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Variant subpopulations of FM3A mouse mammary carcinoma cells that have increased lung-colonizing potential were obtained previously by sequentially harvesting pulmonary metastases, culturing their cells in vitro, and reestablishing the metastases in vivo. In the present study, glycosaminoglycan production by the parental and variant cells was studied after metabolic labeling of cultures by [14C]glucosamine for 24 hr. Analysis of the products indicated that the rate of incorporation of the labeled precursor into hyaluronic acid in the high-metastatic variant cells was 27 to 54 times the rate in the low-metastatic variant cells and that the increase in hyaluronic acid synthesis was not associated with an increase in the rate of synthesis of other glycosaminoglycans. Both the cell layers and media of high-metastatic variants contained a much higher proportion of radioactivity in hyaluronic acid than did the corresponding fractions of low-metastatic cell lines. The results provide a basis for further investigation of the potential role of hyaluronic acid in control of the behavior of epithelial tumor cells during metastasis.

The effect of injection of an inducer and sensitizer on the survival times of syngeneic SL mice inoculated with resistant mouse myeloid leukemia cells (Ml) was examined. In vitro, the resistant Ml cells could not be induced to differentiate into mature macrophages and granulocytes by inducer (certain proteins, bacterial lipopolysaccharides, or glucocorticoids) alone, but could be induced to differentiate by treatment with both the inducer and a sensitizer (actinomycin D). In vivo, lipopolysaccharide alone scarcely affected the survival of SL mice inoculated with the resistant cells, but lipopolysaccharide plus actinomycin D significantly prolonged their survival. Administration of both lipopolysaccharide and actinomycin D also prolonged the survival of athymic nude mice inoculated with resistant Ml cells. These results suggest that prolongation of the survival of SL mice inoculated with resistant Ml cells is associated with the induction of differentiation of the cells.

Purines such as hypoxanthine and 6-thioguanine have the capacity to induce the differentiation of human myeloid leukemia HL-60 cells in culture. Several nucleoside analogs were synthesized and their effects on cell proliferation and differentiation of HL-60 cells were examined. On incubation with these compounds, proliferation of HL-60 cells was inhibited and the cells were induced to differentiate into morphologically and functionally mature granulocytes. Among the compounds we tested, 2,4-diethyl-7,7,8,8-tetramethyl-cis-2,4-diazabicyclol [4.2.0] octane-3,5-dione was the most effective in inducing differentiation of HL-60 cells. This compound was approximately 100 times more potent on a molar basis than hypoxanthine. The compounds reacted synergistically or additively with a typical antileukemic drug (daunomycin) or another potent differentiation inducer (retinoic acid).

Conditioned media of mitogen- or antigen-stimulated spleen cells have been found to contain various lymphokines including macrophage activating factor (MAF), immune interferon (IFN-gamma) and a factor inducing differentiation of mouse myeloid leukemic M1 cells into macrophages and granulocytes (D-factor). We examined the properties and mutual relations of these lymphokines. Conditioned media of concanavalin A (Con A)-stimulated spleen cells and of purified protein derivative (PPD)-stimulated Bacillus Calmette-Guérin (BCG)-immune spleen cells contained the activities of D-factor, MAF and IFN. These lymphokines were similarly eluted on Sephadex G-100 in a peak corresponding to a molecular weight of 40,000 approximately 60,000. However, a rapidly eluted fraction contained MAF but not activities of D-factor and IFN. Treatment of conditioned medium of Con A-stimulated spleen cells at pH 2 abolished the activities of MAF and IFN but did not affect the activity of D-factor. Moreover, addition of cytochalasin B suppressed the productions of MAF and IFN but not that of D-factor by Con A-stimulated spleen cells. Antiserum against mouse IFN-gamma neutralized IFN activity but not MAF activity in the conditioned medium of Con A-stimulated spleen cells. These results indicate that D-factor, MAF and IFN-gamma are all distinct substances.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor), which is suggested to be a glycoprotein. On the other hand, growth and differentiation of normal precursor cells of macrophages and granulocytes can be stimulated by a glycoprotein termed colony-stimulating factor (CSF). Mouse fibroblast L929 cells were found to produce both the D-factor and CSF. The properties of the D-factor and CSF and the roles of carbohydrates in the molecules of these factors were examined using tunicamycin, a specific inhibitor of asparaginase-linked glycosylation. Although both the D-factor and CSF were produced by L-cells in usual medium containing fetal calf serum, production of D-factor, but not CSF, was reduced by omission of serum from the medium. The activity of the D-factor was slightly decreased by treating the L-cells with tunicamycin (0.5 microgram/ml) in the presence of 2% fetal calf serum, without any decrease in CSF activity. Conditioned medium of L-cells incubated with or without tunicamycin was fractionated by gel filtration on a Sephadex G-200 column. Normal D-factor appeared as 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. On the other hand, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced in the presence of tunicamycin was more sensitive than normal D-factor was to trypsin or heat treatment at 70 degrees. The CSF produced in the presence of tunicamycin was resistant to these treatments. These results indicate that the D-factor is distinct from CSF. Furthermore, the results suggest that the D-factor produced by L-cells is also a glycoprotein and that, although carbohydrate is not essential for production or activity of the D-factor, it contributes to stabilizing the protein portion of D-factor.

The effects of conditioned media (CM) of various clones of mouse myeloid leukemia cells (M1) on colony formation of normal mouse bone marrow cells were examined under various conditions. The CM of sensitive M1 cells, which could be induced to differentiate into macrophage-like and granulocyte-like cells by various factors stimulating the differentiation (D-factor), slightly inhibited colony formation of normal bone marrow cells by colony stimulating factor (GM-CSF). The CM of resistant M1 cells, which were resistant to the induction of differentiation even with high concentrations of the D-factor, significantly inhibited the colony formation of bone marrow cells by GM-CSF. The inhibitory activity of the CM from resistant cells decreased on treatment of the cells with a low concentration of actinomycin D, which could sensitize the resistant cells to induction of differentiation. The CM of differentiated sensitive M1 cells induced by dexamethasone stimulated the colony formation of bone marrow cells without exogenously added GM-CSF. Most of the GM-CSF activity in the CM was separated from the D-factor of M1 cells by Sephadex G-75 gel filtration.

Herbimycin A, a benzoquinonoid ansamycin antibiotic, is found to reduce intracellular phosphorylation by tyrosine protein kinase. The human chronic myelogenous leukemia cell line K562 expresses a structurally altered c-abl protein with tyrosine kinase activity. When K562 cells are induced to undergo erythroid differentiation by hemin, reduction in the intracellular level of tyrosine phosphorylation occurs. In order to understand the relationship between induction of differentiation and reduction of tyrosine phosphorylation by the c-abl gene product, the effect that herbimycin A, a selective inhibitor of intracellular tyrosine kinase activity, exerts on the differentiation of K562 cells was examined. Reduction of tyrosine phosphorylation in K562 cells by herbimycin A was observed within 1 h. Noncytotoxic concentrations of herbimycin A induced erythroid differentiation of K562 cells but not of murine erythroleukemia 745A cells. The other human myeloid leukemia cell lines (HL-60, THP-1, and U937) tested were not induced to undergo cell differentiation by this antibiotic. Herbimycin A and the other well-known inducers such as hemin, butyric acid, Adriamycin, and 1-beta-D-arabinofuranosylcytosine had additive or more than additive effects on induction of erythroid differentiation of K562 cells. With respect to inhibition of cell growth, the sensitivity of K562 cells to herbimycin A was highest in the human leukemia cell lines we tested. Noncytotoxic concentrations of herbimycin enhanced the antiproliferative effect of Adriamycin or 1-beta-D-arabinofuranosylcytosine on K562 cells. Combination therapy with herbimycin A and its derivatives may be considered for use in the treatment of some types of leukemia where tyrosine kinase activities are implicated as determinants of the oncogenic state.

Retinoic acid, retinol, retinyl acetate, and retinal induced activities of lysosomal enzymes, such as lysozyme, acid protease, and acid phosphatase, in mouse myeloid leukemia cells (M1), while the pyridyl analog of retinoic acid had no effect. Retinoic acid was the most potent inducer of lysosomal enzyme activities. The induction of lysozyme activity by retinoic acid was inhibited by treatment with puromycin. The retinoids did not induce phagocytic and locomotive activities or morphological changes in M1 cells, and they inhibited the induction of these differentiation-associated properties by various inducers without inhibiting cell growth. Retinoic acid was the most potent inhibitor of induction of these differentiation-associated properties. The inhibitory effect of retinoic acid was found to be reversible. These results suggest that distinct mechanisms exist for control of induction of lysosomal enzyme activities and of other differentiation-associated properties of M1 cells, such as phagocytosis, morphological changes, and migration.

Experimental evidences were presented to show induction by cancer chemotherapeutic agents of terminal differentiation of several cultured myeloid leukemia cell lines and fresh leukemia cells from patients with myelogenous leukemia. A description was also given of recent clinical trials to improve the therapy of patients with refractory acute myelogenous leukemia by administration of small doses of cancer chemotherapeutic agents resulting in enhanced populations of mature granulocytes in the peripheral blood of patients. These cancer chemotherapeutic agents are suggested to induce differentiation of several cultured leukemia cell lines both in vitro and in vivo by mechanisms distinct from those of their cytotoxic actions. Based on these findings, possible therapeutic implications of the differentiation-stimulating actions of the cancer chemotherapeutic agents on host response-modifying actions are discussed.

Although the ascitic fluid of animals bearing various tumors and that of mice induced by complete Freund's adjuvant had high activity for inducing differentiation of myeloid leukemic cell line (M1) from an SL mouse to macrophages and granulocyte-like cells, the activity in the ascitic fluid of syngeneic mice bearing the M1 cells was markedly reduced. Macrophages and granulocytes were abundant in the active ascites of animals bearing tumors (8 to 12% of the total ascites cells) while in the ascites of syngeneic mice bearing the M1 cells they were not (0.1 to 0.7% of the total ascites cells). Appearance of lymphocytes in the ascites of both types was not significantly different. Although the conditioned media of the Ehrlich tumor cells, M1 cells, and whole ascites cells with the M1 cells were not active in inducing differentiation of the M1 cells, the conditioned media of all the ascites cells with Ehrlich tumor cells and those of peritoneal macrophages and granulocytes in mice did show a high activity. These results indicate that the peritoneal macrophages and granulocytes in the ascites are responsible for the production of factors stimulating differentiation of the M1 cells.

The tumor promoter TPA inhibited both functional and morphological differentiation of mouse myeloid leukemia M1 cells cultured in medium containing calf serum or horse serum, but enhanced these inductions in medium containing fetal calf serum. The metabolic processes of prostaglandin E2 synthesis were associated with modification by TPA of differentiation of M1 cells. The factor(s) in the sera affecting the differentiation of M1 cells with TPA was nondialyzable and macromolecular. Upon 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.

Mouse myeloid leukemia cells (Ml) could be induced to differentiate into mature macrophages and granulocytes by treatment with dexamethasone or a protein induced in ascitic fluid from tumor-bearing rats. The effects of antioxidants (butyrated hydroxyanisole, butyrated hydroxytoluene, alpha-tocopherol, propyl gallate, disulfiram, cysteamine, ascorbate, selenite and glutathione) on differentiation of the cells were examined. Butyrated hydroxyanisole, butyrated hydroxytoluene and alpha-tocopherol significantly inhibited the differentiation of the cells induced by dexamethasone or a protein inducer. Other antioxidants had little or no inhibitory activity. Among the antioxidants tested, butyrated hydroxyanisole was the most potent inhibitor. The inhibition by butyrated hydroxyanisole was the most potent inhibitor. The inhibition by butyrated hydroxyanisole was not due to cytotoxicity and was reversible. The butyrated hydroxyanisole-mediated inhibition was counteracted by prostaglandin E1 or E2 but not F1 alpha. Moreover, butyrated hydroxyanisole inhibited the production of prostaglandin E2 by M1 cells treated with dexamethasone. These results suggest that the inhibition by butyrated hydroxyanisole of differentiation of M1 cells may be due to the inhibition of synthesis of prostaglandin E2.

Alkyl-lysophospholipids are synthetic analogs of naturally occurring lysophospholipids. The effects of these compounds on cell proliferation and differentiation of cultured human (HL-60) and mouse (M1) myeloid leukemia cells were studied. Both cell lines were induced to differentiate into morphologically and functionally mature granulocytes and macrophages by incubation with a wide variety of these compounds. Some alkyl-lysophospholipids induced differentiation (judged morphologically and by the appearance of abilities to reduce nitro blue tetrazolium, to phagocytize latex particles, and to induce lysozyme activity) of both the cells lines at concentrations of 1 microgram/ml. However, these compounds did not affect colony formation of normal mouse bone marrow cells even at a higher concentration, 20 microgram/ml. These results suggest that alkyl-lysophospholipids induce cell differentiation of myeloid leukemia cells without affecting proliferation and differentiation of normal bone marrow cells. Thus, these compounds could be useful in therapy of myeloid leukemia.

Mouse myeloid leukemic MI cells can be induced to differentiate into mature macrophages and granulocytes by differentiation-stimulating factor (D-factor) in conditioned medium of mouse peritoneal macrophages. Double-stranded RNA's, such as the copolymers of polyinosinic and polycytidylic acids and polyadenylic and polyuridylic acids, could not alone induce differentiation of the cells, but enhanced induction of differentiation by low concentrations of the D-factor and induced a significant amount of interferon. Rabbit antiserum to purified L-cell interferon neutralized the antiviral activity of interferon of MI cells. Simultaneous treatment of MI cells with the anti-interferon serum and copolymer of polyinosine and polycytidylic acids and D-factor abolished the enhancing effect of copolymer of polyinosine and polycytidylic acids on the action of the D-factor. These results suggest that the effect of double-stranded RNA's on induction of differentiation of MI cells is mediated by interferon produced by the cells.

Recently, it has been shown clearly that various tumor cells can be induced by differentiation inducers including biological response modifiers, synthetic chemicals and conventional anticancer drugs to differentiate terminally both in vitro and in vivo into cells with normal characteristics. On differentiation, the cells cease to proliferate and lose their transplantability in either nude mice or syngeneic animals. Furthermore, prolongation by the differentiation inducers of survival times of animals inoculated with various tumors was confirmed. These findings suggest that induction of terminal cell differentiation by differentiation inducers is another approach to tumor therapy, that is "differentiation therapy" of tumors. In this review, recent results of basic and clinical studies on the differentiation therapy of tumors are described. The problems and perspectives of differentiation therapy are also discussed.

Mouse monocytic Mm-A cells are a highly leukemogenic variant line of the monocytic and non-leukemogenic cell line Mm-1, which developed spontaneously from mouse myeloid leukemia M1 cells. Studies were made on whether Mm-A cells could be induced to differentiate further by agents that were effective for inducing differentiation of the parent M1 cells and other leukemic cells. Of the agents tested, butyrate, conditioned medium from concanavalin A-stimulated spleen cells, lipopolysaccharide (LPS) and N6,O2-dibutyryl adenosine 3'5'-cyclic-monophosphate (dbcAMP) significantly stimulated the lysozyme activity of Mm-A cells, which is one of the most characteristic biochemical markers of monocytes and macrophages. Butyrate was the most effective agent for increasing lysozyme production by Mm-A cells; culture with 0.5mM butyrate for 3 days increased lysozyme production by Mm-A cells about 50-fold. Inducers of M1 cell differentiation such as dexamethasone, 1 alpha,25-dihydroxyvitamin D3, arginase, and proteinous inducer did not increase the lysozyme activity. Butyrate also induced NBT reduction and stimulated other differentiation-associated functions, such as expressions of Fc receptors on the cell surface, immune phagocytosis and production of inducer for M1 cell differentiation. Its effect in stimulating differentiation of Mm-A cells was synergistic with that of dbcAMP or LPS. Incubation with butyrate inhibited the proliferation of Mm-A cells, about 0.3mM butyrate causing 50% inhibition. These results indicate that monocytic, leukemogenic Mm-A cells can be induced to differentiate further by butyrate and that the inducers of differentiation of Mm-A cells are markedly different from those of the parent myeloblastic M1 cells.

Growth inhibition of murine and human myeloid leukemia cells by differentiation inducers during long-term culture was examined to improve the strategy for therapy of myeloid leukemia by differentiation inducers. When the effect of 1 alpha,25-dihydroxyvitamin D3, a typical differentiation inducer, on proliferation of mouse myeloid leukemia M1 cells was examined at a constant product of time and concentration (480 nM in 20 days), the continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3 was the most effective for inhibition of cell proliferation. After 20 days, the cumulative cell number was reduced about 3 X 10(5) times by continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. Similar results were obtained when M1 cells were treated continuously with dexamethasone. M1 cells resistant to 1 alpha,25-dihydroxyvitamin D3 appeared about 25 days after the start of continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. On the other hand, when M1 cells were treated continuously with 1 alpha,25-dihydroxyvitamin D3 and noncytotoxic doses of antileukemic drugs such as 1-beta-D-arabinofuranosylcytosine and daunomycin, resistant cells did not appear for at least 35 days. A similar effect of 1 alpha,25-dihydroxyvitamin D3 and antileukemic drugs on cell proliferation was observed with the human monoblast-like cell line U937. The survival of syngeneic SL mice inoculated with M1 cells was prolonged more by treatment with both 1 alpha-hydroxyvitamin D3 and daunomycin than by treatment with either drug alone. These results suggest that continuous treatment with both differentiation inducers and certain antileukemic drugs may be more effective therapeutically than treatment with a differentiation inducer alone.

Mouse myeloid leukemia line cells, M1, could be induced to differentiate in vitro into macrophages and granulocytes with ascitic fluid of animals bearing various tumors. M1 cells could not be induced to differentiate with dimethyl sulfoxide alone. During the culture of M1 cells, spontaneously appearing cells resistant to factors stimulating differentiation (D-factor) in ascitic fluid were isolated. These resistant cells were more refractile to the toxic action of dimethyl sulfoxide than sensitive cells and grew in culture medium with 1% dimethyl sulfoxide. Although the resistant cells were not induced to differentiate with dimethyl sulfoxide alone, they were sensitized with the aid of dimethyl sulfoxide to undergo differentiation with the D-factor in ascitic fluid.

The sensitivity of human leukemia K562 cells to cancer chemotherapeutic drugs during induction of erythroid differentiation of the cells by hemin was examined. Treatment with hemin greatly increased the sensitivity of the cells to 1-beta-D-arabinofuranosylcytosine (ara-C) but did not affect their sensitivities to other chemotherapeutic drugs, including Adriamycin, daunomycin, hydroxyurea, methotrexate, and vincristine. Thymidine and deoxyguanosine, which are known to potentiate the antileukemic effects of ara-C in K562 cells, also induced erythroid differentiation of K562 cells, but other inducers, such as sodium butyrate and delta-aminolevulinic acid, did not increase the sensitivity of K562 cells to ara-C. Hemin did not enhance the sensitivity to ara-C of other leukemia cell lines (Friend erythroleukemic cells, myeloid leukemic M1 cells, and promyelocytic leukemia HL-60 cells). These results indicate that some inducers of erythroid differentiation of K562 cells potentiate the antileukemic effect of ara-C on K562 cells.

Mouse myeloid leukemia cells (M1) could be induced to differentiate into mature macrophages and granulocytes with dexamethasone or proteinaceous inducer. Retinoic acid inhibited functional and morphological differentiation of M1 cells, but the pyridyl analog of retinoic acid had no effect. M1 cells could be induced to produce a factor(s) inhibiting their own differentiation to macrophage- and granulocyte-like cells by retinoic acid but not by its pyridyl analog. This factor(s) inhibited induction by inducers of phagocytic activity, locomotive activity, lysozyme activity, and morphological changes in M1 cells. The production of the inhibitory factor(s) by M1 cells incubated with retinoic acid was inhibited by a low concentrations (5--10 ng/ml) of actinomycin D. The inhibitory factor seemed to be a protein(s), since it was susceptible to heat treatment and proteases. The effect of retinoic acid in inducing production of the inhibitory factor(s) by M1 cells seemed to be reversible, since it was low on washing the cells with fresh medium. Therefore, induction of this inhibitory factor may be involved in the mechanism of inhibition of functional and morphological differentiation of M1 cells by retinoic acid.

In medium with serum, 12-O-tetradecanoylphorbol-13-acetate (TPA) induced alpha-naphthyl acetate esterase activity in human promyelocytic leukemia cells (HL-60), adherence of the cells to the culture dish, and their change into forms that were morphologically similar to macrophages. HL-60 cells grew in the absence of serum in synthetic medium supplemented with insulin, transferrin, and several trace elements, and could be maintained for more than 6 months in this medium. Induction of differentiation by TPA was observed with cells grown in serum-free medium. Human myeloid leukemia cells (K562-4) cultured in medium with serum could not be induced to differentiate even in the presence of TPA, but their differentiation into macrophages in the presence of TPA, arginase or actinomycin D was observed after they had been grown in serum-free medium for 4 months. Addition of serum inhibited the induction of differentiation of HL-60 and K562-4 cells that had been grown in serum-free medium. 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 results suggest that the different responses in media with different sera may be specific to TPA. Induction of adhesiveness of K562-4 cells by TPA required some unknown serum factor(s), although addition of serum inhibited the inductions of morphological and functional differentiation. The relation between the ability of K-562-4 cells to be induced to differentiate into macrophages and long-term cultivation in serum-free medium is discussed.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a protein inducer, differentiation-stimulating factor (D-factor), and by various other compounds. Mouse spleen cells produced D-factors when treated with various mitogens, such as concanavalin A, phytohemagglutinin, pokeweed mitogen, lipopolysaccharide, and synthetic double-stranded polyribonucleotide copolymer of polyinosinic and polycytidylic acids. Concanavalin A, phytohemagglutinin, and pokeweed mitogen stimulated spleen lymphocytes, but not spleen macrophages, to produce a D-factor with an apparent molecular weight of 40,000 to 50,000. On the other hand, lipopolysaccharide and copolymer of polyinosinic and polycytidylic acids stimulated both spleen lymphocytes and spleen macrophages to produce D-factors. Spleen macrophages produced D-factors with molecular weights of 40,000 to 50,000 and 20,000 to 25,000, whereas spleen lymphocytes produced only the larger molecules. In addition to D-factor, colony-stimulating factor (CSF), which stimulates growth and differentiation of normal bone marrow cells, and interferon, were detected in conditioned medium of spleen cells treated with concanavalin A or lipopolysaccharide. On gel filtration of the conditioned medium with Sephadex G-100, CSF was eluted between the larger D-factor and the smaller one. The fraction with interferon activity overlapped that of the larger D-factor. Incubation of the conditioned medium at pH 2 abolished the activity of interferon but did not affect the activity of either D-factor or CSF. The addition of cytochalasin B suppressed the production of interferon but not of D-factor or CSF by the spleen cells. These results indicate that the D-factor is a different substance from CSF or type II interferon.

The effects of six nitrofuran derivatives (including a formerly used food preservative) on mouse skin sebaceous glands were investigated. A close correlation was found between the carcinogenicities and destructive activities of nitrofuran derivatives on the sebaceous glands. 5-Nitro-2-furaldehyde semicarbazone and 4-methyl-1-[(5-nitrofurfurylidene)amino]-2-imidazolidinone, which are carcinogenic, caused marked destruction of the glands at a dose of 1-5 mg/mouse. 2-(5-Nitro-2-furfurylidene)-aminoethanol almost completely destroyed the glands at a dose of 5 mg/mouse; its carcinogenicity has not yet been investigated. 1-[(5-Nitrofurfurylidene)amino]-carcinogenic, did not affect the glands, even at a dose of 5 mg/mouse. 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, which is a potent mutagen but not carcinogenic, had no effect on the glands at a dose of 5 mg/mouse. Under similar conditions, the potent carcinogen 7,12-dimethylbenz(alpha)athracene almost completely destroyed the sebaceous glands at a dose of 0.05 mg/mouse, but dimethyl sulfoxide (used as solvent for the test compounds) had no effect.

Although various substances are involved in modification of chemical carcinogenesis, prostaglandins have been known to play an important role in the modification of the carcinogenesis. In this report, recent advances in the modification of chemical carcinogenesis by prostaglandins are reviewed. In addition, recent experimental results on the modification of cell differentiation by prostaglandins and some inhibitors of chemical carcinogenesis are described. Some inhibitors of chemical carcinogenesis are found to modify differentiation of mouse myeloid leukemia cells (M1) into macrophages and granulocytes by perturbation of syntheses of prostaglandins. These experimental results suggest that the modification by the inhibitors of carcinogenesis of differentiation of cells by perturbation of syntheses of prostaglandins is involved in the mechanisms of inhibition of chemical carcinogenesis.

Cell line R453, established from a Rauscher virus-induced myeloid leukemia in a C57BL/6 mouse, was induced to differentiate in vitro into macrophages and granulocytes with ascitic fluids from animals bearing various ascites tumors or from mice treated with complete Freund's adjuvant, conditioned media from various cell lines, and glucocorticoid hormone. Differentiated R453 cells had a morphology similar to that of macrophages and granulocytes in normal hematopoietic organs, and they phagocytized small paricles such as latex particles, moved in soft agar showing locomotive activity, and had Fc and C3 receptors on the cell surface. This induction of differentiation of R453 cells was markedly enhanced by addition of inhibitors of RNA synthesis (actinomycin D, nogalamycin, or chromomycin A3), protein synthesis (puromycin or cycloheximide), or DNA synthesis (methotrexate, hydroxyurea, 5-fluorodeoxyuridine, or 1-beta-D-arabinofuranosylcytosine) in the presence of ascitic fluid. Of the inhibitors, actinomycin D was the most effective at a low concentration (5 ng/ml) in stimulating induction of differentiation of R453 cells. However, these inhibitors alone did not induce differentiation of R453 cells. The factor(s) in ascitic fluid that stimulates differentiation of R453 cells was heat labile, nondialyzable, and inactivated by trypsin.

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-activity) has been found in conditioned medium of variant M1 cell clones resistant to differentiation inducers, and this I-activity has been shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-activity in the conditioned medium of the resistant M1 cells was shown to bind to Carboxymethyl-Sepharose CL-6B and to be eluted with 0.27-0.4 M NaCl. The profile of gel filtration of I-activity from Sephadex G-200 indicated considerable heterogeneity in molecules with I-activity; the apparent molecular range of the main I-activity was 60,000-80,000. On chromatofocusing, the I-activity was eluted with Polybuffer 96-acetic acid at pH 8.8-9.0. The I-activity was inactivated by treatment with trypsin or by heating at 75 degrees C for 30 min. Therefore, the main I-activity seemed to be due to a basic protein(s).

The effect of chloroquine on differentiation of cultured mouse myeloid leukemia Ml cells was examined. On treatment with 5 approximately 25 microgram/ml of chloroquine diphosphate for 1 approximately 4 days, the cells were induced to phagocytize latex beads, to form Fc rosettes, to form dispersed colonies in soft agar, and to synthesize lysozyme, unlike untreated cells. The morphology of about 40% of the cells changed during treatment with 20 microgram/ml of chloroquine diphosphate for 4 days; some cells developed small eccentrically located nuclei, and others ring-shaped or segmented nuclei. These results show that Ml cells differentiate into cells resembling macrophages or granulocytes on treatment with chloroquine.

Studies were made on the effect of cancer chemotherapeutic drugs on in vitro differentiation of a clone (R4) of mouse myeloid leukemic cells (MI) that is resistant to inducers. Treatment of the cells with 50% ascitic fluid (an inducer) plus 0.25 microgram/ml of adriamycin or 0.3 microgram/ml of daunomycin induced phagocytic activity and suppressed cell growth, but had little effect on cell viability; treatment with ascitic fluid or the drugs alone had no effect. In combination with ascitic fluid, mitomycin-C, hydroxyurea, 5-fluorouracil, or bleomycin also induced phagocytic activity, but 6-mercaptopurine, amethopterin, or aminopterin did not. These drugs also induced other differentiation-associated properties, lysozyme activity, and locomotive activity. The present results indicate that some cancer chemotherapeutic drugs sensitive resistant leukemic cells to an inducer of cell differentiation.

Leukemia cells from patients with acute myeloid leukemia in relapse were treated with various inducers of differentiation of human myeloid leukemia cell lines. Leukemia cells in primary culture from most, but not all, patients underwent morphological, cytochemical and biochemical changes after treatment with inducers of differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid, actinomycin D, aclarubicin, and alkyl lysophospholipid. The most effective inducer varied from specimen to specimen. Leukemia cells from patients in relapse were compared with those from untreated patients. The responsiveness to TPA of leukemia cells from patients in relapse was similar to that of leukemia cells from untreated patients. However, retinoic acid or actinomycin D resistance was more frequently observed in leukemia cells from patients in relapse than in those from patients before initial therapy. This is the first report to indicate that leukemic cells from relapsed patients who are resistant to cytotoxic chemotherapeutic drugs can be induced to differentiate into mature cells by appropriate inducers. However, the responsiveness to inducers of leukemia cells from patients in relapse is not the same as that of leukemia cells before therapy.

Antiserum to a synthetic peptide that defines a hydrophilic region within the putative c-myb translation product was prepared in the rabbit. In lysates from exponentially growing ML-1, human myeloblastic leukemia cells, the antiserum ("anti-myb") reacted with five proteins of Mr 58,000, 75,000, 85,000, 90,000 and 105,000. Of these, only p75 and a trace of p85 were detected, by immunoblotting, in extracts derived from ML-1 cell nuclei. The proteins p58, p75 and p90 were present in readily detectable amounts only in the relatively immature myeloid cell lines ML-1 and HL-60, whereas in the more mature myeloid cell line THP-1 and in the lymphoid line BALL-1 only traces of these proteins were found. p85 and p105 were detected in lysates from all cell lines tested, including myeloid and lymphoid leukemia cells and mouse 3T3 cells. In lysates from ML-1 cells induced to differentiate to monocyte/macrophages or to granulocytes, the concentrations of p58 and p75 decreased in parallel with the cell population moving to maturity; in completely mature populations these two proteins were no longer detectable. In ML-1 cells arrested in G1 by serum depletion, the amount of p58 and p75 and to a smaller extent that of p90 was decreased, whereas the concentration of p85 and p105 remained unchanged. In nuclei from exponentially growing ML-1 cells, the antiserum or its derived immunoglobulin fraction ("anti-myb IgG") inhibited mRNA transcriptional activity by 30%. DNA synthesis was not affected. In contrast, in nuclei from differentiated ML-1 cells, the mRNA transcriptional activity was not significantly inhibited by anti-myb IgG. Similarly, in nuclei from ML-1 cells arrested largely in G1 by serum depletion for 2 days, mRNA transcriptional activity was inhibited by only 11%. Upon supplementation with serum, the mRNA transcriptional activity inhibitable by anti-myb IgG increased in parallel with the increasing rate of cell growth. The difference in total mRNA transcriptional activity observed in nuclei from cells of different growth rate was accounted for by the difference in transcriptional activity inhibitable by anti-myb IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

Addition of a potent tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), to mouse myeloid leukemia line cells (Ml) in suspension cultures inhibited both functional and morphological differentiation of the cells induced by dexamethasone or protein inducer. A positive correlation was found between the tumor-promoting activities of several plant diterpenes and their inhibition of cell differentiation. The inhibition of cell differentiation by TPA was reversible and was unrelated to its cytotoxic action.

Leukemogenicity was studied in sensitive mouse myeloid leukemia cells that could be induced to undergo cell differentiation in vitro into mature granulocytes and macrophages by incubation with the inducer (certain proteins or glucocorticoids) and in resistant myeloid leukemia cells that could not be induced to differentiate into mature cells. Three sensitive and five resistant clones were tested. The resistant cells were much more leukemogenic than the sensitive cells, and the survival time of syngeneic mice inoculated with the sensitive cells. In a diffusion chamber in a syngeneic, inbred SL mouse without any additional manipulation or injection of inducer, the resistant cells remained undifferentiated, but the sensitive cells were induced to differentiate into mature granulocytes and macrophages and their proliferation rate decreased. These results suggested that the greater leukemogenicity of resistant cells is associated with some defect in inducibility of cell differentiation.

Pulmonary colonizing cells were selected by an in vivo-in vitro selection method from cultured mouse mammary carcinoma FM3A cells. When 10(6) parent cells (P-O) were injected iv into syngeneic C3H/He mice, no tumor nodules were reduced. However, when the selected variant cells (P-15) were injected iv, tumor nodules formed predominantly in the lungs within 2 weeks. Mice inoculated iv with 10(6) P-15 cells began to die on day 22, but mice inoculated with P-O cells survived for more than 6 months. When injected sc into C3H/He mice, P-O cells were as tumorigenic as P-15 cells to the syngeneic mice. There was no significant difference between P-O and P-15 cells in their proliferation in vitro or in the inducibility of alkaline phosphatase activity. During successive selection of colonizing variants, the proportion of adherent cells increased. Adherent P-15 cells had a higher colonizing potential than nonadherent P-15 cells. The correlation of the adhesive property of the cells and their colonizing potential is discussed.

Mouse myeloid leukemia M1 cells were induced to differentiate in vitro into macrophages and granulocytes by various inducers including ascitic fluid. Differentiated M1 cells induced with ascitic fluid expressed a differentiation-associated cell surface glycoprotein with a molecular weight of 180,000 (p180), which can be labeled by lactoperoxidase-catalyzed radioiodination or metabolic labeling with L-[14C]fucose. p180 was also induced by treatment with conditioned medium of hamster embryo cells, dexamethasone, dibutyryl cyclic adenosine 3':5'-monophosphate, and prostaglandin E1. Ascitic fluid, conditioned medium of hamster embryo cells, and dexamethasone induced all the differentiation-associated properties tested, whereas dibutyryl cyclic adenosine 3':5'-monophosphate and prostaglandin E1 induced lysozyme activity and adhesiveness to the substratum but not phagocytosis, locomotive activity, Fc receptors, or morphological changes. The adherent cells induced by dibutyryl cyclic adenosine 3':5'-monophosphate produced a large amount of p180, while the floating cells produced very little, but no difference was detected in the lysozyme activities of the two cell types. These results suggest that p180 is associated with cell-substratum adhesion of differentiated M1 cells.

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by a differentiation-inducing factor (D-factor) and various agents. IFN-gamma alone did not induce differentiation of clone T22-3 of M1 cells but inhibited their differentiation by D-factor. That is, IFN-gamma at 4 U/ml inhibited 50% of phagocytic activity of T22-3 cells induced by 7 x 10(-11) M D-factor. In addition, it inhibited the induction of lysozyme activity and morphological differentiation of these cells by D-factor. IFN-gamma also inhibited dexamethasone-induced differentiation of T22-3 cells. Previously interferon-alpha/beta was shown not to induce differentiation of M1 cells itself, but to enhance induction of their differentiation by D-factor. The present study showed that IFN-alpha/beta and IFN-gamma had opposite effects on induction of differentiation of T22-3 cells by D-factor. The effect of IFN-gamma on the differentiation of M1 cells varied with the clone of M1 cells used: IFN-gamma inhibited D-factor-induced differentiation of cells of clones T22-3 and S2, but induced differentiation of cells of clones B24 and S1.

A new human cell line, Br-NHF-1, was established from the pleural effusion of a patient with advanced mammary carcinoma. The cells survived 52 subcultivations during more than 33 months. The cells possess epithelial features, showing rosettes, acinar formation and domes in or among the compact colonies. The modal chromosome number is 51 with some marker chromosomes. Xenografted tumors retained a similar histology to the original tumor. No estrogen-binding protein was detectable in the culture, but there was a significant amount of basic fetoprotein. Production of casein in the cells was detected by immuno-fluorescence testing.

Glucorticoid hormones induced differentiation of mouse myeloid leukemia cells. From comparison of the structure of steroids with their ability to induce phagocytic and locomotive activities, typical characters of mature macrophages and granulocytes, the simplest steroid with inducing ability was concluded to be a steroid with the structure of progesterone and one hydroxyl group at 11beta- or 21-position. The maximum induction ability seemed to require the structure of progesterone and three hydroxyl groups (at 11beta-, 17alpha-, and 21-positions). A single, 30-min pulse treatment with glucocorticoid was sufficient to induce differentiation of leukemia cells. Glucocorticoid receptors were detected in mouse myeloid leukemia cells. The binding affinity of various steroids for the cytoplasmic receptors was closely correlated with the activities of these compounds to induce differentiation of leukemia cells, suggesting that these receptors may be involved in hormonal induction of differentiation of various cells. This suggests that the binding reaction is important for differentiation of myeloid leukemia cells.