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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.

Mouse myeloid leukemic Ml cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor) or by various chemical compounds. Spleen lymphocytes and spleen macrophages have been shown to produce D-factor when treated with various mitogens including lipopolysaccharide or synthetic double-stranded polyribonucleotide, poly(I) . poly(C). Several immunostimulants from microorganisms were tested for ability to induce differentiation of Ml cells and to stimulate production of D-factor by spleen cells. Mycobacterium bovis BCG (500 micrograms/ml) and the cell wall skeleton of Nocardia rubra (50 micrograms/ml) induced differentiation of Ml cells; but Corynebacterium parvum CN6134 and the cell wall skeleton of Propionibacterium acnes C7 had no effect on Ml cells. On the other hand, all immunostimulants tested at concentrations of more than 10 micrograms/ml were found to stimulate production of D-factor by both spleen lymphocytes and spleen macrophages. Synthetic derivatives of N-acetylmuramyl dipeptide(MDP), the minimal adjuvant-active subunit of the bacterial cell wall, had no direct effect on the differentiation of Ml cells and only slightly stimulated the production of D-factor by spleen cells. Therefore, the structure of microorganisms required for induction of differentiation of Ml cells and for stimulation of production of D-factor are suggested to be different from that of MDP.

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.

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.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibited the induction of both functional and morphological differentiation of mouse myeloid leukemia cells (Ml) cultured in medium containing 10% calf serum, but enhanced these inductions in medium containing 10% fetal calf serum and several inducers. These results suggest that some factor(s) in sera modifies the differentiation-inducing action of TPA on leukemia Ml cells.

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.

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.

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.

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.

Mouse myeloid leukemia cells were induced by some corticoid hormones to migrate in agar, phagocytize, and change into forms which were morphologically similar to macrophages and granulocytes. Inducing ability of corticoids was correlated with glucocorticoid activity, not mineralocorticoid activity. Other steroids were ineffective to induce differentiation of myeloid leukemia cells. Glucocorticoid-resistant cells, which could not differentiate even in a high concentrations of dexamethasone, were selected from steroid-sensitive cells by stepwise increase in concentrations of dexamethasone in culture medium.

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.

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.

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.

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 leukemia M1 cells were induced to differentiate in vitro into macrophages and granulocytes by various inducers, including dexamethasone. Prostaglandin F2 alpha inhibited the inductions by dexamethasone of phagocytic and lysozyme activities in M1 cells. Prostaglandin F2 alpha stimulated the production of differentiation-inhibiting activity (I-activity) in M1 cells. I-activity production by prostaglandin F2 alpha was decreased by simultaneous treatment with actinomycin D (5 ng/ml) but not with 5-fluoro-2'-deoxyuridine (10 ng/ml). The I-activity was inactivated by heating (70 degrees, 20 min) or by treatment with trypsin but not with mixed glycosidases or ribonuclease, suggesting that I-activity was due to a proteinous substance(s). B-Type prostaglandins also stimulated I-activity production, whereas A-, E- and D-type ones did not. Induction of prostaglandin E2. Retinoic acid stimulated the synthesis and release of prostaglandin F2 alpha and production of I-activity in M1 cells. Indomethacin completely inhibited induction of I-activity by retinoic acid. On the basis of these results, the relationship between I-activity production and prostaglandin F2 alpha production is discussed.

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.

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.