Fundamentals of Chemotherapy of Myeloid Leukemia by Induction of Leukemia Cell Differentiation

FUNDAMENTALS OF CHEMOTHERAPY OF MYELOID LEUKEMIA BY INDUCTION OF LEUKEMIA CELL DIFFERENTIATION Motoo Hozumi Department of Chemotherapy, Saitama Cancer Center Research Institute, Saitama. Japan

I. Introduction ....................... ............................................ 11. Myeloid Leukemia Cells U A. Animal Cells............................................................................................................. B. Human Cells............................................................................................................. 111. Induction of Differentiation of Cultured Mouse Myeloid Leukemia Cells ................ A. Inducers of Cell Differentiation............................................................................... B. Stimulators of Production by Leukocytes of D-Factor for Myeloid Leukemia Cells ................................................................................... C. Biochemical Phenotypic Changes Associated with Myeloid Leukemia Cell Differentiation................................................................................ D. Changes in Proliferation Potential during Differentiation of Myeloid Leukemia Cells..................................................................................... E. Positive Feedback Control Mechanisms of Cell Differentiation by Protein Inducer Produced by Differentiating Myeloid Leukemia Cells............................. F. Inhibitors and Their Mechanisms of Inhibition of Differentiation ............................................................... of Myeloid Leukemia Cells..... G. Properties of Myeloid Leukemia Cells Resistant to Inducers of Cell Differentiation .............................................................................................. H. Sensitization of Myeloid Leukemia Cells Resistant to Inducers of Cell Differentiation.............................................................................................. IV. In Vivo Induction of Differentiation of Mouse Myeloid Leukemia Cells and Therapy of Animals Inoculated with Myeloid Leukemia Cells................................... A. In Vrvo Induction of Differentiation of Mouse Myeloid Leukemia Cells............. B. Therapy of Mice Inoculated with Myeloid Leukemia Cells by Induction of Differentiation............................................................................... V. Induction of Differentiation of Cultured Human Myeloid Leukemia Cells............... A. Cultured Myeloid Leukemia Cell Lines.................................................................. B. Primary Cultured Myeloid Leukemia Cells............................................................ VI. Summary ........................................................................................................................ References....................................................................................................................... I.

121 I22 123 123 I23 I23

132 I34 138 141

142 145 I47 148 148 I49 152 153 16 I 162 164

Introduction

There is accumulating evidence that various myeloid leukemia cells can be induced to differentiate into cells with the normal characteristics of macrophages, granulocytes, or erythrocytes. On differentiation the cells cease to proliferate and lose their transplantability into animals. These findings suggest that induction of terminal cell differentiation by certain inducers is 121 ADVANCES IN CANCER RESEARCH, VOL. 38

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-006638-6

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another approach to therapy of myeloid leukemia and that some myeloid leukemia cells are formed by impairment of a certain stage of differentiation of the normal hematopoietic process (Anderson et af.,1979; Bodner et af., 1981;Breitman and Gallo, 1981;Breitman et al., 1981; Burgess and Metcalf, 1980b; Collins et af.,1980; Gallo et af., 1979; Honma et af., 1982a, 1983; Hozumi et af.,1979a,b; Hozumi, 1982; Ichikawa, 1969, 1970; Ichikawa et al., 1976; Koeffler and Gold, 1980; Koeffler et al., 1981;Lozzio and Lozzio, 1975; Lozzio et af., 1981; Marks and RiWnd, 1978; Marks et af., 1978; Metcalf, 1979, 1980 PalC et af., 1979a,b; Rutherford et al., 1979; Sachs, 1978a,b, 1980, 1981; Sugiyama el af.,1979a; Takeda et af.,1982). We have been trying to establish a new method ofcancer chemotherapy by controlling the malignancy of tumor cells by induction of terminal cell differentiation. As a model for this purpose, we examined the in vitro and in vivo effects of various compounds on induction of differentiation of the mouse myeloid leukemia cell line, MI, which was established from a spontaneous myeloid leukemia in an SL strain mouse and which can be induced to differentiate into macrophages and granulocytes with loss of leukemogenicityto syngeneic mice (Hozumi et af.,1979a,b;Hozumi, 1982). Recent results of our experiments and those of others on induction of differentiation of M 1 cells are described in this article. During studies on differentiation of M 1 cells, other myeloid leukemia cell lines from mice (R453, Ichikawa et af.,1976; WEHI-3B, Metcalf, 1979)and humans (HL-60, Collins et af.,1977;K562, Lozzio and Lozzio, 1975;KG- 1, Koeffler and Gold, 1978; ML-1 and ML-3, Minowada, 1981) were also found to be induced to differentiate into macrophages, granulocytes, or erythrocytes by various compounds. Furthermore, the effects of inducers of differentiation of leukemia cell lines were examined on leukemic cells in primary culture from patients with myeloid leukemia to develop a method of therapy by induction of terminal cell differentiation. The compounds and endogenous factors affecting differentiation of these leukemic cells, mechanisms of induction of differentiation of the cells, and perspectives of therapy of myeloid leukemia by induction of terminal cell differentiation are described in this article. II. Myeloid Leukemia Cells Used for Experiments

Most studies on induction of differentiation of myeloid leukemia cells have been conducted with the cultured cell lines described in this section. Primary cultured human leukemic cells began to be employed only recently to obtain information on the differentiation of cultured leukemic cell lines for therapy of human leukemia.

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A. ANIMALCELLS A myeloid leukemia cell line, M 1, was established from a spontaneous myeloid leukemia in an SL mouse (Ichikawa, 1969) and another leukemia cell line, R453, was established from a C57BL/6 mouse with a Rauscher virus-induced leukemia (Ichikawa et al., 1976). WEHI-3B myelomonocytic leukemia was induced in a BALB/c mouse injected with mineral oil (Warner et al., 1969).The myelomonocytic leukemia cell line WEHI-3B was found to form colonies in semisolid agar culture (Metcalf et al., 1969) and it was established in 1972 by Dr. C. Wyss (unpublished data) as a cloned continuous cell line in liquid culture (Metcalf, 1979). B. HUMANCELLS A human promyelocytic cell line, HL-60, was established from peripheral blood leukocytes of a patient with acute promyelocytic leukemia (Collins et al., 1977). Lozzio and Lozzio (1 975) established a human myelogenous leukemia cell line, K562, from the pleural effusion of a patient with chronic myelogenous leukemia in terminal blast crisis. Koeffler and Gold (1978) established a human myelogenous leukemia cell line, KG- 1, from a patient with acute myelogenous leukemia. KG-1 cells are at the myeloblast and promyelocyte stages of differentiation and retain the morphological and cytochemical characteristics of acute myelogenous leukemia cells. The human myeloid leukemia cell lines ML- 1 and ML-3 were established from a patient with acute myelogenous leukemia by Minowada ( 1982). Leukemic cells from patients with myeloid leukemia were obtained from peripheral blood or bone marrow of the patients. Leukemia cells in primary culture were used for experiments. 111. Induction of Differentiationof Cultured Mouse Myeloid Leukemia Cells

A. INDUCERS OF CELLDIFFERENTIATION

Mouse myeloid leukemia cell lines such as M 1, R453, and WEHI-3B can be induced to differentiate both in vitro and in vivo into macrophages and granulocytes by treatment with various compounds. Differentiated cells mainly express properties similar to normal macrophages and granulocytes. These properties or markers of differentiated cells are Fc, C, receptors, motility, phagocytosis, lysosomal enzymes, and morphological changes to macrophages and granulocytes (Fig. 1). Furthermore, differentiated M 1 cells express glycoprotein with a molecular weight of 180,000 in their plasma

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FIG. 1. Typical morphology of differentiated M 1 cells (Okabeet a/., 1979). (a) A subclone of M 1 cells (MI -R 1) cultured in a diffusion chamber in an syngeneic SL mouse for 4 days. (b-d) MI-Rl cells were treated with actinomycin D (5 ng/ml) for 2 days in vitro. Then the cells were cultured in diffusion chambers in SL mice. May-Griinwald-Giemsa stain.

membrane, and lose proliferating capacity in vitro and leukemogenicity to syngeneic mice. The main properties of these inducers are described below. 1. Proteins

a. D-Factor and Colony-Stimulating Factor. Induction of differentiation of M1 cells into macrophages and granulocytes was first demonstrated by Ichikawa (1 969)withconditioned medium (CM) ofmouse embryo cells. The factods) stimulating differentiation of MI cells (D-factor) in the CM of embryo cells was found to be a glycoprotein with a molecular weight of 40,000 to 50,000, differing in nature from the colony-stimulating factor (CSF). The D-factor was detected in the CM of embryo cells of various animals and in the CM of established lines of various cells. One of the D-factors in the CM of the established cell line E 1 of mouse embryo cells was

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named MGI, macrophage-granulocyte inducer, by Guez and Sachs ( 1973). It had a molecular weight of 68,000, CSF activity, and no detectable sugar component. Later, Lotem and Sachs (1978a) reported that CSF purified from mouse lung CM could induce differentiation of M 1 cells. We found that a clone, YS-T22, of cells from the Yoshsida sarcoma cell line YSSF-2 12 grown in serum-free culture medium could produce D-factor (Hozumi et al., 1979c)with or without CSF activity. The D-factor and CSFin CM of spleen cells stimulated with mitogens or copolymer of polyinosinic and polycytidilic acids, poly(1) - poly(C), could be separated by gel filtration (Yamamoto et al., 1979). Additional evidence that the D-factor and CSF are distinct substances was obtained by experiments with CM of the mouse mammary carcinoma cell line, FM3A, (Ayusawa et al., 1979;Hozumi et al., 1981) and mouse fibroblast L929 cells (Yamamoto et al., 198Ib). We examined the molecular sizes of the CSF and the D-factor produced from FM3A cells by gel filtration. The CSF consisted of a major and a minor component with apparent molecular weights of 80,000 and 30,000, respectively, while the D-factor was a single component with an apparent molecular weight of 60,000 to 70,000. Although the molecular size of the D-factor from FM3A cells was scarcely affected by treatment of the cells with tunicamycin, a specific inhibitor of asparagine-linked glycosylation, the CSF produced by the cells had a lower molecular weight of 30,000 and was homogeneous with noaffinity toconcanavalin A (Con A)-Sepharose(Hozumi etal., I98 1). The cells with or without properties of CSF and D-factor produced by b29 tunicamycin were also examined by gel filtration (Yamamoto et al., 1981b). Although D-factor produced by untreated L929 cellsgave a single peak with an apparent molecular weight of 67,000, D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. In contrast, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced from tunicamycin-treated L929cells was more sensitive to trypsin or heat treatment than the D-factor from untreated L929 cells, but the CSF produced in the presence of tunicamycin was resistant to these treatments. These results show that the D-factor is distinct from CSF and that carbohydrate is not essential for production of the activity of the D-factor, although it may stabilize the D-factor. Although Guez and Sachs ( I 973) and Lotem and Sachs ( 1978a) reported previously that CSF from CM of a mouse fibroblast cell line, E 1, and mouse lung had the activities of both D-factor and CSF, Lotem et al. ( 1980)recently demonstrated that D-factor (MGI-2) and CSF (MGI-1) present in serum of mice injected with endotoxin and in CM of lung or macrophages were separable by gel filtration. Furthermore, Lipton and Sachs ( 198 1) reported that CM of serum-free cultures of Krebs ascites tumor cells contained two different molecular species of CSF (MGI- 1) and D-factor (MGI-2). The CM

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of the Krebs ascites tumor cells contained one species of MGI-1 and two species of MGI-2, MGI-2A, and MGI-2B. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the MGI- 1 and MGI-2A activities were associated with a substance of similar molecular weight and each activity gave two bands, one of 23,000 and the other of 25,000. MGI-2B activity was associated with one band with a molecular weight of45,OOO. The production of MGI- 1 and MGI-2 did not appearto involveglywsylation and these compounds did not bind to lectins such as Con A, soybean agglutinin, or wheat germ agglutinin (Lipton and Sachs, 1981). MGI-2 was more readily inactivated by proteases and was more labile at high temperature and low pH than MGI-1 (Lipton and Sachs, 1981). These findings suggest that the general properties of MGI- 1 (CSF) and MGI-2 (D-factor) are similar to those of CSF and D-factor produced by L,,, cells (Yamamoto et al., 1981b). Furthermore, the results suggest that D-factor is not always associated with CSF and that there are at least two types of CSF with or without D-factor activity. However, the chemical properties of D-factor without CSF activity remain to be determined since the D-factor has not yet been purified. D-Factors for M 1 cells were also found in various body fluids containing CSF activity such as ascitic fluid (Hozumi et al., 1979a,b), serum of mice injected with bacterial endotoxin2 (Sachs, 1978a), saliva (Nakayasu et al., 1978),urine(Nakayasuetal., 1978),andamnioticfluid(Nagataetal., 1977). The presence of the D-factor in these body fluids is of special interest in connection with in vivo regulation of proliferation and differentiation of leukemia cells. Therefore, we examined the properties and origin of the D-factor in ascitic fluids of animals bearing tumors. Results showed that the D-factor is a heat-labile protein of heterogeneous molecular size ranging from about 10,000to 400,000, but about halfthe total activity was recovered in the fraction with a molecular weight of less than 50,000 containing a subfraction with an apparent molecular weight of 20,000 to 25,000 and a specific activity 300 times that of the original ascitic fluid (Hozumi et al., 1979a).We examined the mechanisms of production of D-factors in ascites and found that macrophages and granulocytes in the ascites, but not lymphocytes, were mainly responsible for production of the D-factor (Hozumi et al., 1979a,b). Other mouse myeloid leukemia cells, such as R453 cells and WEHI-3B cells, were also found to be induced to differentiate into macrophages and granulocytes by CM of various cells or body fluids containing CSF and D-factors for M1 cells (Ichikawa et al., 1976; Sugiyama et al., 1979a; Metcalf, 1980; Burgess and Metcalf, 1980a,b). The D-factors for R453 cells in ascitic fluid of rats were also proteinous substanceswith properties similar to those of the D-factors for M 1 cells (Sugiyamaet al.,1979a).However, it is still unknown whether the factors from the two sources are identical.

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Although differentiation of WEHI3B cells was induced by purified CSF from CM of lung (Metcalf, 1979), an active factor stimulating differentiation of WEHI3B cells in serum of mice injected with endotoxin had an apparent molecular weight of 23,000 and it was separated from most other components of CSF by several fractionation procedures (Burgess and Metcalf, 1980a;Nicolaand Metcalf, 1981).Part ofthe activefactor, however, was found to be associated with a subset of residual CSF molecules with a selective capacity to stimulate granulocyte colony formation by normal hematopoietic stem cells (Burgess and Metcalf, 1980a,b; Nicola and Metcalf, 1981). Sera from patients with acute myeloid leukemia were recently found to have activity to induce differentiation of WEHI3B cells (Metcalf, 1981). Although the activity was higher in infected patients, the activity was also detected in sera from uninfected patients. 6. Arginase and Histones. During investigations of the mechanisms of induction of differentiation of MI cells by D-factor, we found that two proteins with known chemical structures, arginase and a fraction of histone, H1, were inducers of MI cells (Okabe et al., 1979,1981). The effect of arginase on the induction ofdifferentiation of M 1 cells was found to be due to arginine depletion of the culture media: the leukemia cells did not differentiate in cultcre media containing arginine, but did differentiate into macrophages and granulocytes in arginine-deficient culture media (Okabe et al., 1979).Growth of M 1 cells was significantlyinhibited by treatingthe cells with arginase or culturing them in arginine-deficient medium, but the cells could not be induced to differentiate simply by inhibition of their growth with an inhibitor such as 5-fluorodeoxyuridine (FUdR) (Okabe et al., 1979). We found that M 1 cells could be induced to differentiate into macrophages and granulocytes by lysine-rich, histone H 1 fractions ( 10 to 100pg/ml) isolated from calf thymus, rat liver, and M 1 cells (Okabe et al., 1981). Histone H2A and H2B fractions did not induce differentiation of M 1 cells at concentrations of 10 to 100pg/ml but did induce differentiation at a high concentration (200pg/ml). Other basic polypeptides, such as the histone H3 fraction, poly-L-lysine, and poly-L-arginine, significantly inhibited induction of differentiation of M 1 cells (Okabeet al., 1981). The mechanismsof these effits of histones on induction of differentiationof M 1 cells are unknown, but may be due to the complex structural features of the histones, because, for instance, lysine-rich histone H 1 is an inducer of M 1 cell differentiation, but poly-L-lysine is not. 2. Lipids Both lipopolysaccharide (LPS)and lipid A from various bacteria could inducedifferentiationof M 1 cellsinto macrophagesand granulocytes(Sachs, 1978a). Although their mechanisms of induction were unclear, these com-

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TABLE I

INDUCTION OF DIFFERENTIATION OF M 1 CELLS AND

HL-60 CELLS BY SYNTHETIC ALKYLLYSOPHOSPHOLIPIDS~

Differentiation Alkyllysophospholipid chemical name ~

~~

(3-Tetradecyloxy-2-methoxy)propyl-2trimethylammonioethylphosphate (3-Octadecyl-2-methoxy)propyl-2trimethylammonioethylphosphate 3-Octadecyloxypropyl-2trimethylammonioethylphosphate (3-Tetradecyloxy-2-methoxy)propyl-2aminoethylphosphate (3-Tridecyloxy-2-methoxy)propyl-2trimethylammonioethylphosphate 3-Tetradecyloxypropy1-2trimethylammonioethylphosphate

MI cells

HL-60 cells

ST-024

+ + + +

ST-040

NTb

ST-04 1

NT

+ + + + + +

Abbreviation ~

ST-023 ST-008 ST-00 I

" From Honma el a/. ( I98 I b). NT, not tested.

pounds were found to induce differentiation of M1 cells indirectly by inducing protein inducer, MGI, in the cells (Sachs, 1978a). We examined the effects of alkyllysophospholipids, synthetic analogs of naturally occurring lysophospholipids,on induction of differentiation of M 1 cells and HL-60 cells. Both types of cells were induced to differentiate into morphologicallyand functionally mature granulocytes and macrophages by treatment with a wide variety of these compounds, as shown in Table I (Honma et al., 198 1b). Among these compounds, (3-tetradecycloxy-2methoxy)propyl-2-trimethylammonioethylphosphate(ST-023) was the most effective for induction of differentiation of both M 1 cells and HL-60 cells. This compound and some other alkyllysophospholipids induced differentiation of both M1 and HL-60 cells at concentration of 1 pg/ml. However, these compounds did not affect colony formation of normal mouse bone marrow cells even at a higher concentration of 20pg/ml. These results suggest that alkyllysophospholipids induce cell differentiation of myeloid leukemia cells without affecting growth or differentiation of normal bone marrow cells. Other lysophospholipids such as acyllysophospholipidshad no influence on cell growth or differentiation at the effective concentrations of their alkyllysophospholipid analogs (Honma et al., 198lb). On the other hand, 0-alkyllysophosphatidylethanolamineanalogs were as active as O-alkyllysophosphatidylcholine analogs on M 1 cells, but less active than the latter

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compounds on HL60 cells. Therefore, 0-alkyllysophosphatidylcholineanalogs are suggested to be the most suitable for inhibition of cell growth and induction of cell differentiationof myeloid leukemia cells. 0-Alkyllysophosphatidylcholineanalogs with a short aliphaticside chain had weak inhibitory effects on cell proliferation, but 0-alkyllysophospholipid with a C,,H,, aliphatic side chain had a marked effect on cell proliferation. These results show that the cytotoxic and growth-inhibitoryeffects of alkyllysophospholipids are separable from their effects in inducing cell differentiation. 3. Glucocorticoid Hormones, Prostaglandins,and CAMP Some glucocorticoid hormones also induced differentiation of M 1 cells into macrophages and granulocytes (Sachs, 1978a; Hozumi et al., 1979a). Dexamethasone, prednisolone, and hydrocortisone (optimal inducers) M, whereas another group caused maximal induction at 5 X lO-'-2 X of steroid suboptimal inducers including corticosterone, aldosterone, 1 1jlhydroxyprogesterone, 1 I -deoxycortisol,and 1 1-deoxycorticosteronedid not cause maximum induction, even when added at very high concentrations, but each induced a submaximal level (Hozumi et al., 1979a). All steroids with optimal inducer activity had three functional groups (1 ljl-OH, 17aOH, 2 1-OH), and the simplest optimal inducer was hydrocortisone. The inducibility of cell differentiationof the steroidswas closely associated with their effects on the specific cell-free cytoplasmic binding of ['Hldexamethasone (Hozumi et al., 1979a). Dexamethasone also induced differentiation of mouse myeloid leukemia R453 cells into mainly granulocytes (Sugiyama et al., 1979a), but not differentiation of human promyelocytic leukemia HL-60 cells (Y.Honma, K. Kasukabe, and M.Hozumi, unpublished data). On the other hand, glucocorticoid hormones such as hydrocortisone and dexamethasone (Scher et al., 1978, 1980; Santoro et al., 1978) and, to a lesser extent, aldosterone, corticosterone,and deoxycorticosterone inhibited induction of differentiation of Friend erythroleukemiacells (Scher et al., 1978, 1980). Analysis of glucocorticoid-mediatedinhibition revealed that both hemoglobin and globin mRNA synthesis were markedly inhibited without any cytotoxic effect (Scher et al., 1978). These results suggest that responses to glucocorticoid hormones differ in different types of leukemia cells. ProstaglandinE, ,D, ,and F, were all produced from [14C]arachidonatein an early stage of differentiation of M1 cells, but mature cells produced predominantly prostaglandin E, (Honma er al., 1980d).Indomethacincompletely inhibited the productions of these prostaglandins and markedly inhibited the induction of differentiation of M 1 cells by dexamethasone or D-factor from ascitic fluid of rats (Honma et al., 1980d). The indomethacinmediated inhibitionofcell differentiationwas counteracted by prostaglandin

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E, or E2,but not by prostaglandin F, or Fk (Hozumi et al., 1979a). Prostaglandin E stimulated differentiation of MI cells induced by a suboptimal concentration of dexamethasone, but prostaglandin F did not. Moreover, prostaglandins such as E, and E2and, to a lesser extent, A, and A2when added alone induced lysozyme activity in M1 cells (Hozumi et a/., 1979a). These findings show that the induction of prostaglandin synthesisis involved in the mechanism of differentiation of M 1 cells. Although cAMP markedly induced lysozyme activity in MI cells, dbcGMP or butyric acid did not (Hozumi et al., 1979b). cAMP or prostaglandin E alone had no effect on other differentiation-associated properties of M 1 cells, such as phagocytosis, migrating activity, and the morphology of the cells. Prostaglandin E stimulated phagocytosis, migrating activity, and changes in morphology induced by dexamethasone, but cAMP did not (Hozumi et al., 1979b). 4. Vitamins

The active form of vitamin D,, la,25-dihydroxyvitamin D,,was recently found to induce differentiation of M1 cells into macrophages (Abe et af., 1981) and HL-60 cells into granulocytes (Miyaura et al.. 1981). In MI cells, 1a,25-dihydroxyvitamin D, also induced several markers of differentiated cells, such as phagocytic, migrating, and lysozyme activities. la-Hydroxyvitamin D, had similar inducing activity, but 25-hydroxyvitamin D, and 24R,25-dihydroxyvitamin D, showed very weak activity (Abe et al., 1981). The relations between the mechanisms involved in induction of leukemia cells by these vitamin D, analogs and their well-known biological activities in enhancing intestinal calcium transport and bone mineral mobilization are unknown. Other vitamins, such as vitamin A and vitamin A analogs (retinoids) (Takenaga et al., 1980, 1981a), vitamin C (Takenaga et al., 1981a), and vitamin E (Sakagami ef al., 1981; Takenaga et al., 1981a) did not induce differentiation of M 1 cells but rather inhibited cell differentiation. 5 . Synthetic Polyribonucleotides and Interferons Some synthetic polyribonucleotides, such as poly(1) and poly(C), but not poly(A) and poly(U), induced differentiation of MI cells (Hozumi et al., 1979b).The inducing effects of these single-stranded RNAs were influenced not only by the specificity of nucleotides but also by their chain length. ,~ of 10 or 12 was active as an inducer, but poly(1) of Poly(1) with an s ~ , value 6.9 S was less active (Hozumi et al., 1979b). Double-stranded RNAs such as poly(1) poly(C) or poly(A) * poly(U) could not alone induce differentiation of M1 cells, but markedly enhanced induction of differentiation of M 1 cells by low concentrations of D-factor from mouse peritoneal macrophages and induced a significant amount

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of interferon in M1 cells (Hozumi et al., 1979b, 1982). Although slight interferon activity was detected in medium of poly(1)-treated M1 cells, no activity was induced by other single-stranded RNAs (Yamamoto et al., 1979). Simultaneous treatment of M 1 cells with anti-interferon serum, poly(1) poly(C), and D-factor abolished the enhancing effect of poly(1) * poly(C) on the action of the D-factor (Yamamoto et al., 1979). These results suggest that interferon produced from MI cells mediated the effect of the polynucleotide. We have confirmed that interferon, which was prepared from M1 cells treated with poly(1) poly(C) and purified by anti-interferon antibody column chromatography, did not itself induce differentiation of M 1 cells, but enhanced the induction ofdifferentiation by D-factor from mouse peritoneal macrophages, LPS, or poly(1) (Tomida et al., 1980a). Interferon did not stimulate differentiation of the cells by dexamethasone. Interferon alone could induce lysozyme activity in M 1 cells and the effects of interferon and D-factor or dexamethasone on induction of the lysozyme activity were synergistic (Tomida et al., 1980a). On the other hand, sera from mice given injections of poly(1) poly(C), containing interferon and D-factor, induced differentiation of all M 1 clone cells tested, including resistant clones (R-4and DR-3) that could not be induced to differentiate by D-factor alone (Tomida et al., 1980a). Lotem and Sachs ( 1978b)reported that interferon enhanced the induction of lysozyme in MI cells by dexamethasone or protein inducer, but had no effect on induction of C, rosettes, immune phagocytosis, or differentiation to mature macrophages or granulocytes. In contrast, we observed that interferon stimulated induction ofdifferentiation of our various clones of M 1 cells with different sensitivities to inducers. The reasons for this discrepancy between our results and those of Lotem and Sachs (1978a) are unknown, but may be mainly due to differences in the cell clones used. A polymer of adenosine diphosphate ribose, poly(ADP-ribose), could induce differentiation of M 1 cells into macrophages and granulocytes (Yamada et al., 1978). Although the mechanism of induction of differentiation of the cells by poly(ADP-ribose)is unknown, the polymer may change some physiological functions of the nuclei of the cells, since it has been detected in the nuclei and nuclear membranes ofthe cells by autoradiography (Yamada et al., 1978).

-

6 . Tunicamycin, Chloroquine,Bacillus Calmette-Guirin (BCG),and

Bacterial Cell Wall Skeletons Tunicamycin, a specific inhibitor of asparagine-linked glycosylation,was found by Nakayasu et al. (1 980) to induce functional and morphological differentiation of both MI cells and HL-60 cells. The inducing effect of tunicamycin on MI cells, however, may vary in different clones since

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tunicamycin could not induce differentiationof clone T-22 of M 1 cells even at a concentration of 1 ,ug/ml (Yamamoto et al., 1981b) although Nakayasu et al. ( 1980)reported that it was effectiveon M 1 cells at a concentration ofO. 1 to 1.Opg/ml. Chloroquine [7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline] has antiinflammatory activity, forms complexes with DNA, and inhibits DNA-dependent nucleic acid polymerase reactions. We found that chloroquine was an inducer of differentiation of M1 cells (Takenaga and Hozumi, 1980). Immunostimulants such as Mycobacterium bovis BCG, but not Corynebacterium parvum, and cell wall skeletons from Mycobacterium bovis and Nocardia rubra,but not from Propionibacteriumacnes,were also inducersof differentiation of M 1 cells (Maeda et al., 1980). 7. Other Compounds The effects of various other compoundson the induction ofdifferentiation of M1 cells have been examined by many investigators. Sachs (1978a) reported that dimethyl sulfoxide (DMSO), lectins, various compounds interacting with DNA, and X-ray irradiation induced some phenotypes of macrophages or granulocytes in some clones of M1 cells. Among the compounds tested, the protein inducer MGI was the only compound that induced all the changes to mature macrophages and granulocytes. Sachs and his collegues isolated MI cell clones responding differently to different inducersand showed that they had different cellular sites for MGI and other inducers (Sachs, 1978a). Ichikawa et al. (1975) and Nagata and Ichikawa ( 1979)detected no effect of various inhibitorsof DNA synthesison induction of differentiation of M 1 cells. We observed that W i n s (Con A), wheat germ agglutinin, Ricinus communis agglutinin, phytohemagglutinin (Yamamoto et al., 1980), DMSO (Hozumi et al., 1979b;Hozumi, 1982),and some chemicalsinteracting with DNA or inhibiting synthesis of DNA (Hozumi et al., 1979b;Hozumi, 1982) did not induce significantdifferentiation of some clones of M 1 cells that were sensitive to protein inducers. However, DMSO and some chemicals interacting with DNA (Hozumi et al., 1979b Hozumi, 1982) sensitized some clonesof M 1 cells that were resistantto inducers, causingdifferentiationwith these inducers. The compounds and the mechanisms of sensitization of the resistant M 1 cells will be described later. B. STIMULATORS OF PRODUCTION BY LEUKOCYTES OF D-FACTOR FOR MYELOID LEUKEMIA CELLS For induction of differentiation of myeloid leukemia cells in vivo, compounds stimulating production of D-factors and other modifiers of differen-

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tiation of leukemia cells seem of potential value as indirect inducers of cell differentiation in vivo. Some compounds were found to stimulate production of D-factor from leukocytes, although alone they could not induce differentiation of M 1 cells. Mouse peritoneal macrophages produced D-factor as described previously (Hozumi et al., 1979a) and treatment of the macrophages with either poly(1) * poly(C) or poly(A) * poly(U) enhanced their release of D-factor into the culture medium significantly, but treatment with poly(1) or poly(C) did not (Hozumi et al., 1979b; Hozumi, 1982). Synthetic N-acetylmuramyldipeptide(MDP), with the minimal structure required for adjuvant activity of bacterial cell peptidoglycan for increasing both humoral and cell-mediated immune responses (Azuma et al., 1976; Ellouz et a/., 1974; Taniyama and Holden, 1979), also enhanced the production of D-factor by a macrophage-like cell line, 5774.1, and differentiation of M 1 cells morphologically similar to macrophages, although MDP alone did not induce differentiation of M 1 cells (Akagawa and Tokunaga, 1980). We examined the effects of various compounds on production of D-factor from spleen cells, which are mostly lymphocytes and macrophages (Yamamot0 ef al., 1980,198la). Lectins (Con A, pokeweed mitogen, and phytohemagglutinin) stimulated spleen lymphocytes, but not spleen macrophages, to produce a D-factor with an apparent molecular weight of 40,000 to 50,000. In contrast, LPS and poly(1) poly(C) stimulated both spleen macrophages and spleen lymphocytes to produce D-factors. Although spleen macrophages produced D-factors with apparent molecular weights of both 40,000 to 50,000 and 20,000 to 25,000, spleen lymphocytes produced only the larger molecules. Immunostimulants such as the cell wall skeletons of Nocardia rubru and Propionibacterium acnes C7 and Corynebacterium parvum CN6 134 stimulated production of both spleen lymphocytes and spleen macrophages but none of these immunostimulants alone except Nocardia rubra and Mycobacterium bovis BCG could induce differentiation of M 1 cells, as described previously (Yamamoto et al., 198la). Furthermore, synthetic derivatives of N-acetylmuramyldipeptide (N-acetylmuramyl-L-valyl-D-isoglutamine and benzoquinon yl derivatives of N-acetylmuramyldipeptide), the minimal subunit of the bacterial cell wall with adjuvant activity, had no direct inducing effect on the differentiation of M1 cells and only slightly stimulated the production of D-factor by spleen cells (Yamamoto et al., 198la). The compounds that stimulated production of D-factor from spleen lymphocytes and spleen macrophages were also reported to induce production of various substances including interferon, CSF, and macrophage-activating factor (MAF) which might affect proliferation and differentiation of M 1 cells. We found that spleen lymphocytes treated with poly(1) poly(C) or Con A produced D-factor, CSF, and interferon (Yamamoto et a!., 1980). Although the activity of D-factor overlapped that of interferon with the same

-

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molecular size of about 40,000 to 50,000 on Sephadex G-75 or G-100 gel filtration, most of the CSF activity did not. The activity of D-factor was separated from that of interferon by treatment of the CM of lymphocytes with acid at pH 2.0, a treatment that reduced the activity of interferon only (Yamamoto el al., 1980). C.

BIOCHEMICAL PHENOTYPIC CHANGES ASSOCIATEDWITH MYELOID LEUKEMIA CELLDIFFERENTIATION 1. Membrane Components

During induction of differentiation of the mouse myeloid leukemia cell lines M 1, R453,and WEHI-3B, their morphological and functional phenotypes change into those of normal macrophages and granulocytes. Among these phenotypic changes in M 1 cells, changes such as migrating activity in soft agar, phagocytosis, adhesion to the substratum,agglutinabilityby Con A and Fc and C3receptors are suggested to be due to changes in membrane structure of the cells (Sachs, 1978a; Hozumi, 1982). We found that a glycoprotein with a molecular weight of 180,000(pl80) was induced in the cell surface of differentiated M 1 cells by protein inducers, dexamethasone, dbcAMP, or prostaglandin E, (Sugiyama et al., 1979b, 1980). The role ofp 180in the expressionofother phenotypesofdifferentiatedM 1 cells was examined. It was suggested that p 180 was involved in the mechanisms of cell-substrate adhesion and increase in agglutinability by Con A during differentiation of M 1 cells (Sugiyama et al., 1980). Pearlstein et al. (1978) found that an external membrane protein with a molecular weight of 195,000 on mouse peritoneal macrophages was related to cell-substrate adhesion. In addition, Trowbridge and Omary (198 1) reported that leukocyte surface glycoproteins bearing the macrophage differentiation antigen Mac- 1 could be detected on other murine hematopoietic cell types, including differentiated M 1 cells with Mac- 1 monoclonal antibody. They suggested that some of these surface glycoproteins were also involved in cell - cell or cell - substrate interactions. We examined the changes in the phospholipid compositions of two types of M 1 cells: sensitive M 1 cells that could be induced to differentiate into macrophages and granulocytesby various inducers, and resistant M 1 cells that could not differentiate even with high concentrations of the inducers (Honma et al., 1980~).Although there was no significant difference in the phospholipid compositions of these two types of cells, the percentage of phosphatidylcholine in total membrane phospholipids was less and the percentage of phosphatidylethanolamine was more in differentiated M 1 cells. Changes in the percentages of other phospholipids, such as lysophos-

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phatidylcholine and sphingomyelin,during differentiation were slight. The phospholipid composition of differentiated M 1 cells was similar to that of macrophages. Saito et al. (1980) reported that during differentiation of M 1 cells into macrophages induced by LPS, phospholipidssuch as sphingomyelin and phosphatidylserinedid not change significantly,but the amounts of phosphatidylcholine,phosphatidylethanolamine, and phosphatidylinositol increased markedly. We examined the change in phospholipid methylation during differentiation of M l cells (Honma ef al., 198l a). When M l cells were cultured with inducer, the incorporation of methyl groups into phosphatidylethanolamine decreased while the incorporation of choline into phosphatidylcholine increased significantly. The decrease of phospholipid methylation seemed to be partly due to a decrease of methyltransferase activity. These changes in phospholipid metabolism could alter the structure and function of the cell membrane (Hozumi, 1982). Saito et al. ( 1980) detected three major gangliosides in M 1 cells. During induction of differentiation of M 1 cells into macrophages with LPS, one ganglioside, a monosialoganglioside(GM 1b), increased markedly while the other two gangliosides, which were chromatographicallysimilar to GD3 and GD2 or GTla, decreased. The relative total amount of these major gangliosides remained unchanged. Akagawa et al. ( 1981) also reported that asialo GM 1, gangliotetraosylceramide, appeared on the cell surface of M 1 cells during differentiation induced by lymphokine-rich mouse sera. These changes in ganglioside composition of differentiated M1 cells might be related to some structural and functional alterations associated with cell differentiation.

2. Enzymes a. Lysosomal Enzymes. The activities of various lysosomal enzymes, such as lysozyme, acid protease, acid phosphatase, and 8-glucuronidase, were induced during induction of differentiation of M1 cells by various inducers (Sachs, 1978a; Kasukabe et al., 1979a; Hozumi, 1982). Of these lysosomal enzymes, lysozyme showed the most pronounced increase in activity. However, the activities of other lysosomal enzymes, such as acid DNase and acid RNase, in M1 cells were not induced significantly by dexamethasone (Kasukabe et al., 1977a). Although acid protease in M 1 cells was induced during differentiation,an alkaline protease, the main protease in untreated M 1 cells with maximum activity at pH 1 1.O, disappeared concomitantly (Oshima et al., 1979). This alkalineprotease could not be detected in a macrophage-likecell line (Mm-1) established from spontaneously differentiated macrophage-like cells from M 1 cells (Oshima et al., 1979).

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The biochemical properties of lysozyme produced by differentiated M 1 cells were compared with those of lysozymes produced by normal cells and tissues (Kasukabe et al., 1979a). Lysozyme purified from the culture medium of the macrophage-like cell line Mm- 1 cells had a molecular weight of 15,000 with an optimum pH of 6.6. The electrophoretic mobility of this lysozyme was distinctly lower than those of lysozymes from hen egg white and human urine. On the other hand, rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-inducedM 1 cells, but did not inhibit the activities of hen egg white or human preparations (Kasukabe et al., 1979a).The biochemical and immunochemical properties of lysozyme purified from normal mouse lung, which is rich in alveolar macrophages,were also similar to those of the purified lysozyme from Mm- 1 cells (Kasukabe et al., 1979a). These results show that the molecular structure of the lysozyme induced in differentiatedM 1 cells is similar to that of the lysozyme produced by normal cells. Use of different inducers showed that the induction of lysozyme was under separate control from those of other phenotypes, such as Fc and C, receptors and morphological changes to mature macrophages and granulocytes, in mutant clones of M1 cells (Sachs, 1978a; Hozumi, 1982). b. Prostaglandin Synthetases. We examined the activity of prostaglandin synthetase in M1 cells with [14C]arachidonate(Honma et al., 1980d). Although untreated M l cells could not convert arachidonateto prostaglandins, dexamethasone-treatedM 1 cells produced prostaglandin E2,D, ,and F, in an early stage of differentiation, whereas mature cells produced mainly prostaglandin E, , In differentiated M 1 cells, prostaglandin F, isomerase activity was suggested to be repressed in the process of prostaglandin synthesis (Honma et al., 1980d). The conversion of arachidonate to prostaglandins might be catalyzed by fatty acid cyclooxygenasesince it was completely inhibited by incubating an homogenate of dexamethasone-treated M 1 cells with indomethacin. Homogenates of LPS-treated MI cells also converted arachidonate to prostaglandin F,, E,, and D, ,whereas the particulate fraction of dexamethasonetreated M 1 cells produced prostaglandin F, and E, . These results suggest that prostaglandin D isomerase is present as a cytosolic component and that induction of prostaglandin synthesis during differentiation of M 1 cells results from induction of the activities of prostaglandin synthetases and stimulation of arachidonate from cellular phospholipids. The pattern of prostaglandin production by differentiated M1 cells was similar to that by mouse macrophages, except that the macrophages also released a little 6-oxoprostaglandinF,, whereas differentiated M 1 cells did not. c. Reverse Transcriptase. Dexamethasone induced differentiation of

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some clones of M 1 cells, but it did not induce an increase in the amount of reverse transcriptase activityin the culture medium or in the virus fraction in the culture medium of the cells. Since the cells produced a detectable level of the enzyme activity continuously (Kasukabe et al., 1977b), virus production in M 1 cellsdoes not seem to be associated with induction ofdifferentiationby dexamethasone. Liebermann and Sachs (1977) found that some clones of MI cells that could be induced by MGI to differentiate into mature macrophages and granulocytes (MGI+D+cells) produced a higher activity of reverse transcriptase in the culture medium, indicatingproduction of a higher amount of type c virus than clones in which induction ofdifferentiationby MGI was partially (MGI+D-) or almost completely (MGI-D-) blocked. They further indicated a specific pattern for ecotropic virus production during MGI-induced differentiation of MGI+D+-typeM 1 cells, involving enhanced virus production at an early stage of differentiation and interruption of Virus production in mature cells (Lieberman and Sachs, 1978). They also showed that infection of normal myeloblasts with ecotropic virus from MGI+D+-typeMI cells promoted proliferation of these myeloblasts, which could then still be induced to differentiate normally (Liebermann and Sachs, 1979), d. Cytochrome Oxidase, Glucose-6-phosphatase, and Lipogenic Enzymes. In differentiatedM1 cells induced by CM from secondary embryo cells, or an LPS, the number of mitochondria increased markedly with increase in activity of cytochrome oxidase per cell, although the activity per mitochondrion remained unchanged (Hirai et al., 1979). The rough-surfaced endoplasmic reticulum elongated and the activity of a marker enzyme of the reticulum, glucose-6-phosphataseY also increased in differentiatedM 1 cells. Furthermore, primary lysosomes with histochemically demonstrable acid phosphatase activity were found to be newly formed in the M1 cells (Hirai et al., 1979). Okuma et al. ( 1976) examined the synthesis of phosphatidic acid, a key intermediate in the synthesis of phospholipids and triglycerides in most animal tissues, from sn-glycerol3-phosphatein M 1 cells. They found that the microsomal fraction of M1 cells could catalyze acylation of sn-glycerol 3-phosphate by long-chain fatty acyl-CoA thioesters and could produce phosphatidic acid. But since M 1 cells and macrophages differentiated from M 1 cells had similar levels of sn-glycerol3-phosphate-acylatingactivity and of acetyl-CoA carboxylase activity, differentiation of MI cells is not associated with changes in the activities of these lipogenic enzymes.

3. Cytoplasmic Proteins Liebermann et al. (1980) analyzed changes in cytoplasmic proteins of various clones of mouse myeloid leukemia cells, including M 1 cells, in total

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cell extracts pulse-labeled with [35S]methioninefrom 1 hr to 6 days after addition of a protein inducer, MGI. Results showed that the sequence of protein changeswas similar in MGI-inducednormal and MGI+D+leukemic cells. Many proteins decreased before the appearance of de novo-synthesized proteins and differentiation of the cells was suggested to involve multiple, parallel, separately programmed pathways of gene expression that could be induced separately. These findingssuggest that there is a relation between the constitutive expression of certain pathways of genes in myeloid leukemia cells and cell competence for growth and differentiation in myeloid leukemia cells (Sachs, 1980, 1981). D. CHANGES IN PROLIFERATION POTENTIAL DURING DIFFERENTIATION OF MYELOID LEUKEMIA CELLS 1. Relation between the Cell Cycle and Commitment to Cell Diferentiation

The relation between differentiation and the cell cycle of M1 cells was examined with a protein inducer, CM of hamster embryo cells (Hayashi et al., 1982). A clone of M1 cells, B24, was induced to differentiate into macrophages by the protein inducer. When the M 1 B24 cells were treated with the protein inducer, the cells traversed the S phase of the cell cycle at least once. Then a fraction of the cells lost the ability to enter the S phase and accumulated in the G , phase. Incorporation of [3H]thymidinein phagocytosis-induced cells decreased after treatment with the inducer for 12 - 18 hr and the morphology of the cells changed in association with a significant decreasein the nucleus - cell ratio (NCR)of individual cells during treatment with inducer for 24 hr. The NCR was determined in M 1 B24 cells that had been prelabeled with [3H]thymidine,chased for various periods, and treated with the protein inducer for 24 hr. No significant difference was found between the NCRs of labeled and unlabeled cells. These results suggest that M 1 B24 cells in any phase ofthe cell cycle can respond to the protein inducer and can be initiated to differentiate. M 1 B24 cells treated with the protein inducer showed a lag time of about one cell cycle before arrest in G, (Hayashi et al., 1982). The kinetics of decreasein proliferation of M 1 cells differs from that of HL-60 cells or Friend cells. When HL-60 cells were induced to differentiate into macrophages by phorbol ester, most ofthe cells that were not in the S phase never entered the S phase, and there was a slight increase in cell number shortly after treatment with inducer (Rovera el al., 1980). In contrast, Friend erythroleukemiacells, which also had limited ability to proliferate after inducer treatment, showed four or five additional mitoses after treatment with inducer (Gusella et al.,

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1976; Friedman and Schildkraut, 1977). The discrepancies in these results may be due to differences in the cells, inducers, and differentiation pathways, as well as to differences in variety in the states in which cellulardifferentiation is blocked during leukemic transformation. The finding that there is no specific phase of the cell cycle at which induction of M1 B24 cell differentiation occurs seems consistent with previous observations by Ichikawa et al. (1975) that M 1 cells could be induced to differentiate under conditions when DNA synthesiswas inhibited by FUdR. In the human myeloid leukemia cells HL-60 cells and KG- 1 cells DNA synthesis was also shown to be unnecessary for development of phenotypic properties of macrophages such as phagocytic activity and a-naphthyl acetate esterase and acid phosphatase activities (Rovera et al., 1980; Territo and Koeffler, 1981).

2. Kinetics of Changes in Proliferation and Differentiationof Populations of Myeloid Leukemia Cells Regulation by humoral factors of growth and differentiation of various human and myeloid leukemic cells has been studied by quantitative determinations of biochemical and functional properties during differentiation of the cells (Sachs, 1978a; Burgess and Metcalf, I980b; Hozumi, 1982). These studies showed that the proportion of differentiating cells among the total cells depended on the concentration of inducer and that the cell population did not differentiate synchronously. Similar asynchronous differentiation was also observed in colonies of normal hemopoietic cells in semisolid agar as well as in clonal leukemic cell populations (Metcalf, 1977).However, the details of the kinetics of proliferation and differentiation of the myeloid leukemia cells and the mechanisms of induction of this asynchronous differentiation in the cells remain to be examined. We investigated the mechanisms regulating the kinetics of proliferation and differentiation of M 1 B24 cells by quantitative determination of cellular morphology (Hayashi et al., 1981). Results showed that the process of differentiation of M 1 B24 cells was promoted by increasing the concentration of inducer, protein inducer in CM of embryo cells, and that the transition of M1 B24 cells from the undifferentiated state to the differentiated state occurred in a stochastic manner and the proportion of well-differentiated cells in the whole cell population WPF higher at higher concentration of the inducer. The proliferative activity of Individual M 1 B24 cells, the labeling index of the cells with [3H]thymidine,decreased at a specificstage of differentiation at which the NCR of the cells was between 50 and 30Y0,and this decrease was independent of the culture time of the cells and the concentration of the inducer. No proliferative activity was observed in cells

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day 0

r J

0

2 la.

day 1

20

day 2

day 4

- 5%CM *

60 50 40 30

60 50 40 30 20

60 50 40 30 20

Nucleus - cell ratio ( */.

FIG. 2. Decrease in synthesis of DNA in MI B24 cells during morphological differentiation (Hayashi ef al., 1981). MI B24 cells were cultured with 2.5%conditioned medium (CM) of hamster embryo cells (upper figures) or 5% CM (lower figures) for the indicated times. The ratio of the area of the nucleus to that of the cell was determined on 100 randomly selected [3H]thymidine-labeled (stippled histogram) and unlabeled (open histogram) cells in each specimen.

in which the NCR had decreased below 30% (Fig. 2). These results suggest that the production of differentiated cells is controlled by a balance between proliferation and differentiation of the cells that is dependent on the concentration of the inducer. Ichikawa et al. (1969,1975) previously reported that high concentrations of protein inducer in CM from embryo cells or spleen macrophages suppressed the formation of colonies of M1 cells in agar medium. Recently, Metcalf ( 1980)found that the fraction of colony-formingcells in a WEHI-3B cell population gradually decreased and completely disappeared during serial recloning in the continuous presence of a protein inducer, postendotoxin mouse serum. In principle, the mechanisms controlling the kinetics of proliferation and differentiation of this cell population of leukemic cells may be similar to those of M1 B24 cells. Postendotoxin serum or CM of embryo cells, however, contain various factors affecting growth and differentiation of WEHI-3B or MI cells, as described previously. Therefore, further detailed studies are needed on the mechanisms controlling the proliferation and differentiation of these leukemic cells with purified factors for proliferation and differentiation ofthe cells.

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E. POSITIVEFEEDBACK CONTROLMECHANISMS OF CELL DIFFERENTIATION BY PROTEIN INDUCER PRODUCED BY DIFFERENTIATING MYELOIDLEUKEMIA CELLS M 1 cells could be induced by glucocorticoidsto produce a glycoprotein(s) with a molecular weight of 20,000 to 40,000 that stimulated induction of differentiation of M 1 cells into macrophages and granulocytes (Hozumi, 1982). Although glucocorticoids induced production of the glycoprotein inducer in M 1 cells that could be induced to differentiate by glucocorticoids, they could not stimulate the production of the glycoprotein inducer in dexamethasone-resistant M 1 cells that could not differentiate even with a high concentration of dexamethasone (Hozumi, 1982).Thus, production of the glycoprotein inducer was associated with differentiation of M 1 cells. We recently found that dexamethasone could also induce CSF for mouse bone marrow cells (Okabe-Kado et al., 1982). Induction of differentiation of M 1 cells by glucocorticoidswas suppressed by treatment with actinomycin D or puromycin, but not with FUdR or cytosine arabinoside (Ara C) (Hozumi, 1982).These findings suggest that the synthesis of some species of RNA and proteins may be required for the induction of differentiation of M 1 cells, although it is unknown whether the proteins synthesized mediate glucocorticoid-induced differentiation of M 1 cells. Some clones of M 1 cells were also recently found to produce a factor(s) inducing differentiation of M 1 cells, MGI, during differentiation of the cells by treatment of the cells with various compounds such as LPS (Sachs, 1978a), phorbol esters (Lotem and Sachs, 1979), and N-methyl-N-nitro& nitrosoguanidine (NMNG) (Falk and Sachs, 1980). The regulation of induction of two activities, MGI- I (CSF) and MGI-2 (D-factor), during differentiation ofM 1 cells induced by NMNG or LPS was examined (Falk and Sachs, 1980). Experiments on the time courses of induction of the activities of MGI-1 and MGI-2 by NMNG or LPS showed that MGI-1 was induced before MGI-2. Dexamethasone, however, did not induce either MGI- 1 or MGI-2, contrary to our findings (Honma ez al., 1982a; Hozumi, 1982), although a clonal variation in the inductions of MGI-1 and MGI-2 was found. These results show that the regulations of the inductions of MGI- 1 and MGI-2 in M 1 cellsare different and that the inducibilities ofthe two MGI activities also vary in different clones of M 1 cells. Maeda and Ichikawa ( 1980) also reported that CSF was produced by M 1 cells during differentiation induced by bacterial LPS, although no significant amount of D-factor was produced. Furthermore, they showed that the continued presence of LPS was necessary to stimulate the differentiated M 1 cells, macrophages, to release CSF, whereas a macrophage-like cell line

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(Mm-1) derived from the M1 line produced CSF without stimulation by LPS. F. INHIBITORS AND THEIR MECHANISMS OF INHIBITION OF DIFFERENTIATION OF MYELOID LEUKEMIA CELLS

1. ThymidineAnalogs, Actinomycin D, and Puromycin 5-Bromodeoxyuridine(BUdR) was reported to induce some phenotypes of a certainclone of M 1cells (Sachs, 1978a),but Nagata and Ichikawa (1979) showed that BUdR, 5-bromodeoxycytidine, and 5-iododeoxyuridine blocked the induction of phagocytosis and motility of M1 cells without affectingthe induction of Fc receptor. The blocking effect of BUdR on the induction of cellular phagocytosis and motility was prevented by the addition of excess thymidine and BUdR had no effect in a BUdR-resistant cell line (Nagata and Ichikawa, 1979). Therefore, the inducibilitiesof phagocytosis and motility of M 1 cells, but not the induction of the Fc receptor, are suggested to be controlled genetically. Actinomycin D at 30- 50 pg/ml markedly inhibited the induction of phagocytosis, but not that of the Fc receptor in M 1 cells, while puromycin at a concentrationof more than 2.5 X 10-6Mmarkedlyinhibited the induction of phagocytosis but slightly enhanced the induction of Fc receptor (Nagata and Ichikawa, 1979). These results suggest that new syntheses of messenger RNA and protein are required for induction of new phenotypic markers in M 1 cells and that the mechanisms controlling induction of Fc receptor are different from those controlling induction of phagocytosis. 2. Cytochalasin3 Cytochalasin B reversibly inhibited the induction by CM from mouse embryo cells of phagocytosisand motility of M1 cells (Ichikawa el al., 1975). This suggests that cytochalasin B-sensitive proteins containing actin, such as microfilaments,may be involved in the mechanisms of differentiation of M 1 cells. Nagata et al. ( 1980) found that polymerization of G-actin in the M 1 cells is closely associated with differentiation of the cells. Hoffman-Lieberman and Sachs (1978) also reported that there was a marked increase in the content of actin during differentiation of M 1 cells, although both untreated MI cells and mature macrophages and granulocytes contained actin as a major protein component.

3. Nonsteroidal Antiinfammatory Agents and B- and F-Type Prostaglandins Synthesis of E-type prostaglandins is involved in the mechanisms of differentiation of M 1 cells and nonsteroidalantiinflammatory agents such as

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salicylate, phenylbutazone, and indomethacin, that inhibit synthesis of these prostaglandins, blocked the induction of differentiation of M 1 cells by various inducers (Hozumi et al., 1979a). The inhibition was unrelated to cytotoxicity and was reversible. The inhibition by indomethacin of dexamethasone-induced differentiation was observed only when indomethacin w'as added before the time of commitment of the cells to differentiation. Although prostaglandins E, , E,, and D, induced lysozyme activity and stimulated differentiation of M 1 cells induced by a suboptimal concentraF inhibited the tion of inducer (Honma et al., 1979,1980d),prostaglandin , induction by dexamethasone of phagocytic and lysozyme activities in the F stimulated production of cells (Takenaga et al., 1982). Prostaglandin , differentiation-inhibiting activity (I-activity) in M 1 cells (Takenaga et al., 1982).The I-activity was found to be due to a heat-labile, trypsin-sensitive proteinous substance(s). B-Type prostaglandins also stimulated I-activity production, whereas A-, E-, and D-type ones did not. The induction of I-activity by prostaglandin F, was suppressed by simultaneous treatment with prostaglandin E, (Takenaga et af., 1982). 4. Retinoids and Phorbol Esters

Various retinoids other than the pyridyl analog of retinoic acid induced lysosomal activities in M1 cells (Takenaga et al., 1980). However, the retinoids did not induce phagocytic or migrating activity or morphological changes of MI cells and they reversibly inhibited the induction of these properties by various inducers (Takenaga et al., 1980).These findings suggest that there are distinct mechanisms for control of induction of lysosomal enzyme activities and of other differentiation-associated properties of M 1 cells. The retinoids could induce prostaglandins D, , E,, and F, in M 1 cells and the induction of prostaglandin E, was a prerequisite for increase in lysozyme activity (Takenaga, 198 l), whereas retinoic acid stimulated the productions of prostaglandin F,, and I-activity in M 1 cells, and indomethacin inhibited the I-activity by retinoic acid (Takenaga et al., 1981 b, 1982).It is , alone unknown whether I-activity is actually produced by prostaglandin F in M 1 cells treated with retinoic acid, since prostaglandin E,, which counteracts the production of I-activity in the cells, is also produced by retinoic acid. Furthermore, it is unknown whether the I-activity induced by retinoic acid is actually identical with that induced by prostaglandin F,, although this I-activity is heat labile and protease sensitive (Takenaga et al., 198 1 b). Tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol- 13acetate (TPA) have been reported to modify differentiation of various types of normal and tumor cells and cellular modification by phorbol esters is suggested to be involved in mechanisms of the promotion phase of carcinogenesis(Weinstein etal., 1979;Yamasaki, 1980;Hecker, 198 1). Although we found that TPA and other tumor-promoting plant diterpenes could inhibit

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induction of differentiationof M 1 cells into macrophages and granulocytes by dexamethasone or protein inducer (Kasukabe et al., 1979b), Lotem and Sachs (1979) and Nakayasu et al. (1979) reported that tumor promoters scarcely affected,or rather enhanced the induction of cell differentiation by some inducers. On examination ofthese conflictingresults, we found that the response of M 1 cells to TPA was affected by culture of the cells with different sera (Kasukabe et af., 1981; Hozumi et af., 1982): TPA inhibited both functional and morphological differentiation of MI cells cultured in medium containing calf serum or horse serum, as we reported (Kasukabe et al., 1979b), but it enhanced these inductions in medium containing fetal calf serum, as reported by Nakayasu et al. ( 1979)and Lotem and Sachs ( 1979). Therefore, these discrepant results on responses of the cells to TPA observed in previous studies were partly due to differences in the sera used: other workers used fetal calf serum but we used calf serum. The factor@)in the serum affecting the differentiationof M 1 cells with TPA was a nondialyzable macromolecule. On Sephadex G-200 gel filtration, much more inhibitory activity was found in calf serum than in fetal calf serum and stimulatory activity was found only in fetal calf serum (Hozumi et al., 1982). The modification by TPA of synthesis of prostaglandinE, was found to be closely associated with the effects of different sera on differentiation of M1 cells (Hozumi et al., 1982). Lotem and Sachs ( 1979) reported clonal differences in susceptibility to induction of differentiation by TPA. Hoffman-Liebermann et al. ( 1981) showed that TPA could complement changes in gene expression induced by MGI, and that TPA could regulate gene expression at the level of both mRNA production and mRNA translation. TPA and phorbol 12,13-didecanoateinhibited the induction of lysozyme activityby retinoic acid in M 1 cells, but 4a-phorbol didecanoateand phorbol did not. TPA and phorbol 12,13-didecanoate,but not 4cu-phorbol didecanoate, also inhibited the stimuIation of prostaglandin E, production by retinoic acid, suggestingthat stimulation by retinoic acid of prostaglandin E, production in M 1 cells is a prerequisite for induction of lysozyme activity (Takenaga, 1981). Both TPA and retinoic acid synergistically inhibited the induction of phagocytic activity in M 1 cells by dexamethasone (Takenaga, 1981).

5. Antioxidants Antioxidants, such as phenolic antioxidants, sulfur-containing compounds, a-tocopherol, retinoids, ascorbate, and selenium, have been reported to inhibit chemical carcinogenesis(Wattenberg, 1979;Griffin, 1979). The cellular mechanisms of inhibition of chemical carcinogenesis by these antioxidants are unknown, but have been suggested to involve modification

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of mutation (Batzingeret al., 1978;Calle et al., 1978; Rosin and Stich, 1979) and cellular differentiation (Hozumi, 1982). Therefore, we examined the effects of the antioxidants on differentiation of M1 cells. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and a-tocopherol significantly inhibited differentiation of the cells by dexamethasone or D-factor from ascitic fluid, but other antioxidants had little or no inhibitory activity. Of the antioxidants that were not cytotoxic, BHA was the most potent inhibitor (Takenaga et al., 198la). The inhibition ofdifferentiation of M 1 cells by BHA was reversible and was suggested to be due to inhibition of synthesis of E-type prostaglandins(Takenaga et al., 1981b). Several antioxidants were reported to inhibit prostaglandin synthesis and lipid peroxides formed by lipooxygenase were shown to activate prostaglandin synthesis (Hozumi, 1982). Of the antioxidants tested, hydrophobic antioxidants (BHA, BHT, and a-tocopherol) had more effect than hydrophilic ones (cysteamine, selenite, and glutathione) in inhibiting differentiation of the cells (Takenaga et al., 1981a). These findings suggest that the sites of action of the antioxidants on the cells may be related to their hydrophobicities.

6. Histone H3. Poly-L-lysine. and poly-L-arginine Although histone H 1 could induce differentiationof M 1 cells into macrophages and granulocytes, histone H2A and H2B could not (Okabe-Kado et al., 1981). On the contrary, the histone H3 fraction, poly-L-lysine, and poly-L-arginine markedly inhibited induction of differentiation of M 1 cells by dexamethasone (Okabe-Kado et al., 198I). The differencesin the mechanisms of effects of histone H1 and histone H3 as well as poly-L-lysine and poly-L-arginine require investigation. G. PROPERTIES OF MYELOID LEUKEMIA CELLSRESISTANT TO INDUCERS OF CELLDIFFERENTIATION Some populations of myeloid leukemia cell lines spontaneously become resistant to inducers of cell differentiation during long-term culture of the cells. These resistant cells were isolated by cloning in soft agar medium and their properties were examined. The isolation and characterization of these spontaneous resistant clones from M 1 cells and those ofresistant clones from myeloid leukemiasin X-ray irradiated SJL/J mice were reviewed extensively by Sachs (1978a, 1980, 1981). Results with these resistant clones show that there can be blocks at different stages of differentiation by various inducers and that there are separate controls for the induction of each phenotype of differentiation (Sachs, 1978a). Furthermore, studies on changes in the synthesis of specific proteins in normal myeloblasts, and various resistant

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clones of M 1 cells at different times after treatment with MGI suggested that the constitutive expression of some pathways of gene expression results in leukemia, whereas the constitutive expression of other pathways results in decreased competence for induction of differentiation of the leukemia cells (Sachs, 1980, 198I). Ichikawa er al. ( 1975)also isolated aclone (D-) that was resistant to protein inducer in CM of mouse embryo cells and his group examined its properties. Nagata er al. ( 1980) showed that actin harvested from CM-treated differentiating M 1 cells could polymerize, but could not polymerize with the actin from the D- clone. Moreover, Mae& and Ichikawa (1980) found that bacterial LPS could stimulate production of CSF from differentiation-sensitive M 1 cells but not from the D- clone of M 1 cells. We isolated several variant clones that were resistant to either dexamethasone (DR-1 to DR-6, six clones) or protein inducer (D-factor) in ascitic fluid ofratsbearinghepatoma(R,R1, R2, R15, andRl8)(Hozumier al., 1979a). The dexamethasone-resistant clones did not have a defect in penetration of glucocorticoidsor in cytoplasmic receptor binding in the cells. However, the number of nuclear receptor binding sites of [3H]dexamethasone in the resistant clones was markedly less than in sensitive clones of MI cells (Hozumi et al., 1979a). Sachs and his co-worker also islated dexamethasone-resistant clones of M 1 cells, but found that the lack of response of these resistant clones to dexamethasone was not due to any defects in the binding of dexamethasone to cytoplasmic or nuclear receptor sites (Sachs, 1978a). We examined differences in sensitivitiesof the resistant clones of M 1 cells to glucocorticoids and protein inducer in ascitic fluid from rats (Hozumi et al., 1979a). Although all the dexamethasone-resistant clones showed less response than the sensitivecells to the protein inducer, some clones that were resistant to induction with the protein inducer were induced to differentiate by dexamethasone. Therefore, the steroid and the protein inducer have different targets in the cells. The M 1 cell clones that were resistant to the protein inducer or dexamethasone, but not the sensitive clones, were found to produce an inhibitory activity (I-activity)for induction of differentiation of M 1 cells (Okabe er al., 1978). The I-activity was due to a nondialyzable, heat-labile, and proteasesensitive substance(s) that was precipitated at 30- 50% saturation of ammonium sulfate (Okabe er al., 1978). The I-activity in the culture medium of the resistant cells was decreased by treatment of the cells with a low concentration (5 - 10 ng/ml) of actinomycin D (Okabe er al., 1978). During decrease of their I-activity, the resistant cells became sensitive to the protein inducer in ascitic fluid. Therefore, production of I-activity in the resistant cells was closely associated with resistance of M 1 cells to the D-factor.

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The CM from resistant M1 cells was recently found to inhibit colony formation of bone marrow cells of normal mice by CSF. This inhibitory activity ofthe CM also decreased on treatment ofthe resistant cells with a low concentration of actinomycin D (Okabe-Kado et al., 1982). It is unknown, however, whether the inhibitory activity against normal bone marrow cells is identical with that against leukemia M 1 cells. Normal hematopoiesis has been found to be negatively regulated by inhibitors from various sources (Broxmeyer and Moore, 1978; Broxmeyer et al., 1981;Moore, 1979).It will be interesting to examine the relation between the actions of these inhibitors and that of the I-activities from resistant M 1 cells.

H. SENSITIZATION OF MYELOID LEUKEMIA CELLSRESISTANT TO INDUCERS OF CELLDIFFERENTIATION 1. A41 Cells

We examined the effects of various compounds on sensitization of resistant clones of M1 cells to inducers of cell differentiation. Inhibitors of RNA or protein synthesis were found to induce differentiation of resistant cells in the presence of the protein inducer in ascitic fluid, although they had no effect alone. In contrast, inhibitors of DNA synthesis, such as Ara C and FUdR, had no effect (Hozumi et al., 1979a). Inhibitors of RNA synthesis (actinomycin D, chromomycin A3, nogalamycin, and cordycepin) were effective at low concentrations that scarcely affected cell viability. Of these inhibitors, actinomycin D was the most effective. The sensitizing effect of actinomycin D on the resistant cells (R1 or R2) was found to be roughly parallel to the extent of its inhibition of RNA synthesis in the cells without affecting synthesis of DNA (Hozumi et al., 1979a). We found that the I-activity, protein(s), in CM of the resistant clone of M 1 cells decreased with development of sensitivity to the inducer, suggesting that production of the I-activity in the resistant cells was associated with resistance of the M 1 cells to the inducer (Okabe et a!., 1978). Some cancer chemotherapeutic drugs (adriamycin, daunomycin, mitomycin C, hydroxyurea, 5-fluorouracil, and bleomycin), which might also inhibit the production of I-activity in the resistant M 1 cells, induced differentiation of the resistant cells in the presence of D-factor in ascitic fluid (Hozumi et al., 1979b). The drugs or D-factor alone had no effect. In combination with the D-factor, 6-mercaptopurine, amethopterin, or aminopterin could not induce differentiation of resistant cells. On the other hand, Sachs (1978a) reported that several cytotoxic chemicals, including some anticancer drugs, and X irradiation alone could induce

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some phenotypic markers in differentiated cells from some sensitive clones of M 1 cells. These results suggest that some of the cytotoxic anticancer drugs alone or in the presence of an inducer of cell differentiation control tumor growth not only by their cytotoxic effect, but also by their ability to induce differentiation of the cells. Poly(1) poly(C), which could induce interferon in M1 cells, markedly stimulated differentiationof a resistant clone of M 1 cells, R4,in the presence of CM of macrophages, although CM or poly(1) - poly(C) alone induced scarcely any differentiation of the cells (Yamamoto et al., 1979). Sera obtained from SL strain mice injected with poly(1) poly(C) could induce differentiation of both sensitive and resistant clones of MI cells, since the sera contained both D-factor and interferon that could sensitizethe resistant M 1 clone cells to the D-factor (Tomida et al., 1980a,b).

-

2. R453 Cells R453 cells were induced to differentiate into macrophages and granulocytes by protein inducer in ascitic fluids, CM from various cell lines, and glucocorticoid hormone (Sugiyama et al., 1979a). This induction of differentiation of R453 cells was markedly enhanced by addition of inhibitors of the synthesis of RNA (actinomycin D, chromomycin A3, nogalamycin), protein (puromycin),or DNA (Ara C, FUdR, methotrexate, hydroxyurea)in the presence of ascitic fluid (Sugiyama et al., 1979a). Of the inhibitors, actinomycin D was the most effective, although the inhibitors alone had no effect. IV. In Vivo Induction of Differentiation of Mouse Myeloid Leukemia Cells and Therapy of Animals Inoculated with Myeloid Leukemia Cells

A. In Vivo INDUCTION OF DIFFERENTIATION OF MOUSE MYELOID LEUKEMIA CELLS

Based on the results of in vitro experiments on induction of differentiation of myeloid leukemia cells, we tried to induce in vivo differentiationof mouse myeloid leukemia M 1 cells. We used two types of clones of M 1 cells, clones that were sensitive and resistant to inducers of differentiation, to investigate the relationshipbetween cell competencefor induction ofdifferentiationand changes in leukemogenicity (Honma et al., 1978). Although no detectable difference in the saturation densities or growth rates ofsensitive and resistant cells in culture was observed in vitro, the sensitivecellsgrew more slowly than the resistant cells in diffusion chambers in syngeneic SL mice. The sensitive cells were induced by some endogenous factors to differentiate into mature macrophages and granulocytes losing their proliferation potentials while the resistant cells in the diffusion chambers remained undifferentiated (Honma et al., 1978).

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The leukemogenicities of resistant clones and sensitive clones of M 1 cells were examined in syngeneic SL mice. All the resistant clones were much more leukemogenic than the sensitive cells, and the survival times of syngeneic mice inoculated with them were less than those of mice inoculated with the sensitiveclones (Honma et al., 1978).These findings suggest that the leukemogenicityof M 1 cells in syngeneicmice is related to in vitro and in vivo inducibility of differentiation of the cells. Lotem and Sachs (1978a) also examined the in vivo inducibility of differentiation of several clones of mouse myeloid leukemia cells that differed in their competence to differentiate into macrophages and granulocytes with protein inducer. A sensitive clone, MGI+D+,as well as a resistant clone, MGI+D-, that did not form mature cells with protein inducer in vitro were both induced to differentiate into mature cells in diffusion chambers in normal syngeneic or allogeneic mice. Another resistant clone, MGI-D-, which could not be induced by the protein inducer to develop any differentiation-associated properties in vitro, was also induced in vivo to show C3and Fc receptors, lysozyme, and intermediate stages of morphologically differentiated cells, but not to differentiate into mature cells. The reasons for these differences between the inducibilities of cell differentiation in vitro and in vivo remain to be examined. Although diffusion chambers are useful in studies on differentiation in vivo, they prevent cell-to-cell interaction of inoculated leukemia cells with host immunocompetent cells. To examine the relation between leukemogenicity and in vivo inducibility of differentiation of the cells under natural conditions without any artificial bamers we labeled MI cells in vitro with [3H]thymidine and injected them into the peritoneal cavity of a syngeneic mouse. After several days the peritoneal cells were harvested and the 3H-labeledcells were determined by autoradiography. Results showed that the inoculated isotope-labeled M 1 cells differentiated into macrophages and granulocytes in the peritoneal cavity of the syngeneic mouse (Honma et al., 1982b). An inducer of cell differentiation, LPS, significantly stimulated differentiation of the isotope-labeled cells inoculated into the peritoneal cavity. These results provide direct evidence that M 1 cells can be induced to differentiate in vivo under conditions in which leukemia can develop.

B. THERAPY OF MICEINOCULATED WITH MYELOID LEUKEMIA CELLS BY INDUCTION OF DIFFERENTIATION I . Sensitive M I Cells The effects of LPS and glucocorticoids,two potent inducers of differentiation of M l cells, on the leukemogenicities of sensitive or resistant M l cells were examined (Honma et al., 1978, 1979).The inducers of cell differentia-

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TABLE I1 RELATIONSHIP BETWEEN in Vivo INDUCTION OF DIFFERENTIATION OF MI CELLS AND PROLONGATION OF SURVIVAL TIMES OF SYNGENEIC SL MICE INOCULATED WITH M 1 CELW

M I cell clone

Differentiation

Prolongation of survival times of mice

Protein inducer (MGI)

Clones sensitive to inducer DS4 DS4 T-22 MGI+D+

+ + + +

+ + + +

Lipopolysaccharide Lipopolysaccharide Actinomycin D POlY(1) * PolY (C)

Clones resistant to inducer DR3 R- 1 R- I R-4

-

-

+

+

Compound

Lipopolysaccharide Dexamethasone POlY(1)

*

POIY (C)

+

+

,,FromHonmaelal. (1978,1979,1980b),LotemandSachs(1978,198l),Okabeefal.(1979), and Tomida et al. ( i980b).

tion significantly enhanced the survival times of mice inoculated with sensitivecells, but scarcely affectedthe survivaltimes of mice inoculated with resistant cells (Table 11). These effects are consistent with the effects of the inducers on induction of differentiation of sensitiveand resistant cells in vivo and in vitro, suggesting that prolongation of the survival time may be associated with stimulation by the inducers of in vivo cell differentiation. The sensitive and resistant M1 clone cells contained similar common tumor-related surface antigens (Honma et al., 1978, 1979). LPS enhanced the survival times of immunodeficient newborn syngeneic SL mice and athymic nude mice inoculated with the sensitive M 1 cells to similar extents (Honma et al., 1978, 1979). Therefore, T-lymphocyte-mediated immune mechanisms may not be directly involved in the effect of LPS on prolongation of the survival time. The survival times of syngeneic SL mice were prolonged by dexamethasone at an optimal dose (20pg/mouse) but shortened by dexamethasone at a high dose (400 &mouse) (Table 11). Similar results were obtained with prednisolone, indicating that glucocorticoids, at an optimal dose, prolonged the survival times of syngeneic SL mice inoculated with sensitive M 1 cells (Honma et al., 1978, 1979).

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Intraperitoneal injections of protein inducers from various CMs or cells producing protein inducers into syngeneic SL mice were found to induce differentiation of sensitive M 1 cells, MGI+D+clone 9, in diffusion chambers in the mice (Lotem and Sachs, 1978a).On the other hand, Lotem and Sachs ( 1981) showed that CM from Krebs ascites tumor cells, which contains both inducer for differentiation of M 1 cells and inducer for colony formation of normal myeloblasts, CSF, significantly inhibited the development of myeloid leukemia by M 1 cells and stimulated normal myelopoiesis (Table 11). The CM could enhance the antitumor effect of cyclophosphamide (Lotem and Sachs, 1981)suggestingthat the protein inducer and cytotoxic antitumor drug had synergistic therapeutic effects on myeloid leukemia. 2. Resistant MI Cells Although sensitive M1 cells could be induced to differentiate in vivo by inducer alone, resistant M 1 cells could not. Therefore, we examined the in vivo effect of actinomycin D on differentiation of the resistant M 1 cells since actinomycin D was most effective in vitro in sensitizing the resistant cells to inducers of differentiation. Resistant clone R- 1 cells were treated in vitro with actinomycin D, and then washed and introduced into diffusion chambers (Okabe ef af.,1979). When the chambers were implanted into syngeneic SL mice, the untreated resistant cells remained undifferentiated, whereas the actinomycin D-treated cells differentiated into macrophages and granulocytes (Fig. 1 and Table 11). Then, we examined the effect of actinomycin D on differentiation of resistant M 1 cells in diffusion chambers implanted into syngeneic SL mice (Okabe et al., 1979). As expected, the cells in the chambers in mice treated with low doses of actinomycin D were induced to differentiate, but most of the resistant cells in the chambers in untreated mice remained undifferentiated (Table 11). These findings show that treatment with actinomycin D either in vitro or in vivo sensitizes resistant cells to endogenous inducers. Next, we examined the in vivo effects of actinomycin D (sensitizer) and LPS (inducer) on differentiation of resistant M 1 cells in diffusion chambers in syngeneic SL mice (Honma et al., 1980b). The resistant M 1 cells showed marked induction of differentiation on these treatments. Lipopolysaccharide alone had scarcely any effect on the survival of syngeneic SL mice inoculated with resistant cells, but LPS plus actinomycin D significantly prolonged their survival time (Honma et al., 1980b).Administration of LPS plus actinomycin D also prolonged the survival of athymic nude mice inoculated with resistant M1 cells (Honma et al., 1980b). These findings suggest that the effects of the drugs may not be directly related to T-lymphocyte-mediated immune responses and that combination therapy with the

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inducer and its sensitizer for cell differentiation is definitely more effective than therapy with the inducer alone. Double-stranded RNA, poly(1) poly(C), not only enhanced the sensitivity of M1 cells to D-factor (Yamamoto et al., 1979), but also stimulated production of D-factor by mouse peritoneal macrophages (Hozumi et al., 1979a). We examined the effects of poly(1) poly(C) and other doublestranded RNAs on the survival times of syngeneic SL mice inoculated with sensitive and resistant clones of MI cells (Tomida et a)., 1980b).A11 the mice inoculated with one sensitive (T-22)and two resistant clones (R-4, DR-3) of M1 cells died 20 to 40 days after inoculation. However, treatment with poly(1) poly(C)greatly increased the survival times of mice inoculated with these three clones ofMl cells (Table 11). Treatment with poly(A) poly(U) or poly(1) was far less effective than treatment with poly(1) poly(C) in suppressing leukemia but slightly prolonged the survival time of mice inoculated with the sensitive M1 cells (Tomida et al., 1980b). On injection of poly(1) poly(C) into mice, their serum levels of interferon activity and D-factor activity increased markedly within 3 hr. Interferon disappeared more rapidly than D-factor activity, which remained at a significant level for 72 hr after treatment. The effect of poly(1) poly(C) on in vivo induction of differentiation of MI cells was examined by the diffusion chamber method. Although the induction of differentiation of the sensitive and the resistant clones of MI cells in untreated mice varied, all the clonal cells in mice treated with poly(1) poly(C) differentiated markedly into mature macrophages and granulocytes(Tomida et al., 1980b).These results suggest that stimulation of differentiation of M1 cells is one mechanism of inhibition of leukemogenicity of the cells, but that the cytotoxic activities of poly(1) poly(C) and interferon on leukemia cells and the immunopotentiating activity of poly(1) poly(C) may also be involved in the effect (Hozumi, 1982).

-

-

V. Induction of Differentiationof Cultured Human Myeloid Leukemia Cells

Several cultured human myeloid leukemia cell lines, such as HL-60, K562, KG- 1, ML-1, and ML-3 cells, were recently found to be induced by various compounds to differentiate into macrophages, granulocytes, or erythroid cells. Some primary cultured leukemic cells from patients with myeloid leukemia were also induced by some compounds to differentiate into mature leukocytes. The inducers, phenotypes of the differentiated leukemic cells, and mechanisms of differentiation of the leukemic cells are described below.

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A. CULTURED MYELOID LEUKEMIA CELLLINES 1. HL-60 Cells

a. Inducers of Cell Diflerentiation. i. Proteins. The presence of protein inducers of differentiation of HL-60

cells and the relationship between the protein inducer and CSF were examined recently. Although HL-60 cells proliferated with CSFs from various sources, they were not induced to differentiate by CSFs (Gallo et al., 1979; Ruscetti et al., 1981). On the other hand, Olsson et al. (1981) reported that human mononuclear blood cells stimulated with various mitogens such as Con A, pokeweed mitogen, or protein A produced CSF and protein inducer@) for HL-60 cells with apparent molecular weights of 40,000 and 25,000. However, at least the protein inducer with the molecular weight of 40,000 was distinct from the CSF, which was produced simultaneously. The protein inducer could induce differentiation of HL-60 cells into cells with phagocytic activity, ability to reduce Nitro Blue Tetrazolium, and the morphologic characteristic of granulopoietic or myelomonocytic cells. Conditioned medium from the 2-mercaptoethanol-treated mononuclear leukocyte fraction of normal human peripheral blood (Elias et al., 1980)and CM from human allogeneic lymphocytes (Chiao et al., 1981) or phytohemagglutinin-stimulated lymphocytes (Lotem and Sachs, 1979; Chiao et al., 1981) also induced HL-60 cells to differentiate into macrophage-like cells. Although inducers for HL-60 cells have mainly been found in CM of human leukocytes, we recently found that CM of mouse myeloid leukemia M 1 cells treated with phytohernagglutinin and 2-mercaptoethanol could induce HL-60 cells to differentiate into monocytes or macrophages (Tomida et al., 1982).The factorsin the CM inducingdifferentiation have not yet been characterized. Conditioned medium of HL-60 cells has no activity to induce differentiation or stimulate growth of colonies ofthe same HL-60 cells in medium with agar or methylcellulose (Ruscetti et al,, 1981), but the CM from cultures of HL-60 cells at high cell density stimulates growth of the cell in liquid culture medium (Brennan et al., 1981). The material with this activity for autostimulation of growth of HL-60 cells gave a single peak on Ultrogel AcA.54 with an apparent molecular weight of 13,000. Arginase, a protein inducer for differentiation of MI cells, also induced HL-60 cells to differentiate into macrophages and granulocytes (Honma et al., 1980a). The induction of differentiation by arginase was significantly inhibited by excess arginine, but not by lysine or leucine, suggesting that the effect of argmase may be due to arginase-mediated arginine depletion.

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Histone H 1, another protein inducer of M1 cells, could not induce differentiation of HL-60 cells (J. Okabe-Kado, Y. Honma, and M. Hozumi, unpublished data). ii. Inducers of diferentiation of murine leukemic cells and cancer chemotherapeutic compounds. The effects of various other inducers of differentiation of murine leukemia cells besides proteins on differentiation of HL-60 cells were examined. Collins et af.(1980) found that polar compounds, such as hexamethylene bisacetamide and DMSO, certain purines, particularly hypoxanthine, and actinomycin D were potent inducers of differentiation of HL-60 cells. However, no significant induction of differentiation of the leukemic cells was observed with hemin, ouabain, prostaglandin E, , X irradiation, dexamethasone, or some other antitumor drugs, such as adriamycin, daunomycin, Ara C, vincristine, and hydroxyurea. They suggested from these results that human HL-60 cells have common cellular target sites with Friend erythroleukemia cells for the inducing action of the polar planar compounds hypoxanthine and actinomycin D, and that the other compounds tested might be specific to murine leukemia cells such as Friend erythroleukemia cells or MI cells, but not to HL-60 cells (Collins et al., 1980). L-Ethionine, another inducer of differentiation of Friend erythroleukemia cells, was reported to induce differentiation of HL-60 cells into granulocytic cells (Mendelsohn et al., 1980). Lotem and Sachs (1980) also examined the effects of various cancer chemotherapeutic agents on induction of differentiation of HL-60 cells. They found that the properties of these compounds in inducing Fc and C, receptors on HL-60 cells were in the following order: actinomycin D > Ara C > mitomycin C > adriamycin > BUdR > hydroxyurea. Furthermore, all the compounds except BUdR induced lysozyme with the same order of effectiveness as Fc and C3 receptors, but only actinomycin D and BUdR induced differentiation of HL-60 cells into mature granulocytes. Bodner et al. (198 1) tested the abilities of various purine and pyrimidine analogs and methotrexate to induce differentiation of HL-60 cells. They found that during 6 days treatment, 3-deazauridine induced nearly all the cells to differentiate. Pyrazofurin, virazole, puromycin aminonucleoside, and the tricyclic nucleoside 3-amino- 1,5-dihydro-5-methyl-1-P-Dribofuranosyl1,4,5,6,8-~entaazaacenaphthylene induced differentiation of 44 - 64%of the cells, whereas 5-azacytidine, BUdR, 5-iododeoxyuridine, thymidine, and methotrexate induced differentiation of 28 - 36Yo of the cells. Of these inducers, methotrexate (IO-*M) was the most potent in terms of its effective concentration. After treatment with all ofthese compounds the predominant cell types were metamyelocytes and banded neutrophilic granulocytes. Inducers of differentiation of M 1 cells, such as tunicamycin (Nakayasu et al., 1980), alkyllysophospholipids (Honma et af., 198lb), and la,25-dihy-

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FIG. 3. Morphology of differentiatedHL-60 cells induced by treatment with alkyllysophospholipid (ST-023) (Honma et al., 1981b). (a) Untreated cell. (b-d) HL-60 cells treated with ST-023 (4 pg/ml) for 7 days. May-Griinwald-Giemsa stain.

droxyvitamin D, (Miyauraet al., 1981) also induced differentiationofHL-60 cells (Fig. 3 and Table I). Tunicamycin and la,25-dihydroxyvitamin D, induced the leukemia cells to differentiate mainly into mature granulocytes, but alkyllysophospholipidsinduced the cells to differentiate into both mature macrophages and granulocytes. Retinoids, which induced various lysosomal enzymes in M 1 cells and inhibited differentiation of M 1 cells by other inducers (Takenaga et al., 1980, 1981b), were found to be potent inducers of differentiation of HL-60 cells (Breitman et al., 1980; Honma et al., 1980e).Retinoic acid and related retinoids, but not the pyridyl analog of retinoic acid, induced HL-60 cells to phagocytize, reduce Nitro Blue Tetrazolium, and change into forms that were morphologically similar to mature granulocytes. iii. Tumor promoters. Huberman and Callahan (1 979) observed, on the basis of morphological and functional changes, that HL-60 cells were terminally differentiated by tumor-promoting phorbol esters. Rovera et al. ( 1979a,b, 1980)and Lotem and Sachs ( 1979)showed that TPA could induce HL-60 cells to differentiate into cells with macrophage-like morphology, ability to adhere to plastic, increased activities of NADase and a-naphthyl

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acetate esterase, decreased activities of peroxidase and chloroacetate esterase, and synthesis of acid phosphatase with the typical isozyme pattern of monocytes. Furthermore, Todd et al. (198 1) recently reported that TPA could induce normal monocyte -macrophage differentiation antigens (Mo 1 and Mo2) in HL-60 cells. In HL-60 cells, TPA was also found to induce several properties conimon to both granulocytes and macrophages, such as phagocytosis of IgG-coated erythrocytes, increased activity of lysozyme, and decreased activity of myeloperoxidase (Lotem and Sachs, 1979; Rovera et al., 1979a,b). Although TPA-induced HL-60 cells developed the morphological appearance and enzymatic characteristics of macrophages, they did not show increase in hexose monophosphate shunt activity, superoxide generation, Nitro Blue Tetrazolium reduction, bacterial ingestion, or complement secretion above the uninduced levels (Newberger et al., 1981). New tumor promoters, such as teleocidin from Streptomyces mediocidicus, its catalytically hydrogenated compound dihydroteleocidin B, and lyngbyatoxin A isolated from the marine blue-green alga Lyngbya majuscula, as well as its hydrogenated product, tetrahydrolyngbyatoxin A, were found to induce differentiation of HL-60 cells into macrophage-like cells (Nakayasu et al., 1981). As described before, the effect of TPA on differentiation of mouse myeloid leukemia M1 cells was affected significantly by the type of serum in the culture medium of the cells. Therefore, we examined the effect of serum on differentiation of HL-60 cells (Honma et al., 1982a).HL-60 cells which grew in serum-free synthetic medium supplemented with insulin, transferrin, and several trace elements were induced by TPA to differentiate into macrophage-like cells. Addition of serum inhibited the induction of differentiation of HL-60 cells that had been grown in serum-free medium. Calf serum was more inhibitory than fetal calf serum on TPA-induced differentiation, but it had an effect similar to the latter on the inductions by actinomycin D or arginase. These results suggest that different responses in media with different sera may be specific to TPA. iv. Enhancement of inducer activity on HL-60 cells by other compounds. Although inhibitors of prostaglandin synthesis such as indomethacin and aspirin had no inhibitory effect on retinoic acid-induced differentiation of HL-60 cells, addition of prostaglandin E2 or El was found to stimulate induction of differentiation of HL-60 cells by retinoic acid (Breitman, 1982). The prostaglandin E2and El alone did not induce differentiation of the cells, and other prostaglandins, either alone or in combination with retinoic acid, were much less active than E-type prostaglandins in inducing differentiation of HL-60 cells (Breitman, 1982). Since CAMP is reported to be involved in the mechanisms of various

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157

cellular actions by prostaglandin E, the effects of CAMP and theophylline, an inhibitor of CAMP phosphodiesterase, on differentiation of HL-60 cells were examined (Breitman, 1982). Addition of theophylline alone or in combination with retinoic acid or dbcAMP increased intracellular cAMP to a relatively small extent in HL-60 cells. Treatment ofthe cells with dbcAMP in combination with retinoic acid or prostaglandin E2 resulted in increase in differentiation of the cells that appeared to be synergistic in the case of retinoic acid and additive in that of prostaglandin E,. Furthermore, the intracellular level of cAMP in HL-60 cells was found to be markedly enhanced by addition of prostaglandin E. These findings suggest that cAMP is involved in the mechanisms of stimulation of differentiation ofHL-60 cells by retinoic acid and prostaglandin E. Interferon alone did not induce differentiation of M 1 cells, but it markedly enhanced induction of differentiation by various inducers (Tomida et al., 1980a). Therefore, we examined the effect of interferon on differentiation of HL-60 cells. We found that human interferon-a and interferon-/Icould enhance induction of cell differentiation by the CM of phytohemagglutinin-treated M1 cells, TPA, or retinoic acid, although the interferons alone did not induce differentiation of HL-60 cells (Tomida et af., 1982). Glucocorticoids did not induce differentiation of HL-60 cells (Collins et af., 1980; Brandt et af., 1981; Y . Honma, K. Kasukabe, and M. Hozumi, unpublished data), but glucocorticoids enhanced the number of N-formylated chemotactic peptide receptors on differentiatingHL-60 cells induced by DMSO (Brandt et al., 1981). This steroid effectwas dose dependent, proportional to glucocorticoid activity, and abolished by cycloheximide. Furthermore, this effect was scarcely observed unless the cells were first induced to differentiate by DMSO. b. Phenotypic ChangesAssociated with Cell Diflerentiation.As described before, HL-60 cells could be induced by various compounds to differentiate into macrophages, granulocytes, or macrophage-like cells and granulocytic cells although the type of differentiated cells differed with different inducers. The phenotypes of some of the differentiated cells were examined in detail. HL-60 cells, which were induced to differentiate into morphologically similar cells to granulocytes by various compounds (butyrate, hypoxanthine, actinomycin D, DMSO, and hexamethylene bisacetamide) also had many of the functional characteristics of normal peripheral blood granulocytes, such as phagocytic and chemotaxic activities, complement receptors, and the ability to reduce Nitro Blue Tetrazolium. Furthermore, the differentiated cells lost activities to synthesize DNA, proliferate in both liquid culture medium and sofi agar medium, and form tumors in nude mice (Breitman and Gallo, 1981). On the other hand, differentiated HL-60 cells induced by DMSO were

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found to express various membrane components like those of mature granulocytes, such as surface glycoprotein with a molecular weight of 130,000 (Gahmberg et al., 1979), surface antigens of mature granulocytes (Perussia et al., 198I), cytoskeletal elements with ability to cap fluorescent Con A (Brown et al., 1981), and sterol and phospholipids (Cooper et al., 1981). Several other biochemical changes were also associated with induction of differentiationof HL-60 cells into granulocytesby DMSO. The levels of a histone-2A related polypeptide with an apparent molecular weight of 12,500 (Pantazis et al., 1981),poly(ADP-ribose)(Kanai et al., 1982), and sialidase activity (Nojiri et al., 1982)were significantly increased in the differentiated cells. Although the differentiated HL-60 cells showed various normal morphological and functional characteristics, the compositions of the cytoplasmic granules of both undifferentiated and differentiatedcells were reported to be abnormal (Olsson and Olofsson, 1981).Untreated HL-60 cells had a higher content of myeloperoxidase than normal neutrophils, but had only a low content of other enzymes associated with primary granules formed in promyelocytes. Enzymes in the primary granules decreased, but the amount of lysozyme increased during differentiation of the cells induced by DMSO. Lactofemn was not produced during differentiationof the cells, indicating that the composition of the secondary granules was abnormal or that these granules were produced abnormally. Phenotypic changes associated with induction of differentiation of HL-60 cells into macrophage-like cells by phorbol esters have been investigated extensively. Phorbol esters induced various morphological and functional changes to macrophage-like cells, as described before, and on differentiation the cells were found to stop proliferationand synthesisofDNA (Rovera et al., 1979b). Furthermore, various membrane components and some biochemical characteristics were recently found to change during differentiation of HL-60 cells. These included binding of phorbol esters to specific membrane receptors (Solanski et al,, 198I ) and modifications of membrane phospholipid synthesis (Cabot et al., 1980; Cassileth et al., 1981) and glycoproteins (Cossu et al., 1982). Some specific cytoplasmic proteins that might be associated with the regulation of differentiation of HL-60 cells into macrophages were expressed in TPA-treated HL-60 cells (Liebermann et al., 1981). Polyamine levels were also increased in the differentiatedHL-60 cells by treatment with TPA (Huberman et al., 1981). Although these phenotypic changes in the differentiated HL-60 cells induced by phorbol estersshowedthat the cells acquiredthe characteristicsof macrophages, the macrophage-like cells induced by TPA did not show increases above the uninduced levels of hexose monophosphate shunt activity, superoxidegeneration, Nitro Blue Tetrazoliumreduction, bacterial

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ingestion, or complement secretion. This finding suggests that the cells did not meet several important functional criteria of macrophages (Newberger et al., 1981). The relationship between the cell cycle of HL-60 cells and induction by TPA of differentiation of the cells into macrophages was examined by Rovera et al. (1980). They found that TPA-induced macrophage differentiation was independent of a round of DNA synthesis, and that differentiating HL-60 cells accumulated in the G, phase.

2. K562 Cells a. Inducers of Cell Diferentiation into Erythroid Cells. K562 cells were reported to differentiate into erythroid cells that synthesize either adult (Anderson et al., 1979)or embryonic (Rutherford et al., 1979) hemoglobin on treatment with sodium butyrate and hemin, respectively. Fuhr et al. (198 1) also reported recently that the original K562 cell line established in Lozzio’s laboratory could be induced to synthesize hemoglobin by a low concentration of hemin (0.05 mM) although no indication of normal maturation or of terminal differentiation into reticulocytes or erythrocytes was observed. Rowley et al. (1 98 1) examined the effects of various compounds including many inducers of differentiation of Friend mouse erythroleukemia cells on induction of erythroid differentiation in K562 cells. They found by benzidine staining that 19 of 39 compounds tested were inducers of differentiation of K562 cells, and that most of the compounds inducing differentiation in medium containing fetal calf serum showed little activity in medium containing newborn calf serum. Among these inducers, actinomycin D had the lowest optimal concentration of 2.4 X 10-9M.The optimal concentration for induction of differentiation of K562 cells was higher than that for Friend erythroleukemia cells for Ara C , but lower for Gaminolevulinic acid, bleomycin, butyric acid, cycloheximide, mitomycin C , ouabain, and 6-thioguanine, and the same for actinomycin D, cadaverine, hemin, and 1,6-hexanediamine (Rowley et al., 1981). b. Modijication of Potential of Cell Diflerentiation. Although K562 cells did not differentiate spontaneously when cultured for 7-8 days in liquid medium or for 14- 16 days in soft agar medium, they were induced to differentiate into early precursors of monocytic, granulocytic, and erythrocytic cells by cultivation for 10- 1 1 days in liquid medium that was gradually depleted of the essential nutrients needed for cell proliferation (Lozzio et al., 1981 ). The peroxidase reaction for hemoglobin demonstrated benzidinepositive material only in the region of the Golgi apparatus and this hemoglobin was shown to be the embryonic type. Most cellsgave a strong reaction for a-naphthyl acetate esterase typical of monocytes, and other cells had abundant red cytoplasmic granules characteristic of naphthol AS-D chloroacetate

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esterasein granulocyticprecursors. Some cells had myeloperoxidaseactivity. These results suggest that K562 cells are multipotential hematopoietic malignant cells that differentiatespontaneously into progenitors of erythrocytes, monocytes, and granulocytes (Lozzio et a!., 1981). Inhibition of cell division in K562 cells by glutamine-deficient medium or hydroxyurea was also found to modify the potential of differentiation of the cells and reversibly enhance the amount of hemoglobin in hemin-induced K562 cells up to the level in normal human red cells (Erard et al., 1981). Markers of both granulopoietic (My- 1) and erythropoietic (spectrin) differentiation were detected together in some K562 cells using specificantibodies (Mane et al., 1981). We found that cultivation of K562 cells in serum-free medium for a long period (4 months)or addition ofserum factor could modify their potential of differentiation. Although K562 cells cultured in medium with serum could not be induced to differentiate by TPA, cells that had been grown in serum-free medium for 4 months were induced to differentiate into macrophages by TPA, arginase, or actinomycin D (Honma et al., 1982a).Addition of serum inhibited the induction of differentiation of the cells by TPA or actinomycin D (Honma et al., 1982a).Calf serum was more inhibitory than fetal calf serum on TPA-induced differentiation,but there was no significant difference in the effects of the two sera on induction by actinomycin D or arginase. These findings indicate that various factors in the cultures are involved in the mechanisms of induction of differentiation of K562 cells.

3. KG-I Cells A unique characteristicof KG- 1 cells is their almost complete dependence on CSF for growth in soft agar medium. However, CSF has no effect on differentiation of the cells (Koeffler and Golde, 1980). 12-0-Tetradecanoylphorbol- 13-acetate could induce differentiation of KG- 1 cells into macrophage-like cells. On differentiation the cells became adherent, developed pseudopodia, morphological characteristics of macrophages, phagocytic activity, nonspecific acid esterase, several lysosomal enzymes, and Fc receptors (Koeffleret al., 1979, 1981). Territo and Koeffler (1981) found that TPA-induced differentiation of KG-1 cells did not require DNA synthesis. Proliferation of KG- 1 cells was inhibited by various compounds such as E-type prostaglandins, dbcAMP, theophylline, epinephrine, and other agents known to increase cellular CAMP,although estradiol, thyroxine, and the polypeptide hormones insulin and growth hormone had no effect. Prostaglandins, cyclic nucleotides, thyroxine, and the polypeptide hormones could not induce morphological differentiation of K562 cells (Koeffler and Golde, 1980). KG- 1 cells in diffusion chambers were implanted into mice treated with cyclophosphamide, glucan, or endotoxin to examine the effects

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of humoral factor in vivo on growth and differentiation ofthe cells. Although no differentiation of the cells was observed under these conditions, their proliferation was significantly enhanced by these compounds (Niskanen ef at., 1980). 4. ML-I and ML-3 Cells Human myeloblastic leukemia ML- 1 cells were induced to differentiate into macrophage-like cells by TPA or Ara C, but into granulocytic cells by DMSO (Takeda et at., 1982). Induction of differentiation of the cells was maximal at drug concentrations that inhibited cell proliferation most effectively. Although ML- 1 cells were induced to differentiate into macrophagelike cells by TPA and into granulocytic cells by DMSO like other human leukemic cells, they nearly all differentiated into monocytic cells with Ara C, unlike HL-60 cells, which showed only slight differentiation into granulocytic intermediates with this drug. These results suggest that the process of differentiation may depend, in part, on the stage at which the process of maturation of leukemic cells is blocked (Takeda ef al., 1982). Another human myeloblastic leukemic cell line, ML-3, differentiated into macrophage-like cells with TPA (Koeffler ef al., 1981).

B. PRIMARY CULTURED MYELOID LEUKEMIA CELLS Differentiation of human leukemia cells was examined in primary cultures of cells from patients with various leukemias. Palfi et al. ( 1979a)reported that leukemic cells in primary culture from two patients with acute myelogenous leukemia (AML)differentiated spontaneously into macrophage- or granulocyte-like cells during culture concomitantly with cessation of proliferation. Furthermore, they found that during culture, cryopreserved human AML cells acquired the characteristics of macrophages and lost their tumorigenicity in nude mice, although undifferentiated leukemic cells gave rise to tumors in more than 90% of the animals inoculated (Paltj ef al., 1979b). These results suggest that some human myeloid leukemic cells have the potential to differentiate in some conditions such as in culture in vifro. On the other hand, some inducers of differentiation of myeloid leukemic cells were found also to be effective on human leukemic cells in primary culture. Leukemic cells in primary culture from patients with AML (Pegoraro ef al., 1980; Chang and McCulloch, 1981; Fibach and Rachmilewitz, 1981) or chronic myelogenous leukemia (CML) (Fibach and Rachmilewitz, 1981) were induced by TPA to differentiate into macrophage-like cells with various characteristics of normal macrophages. Breitman el al. ( 1981) examined the effect of retinoic acid on differentiation in primary culture of leukemic cells from 2 1 patients with various types of myelogenous leukemia.

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Of the 21 leukemic specimens, only cells from two patients with acute promyelocytic leukemia (APL) differentiated into granulocytic cells in response to retinoic acid. Although prostaglandin E, induced granulocytic differentiation of HL-60 cells synergistically with retinoic acid (Breitman, 1982), it had no effect on the differentiation of leukemic cells in primary culture either alone or in combination with retinoic acid (Breitman et al., 1981). In primary cultures of several leukemic cells, spontaneous morphological differentiation into monocyte- or macrophage-likecells was observed after a few days, but retinoic acid had essentially no effect on this spontaneous differentiation (Breitman et al., 1981). We examined the effects of various inducers of differentiation of HL-60 cells [actinomycin D, TPA, retinoic acid, arginase, alkyllysophospholipids (ST-023 and ST-OOS), butyrate, and DMSO] and two antitumor drugs (aclacinomycin A and behenoyl cytosine arabinoside) on induction of differentiation ofleukemic cells from 14 patients with acute nonlymphocytic Consistent with previous leukemia in primary culture (Honma et al., 1982~). findings, TPA induced differentiation of leukemic cells from patients with acute nonlymphocytic leukemia, especially those with AML(M2), acute myelomonocytic leukemia (AMMoL), and acute monocytic leukemia (AMoL) into monocyte- and macrophage-like cells. Retinoic acid induced differentiation of cells from three patients with APL into mature granulocytic cells, but had no effect on cells from one patient with APL. The leukemic cells from some patients with AMMoL and AMoL were also induced by retinoic acid to differentiate into monocyte- and macrophage-like cells. Some inducers of differentiation of HL-60 cells induced differentiation of leukemic cells from all the 14 patients tested, and in 12 of 14 cases, more than 50% of the treated cells showed the characteristics of more differentiated cells. Actinomycin D was effectiveon all the leukemia cells, but with some cells TPA or retinoic acid was more effective. These results suggest that most of the acute nonlymphocytic leukemia cells can be induced to differentiate into macrophage-like cells or granulocytic cells by treatment with an appropriate combination of inducers of differentiation of HL-60 cells including actinomycin D. VI. Summary

Various myeloid leukemia cells from both human and experimental animals have been shown to be induced by a variety of compounds to differentiate in vitro into macrophage-like cells, granulocytic cells, or erythroid cells. Some of the differentiated cells from humans and experimental animals were found to stop proliferating in vitro and lose their leukemogenicity in either nude mice or syngeneic mice. Furthermore, some inducers of differentiation were found to induce differentiation of myeloid leukemia

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cells such as M 1 cells in vivo and to prolong the survival of mice inoculated with these cells. Thus induction of terminal differentiation of the myeloid leukemia cells seems to be a possible new method of therapy of myeloid leukemia. Some compounds were found to induce differentiation of myeloid leukemia cells from both human and experimental animals, but differences were also found in the effectiveness of inducers of differentiation of different cell clones of human and murine leukemia, suggesting that the cellular sites responsible for the inducing actions of various inducers differ in different clones. Therefore, appropriate inducers are required to induce differentiation of particular leukemia cells since these cells differ in sensitivity to different inducers. Although different cultured leukemia cell lines and leukemia cells in primary culture from patients with myeloid leukemia also differ in sensitivity to inducers, several inducers are active on a variety of myeloid leukemia cells including leukemia cells in primary culture. These inducers are TPA, actinomycin D, arginase, retinoic acid, (3-tetradecyloxy-2-methoxy)propyl2-trimethylammonioethyl phosphate (ST-023),DMSO, and butyrate. To establish a suitable therapy for induction of terminal differentiation of leukemia cells, we should examine the mechanisms of induction of differentiation of leukemic cells by these inducers and develop inducers with more potent activity and a wider spectrum. Besides direct inducers of differentiation of human myeloid leukemia cells, some compounds, such as prostaglandin E and interferon, enhance the activity of inducers and some sensitize leukemic cells that are resistant to inducers. So far these compounds have mostly been examined with mouse leukemia M 1 cells. Further studies are required to develop compounds that are effective for induction of differentiation of human leukemia cells. Studies have suggested that leukemia cells can be induced or stimulated to differentiate by various biological response modifiers (BRM) that modify a biological host response to a tumor with resultant therapeutic benefit (Carter, 1980a,b). Biological response modifiers affecting differentiation of myeloid leukemia cells, such as immunopotentiators, interferons, hormones, vitamins, and cytokinesincluding protein inducers (D-factor), have mainly been examined with M1 cells, although some BRM have been shown to be involved in the induction of differentiation of human myeloid leukemia cells. Further studies are necessary on the effect on induction of differentiation of human myeloid leukemia cells of BRM affecting host responses to a wide spectrum of tumors. In this way it should be possible to find BRM inducing differentiation of the cells and develop a method of therapy of human myeloid leukemia by induction of terminal differentiation of the leukemia cells. Various cancer chemotherapeutic drugs stimulate differentiation of both

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human and myeloid leukemia cells, although their mechanisms of action are still unknown. Some drugs may control leukemia not only by their cytotoxic effects but also by their ability to induce differentiation ofleukemia cells. We should investigate the particular mechanisms of induction of differentiation of leukemia cells by these anticancer drugs and develop compounds with potent activity to induce terminal differentiation of the myeloid leukemia cells but with less side effects. In conclusion, it seems from available evidence reviewed in this article that it should be possible to establish a suitable therapy for myeloid leukemia by induction of terminal cell differentiation. For this purpose compounds with more potent effects in stimulating cell differentiation must be developed and the effects of various cancer therapeutic agents including BRM must be reevaluated on the basis of cell differentiation. ACKNOWLEDGMENTS The work of our group cited was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare, Japan.

REFERENCES Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S., and Suda, T. (1981). Proc. Natl. Acad. Sci. U.S.A.78,4990-4994. Akagawa, K. S., and Tokunaga, T. (1980). Microbiol. Immunol. 24,1005- 101 I . Akagawa, K. S., Momoi, T., Nagai, T., and Tokunaga, T. (1981). FEBS Letf. 130,80-84. Anderson, L. C., Jokinen, M., and Gahrnberg, G . C. (1979).Nature(London) 278,364-365. Ayusawa, D., Isaka, K., Seno, T., Tomida, M., Yamamoto, Y., Hozumi, M., Takatsuki, A., and Tamura, G . (1979). Biochem. Biophys. Res. Commun. 90,783-787. Azuma, I., Sugimura, K., Taniyama, T., Yamawaki, M., Yamamura, Y., Kusumoto, S., Okada, S., and Shiba, T. (1976). Infect. Immun. 14, 18-27. Batzinger, R. P., Ou, S.-Y., L., and Bueding, E. (1978). CancerRes. 38,4478-4485. Bodner, A. J., Ting, R. C., and Gallo, R. C. ( I98 1). J. Nail. Cancer Inst. 67, I025 - 1030. Brandt, S. J., Barnes, K. C., Glass, D. B., and Kinkade, J. M., Jr. (1981). Cancer Res. 41, 4947-495 I . Breitman, T. R. (1982). In “Expression of Differentiated Functions in Cancer Cells” (R. P. Revoltella and G. Pontieri, eds.), pp. 257-273. Raven, New York. Breitman, T. R., and Gallo, R. C. (1981). Blood C e h 7, 79-89. Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980). Proc. Nail. Acad. Sci. U.S.A. 77, 2936-2940. Breitman, T. R., Collins, S. J., and Keene, B. R. (1981). Blood57, 1000- 1004. Brennan, J. K., Abboud, C. N., DiPersio, J. F., Barlow, G. H., and Lichtman, M. A. (1981). Blood 58,803 - 8 12. Brown, W. J., Norwood, C. F., Smith, R. G., and Snell, W. J. (1981). J. CeU. Physiol. 106, 127-136. Broxmeyer, H. E., and Moore, M. A. S. (1978). Biochim. Biophys. Acta Rev. Cancer 516, 129- 166.

THERAPY O F LEUKEMIA BY CELL DIFFERENTIATION

165

Broxmeyer, H. E., Bognacki, J., Dorner, M. H., and debousa, M. (1981). J. Exp. Med. 153, 1426- 1444. Burgess, A. W., and Metcalf, D. (1980a). Int. J. Cancer 26,647-654. Burgess, A. W., and Metcalf, D. (1980b). Blood 56,947-958. Cabot, M. C., Welsch, C. J., Callahan, M. F., and Huberman, E. (1980). Cancer Res. 40, 3674-3679. Calle, L. M., Sullivan, P. D., Nettleman, M. D., Ocasio, I. J., Blazyk, J., and Jallick, J. (1978). Biochem. Biophys. Res. Commun. 8 5 3 5 I - 356. Carter, S. K. (1980a). Cancer Immunol. Immunother. 8,207-210. Carter, S. K. (1980b). Cancer Treat. Rev. 7,235-238. Cassileth, P. A., Suholet, D., and Cooper, R. A. (1981). Blood58,237-243. Chang, L. J.-A., and McCulloch, E. A. (1981). Blood57,361-367. Chiao, J. W., Freitag, W. F., Steinmetz, J. C., and Andreef, M. (1981). Leukemia Res. 5, 477-489. Collins, S. J., Gallo, R. C., and Gallagher, R. E. (1977). Nature (Lundun)270,347 -349. Collins, S . J., Bonder, A., Ting, R., and Gallo, R. C. ( 1980). Int. J . Cancer 25,2 13 - 2 18. Cooper, R. A., Ip, S. H. C., Cassileth, P. A., and Kuo, A. L. ( I98 I). Cancer Res. 41, I847 - 1852. Cossu, G., Kuo, A. L., Pessano, S., Warner, L., and Cooper, R. A. (1982). Cancer Res. 42, 484-489. Elias, L., Wogenrich, F. J., Wallace, J. M., and Longmire, J. (1 980).Leukemia Res. 4,30 I -307. Ellouz, F., Adam, A,, Ciorbaru, R., and Lederer, E. (1974). Biochem. Biophys. Rex Commun. 59, 1317-1325. Erard, F., Dean, A., and Schechter, A. N. (1981). Blood58, 1236- 1239. Falk, A., andSachs, L. (1980).Int. J. Cancer26, 595-601. Fibach, E., and Rachmilewitz, E. A. (1981). Br. J. Haematol. 47,203-210. Friedman, E. A., and Schildkraut, C. L. (1977). Cell 12,901 -913. Fuhr, J. E., Bamberger, E. G., Lozzio, C. B., and Lozzio, B. B. ( I 98 1). Blood Cells 7,389- 395. Gahmberg, C. G., Nilsson, K., and Anderson, L. C. (1979). Proc. Natl. Acad. Sci. U.S.A.76, 4087-409 1. Gallo, R., Ruscetti, F., Collins, S., and Gallagher, R. (1979).In “Hematopoietic Cell Differentiation” (D. W. Golde, M. J. Cline, D. Metcalf, and C. F. Fox, eds.), pp. 335-354. Academic Press, New York. Griffin, A. C. (1979).Adv. CancerRes. 29,419-442. Guez, M., and Sachs, L. (1973). FEES Lett. 37, 149- 154. Gusella, J., Geller, R., Clarke, B., Weeks, V., and Housman, D. (1976). Cell9,22 1-229. Hayashi, M., Gotoh, O., Kado, J., and Hozumi, M. (1981). 1.Cell. Physiol. 108, 123- 134. Hayashi, M., Okabe-Kado, J., and Hozumi, M. (1982). Exp. Cell Res. 139,422-427. Hecker, E. (1981).J. CancerRes. Clin. Oncol. 99, 103- 124. Hirai, K., Nagata, K., Yamada, M., and Ichikawa, Y. (1979). Exp. Cell Res. 124,269-283. Hoffman-Liebermann, B., and Sachs, L. (1978). Cell 14,825 -834. Hoffman-Liebermann, B., Liebermann, D., and Sachs, L. (198 1). Int. J. Cancer 28,6 15-620. Honma, Y., Kasukabe, T., and Hozumi, M. (1978).J. Natl. Cancerhi. 61,837-841. Honma, Y . , Kasukabe,T., Okabe, J .,and Hozumi, M. (1979). CancerRes. 39,3167-3171. Honma, Y., Fujita, Y., Okabe-Kado, J., Kasukabe, T., and Hozumi, M. (1980a). Cancer Lett. 10,287-292. Honma, Y., Kado, J., Kasukabe, T., and Hozumi, M. (1980b). Gann 71,543-547. Honma, Y., Kasukabe, T., and Hozumi, M. (1980~).Biochem. Biophys. Res. Commun. 93, 927-933. Honma, Y., Kasukabe, T., Hozumi, M., and Koshihara, Y. (198Od). J. Cell. Physiul. 104, 349-357.

166

MOT00 HOZUMI

Honma, Y., Takenaga, K., Kasukabe, T., and Hozumi, M. (1980e). Biochem. Biophys. Res. Commun. 95,507 - 5 12. Honma, Y., Kasukabe, T., and Hozumi, M.(1981a). Biochim. Biophys. Acta 664,441 -444. Honma, Y., Kasukabe, T., Hozumi, M., Tsushima, S., and Nomura, H. (198 1b). Cancer Rex 41,3211-3216. Honma, Y., Fujits, Y., Kasukabe, T., and Hozumi, M. (1982a). Gann 73,97- 104. Honma, Y., Hayashi, M., Kasukabe, T., and Hozumi, M. (1982b).LeukemiaRes. 6,117- 122. Honma, Y., Fujita, Y., Kasukabe, T., Hozumi, M., Sampi, K., Sakurai, M., Tsushima, S.,and Nomura, H. (1983). Europ. J. Cancer Clin. Oncol. (in press). Hozumi, M. (1982). Cancer Biol. Rev. 3, 153-21 1. Hozumi, M., Honma, Y., Okabe, J., Tomida, M., Kasukabe, T., Takenage, K., and Sugiyama, K. (1979a).In “Oncogenic Viruses and Host Cell Genes” (Y. Ikawa and T. Odaka, eds.), pp. 341 -353. Academic Press, New York. Hozumi, M., Honma, Y., Tomida, M., Okabe, J., Kasukabe, T., Sugiyama, K., Hayashi, M., Takenaga, K., and Yamamoto, Y. (1979b). Acta Haematol. Jpn. 42,941 -952. Hozumi, M., Umezawa, T., Takenaga, K., Ohno, T., Shikita, M., and Yamane, I. (1979~). Cancer Res. 39,5 127- 5 131. Hozumi, M., Yamamoto, Y., Tomida, M., Ayusawa, D., Seno, T., and Tamura, G. (198 I). In “Glycoconjugates” (T. Yamakawa, T. Osawa, and S. Handa, eds.), pp. 284-285. Japan Scientific Societies Press, Tokyo. Hozumi, M., Kasukabe, T., and Honma, Y. (1982). In “Carcinogenesis, A Comprehensive Survey” (E. Hecker, N. E. Fusenig, W. Kunz, F. Marks, and H. W. Thielmann, eds.), Vol. 7, pp. 379-384. Raven, New York. Huberman, E., and Callahan, M. F. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 1293- 1297. Huberman, E., Weeks, C., Herrmann, A., Callahan, M., and Slaga,T. ( 1 98 1). Proc. Natl. Acad. Sci. U.S.A. 78, 1062- 1066. Ichikawa, Y. (1969). J. Cell. Physiol. 74,223-234. Ichikawa, Y. (1970). J. Cell. Physiol. 76, 175-184. Ichikawa, Y., Maeda, M., and Horiuchi, M. (1975). Exp. Cell Res. 90,20-30. Ichikawa, Y., Maeda, M., and Horiuchi, M. (1976). Int. J. Cancer 17,789-797. Kanai, M., Miwa, M., Kondo, T., Tanaka, Y., Nakayasu, M., and Sugimura, T. (1982). Biochem. Biophys Res. Commun. 105,404-41 1. Kasukabe, T., Honma, Y., and Hozumi, M.(1977a). Gann68,765-773. Kasukabe, T., Honma, Y., Okabe, J., and Hozumi, M. (1977b). Cancer Lett. 3,333-337. Kasukabe, T., Honma, Y., and Hozumi, M. (1979a). Biochim. Biophys. Acta 586,615-623. Kasukabe, T., Honma, Y., and Hozumi, M.(1979b). Gann 70, 1 19- 123. Kasukabe, T., Honma, Y., and Hozumi, M. (1981). Gann 72,310-314. Koeffler, H. P., and Gold, D. W. (1 978). Science 200, 1 I53 - 1154. Koeffler, H. P., and Golde, D. W.(1980). Blood 56,344- 350. Koeffler, H. P., Bar-Eli, M., and Temto, M.(1979). Blood 54 (Suppl. I), 174a. Koeffler, H. P., Bar-Eli, M., and Tenito, M. (198 I). Cancer Res. 41,9 19-926. Liebermann, D., and Sachs, L. (1977). Nature (London) 269,173- 175. Liebermann, D., and Sachs, L. (1978). Cell 15,823-835. Liebermann, D., and Sachs, L. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,3353-3357. Liebermann, D., Hoffman-Liebermann, B., and Sachs, L. (1980). Dev. Eiol. 79,46-63. Liebermann, D., Hoffman-Liebermann, B., and Sachs, L. ( 1981). Int. J. Cancer 28,285 - 29 1. Lipton, J. H., and Sachs, L. (1981). Biochim. Biophys. Acta 673,552-569. Lotem, J., andSachs, L. (1978a). Proc. Natl. Acad. Sci. U.S.A. 75,3781-3785. Lotem, J., and Sachs, L. (1978b). Int. J. Cancer22,214-220. Lotem, J., and Sachs, L. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,5158-5162. Lotem, J., and Sachs, L. (1 980). Int. J. Cancer 25,5 16- 564.

THERAPY O F LEUKEMIA BY CELL DIFFERENTIATION

167

Lotem, J.,and Sachs, L. (1981). fnl. 1.Cuncer28,375-386. Lotem, J., Lipton, J. H., and Sachs, L. (1980).fnt. J . Cancer 25, 763-77 I . Lozzio, B. M., Lozzio, C. B., Bamberger, E. G., and Feliu, A. S. (1981). Proc. SOC.Exp. Bid. Med. 166,546-550. Lozzio, C. B., and Lozzio, B. B. (1975). Blood 45,321 -334. Maeda, M., and Ichikawa, Y. (1980).J. Cell. Physiol. 102,323-331. Maeda, M., Ichikawa, Y., and Azuma, I. (1980). J. Cell. Physiol. 105,33-38. Marie, J. P., Izaguirre, C. A., Civin, C. I., Mirro, J., and McCulloch, E. A. ( 1 98 1). Blood 58, 708-71 1. Marks, P. A., and Rifkind, R. A. (1978).Annu. Rev. Biochem. 47,419-448. Marks, P. A., Reuben, R., Epner, E., Breslow, R., Cobb, W., Bogden, A. E., and Rifkind, R. A. ( I 978). Antibiot. Chemother. 23, 33 -4 1. Mendelsohn, N., Michl, J., Gilbert, H. S., Acs, G., and Christman, J. K. ( 1980).Cancer Rex 40, 3204- 32 10. Metcalf, D. ( 1977).“Hemopoietic Colonies. In Vitro Cloning of Normal and Leukemic Cells.” Springer-Verlag, Berlin and New York. Metcalf, D. ( I 979). Int. J. Cancer 24,6 I6 -623. Metcalf, D. (1980). fnt. J. Cancer 25,225 -233. Metcalf,D.(l981).Int. J. Cancer27,577-584. Metcalf, D., Moore, M. A. S., and Warner, N. L. ( 1 969). J. Natl. Cancer Inst. 43,983- LOOl. Minowada, J. ( 1 982). In “Immunology of Leukemic Cells” (F. Gunz and E. Henderson, eds.), pp. 119- 139. Grune & Stratton, New York. Miyaura, C., Abe, E., Kuribayashi, T., Tanaka, H., Konno, K., Nishii, Y., and Suda, T. (1981). Biochem. Biophys. Res. Commun. 102,937 -943. Moore, M. A. S. (1979). In “Clinics in Haematology” (L. G. Lajtha, ed.), Vol. 8, pp. 287- 309. Saunders, Philadelphia, Pennsylvania. Nagata, K., and Ichikawa, Y. (1979). J. Cell. Physiol. 98, 167- 176. Nagata, K., Ooguro, K., Saito, K., Kuboyama, M., and Ogasa, K. (1977). Gann 68,757- 764. Nagata, K., Sagara, J., and Ichikawa, Y. (1980). J. CellBiol. 85,273-282. Nakayasu, M., Shimamura, S., Takeuchi, T., Sato, S., and Sugimura, T. ( 1 978). Cancer Res. 38, 103- 109. Nakayasu, M., Shoji, M., Aoki, N., Sato, S., Miwa, M.,and Sugimura, T. (1979).CancerRes. 39, 4668-4672. Nakayasu, M ., Terada, M., Tamura, G., and Sugimura, T. (1980). Proc. Natl. Acad. Sci. U S A . 77,409-413. Nakayasu, M., Fujiki, H., Mori, M., Sugimura,T., and Moore, R. E. (1981). Cancer Lett. 12, 27 1-277. Newberger, P. E., Baker, R. D., Hansen, S. L., Duncan, R. A., and Greenberger, J. S. ( 1 98 1). Cancer Res. 41,186 I - 1865. Nicola, N. A., and Metcalf, D. (1981). J. Ce//.Physiol. 109,253-264. Niskanen, E., Koeffer, H. P., Golde, D., and Cline, M. J. (1980). Leukemia Res. 4,203-208. Nojiri, H., Takaku, F., Tetsuka, T., and Saito, M. (1982). Biochem. Biophys. Res. Commun. 104, 1239-1246. Okabe, J., Hayashi, M., Honma, Y., and Hozumi, M. (1978). fnt. J. Cancer 22,570-575. Okabe, J., Honma, Y., Hayashi, M., and Hozumi, M. ( I 979). Int. J. Cancer 24,87 -9 1. Okabe-Kado, J., Honma, Y., Hayashi, M., and Hozumi, M. (1981). Cancer Res. 41, 1997-2002. Okabe-Kado, J., Honma, Y.,Hayashi, M., and Hozumi, M. (1982). Gann 73,398-402. Okuma, M., Ichikawa, Y., Yamashita, S., Kitajima, K., and Numa, S. (1976). Blood 47, 439-446. Olsson, I., and Olofsson, T. (1981). Exp. Cell Res. 131,225-230.

168

M O T 0 0 HOZUMI

Olsson, I., Olofsson, T., and Mauritzon, N. (198 1). J. Natl. Cancer Inst. 67, 1225 - 1230. Oshima, G., Yamada, M., and Sugirnura, T. (1979). Biochem. Biophys. Res. Commun. 90, 158- 163. Pal& G., Powles, T., Selby, P., Summersgill, B. M., and Alexander, P. (1979a).Br. J. Cancer40, 719-730. Palfi, G., Selby, P., Powles, R., and Alexander, P. (1979b).Br. J. Cancer 40,7 I3 -735. Pantazis, P., Sarin, P. S., and Gallo, R. C. (1981).Int. J. Cancer 27,585-592. Pearlstein, E., Dienstman, S. R., and Defendi, V. ( 1978). J. Cell Biol. 79,263 -267. Pegoraro, L., Abrahm, J., Cooper, R. A., Levis, A., Lange, B., Meo, P., and Rovera, G. (1980). Bl00d55,859-862. Perussia, B., Lebman, D., Ip, S. H., Rovera, G., and Trinchieri, G. ( I98 1 ) . Blood 58,836- 843. Rosin, M. P., and Stich, H. F. (1979).Int. J. Cancer 23,722-727. Rovera, G., OBrien, T. G., and Diamond, L. (1979a).Science 204,868-870. Rovera, G., Santoli, D., and Damsky, C. (1979b).Proc. Natl. Acad. Sci. U S A . 70,2779-2783. Rovera, G., Olashaw, N., and Meio, P. (1980).Nature (London) 284,69-70. Rowley, P. T., Ohlson-Wilhelm,B. M., Farley, B. A., and Labella, S. (198 I). Exp. Hematol. 9, 32-37. Ruscetti, F. W., Collins, S. J., Woods, A. M., and Gallo, R. C. ( 1 98 I). Blood 58,285 -292. Rutherford, T . R., Clegg, J. B., and Weatherall, D. J. (1979). Nature(London) 280, 164- 165. Sachs, L. (1978a).Nature (London) 274,535-539. Sachs, L. (1978b).Br. J. Haematol. 40,509-517. Sachs, L. ( 1980). Proc. Natl. Acad. Sci. U.S.A. 77,6 I52- 6 1 56. Sachs, L. (198 I). Blood Cells 7 , 3 1-44. Saito, M., Nojiri, H., and Yamada, M. ( 1 980). Biochem. Biophys. Res. Commun. 97,452-462. Sakagami, H., Asaka, K., Abe, E., Miyaura, C., Suda, T., and Konno, K. (1981).J. Nutr. Sci. Vitaminol. 27,29 I - 300. Santoro, N. G., Benedetto, A., and Jaffe, B. M. (1978).Biochem. Biophys. Res. Commun. 85, I5 10- 15 17. Scher, W.,Tsuei, D., Sassa, S., Price, P., Gabelman, N., and Friend, C. (1978).Proc. Natl. Acad. Sci. U.S.A. 75,385 1-3855. Scher, W., Tsuei, D., and Friend, C. ( 1980). Leukemia Res. 4.2 17- 229. Solanski, V., Slaga, T. J., Callaham, M.,and Huberman, E. ( I 98 1). Proc. Natl. Acad. Sci. U.S.A. 78, 1722- 1725. Sugiyama, K., Hozumi, M., and Okabe, J. (1979a).Cancer Res. 39, 1056- 1062. Sugiyama, K., Tomida, M., and Hozumi, M. (1979b).Biochim. Biophys. Acta587, 169- 179. Sugiyama, K., Tomida, M., Honma, Y., and Hozumi, M. (1980).CancerRes. 40,3387-3391. Takeda, K., Minowada, J., and Bloch, A. (1982). Cancer Res. (in press). Takenaga, K. (1981).Gann 72,488-497. Takenaga, K., and Hozumi, M. (1980).Gann 71,141 - 145. Takenaga, K., Hozumi, M., and Sakagami, H. (1980). Cancer Res. 40,914-919. Takenaga,K., Honma, Y.,andHozumi,M. (1981a).Gann72, 104-112. Takenaga, K., Honma, Y., Okabe-Kado, J., and Hozumi, M. (1981b). Cancer Ref. 41, 1948- 1953. Takenaga, K., Honma, Y.,Okabe-Kado, J., and Hozumi, M. (1982).Gann 73,175- 183. Taniyama, T., and Holden, H. T. (1979). Cell. Immunol. 48,369-374. Temto, M. C., and Koeffler, H. P. (1981).Br. J. Haematol. 47,479-483. Todd, R. F., 111, Griffin,J. D., Ritz, J., Nadler, L. M., Abrams, T., andschlossman, S. F. (198 I). Leukemia Res. 5,491 -495. Tomida, M., Yamamoto, Y., and Hozumi, M. (1980a).Cancer Res. 40,2919-2924. Tomida, M., Yamamoto, Y.,and Hozumi, M. (1980b).Gann 71,457-463.

THERAPY OF LEUKEMIA BY CELL DIFFERENTIATION

169

Tomida, M., Yamamoto, Y., and Hozumi, M. (1982). Biochem. Biophys. Res. Commun. 104, 30-37. Trowbridge, 1. S., and Omary, B. ( 198 1 ). J. Exp. Med. 154, I5 17 - 1524. Warner, N. L., Moore, M. A. S., and Mendelsohn, N. (1969). J.Natl. Cancerlnst. 43,963-968. Wattenberg, L. (1979). In “Carcinogenesis, Identification and Mechanisms of Action” (A. C. Griffin and C. R. Shaw, eds.), pp. 299-316. Raven, New York. Weinstein, I. B., Lee, L. S.,Fisher, P. B., Mufson, R. A., and Yamasaki, H. (1979).J. Supramol. Strucf. 12, 195-208. Yamada, M., Shimada, T., Nakayasu, M., Okada, H., and Sugimura, T. (1978). Biochem. Biophys. Res. Commun. 83, I325 - 1332. Yamamoto, Y., Tomida, M., and Hozumi, M. (1979). Cancer Res. 39,4170-4174. Yamamoto, Y., Tomida, M., and Hozumi, M. (1980). Cancer Res. 40,4804-4809. Yamamoto, Y., Tomida, M., Hozumi, M.,andAzuma, I. (1981a). Gann72,828-833. Yamamoto, Y., Tomida, M., Hozumi, M., Ayusawa, D., Seno, T., and Tamura, G. (1981b). Cancer Res. 41,2534-2539. Yamasaki, H. ( 1980).h “Molecular and Cellular Aspects of Carcinogen Screening Tests” (R. Montesano, H. Bartsch, and L. Tomatis, eds.), No. 27, pp. 91 - 1 1 I . IARC Scientific Publications, Lyon.