietic cells. Leukemic cells growing in culture behave like relatively homogeneous cell populations and differentiate extensively (>90%) in cells resembling normal blood cells of different phenotypes (orthochromatophilic normoblasts, polymorphonuclear granulocytes, and eosinophils). Despite the limitations the transformed hematopoietic (leukemic) cells have in studying hematopoietic differentiation, these systems were overall fruitful. Depending on the potential each leukemic cell line has to differentiate into 1 or >1 phenotypes (unipotent, bipotent, or multipotent), these models permitted investigation of fundamental questions as follows: How do leukemic cells commit to differentiate into 1 or another phenotype? What major events occur during leukemic cell differentiation? Are leukemic cells suitable to study apoptosis of transformed hematopoietic cells because programmed cell death appears to be part of homeostatic regulation of blood cell development (Domen, 2001)? The most widely studied systems include the MEL, the HL-60, and the K562 cells. 3.1.1. Murine erythroleukemia (or Friend leukemia) cells These cells are murine virus-transformed spleen hematopoietic cells, which grow in suspension culture and differentiate into terminally erythroid differentiated cells resembling orthochromatophilic normoblasts (see Harrison, 1976; Marks & Rifkind, 1978; Housman et al., 1980; Tsiftsoglou & Robinson, 1985; Tsiftsoglou & Wong, 1985; Rifkind et al., 1996 for review). Evidence also exists to indicate that MEL cells undergo megakaryocytic differentiation (Paoletti et al., 1995; Hyman et al., 2001). Differentiating MEL cells produce much hemoglobin and undergo selective activation/repression pattern of gene expression. MEL cells committed to terminal erythroid differentiation exhibit programmed limitation of their proliferation potential to 4– 5 divisions and growth arrest in G1 phase (see Tsiftsoglou & Wong, 1985; Rifkind et al., 1996; Tsiftsoglou et al., 2003 for review). MEL cells, unlike the normal hematopoietic progenitors (CFU-E), fail to respond to erythropoietin (EPO) and give birth to blood cells.

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3.1.2. Human promyelocytic leukemia cells These cells were derived from the peripheral blood of a patient with acute promyelocytic leukemia (APL) by Collins et al. (1978) and grown in culture as an established cell line. HL-60 cells are induced to differentiate into mature functional neutrophils by dimethylsulfoxide (DMSO), retinoic acid (RA), anthracyclines, 6-thioguanine (6-TG), 5-azacytidine, and tunicamycin (Breitman et al., 1980; Nakayasu et al., 1980; Papac et al., 1980; Schwartz & Sartorelli, 1981; Tarella et al., 1982; Christman et al., 1983) or into macrophage-monocyte-like cells by phorbor ester (12-O-tetradecanoylphorbol 13-acetate [TPA]), 1a,25-dihydroxyvitamin D3, and cytosine arabinoside (Ara-C) (Griffin et al., 1982), as described elsewhere. Growing under slightly alkaline culture conditions, HL-60 cells can develop granules characteristic of eosinophils (see Tsiftsoglou & Robinson, 1985 for review). HL-60 cells committed to granulocytic maturation undergo an irreversible differentiation and loss of proliferation like MEL cells (Tsiftsoglou et al., 1985). Moreover, differentiated HL-60 cells exhibit a variety of cellular, biochemical alterations at the level of cell surface, metabolism, and nucleus as reported elsewhere (Tsiftsoglou & Robinson, 1985). Terminally differentiated HL-60 cells fail to form colonies in soft agar. 3.1.3. Human leukemic K562 cells The K562 cells were derived from a patient suffering from CML (Lozzio & Lozzio, 1975). They bear the Philadelphia chromosome t(9;22 chromosomal translocation) and grow well in suspension culture. On treatment with hemin and other inducing agents (like anthracyclines, sodium butyrate, hydroxyurea, and many other agents), K562 cells are differentiated into hemoglobin-producing cells (Rutherford et al., 1979; Hoffman et al., 1980). Differentiated K562 cells synthesize embryonic and fetal but not adult hemoglobin although they bear intact h-globin gene (Fordis et al., 1984). To some respect, K562 cells behave like thalassemic cells and serve as a suitable model for hemoglobin ‘‘switching.’’ Moreover, evidence exists to indicate that K562 cells exposed to phorbor esters (phorbol 12-myristate-13-acetate [PMA]) are differentiated along the megakaryocytic and macrophage-monocytic pathways via activation of mitogen-activated protein kinase (MAPK) pathways (Whalen et al., 1997). Moreover, K562 have been used as a model system for hemin-induced erythropoiesis (Charney & Maniatis, 1983; Dean et al., 1983; Tsiftsoglou et al., 1989). K562 cells show a considerable degree of plasticity in expressing properties related to distinct lineages of differentiation. Although each of these systems presented is suitable for studying various aspects of leukemic cell differentiation and apoptosis in culture, emphasis in this review will be given on MEL cells and to a lesser extent on HL-60 and K562 cells. The possible mechanisms by which hemin selectively induces erythroid differentiation in K562 cells without causing commitment to terminal maturation will be discussed.

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3.2. The developmental programs and subprograms in differentiating leukemic cells Induction experiments with MEL cells growing in liquid cultures were able to determine the likelihood of inducertreated individual cells to commit to erythroid maturation. Exposing cells to an inducer in culture for certain time, removing them out, subcloning in inducer free plasma clots, and examining the phenotype of the colonies outgrown permitted the analysis of the developmental program of MEL cells (Gusella et al., 1976). Plasma clot cloning assays established that MEL exposed to the inducer for >12– 18 hr undergo (latent period) an irreversible decision to commit to erythroid maturation. Committed MEL cells continue to terminally differentiate in the absence of the inducer and form small colonies containing hemoglobin-producing cells in contrast to uncommitted cells that form progressively expanding large colonies without hemoglobin (Tsiftsoglou & Robinson, 1985). Commitment as central event in differentiation leads to limitation of cellular proliferation to a few divisions and to expression of erythroid differentiation markers. Committed cells in addition to producing highly

detectable quantities of globin mRNA and hemoglobin undergo a series of morphological and biochemical events that resemble those seen in differentiation of normal early erythroid progenitors (see Tsiftsoglou & Robinson, 1985; Tsiftsoglou & Wong, 1985 for review). These include reduction in cell size, progressive changes in cell surface architecture (Tsiftsoglou & Sartorelli, 1981b; Arcangeli et al., 1993), condensation of nucleus, transcriptional activation of several genes, and selective repression of many others. Under certain experimental culture conditions, terminally differentiated MEL cells extrude their nucleus and cause enucleated reticulocyte cells (Fig. 2) (Tsiftsoglou et al., 1979; Volloch & Housman, 1982). Induction of differentiation is triggered at the level of the cell plasma membrane via a Ca2 + -mediated process (Levenson et al., 1980; Tsiftsoglou et al., 1981a) and ends up at the transcriptional level in the nucleus presumably via signaling. The observation that committed MEL cells undergo irreversible loss of proliferation while they synthesize erythroid markers has prompted several studies aimed to demonstrate whether commitment can occur in cells without the expression of erythroid markers and vice versa. Use of selective inhibitors

Fig. 2. Schematic presentation of the major events of the developmental program of MEL cells. The inducer (I) interacts selectively with a cytoplasmic receptor (R) and triggers initiation of cell differentiation that occurs with a probability (P). A latent period that varies from clone to clone of MEL cells is needed before cells commit irreversibly to terminal erythroid maturation and limit their potential to proliferation to only 4 – 5 cellular divisions. Each committed MEL cell can yield 32 cells at maximum. Evidence accumulated thus far indicates that agents like cycloheximide, cordycepin, dexamethasone, and N 6mAdo act on the early events of differentiation during latent period and impair the expression of ‘‘memory response’’ (see text for detail). Once the commitment passed, it is accompanied by coordinated expression of erythroid markers (subprogram II), nuclear condensation, limitation of proliferative capacity (subprogram I), and discrete patterns of gene expression. Dissociation of the 2 subprograms of MEL cell differentiation can be achieved in DMSO treated cells with the addition of either imidazole or succinylacetone, whereas hemin can induce only the subprogram II related to coordinated expression of erythroid markers. Finally, MEL terminal maturation is likely to be accompanied by apoptosis (spontaneous and/or DDA?). The numbers indicate the number of cells produced from an individual cell at different stage of erythrocytic differentiation. Recent evidence indicates that MEL cells can also be induced to differentiate into megakaryocytes (see text for detail).

A.S. Tsiftsoglou et al. / Pharmacology & Therapeutics 100 (2003) 257–290 Table 1 Alterations in expression of several genes before and after commitment of MEL cells to terminal maturation1 mRNA

Effect of differentiation on mRNA steady-state level Before commitment

I. Genes exclusively related to erythroid phenotype a1-globin – z2 Sheffery et al., 1984 major -globin – z Curtis et al., 1980 h hminor-globin – z Curtis et al., 1980 e-ALAS – z Fujita et al., 1991 EKLF – – Spadaccini et al., 1998 EpoR # z Green et al., 1992 GATA-1 # z (biphasic) z Green et al., 1992 NF-E2 NR z Nagai et al., 1998 SCL (TAL1) # z (biphasic) z Green et al., 1992 Tf-R – z Klinken et al., 1987 II. Protooncogenes and genes related to cell cycle CDK2, CDK4 – # Hsieh et al., 2000 c-fos z # Ramsay et al., 1986 cK-ras – – Coppola et al., 1989 c-myb #z # Ramsay et al., 1986; (biphasic) Spotts & Hann, 1990 c-myc #z # Lachman & Skoultchi, (biphasic) 1984 Cyclin D3 – # Hsieh et al., 2000 Id # # Shoji et al., 1994 junB z # Francastel et al., 1994 myn (murine max) # z (biphasic) # Dunn et al., 1994 p21 z z Hsieh et al., 2000 p53 # z (biphasic) # Tsiftsoglou et al., 1987 pRB NR z Coppola et al., 1990; Richon et al., 1992 III. Genes encoding ribosomal RNAs, ribosomal proteins, and other housekeeping proteins a-actin – ! # Richon et al., 1992 h-actin – ! # Lachman & Skoultchi, 1984 h2-microglobulin – – Khochbin et al., 1988 GAPDH – ! # Francastel et al., 1994 H2b histones NR # Brown et al., 1985 HSP 70 # # Hensold & Housman, 1988 H10 histone z # Cheng & Skoultchi, 1989 H2a histones # # Brown et al., 1985, 1988 H3.1 histone NR # Brown et al., 1985 H3.2 histone # # Brown et al., 1985, 1988 H3.3 histone NR – Brown et al., 1985 MER5 z # Yamamoto et al., 1989 RpL35a – # Pappas et al., 2001 RpS5 – # Vizirianakis et al., 1999 rRNAs # # Tsiftsoglou et al., 1982 PrP – !z Gougoumas et al., 2001 IV. Genes encoding mitochondrial proteins COX II # z (biphasic) # COX IV z # B22 – #

Table 1 (continued) mRNA

Representative reference

After commitment

Vizirianakis et al., 2002 Vizirianakis et al., 2002 Vizirianakis et al., 2002

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Effect of differentiation on mRNA steady-state level Before commitment

After commitment

V. Genes related to apoptosis Bax NR

z

Bcl-XL

z

NR

Representative reference

VI. Genes encoding other proteins eEF-Tu – # ODC # # PU.1 #3 # Vimentin # #

Tsiftsoglou et al., unpublished results Hafid-Medheb et al., 1999

Roth et al., 1987 Klinken et al., 1987 Schuetze et al., 1992 Ngai et al., 1984

Abbreviations: eEF-Tu: eukaryotic elongation factor Tu; ODC: ornithine decarboxylase. 1 The data included are referring only to changes observed in the mRNA steady-state levels of the genes examined. 2 # = decrease,z = increase, – = not changed, !z = increase only at late stages of differentiation, ! # = decrease only at late stages of differentiation, NR = not referred. 3 Actually, a very modest increase in PU.1 mRNA level between 12 and 24 hr was observed before the final decline afterward (Rao et al., 1997).

of hemoglobin biosynthesis has shown that commitment occurs during cell differentiation separately and independently from hemoglobin biosynthesis (Gusella et al., 1982; Tsiftsoglou et al., 1983a,b). These findings suggested that the erythroid developmental program of MEL cells consists of at least these 2 major subprograms: one being responsible for commitment and the other for the expression of erythroid markers including hemoglobin and other hemoproteins as illustrated in Fig. 2. 3.2.1. Commitment as a central event in leukemic cell differentiation leads to irreversible growth arrest and discrete patterns of gene expression Northern blot hybridization analysis of cytoplasmic RNA derived from committed MEL cells at different times during the erythroid differentiation revealed that the steady-state level of several RNA transcripts begins to decline before and after commitment while that of others is transiently activated and declines thereafter. This kinetic analysis of gene expression in inducer-treated MEL cells indicated that the expression of a large number of genes changes within the latent period before commitment as well as after commitment has been initiated (see Table 1). These changes occur in housekeeping genes, genes encoding oncogene products and transcriptional factors, enzymes involved in cell cycling, mitochondrial hemoproteins, and several other nucleoproteins including members of the histone class. These findings suggest that some gene expression events proceed initiation of commitment and may be responsible for it while others may result from commitment. Moreover, this analysis revealed that changes occur at various cellular levels (plasma membrane, nucleosomes, nucleolus, mitochondria, and cell cycle apparatus).

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At least 5 discrete patterns of gene expression have been recognized to operate during the developmental program of MEL cells along the erythroid maturation (see Fig. 3). These include the following: i. Genes being exclusively responsible for the expression of the erythroid phenotype (a- and h-globin, Tf-R, erythroid form of 5-aminolevulinate synthase [e-ALAS,] EPO receptor [EpoR], erythroid Kru¨ppel-like factor [EKLF], stem cell leukemia [SCL], GATA-1, and erythroid-specific nuclear factor 2 [NF-E2]) are progressively activated along the maturation of committed cells. ii. Genes encoding transcriptional factors (ets-related transcription factor [PU.1,] Id, c-myc, c-fos, c-myb, junB, p53, and myn), cell cycle regulators (cyclin-dependent kinases [CDK] CDK2, CDK4, and cyclin D3), and mitochondrial proteins (mitochondrial cytochrome c oxidase [COX] II and IV, B22 subunit of nicotinamide adenine dinucleotide-ubiquinone oxidoreductase complex of mitochondria [B22], and Bcl-Xc) are inactivated in the terminal differentiation state.

iii. Genes encoding ribosomal RNA, ribosomal proteins (RpS5 and RpL35a), and housekeeping proteins (gene preferentially expressed in MEL cells [MER5], histones, actin, heat shock protein [HSP] 70, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and vimentin) remain consistently expressed for most of the course of differentiation and decline in growtharrested mature differentiated cells. In contrast, iv. Genes like the prion protein gene [PrP], Bax, Bcl-XL, retinoblastoma protein (pRB), and p21 are activated transcriptionally in terminal differentiated cells presumably as a result of differentiation-dependent apoptosis (DDA) occurring in a proportion of differentiated cells. Finally, v. A few genes that encode the transcriptional factor EKLF, cK-ras, H3.3 histone, and h2-microglobulin remain steady in differentiated MEL cells. While a gradual accumulation and/or decline of RNA transcripts has been observed in most cases, a biphasic pattern of expression of genes (SCL, GATA-1, c-myc, c-

Fig. 3. Presentation of discrete gene expression patterns observed in the developmental program of MEL cells. The gene expression data presented in Table 1 are organized into the 5 different expression patterns seen thus far during differentiation of MEL cells in culture, whereas the genes belonging to each category are shown next to each diagram. Note that only published data related to the steady-state expression level of the corresponding mRNA for the referred genes are presented and the related references are shown in Table 1.

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myb, myn, COX II, and p53) has been recorded (Table 1). The reason for this biphasic pattern of expression is still unknown, although one could postulate that some genes that are transiently repressed may facilitate others function during the leukemic cell maturation. Based on these results, we envision the developmental program of MEL cells as a coordinated set of events where some gene products consecutively activate or repress others, selectively and so on. How many biochemical events are needed for MEL cells to commit to erythroid maturation is still unknown. The order by which these events occur prior to commitment is also unknown. Earlier studies with inhibitors of commitment that act at different levels (e.g., Ca2 + transport, protein synthesis, replication and transcription of DNA, methylation, and RNA polyadenylation) have made clear that the events, which contribute to initiation of commitment, are likely to occur in parallel rather than in sequential fashion (Housman et al., 1980) because inhibition of 1 event does not necessarily abrogate others to occur, as expected if the events had functioned in sequential fashion. The ability of commitment as a central event to control specific gene expression patterns has been also observed in differentiating HL-60 cells (Tsiftsoglou et al., 1985). This suggests that regardless of the nature of leukemic cells studied (proerythroid vs. promyelocytic) commitment is the central process in both erythroid and myeloid cell differentiation. An essential question related to these developmental systems is how commitment initiated at the level of individual cells in response to an inducing agent and what processes are required for commitment to occur. Direct answer to this question is still missing although evidence indicates that commitment is likely to be initiated by inducers acting at the level of cell membrane via signaling pathways or intracellularly via binding to putative receptors (see later sections 5.1 and 5.2). Initiation of commitment in MEL cells occurs in absence of cytokinesis (Tsiftsoglou & Sartorelli, 1979) but depends on new RNA and protein synthesis as shown by a series of specific inhibitor studies (Levenson & Housman, 1979a; Levenson et al., 1979). Studies with metabolic inhibitors have shown that the newly synthesized mRNA and protein mediate initiation of commitment, although such molecules have not yet been identified. Earlier work showing that commitment may be initiated by intracellular differentiation proteins (DIF-I and DIF-II) (Watanabe et al., 1988) is very interesting but needs further verification. Initiation of commitment acquires not only new RNA synthesis but also methylation of polyA + RNA in MEL cells as shown in experiments with N6-methyladenosine (N6mAdo) and 3deazaadenosine, 2 adenosine analogues that block RNA methylation and inhibit commitment (Sherman et al., 1985; Vizirianakis et al., 1992; Vizirianakis & Tsiftsoglou, 1995a). The question of whether MEL cell differentiation depends exclusively on DNA replication is still quite controversial (see Tsiftsoglou & Wong, 1985 for review). Transfection experiments have shown that overexpression of c-myc (Lachman et al., 1986; Prochownik &

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Kukowska, 1986) and PU.1 genes in MEL cells (Rao et al., 1997; Yamada et al., 1997) renders them unable to commit to terminal erythroid maturation, suggesting that these 2 genes encode proteins regulating the initiation of commitment. Studies with antisense oligos (Prochownik et al., 1988) have also shown that initiation of commitment depends on transcriptional inactivation of c-myc protooncogene, a gene that plays a vital role in cell proliferation. Construction and analysis of the DNA libraries derived from undifferentiated and differentiated cells (by subtraction hybridization) have failed to identify unique novel genes and proteins regulating initiation of commitment in MEL cells despite this enormous effort (see Leszczyniecka et al., 2001 for review). 3.2.2. Expression of memory proceeds commitment Initiation of commitment is consistent with the ability of inducer-primed MEL cells to ‘‘remember’’ the ‘‘original trigger’’ and enter the committed state of differentiation. MEL cells exposed to an inducer of differentiation for several hours (6– 12 hr) then washed out and grown in inducer-free medium acquire a unique ability of cells to remember the previous exposure and respond rapidly on rechallenge with the same or other inducer without recapitulating the entire latent period (12 hr prior commitment) (Levenson & Housman, 1979b). In other words, the inducer-treated cells acquire a new property to ‘‘remember’’ the first stimulus and to continue to differentiate after a discontinuous exposure to inducer. Expression of ‘‘memory to the previous exposure’’ suggested that the inducing agent triggers ‘‘cellular memory’’ before an individual cell commits irreversibly to terminal maturation. The fact that ‘‘memory’’ can last >1 or 2 cell generations and subclones of inducible MEL cells exhibit diversified ability to remember (having short, medium, or long term memory) (Tsiftsoglou, unpublished data) indicates that the specific ability of inducertreated cells to ‘‘remember’’ the previous exposure to inducer is transmitted into the daughter cells unaffected by cellular replication and DNA synthesis (Tsiftsoglou & Wong, 1985). Therefore, ‘‘memory’’ can be 1 of the initiative events that enable cells to recognize signals promoted by the inducer on its interaction with cellular components. The molecular basis of ‘‘memory’’ is not known. However, experiments with metabolic inhibitors indicated that ‘‘memory’’ is erased by several structurally unrelated agents including inhibitors of new protein and RNA synthesis (Housman et al., 1980), inhibitors of posttranscriptional methylation of RNA (Vizirianakis & Tsiftsoglou, 1995b), and even glucocorticosteroids (Tsiftsoglou et al., 1983a, 1983b). Based on these studies, ‘‘memory’’ must be carried by macromolecules that accumulate into the cells and reach a threshold level just prior to commitment. Both ‘‘memory’’ and commitment are interrelated properties of differentiated MEL cells because both can be initiated by 1 inducer and can be maintained by another. ‘‘Memory’’ and commitment have also been observed in RD/TE-671 cells, a

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human neoplastic cell line of neuroectodermal origin that respond to inducing agents like MEL cells and differentiate into neurons and astrocyte-like cells (Pappas et al., 1996). 3.3. Coordination of cell proliferation and differentiation in murine erythroleukemia cells Because leukemia, like many other malignancies, is characterized by loss of control of cell proliferation, inability of cells to differentiate and apoptose prompted intensive investigation of the relationship between cellular replication and differentiation in vitro. Reprogramming of MEL cells by treatment with several clinical/pharmacological agents from the malignant state into terminal erythroid differentiation state via a few cellular divisions, and an irreversible cell cycle arrest stirred a lot of interest for studies on cell cycle. The use of metabolic inhibitors and cytochalasin B 2 decades ago has shown that cellular proliferation is coupled with the initiation of erythroid differentiation, although MEL cell differentiation can proceed independently of cytokinesis (Tsiftsoglou & Sartorelli, 1979; Tsiftsoglou & Sartorelli, 1981a). Initiation of differentiation is likely to occur within the G1/S interphase of the cell cycle (Friedman & Schildkraut, 1977; Geller et al., 1978; see Marks & Rifkind, 1978 for review) and that terminally differentiated cells are arrested in the G1/G0 phase of the cell cycle. These early observations suggested that cell proliferation and differentiation are highly coordinated processes during erythropoiesis without explaining how this occurs at the level of cell cycle or transcription of DNA. The discoveries in the 1980s, that the cell cycle in eukaryotic cells is primarily regulated by a family of CDKs (serine/threonine protein kinases) (CDK2, CDK4, and CDK6), CDK inhibitors (CDKI), and the key substrate for CDKs, the pRB, sparked new research activities designed to address the following questions: Are the cell cycle regulators responsible for both cell cycle progression needed for initiation of commitment to erythroid maturation and cell cycle arrest seen in terminal stages? Do the cell cycle regulators share dual function as regulators of pRB and differentiation-promoting transcription factors? Exploiting the fact that CDKs consist of a catalytic (CDK) and a regulatory subunit (cyclin A, E, or D) and their enzymatic activities are regulated by binding, phosphorylation, and dephosphorylation as well as by binding of CDKIs, attempts were made to explore the role of these cell cycle regulators in both cell cycle control and differentiation. In a series of elegant experiments published elsewhere, Skoultchi and his colleagues (Hsieh et al., 2000; Matushansky et al., 2000a, 2000b; Zhu & Skoultchi, 2001) explored the role of cell cycle regulators in growth and differentiation by applying different but complementary approaches: (a) Assessment of the steady-state level enzymatic activities of CDK2, CDK4, and CDK6 in hexamethylene bisacetamide (HMBA)-treated MEL cells by

immunoprecipitation and Western immunoblotting analysis. (b) Transfection and overexpression of the CDK2, CDK4, and CDK6 kinases as well as their CDKIs in MEL cells via a tetracycline-inducible promoter and assessment of the ability of terminally differentiated cells to exit G1 phase or to prolong their cellular replication even in terminal differentiated state. These studies have revealed the following: (i) MEL cell differentiation is associated with gradual decline of pRB protein and accumulation of p27KIP1 (a CDKI of the KIP family), (ii) Onset of the cell cycle exit in differentiated MEL cells is regulated by CDKs (CDK2 and CDK4) as well as CDKIs, (iii) CDK4 and CDK6 participate in cell division control at different stages of erythroid maturation and CDK6 may play a key role in the blockade of differentiation because CDK6 overexpression prevents differentiation to occur, and (iv) Accumulation of CDKI p15INK4h, p18INK4C, p21CIP1, and p27KIP1 in the terminal stages of differentiation indicates that phenotypic differentiation and terminal cell division are 2 independent processes of cell differentiation, representing 2 different subprograms as suggested by earlier studies. Now, it is unclear how the CDKs and CDKIs do control cell proliferation during differentiation and reprogramming of MEL cell fate. CDKs phosphorylate several substrates to promote cell cycling. It may be true that CDKs regulate the function of transcription factors that impinge on the genes that initiate differentiation along the erythrocytic pathway (e.g., PU.1, GATA-1, and GATA-2). Within this frame, it is still unknown how do the chemical inducers promote signal transduction processes to activate commitment via transcriptional events. The observation that Id2 factor, a dominant negative antagonist of basic helix-loop-helix proteins and pRB protein, mediates signaling by myc (Lasorella et al., 2000) brings new insights into the mechanics that regulate MEL cell growth and differentiation.

4. Inducers of leukemic cell differentiation Since the discovery of DMSO as an inducer of differentiation of MEL leukemic cells (Friend et al., 1971), a plethora of agents (chemical, natural, pharmacological, and biological) was invented. These include bisacetamides, pyridine derivatives, cyclic ureas and thioureas, benzodiazepines, bis-hydroxamic acids, DMSO, purine and pyridine analogues, substituted amides, and other agents. These substances differ in structure, physicochemical properties, and specificity on the type of leukemic cells that act on at optimum inducing concentration to promote differentiation. Quantitative structure-activity relationship (QSAR) studies have shown that some classes of chemical inducers may share common structural features that enable them to act by common mechanism(s) via receptor-mediated processes or throughout signal transduction pathways. We have grouped (see Tables 2 – 5) the inducers according to specificity in action (type of differentiated progeny in-

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Table 2 Inducers of MEL cell differentiation Inducing agents

Optimum concentration

Benzidine-positive cells1 (%)

Terminal differentiated phenotype

References

Actinomycin D Activin A 5-Azacytidine Benzodiazepines Bis-hydroxamic acids SAHA Cyclic ureas and thioureas DMSO HMBA

1.5 ng/ml 1 nM 0.25 AM 15 – 80 AM 4 – 5 AM 2 AM 1 – 4 mM 210 mM 5 mM

95 80 43 30 – 70 60 – 65 85 61 – 92 95 95

Hypoxanthine Purine analogues Pyridine derivatives Trichostatins UDP Sodium butyrate Substituted amides, lactams, and 2-pyridones Xylosyladenosine

3.7 mM 0.1 – 3.7 mM 2 – 16 mM 0.2 Ag/ml 0.075 – 1 mM 1.5 mM 2 – 4 mM

83 70 – 83 43 – 74 75 90 – 95 44 90 – 96

Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic/ megakaryocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic

Terada et al., 1978a, 1978b Yamashita et al., 1990 Creusot et al., 1982 Wang et al., 1984 Breslow et al., 1991 Richon et al., 1996 Li et al., 1981a Friend et al., 1971 Reuben et al., 1976; Green et al., 1993 Gusella & Housman, 1976 Gusella & Housman, 1976 Li et al., 1978 Yoshida et al., 1987 Pappas et al., 1992 Malinin & Ebert, 1980 Li et al., 1981b

0.1 AM

83

Erythrocytic

Sherman et al., 1988

1

Cells producing hemoglobin detected by benzidine-H2O2 staining (Orkin et al., 1975).

duced), potency at optimum inducing concentration, structure, and possible mechanism(s) of action, although direct comparison cannot be done in many cases because different cell lines of leukemic cells or subclones with diversified inducibility have been employed for evaluation of their potency.

Inducing agents

Terminal differentiated phenotype

References

Antifolates Anthracyclins Aphidicolin 5-Azacytidine Bis-hydroxamic acids Bryostatin 1 Butyric acid Camptothecin Ara-C 3-Deazauridine dbcAMP 1a,25-Dihydroxyvitamin D3

Granulocytic Granulocytic Megakaryocytic Granulocytic Granulocytic Monocytic Monocytic Granulocytic Monocytic Granulocytic Granulocytic Monocytic/ macrophage Granulocytic Granulocytic Granulocytic Granulocytic Monocytic/ macrophage

Sokoloski et al., 1989a Schwartz et al., 1983a Griffin et al., 1982 Christman et al., 1983 Breslow et al., 1991 Stone et al., 1988 Zucker et al., 1983 Pantazis, 1995 Griffin et al., 1982 Bodner et al., 1981 McCachren et al., 1986 Miyaura et al., 1981

Retinoids Tiazofurin 6-TG Tunicamycin

Granulocytic Granulocytic Granulocytic Monocytic

All-trans-RA (ATRA) is a well-known inducer of differentiation of HL-60 and plays an important role in the regulation of growth and differentiation in many tissues Table 4 Inducers of human erythroleukemia K562 cell differentiation

Table 3 Inducers of HL-60 cell differentiation

DMSO HMBA Hypoxanthine PA Phorbol esters

4.1. Retinoids

Collins et al., 1978 Haces et al., 1987 Collins et al., 1980 Samid et al., 1992a Huberman & Callaham, 1979; Rovera et al., 1979a, 1979b Breitman et al., 1980 Lucas et al., 1983 Schwartz et al., 1983b Nakayasu et al., 1980

Inducing agents

Terminal differentiated phenotype

References

Anthracyclins

Erythrocytic

Apicidin 5-Azacytidine Butyric acid Chromomycin cis-platin Ara-C 5-FU Hemin Herbimycin HMBA

Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic Erythrocytic/ megakaryocytic Erythrocytic Erythrocytic Erythrocytic Megakaryocytic

Steinheider et al., 1986; Tsiftsoglou et al., 1991 Witt et al., 2003 Gambari et al., 1984 Cioe` et al., 1981 Bianchi et al., 1999 Bianchi et al., 2000 Bianchi Scarra` et al., 1986 Yang & Chang, 1995 Rutherford et al., 1979 Honma et al., 1989 Green et al., 1993

Hydroxyurea Mithramycin PMEA Phorbol esters (PMA, TPA) PA Resveratrol Sodium butyrate Tiazofurin

Erythrocytic Erythrocytic Erythrocytic/ megakaryocytic Erythrocytic

Tallimustine Vitamin B12

Erythrocytic Erythrocytic

Erard et al., 1981 Bianchi et al., 1999 Hatse et al., 1996 Whalen et al., 1997 Samid et al., 1992b Rodrigue et al., 2001 Lozzio et al., 1979; Sutherland et al., 1986 Olah et al., 1988; Hatse et al., 1996 Bianchi et al., 2001 Rowley et al., 1981

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Table 5 Cytokines as inducers of leukemic cell differentiation Cytokine

Cell type

Phenotype

Reference

J2E UT-7/GM UT-7/GM M1 B1 KU812 M1 U937 IL-1, G-CSF M1

Erythrocytic Erythrocytic Megakaryocytic Macrophage Myeloid Basophilic Macrophage Macrophage Macrophage

LIF

M1

Macrophage

TGF-h1 TNF-a TNF-a

U937 Macrophage KU812 Basophilic WEHI-3B-JCS Macrophage

Busfield & Klinken, 1992 Komatsu et al., 1997 Komatsu et al., 1997 Metcalf, 1989 Cohen et al., 1992 Nilsson et al., 1994 Miyaura et al., 1988 Maekawa et al., 1990 Yamamoto-Yamaguchi et al., 1989 Hoffman-Liebermann & Liebermann, 1991 Kamijo et al., 1990 Nilsson et al., 1994 Leung et al., 1994

EPO EPO TPO G-CSF IL-6

including bone marrow. It induces granulocytic differentiation of HL-60 (Breitman et al., 1980) via the RA receptors (RAR) (Table 3) (Collins et al., 1990). Combination of ATRA with other inducing agents such as the inhibitors of protein kinase C (PKC), 1a,25(OH)2D3, butyrate, and phenylbutyrate (PB) have shown to enhance the ability of leukemic cells to differentiate (Yu et al., 1999; Zheng et al., 2000). Retinoid-induced differentiation of HL-60 is associated with commitment to granulocytic maturation, cell surface changes, and down-regulation of oncogene expression (Tsiftsoglou & Robinson, 1985). Currently, ATRA is used as an effective antineoplastic agent in humans suffering from APL (Degos & Wang, 2001). Retinoids act by interacting with 2 classes of nuclear receptors, the RARs and the retinoid X receptors (RXR), each 1 of which consists of 3 isotypes (a, h, and g) encoded by separate genes (Chambon, 1996). ATRA is only capable of activating RARs, although its stereoisomer 9-cis-RA binds to RXRs as well as to RARs (Levin et al., 1992). On dimerization with RXRs, RARs interact with specific DNA sequences, the so-called RA response elements (RARE), resulting in transcriptional activation of target genes in the presence of RA. These response elements are composed of 2 hexameric half sites organized as direct repeats and separated by a spacing of 2 or 5 nucleotides for RARE (DR2 and DR5) or 1 for retinoid X response elements (RXRE) (DR1) (Mangelsdorf & Evans, 1995). Nonrandom chromosomal translocations detected have shown that RARa is fused to the genes encoding promyelocytic leukemia (PML), zinc finger protein (PLZF), nucleophosmin (NPM), nuclear mitotic apparatus protein (NuMA), and signal tranducer and activator of transcription 5b (STAT5b). In the absence of ligand, the RXR/RAR heterodimer binds to corepressors like nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT). These proteins recruit Sin3 and histone deacetylase (HDAC) resulting in histone deacetylation, an event that leads to chromatin remodeling and does not allow transcriptional

activators to bind to DNA to initiate transcription (Melnick & Licht, 1999; Breems-de Ridder et al., 2000). Ligand binding to HDAC has been shown to induce conformational changes in the corepressor binding domain of the receptor (Renaud et al., 1995), causing a dissociation of the corepressor complex and allowing binding of coactivation proteins with histone acetyltransferase (HAT) activity. Acetylated histones relax the tight binding of DNA allowing gene transcription. Fusion protein PML-RARa binds to retinoids with the same affinity and specificity but shows an enhanced binding with the corepressor proteins (Nervi et al., 1992; Kastner et al., 1995; Benedetti et al., 1997). At physiological concentrations (1– 10 nM), ATRA binds to RARa receptor in normal hematopoietic cells. This event leads to its dissociation from a HDAC corepressor complex and results in transcriptional activation of a variety of genes involved in hematopoietic cell maturation (Grignani et al., 1998). This process is disrupted by PML protein but can be activated once again at higher concentrations of ATRA (0.1 – 1 AM), where the corepressors are replaced by coactivators and HATs, allowing transcriptional activation to occur (Grignani et al., 1998). 4.2. Vitamin D3 and its analogues The naturally active form of vitamin D3, 1a,25(OH)2D3, induces differentiation of normal and leukemic myeloid cells like HL-60 along the monocyte/macrophage lineages (Abe et al., 1981; McCarthy et al., 1983) by binding to its receptor (vitamin D response [VDR]) that is present in myeloid cells (Table 3) (Lee et al., 1989; Kizaki et al., 1991). The VDR interacts with specific DNA sequences, the so-called VDR elements (VDRE), to transcriptionally active target genes in the presence of 1a,25(OH)2D3 (Baker et al., 1988). Each VDRE is composed of 2 hexameric half sites arranged as a direct repeat and separated by a nucleotide spacing of 3 (DR3) (Umesono et al., 1991). VDRs form heterodimers with RAR or RXR to initiate transcription of the responsive genes. HL-60 cells are induced to differentiate along the granulocytic pathway by dibutyryl cAMP (dbcAMP) and inhibitors of cAMP phosphodiesterase (McCachren et al., 1986) that cause an arrest in G0/G1 phase of the cell cycle. Elevation of cAMP stimulates the cAMP-dependent protein kinase A (PKA) allele (Fontana et al., 1984). Combination of dbcAMP and 1a,25(OH)2D3 significantly augmented the differentiation-inducing effect of 1a,25(OH)2D3 along the monocyte/macrophage pathway, suggesting that the levels of cAMP are crucial for differentiation (Inaba et al., 1992). In contrast, elevation of cAMP levels in MEL cells or activation of PKA with N 6 -phenylisopropyladenosine (PIA) caused inhibition of differentiation induced by DMSO and xylosyladenine (Sherman et al., 1988). The major limitation of the vitamin D3 clinical use has been its potent calcemic activity. Thus, intensive research has led to the development of analogues of 1a,25(OH)2D3,

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which are characterized by a clear dissociation of their antiproliferative and differentiating potency from their calcemic effects. The vitamin D3 analogues, 22-oxacalcitriol, Ro 23-7553, and Ro 24-5531 are more potent inducers of differentiation of HL-60 cells (Abe et al., 1987; Zhou et al., 1990, 1991). In in vitro studies, these compounds have shown to prolong survival of mice inoculated with myeloid leukemia cells or improve primary immune response in mice (Abe et al., 1989; Zhou et al., 1990). 1,25-Dihydroxy-24homo-vitamin D3 and 1,15-dihydroxy-22-ene-24-homo-vitamin D3 are more potent as inducers of differentiation of HL-60 cells than vitamin D3 itself (Ostrem et al., 1987). Agents CB1093 and EB1089 are potent inducers of differentiation that inhibit cell proliferation more extensively than 1a,25(OH)2D3 (Pakkala et al., 1997). The 24-oxo-vitamin D3 analogues that inhibit proliferation of HL-60, NB4, and U937 leukemic cell lines are very potent inducers of differentiation with weak calcemic activity (Shiohara et al., 2001). Finally, the 2-methyl analogues of 20-epi-2,22dimethyl-1a,25-dihydroxyvitamin D3 induce HL-60 cell differentiation at concentrations 40-fold lower than that of natural vitamin D3 (Fujishima et al., 2001). 4.3. Agents that remodel chromatin Over the past several years, experiments have established that agents, which hypomethylate DNA in CpG islands, alter the superfine structure of chromatin. Moreover, chromatin structure is affected by histone acetylation as well as by inhibition of topoisomerases, resulting in the regulation of gene transcription. Hypomethylating agents, HDAC inhibitors (HDACI), and inhibitors of topoisomerases are presented below since throughout their effects on chromatin structure and conformation regulate cell growth and differentiation. 4.3.1. Hypomethylating agents Hypomethylating agents represent a class of agents that directly or indirectly inhibit methylation of DNA at the cytosine residues. The hypomethylation of CpG islands remodels chromatin locally and leads to activation of several protooncogenes, including that of Harvey-ras (Hras) and c-myc, which contribute to abnormal cell growth. There are 2 discrete ways to inhibit DNA methylation: one is to block methylation by incorporating nucleotide analogues into DNA and the other is to purturbate DNA methylation by inhibiting the activity of DNA methyltransferases (Attwood et al., 2002). Cytosine analogues modified in the 5V position by substitution of hydrogen with fluorine or by replacing of 5-carbon of the pyrimidine ring with nitrogen markedly reduced the proportion of methylated cytosine residues in DNA. Hypomethylating agents include 5-azacytidine (commercially known as azacytidine), 5-aza2V-deoxycytidine (commercially known as decitabine), and 5-fluoro-2V-deoxycytidine. In addition to these agents, the new DNA methylation inhibitor zebularine [1-(h-D-ribofur-

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anosyl)-1,2-dihydropyrimidin-2-one] has been recently developed as a cytidine analogue with increased chemical stability, a property that allow oral delivery in clinical studies (Cheng et al., 2003). 5-Azacytidine triggers differentiation of HL-60 cells (Table 3) (Christman et al., 1983), K562 cells (Table 4) (Gambari et al., 1984), and MEL cells to a lesser degree (Table 2) (Creusot et al., 1982). Combination of 5-azacytidine with RA (Momparler et al., 1990) and vitamin D3 analogues caused extensive cell growth arrest and induction of differentiation of HL-60 and NB4 cells (Dore et al., 1998). Inhibitors of S-adenosylhomocysteine (SAH) hydrolase such as 3-deazaadenosine, aristeromycin, 3-deaza-( F )-aristeromycin, neplanocin A, and MDL 28.842 have been reported to induce differentiation of various cell lines (Niitsu & Honma, 1999). Such agents in addition to their ability to inhibit DNA methylation at specific sites perturb the entire methylation cycle and abrogate initiation of MEL cell differentiation (Vizirianakis & Tsiftsoglou, 1996). Treatment of MEL cells separately with neplanocin A, 3-deazaneplanocin A, and/or cycloleucine (3 known inhibitors of active methylation cycle) led to blockade of differentiation irrespectively of the inducer used (DMSO, HMBA, butyric acid, hypoxanthine, and ureido derivatives of pyridine (UDP-4)) (Vizirianakis & Tsiftsoglou, 1996). 4.3.2. Histone deacetylase inhibitors Histone acetylation by HATs leads to less tight binding of DNA on histones and promotes initiation of transcription. In contrast, removal of acetyl groups from histones by HDAC allows histones to bind more tightly to DNA and prevents gene transcriptional activation. The changes in gene transcription due to nucleosomal packaging of DNA are 1 of the critical steps in regulation of gene expression that affect cell differentiation, proliferation, and apoptosis (Lin et al., 1998). Human HDACs have been grouped in 2 classes: class 1 consists of Rpd3-like proteins (HDACs 1– 3 and 8) and class 2 is related to the yeast HDA1 protein (HDACs 4– 7 and 9) (Kramer et al., 2001). In acute myelogenous leukemia (AML), where translocations make the RARa gene to fuse with the genes PML, PLZF, NPM, NuMA, or STAT5b, several chimeric proteins are generated. The repression of gene transcription seen in AML is attributed to histone deacetylation by recruitment of nuclear corepressors (N-CoR or SMRT), Sin3A, or Sin3B, which in turn is associated with HDACs (Urnov et al., 2001). However, in the presence of pharmacological doses of RA (0.1 –1 AM), HDACs are released from the complex, thus permitting the transcription of RA-mediated gene expression. In PLZFRARa, where the fusion protein interacts with a ligandindependent site to N-CoR, inhibitors of HDACs act synergistically with RA to reverse the APL phenotype (He et al., 2001). ETO (eight twenty one) is another gene that fuses with acute leukemia-1 (AML-1) gene and especially with AML-1 DNA binding domain and represses transcription of genes that would otherwise be activated by AML-1 (Wang

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et al., 1998). The ETO moiety in the AML-1-ETO fusion protein has been shown to interact with the N-CoR complex, thus resulting in the recruitment of Sin3 and HDAC1 enzyme to AML-1-dependent promoter (Amann et al., 2001). Furthermore, the HDAC1 inhibitor, phenylbutyrate (PB), relieved this ETO-mediated transcriptional gene repression (Gelmetti et al., 1998; Wang et al., 1999). Short-chain fatty acids have shown inhibitory effects on HDAC. The most active member of this group is butyric acid, an inducer of differentiation of MEL, HL-60, and K562 cells (Tables 2 – 4). Fatty acids with higher or lower molecular weight exhibited less to none inducing capacity (Malinin & Ebert, 1980). Sodium phenylacetate (PA) and sodium PB are aromatic fatty acids that promote differentiation in a variety of cell lines including leukemic cells (Samid et al., 1992a, 1992b; Gore et al., 1997; Witt et al., 2000). Induction of differentiation is followed by an increase of cAMP (Tai et al., 1996), activation of PKC cascade (Rickard et al., 1999), and alterations in p21Cip-1/mda-6/sdi-1/Waf-1 gene expression (Archer et al., 1998). Retinoids significantly augment the cytostatic and differentiating activity of PA in ML-1 myeloid leukemia cell line (Yu et al., 1999). Combined treatment with the butyrate derivative tributyrin and RA in vitro led to a 7-fold increase of hemoglobin-F (a2g2) synthesis in K562 cells (Witt et al., 2000). In a clinical study, HDACIs, butyrate, and PB have shown promising results on treatment of RA-refractory PMLs in which HDAC forms complexes with abnormal receptor fusion protein products PML-RAR and PLZF-RAR (Warrell et al., 1998). Furthermore, these compounds are not specific for HDACs because they inhibit phosphorylation and methylation of DNA and proteins (Newmark & Young, 1995). Trichostatin A (TSA), an inducer of MEL cell differentiation (Table 2), has inhibitory activity against HDACs. This molecule possesses a cap group, an aliphatic chain, and a functional group that would chelate metal cation in the active site. The optimal length of the aliphatic chain of TSA to act as inducer ranges from 5 to 6 carbon residues (Jung et al., 1999). The aromatic group serves as a cap necessary for packing the inhibitor at the rim of the tube-like active-site pocket. Exposure of MEL cells to nanomolar concentrations of TSA increases the h-globin gene expression (Yoshida et al., 1987). Moreover, TSA synergizes with RA to stimulate hormone responsive genes and promote differentiation of myeloid leukemia HL-60 cells. Bis-hydroxamic acids are very effective inducers of differentiation of MEL and HL-60 cells at very low concentrations (Tables 2 and 3) (Breslow et al., 1991). These compounds contain an aliphatic chain of 5 – 7 carbon residues and 2 functional groups at both sides. Substitution of aliphatic chain with an aromatic ring regenerated biologically active compounds. Adding an extra methylene group between aromatic and hydroxamic acid groups led to a substantial loss of activity. Substitution of an ethylene group instead of methylene group gave m-carboxycinnamic acid bis-hydroxamide (CBHA), the most active compound of the

bifunctional agents acting at 4-AM concentration. Combining the prototype inducer of MEL cell differentiation HMBA (Table 2) with hydroxamic acid gave birth to a second-generation hybrid polar compounds. The most effective inducer of this series, the suberoylanilide hydroxamic acid (SAHA), promotes the differentiation of MEL cells at 2-AM concentration (Richon et al., 1996). Introducing a very lipophilic group such as biphenyl group in an hydroxamic acid also led to more potent inducer of MEL cell differentiation (Wittich et al., 2002). Induction of MEL cell differentiation by SAHA is accompanied by down-regulation of c-myb (Richon et al., 1996) and phenylalanyl tRNA synthetase regulatory asubunit-like protein (Zhou et al., 1999) as well as induction of CDKI (p21Cip-1/mda-6/sdi-1/Waf-1) gene expression (Richon et al., 1998). In U937 and HL-60 cells, SAHA acts as weak inducer of differentiation but a potent inducer of apoptosis because it down-regulated the expression of c-myc and c-myb genes but activated the expression of p21Cip-1/ mda-6/sdi-1/Waf-1 gene (Vrana et al., 1999). The known HDACIs sodium butyrate, TSA, and SAHA induce a distinct pattern of caspase-dependent apoptosis in primary leukemia cells derived from patients with APL t(15;17) chromosomal translocation. The apoptosis is followed by down-regulation of daxx gene without significant changes in both bcl-2 and bax genes (Amin et al., 2001). MS-27-275 is a newly synthesized benzamide derivative with HDACI activity. Structure-activity relationship has shown that substitution at the 2Vposition with an amino or hydroxy group was indispensable for inhibitory activity. The steric factor in the anilide moiety, especially at 3V and 4V positions, played an important role in the interaction with the enzyme (Saito et al., 1999). Trapoxin (TPX) A and B are cyclic tetrapeptides and irreversible inhibitors of HDAC1. They contain the unusual amino acid, 2-amino-8-oxo-9,10-epoxy-decanoic acid (Aoe), in their core molecule cyclo(Aoe-Phe-Phe-D-Pro). The epoxy-ketone group in the Aoe residue acts as an acetylated lysine mimic and it is a chemically reactive moiety forming a covalent bond between TPX and the enzyme molecule. TPX has poor bioavailability in vivo as well as toxic side effects at high doses (Kramer et al., 2001). 4.3.3. Inhibitors of topoisomerases DNA topoisomerases are very important enzymes that contribute to the organization of DNA and serve as potential molecular targets for many clinically used antineoplastic agents including DNA damaging agents. In particular, topoisomerases alter DNA topology by the generation of single-strand (topoisomerase I [TOPO I]) and double-strand (topoisomerase II [TOPO II]) breaks in DNA. TOPO I exists as a single protein and TOPO II exists as 2 distinct isoforms, a and h. The role of DNA TOPO I and II isoforms has been implicated in leukemia cell differentiation (Larsen, 1994). RA up-regulates TOPO IIh isoform by hyperphosphorylation (Aoyama et al., 1998), while DMSO and phorbol ester

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(TPA) down-regulate TOPO IIa at both mRNA and protein levels in HL-60 cells committed to terminal maturation and growth arrest (Constantinou et al., 1989; Ellis & Zwelling, 1994). Short-chain fatty acids transiently increase TOPO IIa gene promoter activity and protein levels in a way closely related with histone H4 acetylation status (Kurz et al., 2001). Novobiocin, a TOPO II inhibitor, caused differentiation of TPA-sensitive but not TPA-resistant HL-60 cells (Constantinou et al., 1989), suggesting that PKC may be involved in phosphorylation status of TOPO II. PMA downregulates TOPO IIa by a post-transcriptional regulation in K562 cells (Loflin et al., 1996). Etoposide and aclarubicin, 2 cytotoxic drugs by virtue of their ability to stabilize a cellular toxic covalent TOPO II-DNA intermediate (the cleavable complex), induce differentiation of leukemic cells (Larsen, 1994). VM-26, also a TOPO II targeting drug, induces differentiation of erythroleukemia IW32 cells by inhibiting TOPO II activity (Tan et al., 1996). However, an increase in TOPO II mRNA has been observed in VM-26treated IW32 cells and attributed to cell cycle arrest in G2/M phase and prevention of complete chromosomal segregation (Tan et al., 1996). In contrast, TOPO II inhibitor dexrazoxane that is unable to cause DNA strand breaks can induce differentiation and apoptosis of K562 cells (Hasinoff et al., 2001). The last finding suggests that inhibition of TOPO II catalytic activity is sufficient for K562 cells to commit to erythroid differentiation. Induction of differentiation of U937 cells by PMA, HMBA, and RA was followed by a rapid decrease in TOPO I activity without changes in mRNA and protein levels. These data imply involvement of a post-transcriptional mechanism of regulation of this enzyme in these cells (Shayo et al., 1997). Furthermore, camptothecin is a TOPO I inhibitor that triggers differentiation of HL-60, U-937, ML-1, and K562 cells in culture (Pantazis, 1995). 4.4. Purine and pyrimidine analogues It has been established over the years that the proliferative capacity of transformed cells is closely associated with the function of several enzymes involved in pyrimidine and purine nucleotide metabolism during DNA and RNA biosynthesis. These enzymes have attracted a lot of attention as primary targets for the development of purine and pyrimidine antimetabolites as potent differentiation inducers (Hatse et al., 1999a). 4.4.1. Purines Many purine derivatives has been shown to induce differentiation of MEL, K562, and HL-60 cells (Tables 2 – 4) (Gusella & Housman, 1976). This group of compounds bears specific structural features required for potent inducing activity. For example, 6-mercaptopurine is a weak inducer of differentiation. Addition, however, of an amino group in 2V position gave 6-TG that it is a 20-fold more active inducer. In contrast, introduction of a hydroxyl-(6-thioxanthine) or mer-

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capto group (2,6-dithiopurine) into purine led to loss of inducing capacity. Acetylation of 2-amino group of 6-TG or substitution of the 6-mercapto group with a benzyl group lessens its inducing capacity. Replacement of 6-SH group with hydroxyl-one (xanthine) or removal of the substituent in 2Vposition (2-thioxanthine) abrogated the inducing ability. 2,6-Diamino-purine is a very potent inducer in contrast to 2-aminopurine and 6-aminopurine as observed in the MEL cell differentiation system (Gusella & Housman, 1976). Other metabolic products of purines, such as uric acid, allantoin, thioxanthine, and thiouric acid, are biologically inactive as inducing agents. Addition of sugar moiety at 9V position of the purine ring or substitution of h-Dribofuranose with h-D-arabinofuranose did not alter the purine inducing capacity (Gusella & Housman, 1976). The 6-thioanalogues of purines, 6-mercaptopurine and 6TG, are converted into thio-inosine 5V-monophosphate (IMP) and thio-GMP by hypoxanthine-guanine phosphoribosyltransferase (HGPRT), respectively, 2 compounds that inhibit IMP dehydrogenase (IMPDH) (Perigaud et al., 1992). Purine and 8-substituted purine arabinofuranosyl and ribofuranosyl nucleotide derivatives are also potent inducers of MEL cell differentiation (Lin et al., 1985). Finally, 9-(2-phosphonyl-methylethyl)-adenine (PMEA) exhibited strong inducing capacity in K562 cells by acting via inhibition of cellular DNA polymerases a, j, and q. No substantial changes in c-myc mRNA levels and p21, PCNA, cdc2, and CDK2 protein levels were observed in K562 cells exposed to PMEA (Hatse et al., 1999b). 4.4.2. Pyrimidines This class of agents includes 5-fluorouracil (5-FU), AraC (Table 4), and amphidicolin (Table 3), 3 inducers of leukemic cell differentiation. Ara-C is a potent inhibitor of mammalian DNA polymerases a and h that acts through competition with the natural substrate dCTP (Yoshida et al., 1977). On incorporation into nascent DNA chain, Ara-C resulted in accumulation of short strands of DNA (Dijkwel & Wanka, 1978), thus inhibiting cell proliferation and causing expression of differentiation markers (Ross et al., 1990). Ara-C promotes erythroid differentiation of K562 cells by irreversible induction of hemoglobin synthesis, loss of cell renewal capacity, and marked decrease of c-myc expression (Bianchi Scarra` et al., 1986). Furthermore, Ara-C promotes monocytic differentiation of HL-60 cells by inhibiting cell proliferation (Griffin et al., 1982). Aphidicolin, which selectively inhibits DNA polymerase a activity in competition with pyrimidine deoxyribonucleoside triphosphates, induces differentiation of HL-60 (Griffin et al., 1982) and K562 (Murate et al., 1990) cells. 5-FU can be converted to FdUMP by either the subsequent action of dThd/dUrd phosphorylase and dThd kinase or the action of uracil or orotate phosphoribosyl transferase. FdUMP inhibits thymidylate synthase and is incorporated into DNA, causing genetic miscoding, DNA damage (Danenberg et al., 1981; Christopherson & Lyons, 1990), and inhibition of

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rRNA maturation (Heidelberger et al., 1983). Exposure of K562 cells to 5-FU resulted in a marked increase of hemoglobin-producing cells with concomitant reduction in cell proliferation that is not followed by changes in c-myc mRNA or protein levels. 5-FU-induced K562 cell differentiation is associated with a biphasic change of p210bcr/cAbl and the abl-associated kinase activities mediated at the translational level (Yang & Chang, 1995). Combination of 5-FU with chemical inducers such as HMBA, DMSO, and ATRA led to enhancement of differentiation and cell death in both MEL (Waxman et al., 1990) and HL-60 (Waxman et al., 1992) cells. 4.5. Inosine 5V-monophosphate dehydrogenase inhibitors IMPDH catalyzes the rate-limiting reaction of de novo guanosine 5V-triphosphate (GTP) biosynthesis at the IMP metabolic branch point. Inhibitors of IMPDH promote induction of differentiation by reduction of intracellular GTP levels in a variety of cancer cells. Reduced levels of GTP decrease the levels of inositol triphosphate (IP3) Ras-GTP that subsequently down-regulates ras and myc oncogenes and induces IP3 formation (Olah et al., 1988). Tiazofurin (2-h-D-ribofuranosylthiazole-4-carboxamide) converted intracellularly to the active metabolite thiazole4-carboxamide adenine dinucleotide (TAD), a NAD + analogue, inhibits IMPDH through competition for the NAD + cofactor binding site of the enzyme (Lui et al., 1984). Similarly, the agents 5-ethynyl-1-h-D-ribofuranosylimidazole-carboxamide (EICAR) (Balzarini et al., 1998), mycophenolic acid (MPA, a natural compound produced by the fungus Penicillium stoloniferum) (Sintchak et al., 1996), and the antiviral agent ribavirin (Streeter et al., 1973) all inhibit IMPDH. IMPDH inhibitors are known to induce differentiation of K562 (MPA, EICAR, and ribavirin) (Yu et al., 1989; Hatse et al., 1999a), HL-60, and U-937 cells (MPA) (Inai et al., 2000). In addition, tiazofurin induces erythroid differentiation of K562 (Hatse et al., 1996; Olah et al., 1988; Yu et al., 1989) and granulocytic differentiation of HL-60 cells (Lucas et al., 1983). Furthermore, tiazofurin-induced maturation of HL-60 cells is accompanied with modulation of c-myc and c-myb protooncogene expression (Kharbanda et al., 1988) and is mediated during the G1 phase of the cell cycle (Sokoloski et al., 1989b). Because the known inducers of differentiation of HL-60 cells, dimethyl formamide (DMF), hypoxanthine, and RA, decrease guanine nucleotides and IMPDH activity, it was reasonable to propose that alteration in nucleotide levels contribute to the regulation of the terminal maturation of myeloid cells (Lucas et al., 1983). 4.6. Activators and inhibitors of signal transduction pathways Several studies over the years have shown that protein phosphorylation reactions per se are involved in signaling

pathways that modulate cell growth, differentiation, and apoptosis. This include the enzymes PKA and PKC (a family that consists of various isoforms that are responsive to phorbol esters—TPA, phorbol-12,13-dibutyrate [PDBu], and bryostatin 1), the MAPK superfamily of serine/threonine that includes the extracellular signal-regulated kinases (ERK), the c-jun N-terminal kinase/stress-activated protein kinases (JNK/SAPK), and the p38 kinases (Kyriakis & Avruch, 2001). On activation of PKC-h by TPA or bryostatin 1, PKC-h translocates from cytosol to membrane and promotes myeloid cell differentiation (Stone et al., 1988). PKC-h is a critical protein because HL-60 cells deficient in PKC-h expression (MacFarlane & Manzel, 1994) and U937 cells expressing PKC-h in low level (Hass et al., 1993) were found to be resistant to TPA, an agent that down-regulates cmyc during monocytic differentiation and induces the expression of c-jun (Sherman et al., 1990). ERKs are a group of kinases involved in hematopoietic differentiation. For example, differentiation of U937 cells into monocytes/ macrophages induced by TPA was accompanied by activation of ERK1 and ERK2 (Pulverer et al., 1993) like in HL60 cells (Kharbanda et al., 1994) and K562 cells (Whalen et al., 1997) undergoing mononuclear and megakaryocytic differentiation, respectively, induced by this agent. In contrast, inhibition of ERK activation by PD98059 blocked differentiation of ML-1 (He et al., 1999) and U937 (Okuma et al., 2002) cells. Tumor-promoting agents, particularly PMA (also known as TPA), was shown to induce HL-60 cell differentiation at extremely low concentration (6  10  11) as shown early in 1979 (Huberman & Callaham, 1979; Rovera et al., 1979a, 1979b). Detailed analysis of the mechanism of action of PMA since then by Huberman and colleagues (Semizarov et al., 1998; Laouar et al., 2001; Zheng et al., 2002) have shown that PMA-induced macrophage differentiation is initiated by signal transduction processes in which PKC-h plays an essential role as binding site for PMA (Tonetti et al., 1994). As has been shown, PMA-induced HL-60 macrophage as well as granulocytic differentiation is accompanied by unique activation of a lineage-specific protein kinase (PRKX) that acts downstream of PKC isozyme in the same signal transduction pathway (Semizarov et al., 1998). Moreover, evidence has been accumulated to show that PMA triggers exclusive apoptosis in HL-60 cells (Macfarlane & O’Donnell, 1993) and much less in HL-60 subclones resistant to PMA and deficient in PKC-h (Laouar et al., 2001). Although it has not been determined whether PMAinduced apoptosis is DDA or occurs independently in HL60 cells via PKC-h, a5h1-integrin, caspase-1 or caspase-4, PMA is also likely to be an apoptotic stimulus (Macfarlane & O’Donnell, 1993) in addition to acting as inducer of macrophage/monocytic maturation of leukemia cells. Most recent DNA chip analysis of RNA derived from highly inducible and resistant HL-60 cells has revealed that PMAinduced HL-60 differentiation is accompanied by discrete changes in expression of early, intermediate, and late

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response genes that encode various transcription factors, cytokines, and protein markers (Zheng et al., 2002). Moreover, this study has shown that HL-60 cell maturation is accompanied by biphasic expression of several genes (Zheng et al., 2002). To this respect, PMA-induced HL-60 differentiation is a highly coordinated process carried out via discrete patterns of gene expression like MEL cell maturation described in this paper. CML is characterized from a reciprocal translocation between chromosomes 9 and 22 (Philadelphia chromosome) (Rowley, 1973) that causes fusion of the bcr gene and exon1 of c-abl genes. This leads to the production of a 210-kDa Bcr-Abl fusion oncoprotein (Shtivelman et al., 1985) found in 95% of CML patients. A 185-kDa Bcr-Abl protein product derived from fusion of bcr genes and exons 2– 11 of c-abl gene occurs in 5– 10% adult cases of CML and 5 – 10% of children suffering from acute lymphoblastic leukemia (Hermans et al., 1987; Clark et al., 1988; Clarke et al., 1988). The inhibitor of Bcr-Abl tyrosine kinase imatinib mesylate (STI-571, commercially known as Gleevec or Glivec) has been found to enhance the cytodifferentiating, antiproliferative, and apoptogenic activity of ATRA by decreasing the ATRA-induced degradation of RARa and APL specific PML-RARa fusion protein in HL-60, U-937, and NB4 cells (Gianni’ et al., 2001). Another interesting observation that has been emerged recently is that the use of HDACIs like SAHA in combination with STI-571 promotes apoptotic death in STI-571-sensitive and STI-571-resistant Bcr-Abl + human myeloid leukemia cells (Yu et al., 2003). The latter study indicates that the use of tyrosine kinase inhibitors in combination with HDACIs would be beneficial in clinical practice during cancer treatment. 4.7. Polar agents and ureido derivatives of pyridine This class of agents contains several well-known inducers like DMSO, DMF, bisacetamides, benzodiazepines, cyclic ureas and thioureas, pyridine derivatives, and others. 4.7.1. Dimethylsulfoxide DMSO was used extensively since early 1970s for induction of differentiation of MEL and other cells like HL-60 (Friend et al., 1971; Collins et al., 1978). The precise mechanism by which DMSO induces differentiation is not known but may involve perturbation of PKC activity, increase in membrane fluidity, elevation of calcium uptake, and alteration in expression of c-myc and p53 genes (Tsiftsoglou & Robinson, 1985). DMSO-mediated differentiation has been associated with G0/G1 arrest, induction of CDKI p21Cip-1 (Jiang et al., 1994), and generation of DNA strand breaks (Terada et al., 1978a, 1978b; Scher et al., 1982; Pulito et al., 1983; McMahon et al., 1984). 4.7.2. Bisacetamides Diamines containing 5 –6 methylene groups showed low to high level of induction of MEL cell differentiation.

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However, mono- and bis-acetylation of amino groups in particular gave a new class of inducers, the bisacetamides. Bisacetamides bearing 5– 8 methylenes were the most active chemical inducers of differentiation. Members of this class with 7- and 8-methylene groups caused higher degree of induction at concentrations of 3 – 5 mM but were more toxic. Replacement of the acetyl group with a propyl one also yielded potent inducers of differentiation (Reuben et al., 1976). HMBA was also found to induce differentiation of HL-60 and U937 cells in addition to MEL cells (Haces et al., 1987). HMBA-induced differentiation is accompanied by cell cycle arrest, induction of the CDKI p21Cip-1/mda-6/ sdi-1/Waf-1, and pRb underphosphorylation (Kiyokawa et al., 1993), events that are not sufficient on their own to trigger the differentiation program (Zhuo et al., 1995). Activation of PKC (Mallia et al., 1999), perturbation of cellular calcium concentration (Sparatore et al., 1995), and activation of tyrosine phosphatases (Kume et al., 1994) also contribute to leukemic cell differentiation. 4.7.3. Benzodiazepines It was quite surprise at first to observe that benzodiazepines, well-known drugs used as tranquilizers by binding to the peripheral-type benzodiazepine receptor, induce differentiation of MEL cells (Wang et al., 1984). Structureactivity relationships revealed the methyl group at 1Vposition is essential for their inducing activity. Replacement of 1-methyl group with an ethyl or propenyl one eliminated their biological activity. Similarly, substitution at 2V, 4V, or 6V position with halogens decreased their ability to induce differentiation, although their binding affinity on receptors was increased. Insertion of halogens at both 2V and 4V positions or both 2Vand 6Vpositions led to a biologically inactive benzodiazepine. The inducing activity was increased, however, when the 4Vsubstituent was a hydroxyl or methoxyl group. 4.7.4. Cyclic ureas and thioureas Substituted tetramethyl and tetraethyl ureas were found to be potent inducers of MEL cell differentiation (Li et al., 1981b). Bulky ring systems attached covalently to the nitrogen atoms of urea to form bispentamethyleneurea markedly decrease differentiation inducing capacity. Like aliphatic ureas, cyclic ureas are also biologically active inducers (Li et al., 1981a). An increase in the size of alkyl substituents attached on the ring nitrogen led to the decrease in the capacity of cyclic ureas to initiate cellular differentiation. Replacement of oxygen with sulfur in urea moiety produced less biologically active thioureas in promoting differentiation of cultured MEL cells (Li et al., 1981a). 4.7.5. Substituted amides, lactams, and 2-pyridones N-methylacetamide and N,N-bismethylacetamide are potent inducers of MEL cell differentiation (Tanaka et al., 1975; Reuben et al., 1976). Replacement of N-methyl group with a bulkier substituent (e.g., pyridyl, phenyl, and cyclo-

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hexyl) increased the inducing capacity. The cyclohexyl moiety produced an acetamide derivative that was effective inducer as HMBA. The presence of bulky groups as part of the amide structure led to abolishment of inducing capacity. In lactams, N-alkyl substitution in general enhanced the inducing potency. Furthermore, an increase in the size of the lactam ring progressively enhanced the activity of non-Nalkylated lactams as inducers. N-alkyl-pyridones are also potent inducers of MEL and HL-60 cell differentiation. Replacement of hydrogen by methyl, ethyl, and/or isopropyl groups yielded potent inducers of differentiation. In contrast, an increase in the volume of substituent at nitrogen of pyridine produced less active inducers (Li et al., 1981a). 4.7.6. Pyridine derivatives The acetylamino derivatives of pyridine were 1 of the first class of potent inducers of MEL cell differentiation developed (Li et al., 1978). Induction experiments have shown that 2-acetylaminopyridine was more active than 3acetylaminopyridine. N-oxidation led to less active compounds. Addition of an methylene or ethylene group between the pyridine ring and the acetylamino group led to the reduction of inducing capacity of these agents. Addition of a methyl group at nitrogen of the pyridine ring did not change the inducing activity. Conclusively, a structure of -CO-NHR, where R = H or methyl, directly attached to a ring or as a part of a ring itself (pyridones), is necessary for this group of inducers. Having these conclusions in mind, we have developed UDPs as a new class of potent inducers of MEL, K562, and HL-60 cell differentiation (Pappas et al., 1992). The ureido group attached at 2V position of the pyridine ring yielded a very potent agent (2-(3-ethylureido)-6-methylpyridine [UDP-4]) that induced differentiation in leukemic as well as in neuroectodermal neoplastic cells (Pappas et al., 1996). Other hydrophobic substituents (methyl or ethylureido groups) attached at 4V, 5Vor 6Vpositions of the pyridine ring seem to contribute to the inducing activity. Attachment of the ureido group to the pyridine ring via a long methylene bridge abrogates inducing capacity. Addition of the bulkier substitutes at nitrogen of urea yielded biologically inactive inducers. 4.8. Hemin and other agents as inducers of human K562 leukemia cell differentiation In addition to the plethora of inducing agents mentioned above that differentiate MEL, HL-60, or U937 cells, some other structurally unrelated agents were found to induce erythroid differentiation of K562 cells. These include hemin, a natural regulator of erythropoiesis, sodium butyrate, anthracyclines, hydroxyurea, apicidin, 5-FU, HMBA, PMA, resveratrol, tiazofurin, tallimustine, and Ara-C (see Table 4) (Rutherford et al., 1979; Bianchi Scarra` et al., 1986; Olah et al., 1988; Tsiftsoglou et al., 1989; Fibach et al., 1993; Green et al., 1993; Aries et al., 1996; Hatse et al., 1996; Bianchi et al., 2001; Rodrigue et al., 2001; Witt et al., 2003). All of

these inducing agents regardless to their disparate mechanism of action selectively activate the embryonic and fetal globin synthesis without causing any adult hemoglobin synthesis and commitment to terminal maturation. However, cotreatment of K562 cells with hemin and anthracyclines led to the terminal erythroid maturation (Tsiftsoglou et al., 1989; Tsiftsoglou et al., 1991). The precise mechanisms by which these agents act as inducers still remain unclear although evidence exists to indicate that they work by different mechanisms. Hemin is transported into K562 cells by hemopexin and via a cell surface receptor and binds to intracellular and nuclear proteins called hemin binding proteins (HeBP) (Tsiftsoglou et al., 1993). In the nucleus, hemin modulates the interactions of known transcriptional factors like NF-E2, Oct-1, and GATA-1 by acting selectively on the Gg-globin gene promoter (Palma et al., 1994; Aries et al., 1996). 4.9. Cytokines as inducers of leukemic cell differentiation In addition to the chemical inducers described thus far, evidence exists to indicate that several known cytokines under certain culture conditions promote differentiation of various leukemic cell lines into cell lineage restricted phenotypes as illustrated in Table 5. These include EPO that causes erythrocytic differentiation of J2E cells as well as of MEL cells primed with DMSO (Busfield & Klinken, 1992). Moreover, thrombopoietin (TPO) and EPO have been shown to promote differentiation of human megakaryoblastic leukemia UT-7/GM cells (a subline isolated after long-term culture of UT-7 cells with granulocyte-monocyte colony stimulating factor [GM-CSF]) into megakaryocytic and erythrocytic phenotype, respectively (Komatsu et al., 1997). Furthermore, granulocyte colony stimulating factor (G-CSF), interleukin (IL)-1, and IL-6 induce differentiation of mouse myeloid leukemic M1 cells toward the macrophage phenotype (Table 5) (Miyaura et al., 1988; Metcalf, 1989; Yamamoto-Yamaguchi et al., 1989). Leukemia inhibiting factor (LIF) and tumor necrosis factor-a (TNF-a) were found to promote macrophage development in M1 and WEHI-3B-JCS cells, respectively (Hoffman-Liebermann & Liebermann, 1991; Leung et al., 1994), although TNF-a induced basophilic differentiation of KU812 cells (Nilsson et al., 1994). Finally, the list of cytokines that act as inducers of leukemic cell differentiation includes transforming growth factor-h (TGF-h), which promotes differentiation of U937 cells into macrophages (Kamijo et al., 1990).

5. Mechanisms of induced differentiation of leukemic cells More than 30 years have elapsed since the discovery of the Friend cell differentiation system and the precise mechanism(s) by which the inducers initiate commitment of leukemic cells to terminal maturation are still not clear.

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However, real progress in this field of leukemic cell differentiation came from different areas of investigation, which include the following: (a) Membrane-mediated events and signal transduction pathways, (b) Receptor-mediated processes, (c) Remodeling of the superfine structure of chromatin, (d) Alterations in transmethylation of DNA and RNA, (e) Cell lineage restricted transcriptional factors acting as regulators of reprogramming of hematopoietic cell fate under normal and leukemic state, and (f) The potential role of protooncogenes in differentiation and apoptosis. 5.1. Membrane-mediated events and signal transduction pathways Normal hematopoiesis within the bone marrow microenvironment is regulated by a complex interplay of different growth factors that act on stem cells or early progenitors at various stages. By interacting with their own cell surface receptors, growth factors initiate signals transduced from the plasma membrane into the nucleus to promote selective activation and/or repression of genes. Thus, they affect selfrenewal, proliferation and differentiation of various cell types in most cases (Ghysdael et al., 2000). Reversibly, some cells like the macrophage-monocyte cells produce factors (e.g., TNF-a), which cause apoptosis or necrosis in hematopoietic cells via receptor-mediated signaling pathways (Ashkenazi & Dixit, 1998). The fact that most cytokines share intrinsic protein tyrosine kinase activity allowed several investigators to delineate unique downstream signaling pathways comprehensively presented elsewhere (Smithgall, 1998; Ghysdael et al., 2000; Rane & Reddy, 2002). Apparently, this homeostatic regulation of hematopoiesis is disrupted in leukemiogenesis (Zhu & Skoultchi, 2001; Cantor & Orkin, 2002; Graf, 2002; Tenen, 2003) because most leukemic cells do not respond to known growth factors and differentiators (EPO, GM-CSF, M-CSF, and IL-3) to generate mature blood cells. Therefore, our knowledge on signaling transduction pathways functioning during hematopoiesis has been derived mainly from studies with growth factors acting on normal hematopoietic progenitors and in a few occasions on transformed leukemic cells. Priming of MEL cells with DMSO renders them responsive to EPO that promotes erythroid differentiation of MEL cells by interacting with its cell surface receptors (Busfield & Klinken, 1992). These studies have revealed that EPO activates the PKC signaling pathways and activate transcription of c-myc (early response gene) and SCL (TAL1) gene (Mason-Garcia & Beckman, 1991; Nielsen et al., 1996; Hoffman et al., 2002). In addition, EPO was found to promote phosphorylation of SCL-1 gene product (Prasad-Srinivasa et al., 1995), whereas GATA-1 and EPO seems to cooperate in promoting survival of

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erythroid cells by regulating the expression of Bcl-XL (Gregory et al., 1999). The question whether the small-weight chemical inducers initiate commitment to differentiation by activating known signaling pathways or by promoting other events is still unresolved. Evidence exists, however, to indicate that some inducing agents like PMA promote maturation of leukemic cells via PKC-h-mediated signaling pathways as discussed above. Moreover, it has been shown that HMBA, a potent inducer of MEL cell differentiation, activates tyrosine phosphorylation of JAK2 and STAT5 signal transducers (Rane & Reddy, 2002). Signal transduction pathways, however, are not the only membrane-mediated events that occur in leukemic cell differentiation. Polar/apolar chemical inducers cause pulse shift at the level of plasma membrane, alterations in cell surface architecture, reduction in cell size, activation of Ca2+ ion transport early in the precommitment phase, and other biochemical events including shortening of cell cycle G1 phase, as reviewed elsewhere (Tsiftsoglou & Robinson, 1985). 5.2. Receptor-mediated processes The majority of chemical inducers discovered and/or developed via organic synthesis have highly diversified structure and do not permit a direct QSAR study to uncover the mechanisms of induction of leukemia cell differentiation. QSAR studies, however, with certain classes of potent inducers (bisacetamides, hydroxamic acid derivatives, and ureidopyridine derivatives), have been possible and quite fruitful (Gusella et al., 1976; Reuben et al., 1976; Li et al., 1978; Pappas et al., 1992; Wittich et al., 2002). The concept that bisacetamides induce differentiation by interacting via receptor-like cellular components was conceived at first in late 1970s when radiolabeled bisacetyl-diaminopentane (BADP), a potent inducer of MEL cells, was found to form a complex with intracellular components (Tsiftsoglou et al., 1981b). This study suggested that certain class of agents that carry the acetylamino group (CH3-CO-NH-) as part of either the ring structure itself or the aliphatic chain have the potential to induce MEL cell differentiation. Additional studies with hydroxamic acid derivatives led to the detection of the ribosomal protein S3 acting as putative receptor of inducers involved in MEL cell differentiation (Webb et al., 1999). Most recent independent studies with the use of radiolabeled potent inducer UDP-4 (Pappas et al., 1992) led to the discovery of a 40-kDa cytosolic protein acting as binding protein for UDP inducers. Although the entire structure of 40-kDa protein has not been fully established, the primary structure of a portion of this protein was confirmed by conventional protein microsequencing (Pappas et al., 2003). Evidence exists to indicate that 40-kDa protein forms complexes with the inducer UDP-4 both in the cytoplasm and in the nucleus that last for 12 –24 hr as long as the precommitment period. By the end of this time

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and after commitment has been initiated, these complexes are no longer detected. 5.3. Remodeling of the superfine structure of chromatin Nuclear condensation and irreversible cessation of DNA replication are among the most prominent events occurring in terminally committed and differentiated MEL cells (Scher et al., 1983). Previous studies examining the structure of chromatin by digestion with nucleases have shown that induction of MEL erythroid differentiation correlates with an increase in DNAse I hypersensitivity at sequences near the a- and h-globin genes (Sheffery et al., 1982; Yu & Smith, 1985). However, these changes seen in the superfine structure of chromatin have been found not to be directly related with alterations in DNA methylation of these genes at CpG islands. Although the impact of these changes in chromatin structure of the globin genes is still elusive, it is reasonable to postulate that such alterations could reveal cis-acting elements that influence the interactions of cell lineage restricted transcription factors to globin gene promoters (Robertson & Wolffe, 2000; Jones & Baylin, 2002; Laird, 2003). The recent finding that the methyl-CpG binding protein MeCP2 interacts with the HDAC-involved complex provides evidence on the connection among DNA methylation, chromatin structure remodeling, and gene transcription silencing (Ng & Bird, 1999; Robertson, 2002). 5.4. The role of DNA and RNA transmethylation in murine erythroleukemia cell differentiation Investigation of the impact of DNA methylation on chromatin structure remodeling and gene expression patterns seen in differentiating MEL cells can be useful in understanding the mechanisms of leukemic cell maturation. The observations that discrete patterns of gene expression operate in differentiating MEL cells (see Table 1) suggest that the steady-state cytoplasmic accumulation of RNA transcripts result from transcriptional activation, repression, or post-transcriptional processes. In fact, previous studies with inhibitors of DNA methylation (e.g., 5-azacytidine) have considered DNA hypomethylation as being responsible for activation of transcription of globin genes in inducertreated MEL cells (Christman et al., 1977, 1983). Although changes in transcription due to DNA hypomethylation can explain in part the discrete patterns of gene expression observed in differentiating MEL cells, the possibility that such changes may be also due to differential stability of RNA has drawn attention in the past (Volloch & Housman, 1981; Volloch et al., 1987). Biochemical studies with cordycepin (3V-deoxyadenosine) (an inhibitor of polyadenylation and methylation of RNA), and more recently with N6mAdo, have suggested that initiation of commitment of inducer-treated MEL cells to terminal erythroid maturation may depend on both synthesis and post-transcriptional

modifications of RNA, such as methylation (Vizirianakis et al., 1992). These studies have shown that MEL cell differentiation is associated with hypermethylation of RNA (Vizirianakis & Tsiftsoglou, 1996). Treatment of MEL cells with the inducer DMSO led to an increase in methylation of total cytoplasmic and polyA + RNA as well as a transient increase in methylation of ribosomal RNAs at various base residues as well as in mRNA in residues located within the 5V cap structure and in internal sites of the molecules (Vizirianakis & Tsiftsoglou, 1995a; Vizirianakis & Tsiftsoglou, 1996). That RNA transmethylation is considered to be an integral part of the differentiation program of MEL cells was further supported from studies carried out with the use of N 6mAdo, an agent found to block commitment, to erase ‘‘memory,’’ to reduce methylation of total cytoplasmic RNA, and to alter the intracellular pools of SAH and Sadenosylmethionine (SAM) (Vizirianakis & Tsiftsoglou, 1995b). These effects seem to occur via intracellular conversion of N 6mAdo into the S-N 6-methyladenosylhomocysteine (N 6 -SAH), an intermediate that inhibits RNA methyltransferases (Pugh et al., 1977; Hoffman, 1978). Similar observations were obtained with 3-deazaadenosine, 5V-S-isobutylthioadenosine, 5V-methylthioadenosine, neplanocin A, 3-deazaneplanocin A, and cycloleucine, agents that modulate the methylation cycle and inhibit initiation of MEL cell differentiation (Di Fiore et al., 1984; Sherman et al., 1985; Chiang & Miura, 1986; Vizirianakis et al., 1992; Vizirianakis & Tsiftsoglou, 1996). By knowing that commitment of MEL cells depends exclusively on the synthesis of new RNA and proteins as well as on methylation and differential stability of polyA + RNAs, it was reasonable to assume that inhibition of RNA methylation blocks initiation of commitment by affecting stability of critical species of RNA transcripts. Alternatively, the increase in RNA methylation seen in differentiating MEL cells could be attributed to the changes in the ratio SAH/SAM that influences the extent of RNA methylation (Vizirianakis & Tsiftsoglou, 1996). In lower ratio, RNA methyltransferases are no longer inhibited by SAH (Cantoni, 1986). Unfortunately, the precise role of changes in transmethylation of both polyA  and polyA + RNA during MEL cell differentiation is still elusive. Increased methylation of RNA transcripts may affect their physicochemical behavior and facilitate transport from the nucleus into cytoplasm as previously reported (Camper et al., 1984). Otherwise, methylation may alter the configuration of 5V end of mRNA (Kim & Sarma, 1978), thus rendering them less susceptible to ribonucleases that degrade RNA beginning from the 5V end (Goutts & Brawerman, 1993; Goutts et al., 1993). Finally, changes in RNA methylation may contribute to alterations in the tertiary structure and conformation of RNA in a way that enables it to interact with trans-acting factors (Peltz et al., 1991; Belasco & Brawerman, 1993; Klausner et al., 1993; Burd & Dreyfuss, 1994). Overall, changes in methylation of RNA at specific sites may affect RNA

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stability and the steady-state level of RNA transcripts, an event that is developmentally regulated during MEL cell differentiation (Volloch & Housman, 1981; Volloch et al., 1987; Christou & Tsiftsoglou, 1994; Vizirianakis & Tsiftsoglou, 1996). Consequently, the need for further investigation of DNA and RNA transmethylation in leukemic cell differentiation is justified on the light of current evidence showing that chromatin structure remodeling, among other factors, involves DNA methylation to affect gene transcription (Robertson & Wolffe, 2000; Jones & Takai, 2001; Jones & Baylin, 2002). 5.5. Lineage-specific transcriptional factors as regulators of hematopoietic cell fate under normal and leukemic states The discovery of lineage-specific transcriptional factors as regulators of hematopoietic development over the last several years provided new insights into the mechanisms involved in the reprogramming of HSCs into specific cell

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lineages (Orkin, 2000; Graf, 2002). In most cases, these transcriptional factors work at the gene expression level as activators or repressors in a way directed by signals transduced by external stimuli to the nucleus. This class of transcriptional factors includes proteins regulating initiation of differentiation, cell cycling, and apoptosis (see Table 6). Within the class of differentiation-related transcriptional factors belong factors being responsible for the entire course of hematopoiesis like the SCL (TAL1) factor acting as a master switch as well as factors primarily involved in erythropoiesis (GATA-1, GATA-2, EKLF, and Friend of GATA-1 [FOG]) and others being responsible for differentiation into myeloid (PU.1 and MafB) and lymphoid phenotypes (Pax5). A lot of attention was also drawn by cell cycle related factors, which include several proteins encoded by c-myc, c-myb, c-jun, junB, junD, c-fos, p53 genes, pRb protein, CDKs, and CDKIs being involved in cell cycle progression of early erythroid progenitors (see Table 6).

Table 6 Transcription factors regulating hematopoiesis Lineagea

Result

Representative referenceb

Antagonizes GATA-1

Oikawa et al., 1999

MafB

Monocytic, lymphoid, granulocytic Myeloid

Kelly et al., 2000

GATA-1 EKLF NF-E2 FOG Pax-5

Erythroid Erythroid Erythroid Erythroid B-cell lymphoid

SCL (TAL1) LMO2

Master regulator of hematopoiesis Progenitors, erythroid

GATA-2

Progenitors

Represses the activity of Ets-1 on erythroid-specific gene promoters Antagonizes PU.1 Transcription of h-globin gene Transcription of h-globin gene Cofactor of GATA-1 that antagonizes C/EBP-h Suppression of gene transcription involved in non-B-cell lymphopoiesis Transcriptional regulator for several genes that interacts with LMO2 Transcriptional regulator for several genes that interacts with SCL Cell survival and proliferation factor

Cell cycle related p53 pRb p21CIP1, P27KIP1

Progenitors, erythroid Erythroid Erythroid

Regulator of cell growth and apoptosis Regulator of cell cycle progression through G1 phase Inhibitors of G1 phase CDKs

c-jun, junB, junD

Erythroid Many cell types including hematopoietic cells Many cell types including hematopoietic cells Myeloid

c-fos

Myeloid

E2A Ets-1

B-lymphoid Lymphoid cells

Regulators of cell cycle progression through G1 phase Regulator of cell proliferation, differentiation, and apoptosis Regulator of cell proliferation, differentiation, and apoptosis Regulators of cell proliferation, differentiation, and apoptosis through the formation of AP-1 complexes Regulator of cell proliferation, differentiation, and apoptosis through the formation of jun/fos AP-1 complex formation Induction of B-lymphoid-specific gene expression Mainly activates myeloid-like gene promoters

Ryan et al., 1994 Rifkind et al., 1996 Hsieh et al., 2000; Zhu & Skoultchi, 2001 Matushansky et al., 2000a, 2000b Hoffman et al., 2002

Factor Differentiation related PU.1

CDK2, CDK4, CDK6 c-myc c-myb

Abbreviations: C/EBP-h: CCAAT/enhancer binding protein-h. a The information included refers to data obtained by using the cell lineage examined. b Whatever is possible, a review article is cited for further reading.

Nerlov et al., 2000 Cantor & Orkin, 2002 Cantor & Orkin, 2002 Cantor & Orkin, 2002 Nutt et al., 1999 Green et al., 1992; Cantor & Orkin, 2002; Koury et al., 2002 Cantor & Orkin, 2002; Koury et al., 2002 Orkin, 1995; Heyworth et al., 1999

Oh & Reddy, 1999; Chen et al., 2002; Yamamoto et al., 2002 Liebermann & Hoffman, 2002 Liebermann & Hoffman, 2002

Zhuang et al., 1994; Orkin, 1995 Maroulakou & Bowe, 2000; Oikawa & Yamada, 2003

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Lineage-specific transcriptional factors appear to exert dual actions on hematopoietic cell lineages by acting either directly to promote differentiation into specific cell phenotypes or indirectly to antagonize the action of other transcription factors, thus inhibiting alternate cell lineage gene expression programs to occur (reviewed recently by Cantor & Orkin, 2001). Once these processes are being deregulated, normal hematopoiesis is disrupted and a leukemic state has been initiated and established (Tenen, 2003). Cross-talk antagonism between different transcriptional factors has been established especially for PU.1 and GATA-1 as well as for FOG and C/EBP-h. Interestingly, the antagonistic actions between PU.1 and GATA-1 are mediated through a direct physical contact of these transcriptional factors, leading either to inability of PU.1 to interact with its coactivator c-jun or to inefficient GATA-1 binding to its DNA consensus sequence (Rekhtman et al., 1999; Zhang et al., 1999, 2000; Nerlov et al., 2000). Excellent reviews on the interactions of cell lineage transcriptional factors and their impact on disruption of hematopoiesis during leukemiogenesis have been reported elsewhere (Oikawa et al., 1999; Tenen, 2003; Oikawa & Yamada, 2003). Overexpression of PU.1 deregulates the development of erythroid lineage progenitors mediated by GATA-1, whereas overexpression of GATA-1 in myelomonocytic progenitors perturbs their differentiation sustained by the action of PU.1. Another interesting finding related to PU.1 function in erythroid cells is the fact that overexpression of PU.1 induces inhibition of differentiation and apoptosis associated with down-regulation of c-myc transcription in MEL cells treated with DMSO. Most importantly, PU.1-induced apoptosis in MEL cells seems to occur in G0/G1 through S phases with the involvement of c-myc and bcl-2 (KiharaNegishi et al., 1998). Such studies impinge on the important role of specific transcriptional factors on fundamental cellular processes, like differentiation and apoptosis of normal and leukemic hematopoietic cells. 5.6. The role of c-myc and other protooncogenes in murine erythroleukemia cell differentiation and apoptosis Experiments with transfection of genes driven by strong inducible promoters or with antisense oligos have suggested that the products of some protooncogenes including c-myc, c-myb, c-jun, and PU.1 transcription factor (member of the Ets gene family) are involved in initiation or blockade of erythroid differentiation (Dimitrovsky et al., 1986; Clark et al., 1988; Clarke et al., 1988; Smith & Prochownik, 1992; Francastel et al., 1994; Rao et al., 1997; Yamada et al., 1997; Oikawa et al., 1999; Chen et al., 2002). The expression of c-myc, c-myb, p53, and PU.1 is down-regulated on induction of differentiation of MEL cells with chemical inducers whereas that of c-fos is upregulated (Lachman & Skoultchi, 1984; Kirsch et al., 1986; Ramsay et al., 1986; Tsiftsoglou et al., 1987; Rao et al., 1997; Vizirianakis et al., 2002). Interestingly, treatment of

MEL cells with chemical inducers triggers a very rapid decline in c-myc, c-myb, PU.1, and p53 followed by increased expression later on up to a level comparable with that seen in untreated cells by about 18– 24 hr postinduction (Table 1). The increase at this time, however, for PU.1 and p53 is modest and not comparable with that seen with c-myc and c-myb. A second decline in the expression of these genes occurs progressively afterward as the proportion of committed MEL cells increases in the culture. In addition to the expression profiles of oncogenes reported above, the level of junB is increased quite early within the latent period preceding the commitment to erythroid maturation of MEL cells (Francastel et al., 1992; PoindessousJazat et al., 2002). This finding implies that junB is likely to play a role in the modulation of the differentiation process of leukemic cells. This disparate expression pattern of different oncogenes on induction of MEL cell differentiation indicates that they contribute into the regulatory machinery of differentiation at different stages. Likely, the activation of expression of some of these genes is implicated in the initiation of commitment, while suppression of others facilitate the cessation of proliferation in terminally differentiated cells (Smithgall, 1998; Oikawa et al., 1999; Zhu & Skoultchi, 2001; Cantor & Orkin, 2002; Skalnik, 2002). Moreover, it has been shown that the observed transcriptional repression of c-myc seen on overexpression of PU.1 in MEL cells may be mediated through complex formation of PU.1 with HDACs (Kihara-Negishi et al., 2001). Interestingly, a negative cross-talk between c-myb and the CREB-binding protein (CBP), which is a coactivator of hematopoietic Ets transcription factor Spi-B, has been established, providing thus new insights into hematopoietic cell differentiation program (Yamamoto et al., 2002). This finding adds new knowledge, especially in the light of the recently published data, suggesting an involvement of c-myb in maintaining MEL cells in an immature and proliferating state (Chen et al., 2002). c-myc is a transcriptional regulator that controls the expression of distinct sets of several target genes, thus affecting cellular differentiation and apoptosis (Boon et al., 2001; Nasi et al., 2001; Schuhmacher et al., 2001; Hoffman et al., 2002). c-myc is also considered to be a crucial gene involved in reprogramming of hematopoietic progenitors into apoptosis and/or differentiation (McKenna & Cotter, 1997). The biphasic pattern of expression of c-myc gene observed in MEL cells differentiated by several inducers, like DMSO, HMBA, hypoxanthine, and/or UDP-4, taken together with the findings that continued expression of c-myc by stable transfection of MEL cells with the full-length c-myc cDNA impaired differentiation (Lachman & Skoultchi, 1984; Prochownik & Kukowska, 1986; Vizirianakis et al., 2002) indicates that this protooncogene plays a central role in commitment. The interplay between cellular differentiation and c-myc gene expression is also documented in neuronal differentiation of neuroectodermal RD/TE-671 induced by UDP-4, because this

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agent caused a biphasic pattern of accumulation of c-myc RNA transcripts in these cells (Pappas et al., 1996; Vizirianakis et al., 2002). Experimental evidence accumulated over the past years suggests that expression of c-myc sensitizes cells to a variety of apoptotic stimuli (Juin et al., 1999; Nasi et al., 2001). It is not clear whether the functions of c-myc in cell differentiation and apoptosis are mediated by the same or different set of genes. However, the c-myc-mediated sensitization of cells occurs via the release of cytochrome c from mitochondria to cytosol, an event initiated by CD95/Fas ligand. CD95/Fas and p53 are required to activate apoptosis through the involvement of caspases (Juin et al., 1999). Following this, it is of great interest to report that Myc-induced apoptosis is associated with loss of mitochondrial transmembrane potential, an event seen very early in inducer-treated MEL cells (Hotti et al., 2000; Wong et al., 1985) as well as in K562 cells. It is likely in these cases that DDA is associated with changes in mitochondrial transmembrane potential that are independent of Bcl-2 and caspases (Diaz et al., 1999). Therefore, the question whether c-myc controls the expression of mitochondrial function related genes as well that of genes encoding ribosomal proteins during differentiation or DDA is quite challenging (Boon et al., 2001; Schuhmacher et al., 2001; Vizirianakis et al., 2002). Another interesting point that has emerged recently is the fact that c-myc has been involved in chromatin remodeling via HAT ATP-dependent chromatin remodeling complexes (see Amati et al., 2001 for review). These observations taken together with the fact that sodium butyrate, an inhibitor of HDACs, promotes differentiation of MEL cells (Corin et al., 1986) by favoring histone acetylation and histone H1 gene expression is regulated by c-myc on induction of MEL cell differentiation (Cheng & Skoultchi, 1989) tend to suggest that this protooncogene is involved in chromatin remodeling. Detailed analysis, however, of these processes is needed to confirm the regulatory role of c-myc in histone-mediated function of gene transcription in leukemic cell differentiation.

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6. Induction of differentiation and apoptosis in leukemic cells Experiments reported over the last years have shown in several occasions that cultures of terminally differentiated leukemia cells contain a proportion of apoptotic cells. Although it has not been clear whether this apoptosis occurs spontaneously or as ‘‘DDA’’ in terminally differentiated cells, we have thought it is worthy to discuss this issue from biological and pharmacological point of view. 6.1. Is differentiation-dependent apoptosis part of the developmental program of leukemic cells? Studies over the last years with leukemic cell lines (MEL, K562, and HL-60) have indicated that several inducers promote programmed cell death (apoptosis) in addition to causing terminal differentiation (Bianchi et al., 2001; Zheng et al., 2002; Tsiftsoglou et al., 2003). That the proportion of terminally differentiated cells, in most cases, is much higher than that of apoptotic cells recorded in the same culture tend to suggest that a fraction of differentiated cells undergo apoptosis. These findings suggest that this is either an intrinsic property of the inducers or the initiation of differentiation occurs simultaneously with apoptosis in cells undergoing maturation. This phenomenon that we have called DDA may be part of the developmental program of leukemic cells. Whether DDA results from simultaneous activation of differentiation and apoptotic processes during in vitro maturation of leukemic cells or if it is a unique process itself is not clear. 6.2. Coactivation of differentiation and apoptosis programs as potential target for combination chemotherapy of leukemias The early observations of Sachs (1964), Friend et al. (1971), and Pierce (1974) that some neoplastic cells can be

Fig. 4. Diagrammatic representation of the concept of synergistic action between inducers of differentiation and TNF-a on neoplastic cells. This concept lies on the ability of TNF-a (or other apoptotic stimuli) and inducers of differentiation to promote terminal differentiation and apoptosis leading to extensive cell death in mature neoplastic cells. R denotes the putative receptor of inducing agents. * This concept of synergistic action can be applied with TNF-a or other apoptotic stimuli and inducing agents.

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differentiated in vitro and/or in vivo into cells resembling their normal counterparts via treatment with a large variety of agents including growth factors led to a new way of thinking to combat malignancy under relatively less cytotoxic conditions. The so-called differentiation therapy of neoplasms (Lotan et al., 1990) has been already successfully applied as an effective approach of cancer treatment in human APL (Castaigne et al., 1990). This notion is based on the fact that many neoplasms exhibit reversible defects in differentiation, which on appropriate treatment results in tumor cell reprogramming with loss of proliferative capacity and induction of terminal maturation or apoptosis (Leszczyniecka et al., 2001). Moreover, induction of differentiation has been achieved in a large number of neoplastic cell systems (Reiss et al., 1986). In most cases studied, induction of differentiation led to less malignant or nonmalignant cells that fail to proliferate and support malignant growth. Finally, in most of the differentiation systems of neoplastic cells studied thus far, it looks like that the same principles and cellular behavior appear to occur although the precise mechanism(s) has not been fully elucidated (Tsiftsoglou & Robinson, 1985). The recent advances in molecular biology of cancer opened up new ways for developing more powerful and specific anticancer drugs. The process of cell renewal versus that of cell differentiation and/or apoptosis (cell growth restriction and programmed cell death) appears to be linked somehow to each other even under normal hematopoiesis (Domen, 2001; Domen & Weissman, 2000). The observations that spontaneous apoptosis as well as DDA appear to occur in MEL cells as reported (Tsiftsoglou et al., 2003) tend to suggest that the differentiation process and apoptosis can be activated in leukemic cell differentiation and serve as potential platforms for developing combined therapeutic approaches such as the one illustrated in Fig. 4. The fact that TNF-a (a signal for apoptosis) and UDPs (potent inducers of differentiation) act synergistically in promoting extensive cell death in mouse fibrosarcomas WEHI-13Var, MEL-745PC-4A, and K562 leukemia cells (Dretaki et al., 1999) indicates that these processes can turn out to be exploitable targets for novel treatments of leukemia and maybe of solid tumors. These findings are consistent with earlier and most recent observations showing that TNF-a can be combined successfully with retinoic acid (Trinchieri et al., 1987), 1a,25-dihydroxyvitamin D3 (Wang et al., 1991), and other low molecular weight polar differentiation agents in promoting extensive cell death (Nakaya et al., 1990; Depraetere et al., 1995). A critical role in this synergism appears to play c-myc oncogene that sensitizes hepatocytes to TNF-a-induced apoptosis (Klefstrom et al., 1994; Liu et al., 2000). Whether these combinations promote DDA in cell lines examined or differentiation and apoptosis are activated simultaneously in parallel pathways remains to be investigated. Such a combined approach could also find application in solid tumors where differentiation therapy of cancer met with limited success.

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