Search

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.

Alkyl ethyleneglycophospholipids induced differentiation in vitro of mouse myeloid leukemia M1 cells into mature granulocytes and macrophages. The compounds also prolonged the survival of syngeneic SL mice inoculated with M1 cells. Although in mice with florid leukemia these compounds alone scarcely affected survival, administration of dodecyl ethyleneglycophospholipid with pyridinioethyl as a polar group plus actinomycin D significantly prolonged survival.

A rapid decrease of myeloblasts and a remarkable increase of mature neutrophils, mostly with Pelger anomaly, were observed in the peripheral blood of a 51-year-old woman with terminal acute myeloblastic leukemia during 16 days of daily i.v. administration of 20 mg aclacinomycin-A (ACM-A). When the same dose was administered later on three consecutive days each week, a similar hematore neutrophils, mostly with Pelger anomaly, were observed in the peripheral blood of a 51-year-old woman with terminal acute myeloblastic leukemia during 16 days of daily i.v. administration of 20 mg aclacinomycin-A (ACM-A). When the same dose was administered later on three consecutive days each week, a similar hematore neutrophils, mostly with Pelger anomaly, were observed in the peripheral blood of a 51-year-old woman with terminal acute myeloblastic leukemia during 16 days of daily i.v. administration of 20 mg aclacinomycin-A (ACM-A). When the same dose was administered later on three consecutive days each week, a similar hematological change occurred again. An increase of myeloblasts observed between the third and the fifth week of this intermittent schedule was accompanied by that of mature neutrophils. Thrombopenia and anemia did not improve significantly. These findings may indicate the induction of leukemic myeloblasts by ACM-A into mature neutrophils. Administration of relatively small dose anthracyclines including ACM-A may be another potential choice in the treatment of refractory acute non-lymphocytic leukemia (ANLL) or ANLL after relapse.

The effect of L-cell interferon (IFN) preparation on the survival times of mice implanted with two different clones (T-22 and R-4) of mouse myeloid leukemic M1 cells were examined. T-22 cells, but not variant R-4 cells, can be induced to differentiate in vitro or in vivo into macrophages and granulocytes. In vitro growth of R-4 cells was markedly suppressed by IFN, but that of T-22 cells was resistant to IFN. The survival times of mice with implanted T-22 cells were prolonged by treatment with IFN but those of mice with implanted R-4 cells were not.

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.

Various alkyl ethyleneglycophospholipids, i.e., alkyl phospholipids, with ethyleneglycol or its congener in place of glycerol as a molecular backcone, were synthesized and their effects on cell proliferation and differentiation of cultured human (HL-60) and mosue (Ml) myeloid leukemia cells were studied. On incubation with alkyl ethyleneglycophospholipids, proliferation of both cell lines was inhibited and the cells were induced to differentiate into morphologically and functionally mature granulocytes and macrophages. Among the compounds tested, dodecyl ethyleneglycophospholipid with a pyridinioethyl group was the most effective in induction of differentiation of both cell lines.

The differentiation of mouse myeloid leukemia line cells (M1) into macrophages or granulocytes has been reported. Resistant M1 cells which did not differentiate even with a high concentration of inducer of differentiation were isolated from M1 cells. The conditioned medium of the resistant M1 cells (RCM) inhibited the induction of differentiation of M1 cells and the formation of macrophage and granulocyte colonies of normal mouse bone marrow cells. Acidic isoferritins known as negative regulators of normal bone marrow cells (CFU-GM) failed to inhibit the induction of differentiation and growth of M1 cells. The RCM treated with anti-acidic isoferritin serum could inhibit the induction of differentiation of M1 cells as did the untreated RCM. The activities, in RCM, inhibiting growth and differentiation of the normal bone marrow cells were partly neutralized by treatment with the antiserum but most of the activities remained. These results suggest that growth and differentiation of the mouse myeloid leukemia M1 cells are not regulated by acidic isoferritins and other inhibitory activities affecting normal bone marrow colony formation, in addition to acidic isoferritins, are released from M1 cells.

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.

Inhibitors and their mechanisms of inhibition of various processes in chemical carcinogenesis, metabolic activation of chemical carcinogens followed by initiation and promotion in chemical carcinogenesis are reviewed. Furthermore, significance of the inhibitors of chemical carcinogenesis in foods and food additives and problems of side effects of these inhibitors are discussed.

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.

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.

We developed an experimental model to investigate in vivo differentiation and proliferation of leukemia cells using mouse myeloid leukemia aneuploid cells (LL-2) and syngeneic SL mice. The LL-2 cells were near-tetraploid cells isolated from mouse myeloid leukemia cell line M1 (clone D501). In suspension culture, the LL-2 cells were myeloblastic and grew well like parent D501 cells, but were distinct from the parental cells due to the large size of their nucleus, double chromosome number and DNA content. The LL-2 cells as well as D501 cells could be induced to differentiate in vitro into mature macrophage-like cells by a protein inducer of differentiation. After transplantation of 4 X 10(6) LL-2 cells into the intraperitoneal cavity of syngeneic SL mice, most of them died of leukemia within 10 weeks. On microscopic examination of the peritoneal cells of the mice, the transplanted LL-2 cells were clearly distinguishable from normal host cells by the size of their nucleus. We determined the increase in the LL-2 cells in the peritoneal cavity by morphological examination of the large-sized LL-2 cells. Survival times of the mice inoculated with the LL-2 cells were prolonged by administrations of an inducer of differentiation, poly(I).poly(C). We found morphological changes in the peritoneal blastic LL-2 cells to mature macrophage-like cells after the serial administrations of poly(I).poly(C) to the recipient mice. Thus the aneuploid LL-2 cells that grow in syngeneic mice may be useful to study in vivo differentiation and proliferation of leukemia cells, and to develop a therapeutic strategy of leukemia using various treatments including differentiation inducers.

Syngeneic SL mice inoculated with murine myeloid leukemia cells (M1) all died of leukemia within 30 days. Treatment three times a week with 12.5-50 pmol per mouse of either 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], the active form of vitamin D3, or its synthetic analog, 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3], considerably prolonged the survival time of mice inoculated with M1 cells. 1 alpha(OH)D3 was more effective than 1 alpha,25(OH)2D3 in increasing the survival time of the mice. 1 alpha(OH)D3 also increased the survival time of nude mice inoculated with M1 cells. The 1 alpha(OH)[3H]D3 administered intraperitoneally to tumor-bearing mice was converted very rapidly to 1 alpha,25(OH)2-[3H]D3. The chronic administration of 25 pmol of 1 alpha(OH)D3 to tumor-bearing mice for 30 days caused no appreciable hypercalcemia. These results indicate clearly that 1 alpha,25(OH)2D3 is effective not only in inducing differentiation of M1 cells in vitro, as previously reported [Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S. & Suda, T. (1981) Proc. Natl. Acad. Sci. USA 78, 4990-4994], but also in prolonging the survival time of mice inoculated with M1 cells.

Two cases in myeloid blast phase of chronic myelogenous leukemia (CML) responded to treatment with plicamycin alone. Their total white blood cell (WBC) count and immature myeloid cells fell in one case rapidly and in the other gradually. Approximately two to three weeks after initiation of plicamycin their total WBC count began to rise again, whereas the mature myeloid cells remained constant throughout the treatment. These results suggest that plicamycin may be effective in suppressing proliferation of blasts and promoting their maturation in the myeloid blast phase of CML, but that plicamycin alone may not be effective enough to sustain duration of response.

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.

In-vitro studies of leukemic cells with retinoic acid and a therapeutic clinical trial with its derivative, etretinate, in a 58 yr-old male patient with 15;17 translocation-positive acute promyelocytic leukemia (APL) in relapse are reported. Actinomycin D was used in combination. Bone marrow promyelocytes from the patient prior to etretinate and actinomycin D matured morphologically in the liquid culture with retinoic acid; 98% were matured myeloid cells after 6 days at a concentration of 10(-6) M of retinoic acid as compared with 2% in the control culture. Positive NBT reactions were seen in none of the cells in the latter but in 95% of the cells in the former. Actinomycin D, when added alone, only induced NBT positivity, but, when used in combination with retinoic acid, increased both NBT positivity and morphologically matured cells. The patient was treated daily with 2 micrograms/kg of actinomycin D (or 20 mg/m2 or 33 mg/m2 cytosine arabinoside after the 6th day) in 24-h infusions and per oral 90 mg/body of etretinate. No effectiveness was observed both morphologically and clinically. The patient expired 15 days after the initiation of etretinate. Thus, a discrepancy existed in the response of leukemic cells from this relapsed patient with APL to the in-vitro and in-vivo attempts to include differentiation by retinoids and actinomycin D.

The interaction of granulocyte-colony stimulating factor (G-CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G-CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10(-8) M RA induced granulocytic differentiation of APL cells. Although G-CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G-CSF significantly enhanced the RA-induced granulocytic differentiation of APL cells in vitro. Enhancement by G-CSF was not due to the prolongation of survival of RA-induced differentiated cells, but the differentiation-inducing effects of G-CSF might be evident only in the presence of RA. Since G-CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G-CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.

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.

Murine myeloid leukemia cells (MI) are induced to differentiate into macrophages by the metabolically active form of vitamin D3,1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] (E. Abe et al., (1981) Proc. Natl. Acad. Sci. USA 78, 4990-4994). At 0.12-120 nM, 1 alpha,25(OH)2D3 suppressed cell growth in a dose-dependent manner and markedly induced phagocytic activity, lysozyme activity, and C3-receptor formation. The potency of 1 alpha,25(OH)2D3, at 0.12-120 nM, in inducing differentiation was nearly equivalent to that of 10-10,000 nM of dexamethasone, one of the most potent stimulators of Ml cells. Simultaneous treatment with low physiological plasma concentrations of 1 alpha,25(OH)2D3 (0.12 nM) and dexamethasone (10 nM) induced differentiation of Ml cells equivalent to the responses obtained only by using much higher concentrations of the respective steroids when used separately. In addition, two variant clones of Ml cells resistant to either 1 alpha,25(OH)2D3 or dexamethasone were isolated. One was resistant to 120 nM of 1 alpha,25(OH)2D3 but sensitive to 10-1000 nM of dexamethasone. The other was resistant to 1000 nM of dexamethasone but sensitive to 12 nM of 1 alpha,25(OH)2D3. This suggests that the mechanism of action of 1 alpha,25(OH)2D3 in inducing differentiation of Ml cells is different at least in part from that of dexamethasone, and that combination therapy by both steroids may be useful in reducing leukemogenicity of Ml cells in vivo.

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.

New antitumor alkylglycerophospholipids, in which primarily the phosphocholine moiety of the platelet activating factor (PAF) molecule was modified, were synthesized from 1-alkyl-2-substituted glycerols by introducing polar head phosphoryl groups having methylene bridges of various lengths (from 2 to 14 carbons). They were tested for PAF agonistic activity and antitumor properties. In a series of 1-octadecyl-2-acetoacetylglycerophospholipids (1a-f), an increase in the length of the methylene bridge separating the phosphate and trimethylammonio group in the polar head side chain at position 3 of the glycerol backbone resulted in a progressive decrease in PAF agonistic activity and a characteristic change in antitumor activity against human promyelocytic leukemia cells (HL-60). Maximal potency was obtained with the compound having a decamethylene bridge (1e, IC50 value = 1.5 microgram/ml). Thus, alkylphospholipids possessing a decamethylene bridge and a variety of substituents at position 2 (1g-n) were synthesized. They showed potent inhibitory activity with IC50 values ranging from 0.4 to 1.9 micrograms/ml, depending on the nature of the 2-substituent in the phospholipid molecule. In in vivo tests of the present series of alkylglycerophospholipids (1a--n), using mice bearing sarcoma 180 and mice with mammary carcinoma MM46 (both cells and compounds were given i.p.), 1-octadecyl-2-acetoacetyl-3-glyceryl omega-trimethylammoniodecyl phosphate (1e) showed the most potent life-prolonging effect. The structure-activity relationships are discussed.

Several clones of dexamethasone-resistant cells, which could not differentiate even in a high concentration of dexamethasone, were isolated from glucocorticoid-sensitive myeloid leukemic cells. Some of them were shown to be deficient in steroid binding to specific cytoplasmic receptors, while the others contained glucocorticoid-specific cytoplasmic receptors that might be the same as those in sensitive cells. One of the resistant clones was found to be almost completely deficient in nuclear acceptor sites for cytoplasmic steroid-receptor complexes. The remaining clones were also characterized by significantly reduced amounts of nuclear-bound glucocorticoid. These results suggest that resistibility to glucocorticoids in the resistant clones of myeloid leukemic cells is due mainly to a defect in some steps of intracellular transfer of the steroid. Dexamethasone-sensitive cells, which could differentiate in the presence of dexamethasone, could be also induced to differentiate by protein factor(s) in ascitic fluid. Although all the resistant cells showed a low response to ascitic fluid, some of them showed 10-fold enhancement of phagocytic activity which is a typical character of differentiated cells. These results suggest that response to steroids is not directly correlated with that to protein inducer(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.

Treatment with ascitic fluid from animals bearing various tumors, can induce mouse myeloid leukemia line cells, M1, to differentiate in vitro into macrophages and granulocytes. Cells were isolated that were resistant to the ascitic fluid factor(s) stimulating differentiation (D-factor). The resistant cells became sensitive to the D-factor and differentiated after treatment with various inhibitors of RNA synthesis (actinomycin D, nogalamycin, chromomycin A3 or cordycepin) or protein synthesis (puromycin or cycloheximide). The cells could not be induced to differentiate by treatment with the inhibitors alone. The effective doses of the inhibitors of protein synthesis were toxic to the cells. Among these inhibitors actinomycin D (5 ng/ml) was the most effective for sensitizing the resistant cells. Inhibitors of DNA synthesis did not sensitize the resistant cells. Added actinomcyin D was mainly recovered in the nuclear fraction of the cells. The sensitizing effect of actinomycin D on the cells was roughly parallel to the extent of its inhibition of RNA synthesis in the cells. The effective concentration of actinomycin D (5 ng/ml) mainly inhibited it also inhibited alpha-amanitin-resistant RNA synthesis to some extent. These results suggest that alpha-amanitin-sensitive RNA synthesis may be involved in sensitization of the resistant cells to the D-factor.

The effects of some synthetic polyribonucleotides on induction of differentiation of mouse myeloid leukemic M1 cells were examined. Poly(I) was found to be a potent inducer; on treatment with 100--200 microgram/ml of poly(I) for 2--4 days, M1 cells differentiated into cells resembling macrophages and granulocytes and developed phagocytosis and locomotive activities, Fc receptors and lysozyme activity. Poly(C) was less effective than poly(I) for induction of phagocytic activity, while the other single-stranded RNAs, poly(U) and poly(A), had no effect. Double-stranded RNAs, such as poly(I) . poly(C) and poly(A) . poly(U), were cytotoxic to M1 cells, and differentiation of the cells could not be detected even at the highest tolerable concentrations of these double-stranded RNAs.

Differentiation-stimulating factor (D-factor) purified from mouse Ehrlich ascites cells was sequenced partially and found to be almost identical to leukemia inhibitory factor (LIF) from mouse Krebs II ascites cells. In comparison to LIF, D-factor had an additional amino-terminal serine residue. Using synthetic oligonucleotide probes designed from the murine D-factor sequence, we cloned the human gene encoding D-factor. A partial D-factor cDNA was cloned from COS-1 cells transfected with the human D-factor gene under the control of a heterologous promoter. We used this cDNA to construct a vector for direct expression of the protein in Escherichia coli. A mammalian cell expression vector was constructed using the signal sequence of interferon-alpha A linked to the D-factor cDNA. Both forms of recombinant human D-factor were active on the murine myeloid leukemia cell line M1 in a dose- and time-dependent manner for the inhibition of [3H]thymidine incorporation, and also induced phagocytosis, Fc receptor expression, and prostaglandin E2 synthesis by M1 cells.