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Protein kinase activities are involved in cellular proliferation and differentiation, and inhibitors of these activities are useful for studying the mechanisms of induction of differentiation. We found that staurosporine, an inhibitor of protein kinase activities, induced morphological differentiation of human myeloblastic leukemia ML-1 cells along myelomonocytic lineage and also induced functional differentiation (increase in nitroblue tetrazolium-reducing and lysozyme activities) in the cells. Several other protein kinase inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), sphingosine, N-(6-aminoethyl)-5-chloro-1-naphthalenesulfonamide and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) did not induce the differentiation of ML-1 cells. Treatment with staurosporine induced formation of granules in ML-1 cells, and the granules showed metachromasia by toluidine blue staining; however, histamine content did not increase. The "metachromatic" ML-1 cells were positive for CD14, indicating that staurosporine induced the differentiation of ML-1 cells into metachromatic monocytes/macrophages, 1 alpha,25-dihydroxyvitamin D3 (VD3) enhanced appearance of metachromatic granules in staurosporine-treated cells. These results suggest that modulation of protein phosphorylation by a staurosporine-sensitive protein kinase(s) may be associated with differentiation of ML-1 leukemia cells.

We studied the effects of D-factor on the growth of leukemic blast progenitors from 15 patients with acute myeloblastic leukemia and two leukemia cell lines in methylcellulose and suspension cultures. When stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor or interleukin-3, leukemic blast progenitors undergo terminal division with limited differentiation in methylcellulose culture, forming blast colonies. Leukemic blast progenitors can renew themselves. The self-renewal can be detected as secondary colony formation after replating primary blast colonies in fresh methylcellulose media and by the growth of clonogenic cells in suspension culture. D-Factor suppressed primary and secondary colony formation in methylcellulose culture. Furthermore, D-factor suppressed clonogenic cell recovery in suspension culture. The suppression by D-factor of the growth of leukemic blast progenitors was not significantly dependent upon the colony-stimulating factors used as growth-stimulating factors. High concentration of G-CSF did not overcome the suppressive effect of D-factor. The results indicate that D-factor is effective in suppressing not only terminal division but also self-renewal of leukemic blast progenitors.

New pyrimidine nucleoside analogs (18 compounds) were synthesized and their growth-inhibiting and differentiation-inducing activities on human myeloid leukemia HL-60 cells were examined. Some of the analogs were found to induce nitroblue tetrazolium (NBT) reducing activity in the HL-60 cells. The inducing activities of these compounds were compared at their concentrations for 50% inhibition of cell growth. TI-79 (3-benzyl-5-methyl-3-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine- 2,4(1H,3H)-dione) was a very effective inducer of NBT-reduction and of differentiation of the cells into mature granulocytes. The induction of NBT-reducing activity by TI-79 was inhibited by high concentrations of the natural nucleoside, adenosine. Other differentiation inducers, such as retinoic acid, 1 alpha,25-dihydroxyvitamin D-3 and staurosporin markedly enhanced the induction of differentiation of HL-60 cells by TI-79. Nucleoside analogs such as TI-79 should be useful for differentiation therapy of some types of myelogenous leukemia.

We studied differentiation inducing effects of retinoic acid (RA), 1 alpha, 25-dihydroxyvitamin D3 (D3) and interferons (IFNs), alone and in combination, on fresh myeloid leukemic cells from 8 patients. RA not only induced the differentiation of leukemic cells in 5/8 cases, but potentiated differentiation by IFNs either in granulocytic or monocytic pathways. In particular, interferon-alpha enhanced granulocytic differentiation and interferon-gamma induced mono-macrophage differentiation of promyelocytic leukemic cells in the presence of RA. Differentiation induced by D3, alone or in combination with IFNs, was limited in all cases. RA plus IFNs might be an effective combination for differentiation therapy for some types of myeloid leukemia.

Leukemia cells from patients with acute non-lymphocytic leukemia were treated with various inducers of differentiation of the human promyelocytic leukemia cell line HL-60. All cells in 14 specimens tested underwent morphological, functional and histochemical changes after treatment with some inducers of differentiation of HL-60 cells, but the most effective inducer varied for different specimens. These results suggest that treatment with some inducers should be effective for inducing most acute myeloid leukemia cells to differentiate into morphologically and functionally mature granulocytes and macrophages.

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.

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 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.

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).

Resistant mouse myeloid leukemia cells could not be induced to differentiate in vitro into mature macrophages and granulocytes by incubation with ascitic fluid or dexamethasone as inducer. Neither could endogenous inducers acting on resistant cells maintained in a diffusion chamber in syngeneic SL mice induce differentiation. However, when resistant cells were pretreated in vitro with low doses of actinomycin D they became sensitive to inducer in vitro or in vivo. The concentration of actinomycin D effective for this effect did not induce differentiation. The effect of actinomycin D was not due to inhibition of cell growth, since sensitivity was not observed when resistant cells were pretreated with the growth inhibitor 5-fluorodeoxyuridine. When resistant cells were kept in a diffusion chamber in mice injected with low doses of actinomycin D they showed significant differentiation, suggesting that the in vivo effect of actinomycin D is partly attributable to sensitization of the resistant cells to endogenous inducers.

The murine macrophage-like cell line (Mm-1), which is nonleukemogenic to syngeneic SL mice, was originally derived from spontaneously differentiated cells of a clonal line of mouse myeloid leukemia cells (M1). In the present experiment, variant cell lines with a high (Mm-A), moderate (Mm-P), and little or no (Mm-S1 and Mm-S2) leukemogenic potential were obtained from the Mm-1 cells. The mean survival times of syngeneic SL mice inoculated i.p. with 5 X 10(6) Mm-A and Mm-P cells were 17 and 33 days, respectively, whereas almost all the mice inoculated with Mm-S1 or Mm-S2 cells survived for more than 90 days. These variant cell lines did not lose their macrophage-like characteristics in vitro. These variant cell lines phagocytized latex beads and sensitized sheep erythrocytes, produced lysozyme, and adhered to culture dishes. The four variant cell lines showed no significant difference in proliferation rates in vitro in liquid medium containing 10% calf serum, but Mm-A cells could grow both in soft agar medium in the absence of ascitic fluid containing colony-stimulating factor (CSF) and in liquid medium containing 1% serum, whereas Mm-P cells could grow in the liquid medium but not in soft agar medium without ascitic fluid, and Mm-S1 and Mm-S2 cells could not grow in either medium. The ratio of the nuclear area to the cell area (NCR) of Mm-A cells was a high (51%) but those of Mm-S1 and Mm-S2 cells were low (40-41%), and that of Mm-P cells was intermediate (44%). The leukemogenicity of Mm-1 cell lines was roughly correlated with their NCR. The possibility that interactions between Mm-1 variant cells and host immune cells might be involved in the mechanisms of their different leukemogenicities was not supported by results on the in vitro susceptibilities of Mm-1 variant cells to the cytostatic actions by normal macrophages and spleen cells and on leukemogenicities of the Mm-1 variant cells in athymic nude mice. A possible method of control of the leukemogenicity of Mm-1 variant cells is discussed.

Studies were made on whether differentiation and proliferation of antitumor drug-resistant leukemia cells could be controlled by specific inducers of terminal differentiation. Leukemia subclones resistant to daunomycin and/or cytosine arabinoside were isolated from differentiation-inducible mouse myeloid leukemia M1-B24 cells by selection with these antitumor drugs. The drug-resistant cells were found to retain their potential for terminal differentiation induced by various inducers, such as a protein inducer in the conditioned medium of mouse L929 cells, dexamethasone, 1 alpha,25-dihydroxyvitamin D3, 2-[2-(dodecyloxy)ethoxy]ethyl 2-pyridinioethyl phosphate, and poly I. Differentiated cells showed morphological changes to mature macrophage-like cells, increase in phagocytic activity, and decrease in proliferative activity. Clonal analysis of M1-B24 cells showed that the cellular responses to the protein inducer of differentiation were not significantly different between drug-resistant clones selected with anti-tumor drugs and control (drug-sensitive) clones randomly isolated without selection. These results suggest that induction of differentiation of leukemic cells with the specific inducers is another approach to control the drug-resistant leukemia.

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.

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.

Retinoic acid and retinol induced functional and morphological differentiation of human promyelocytic leukemia cells (HL-60) into mature granulocytes, but did not induce functional or morphological differentiation of mouse myeloid leukemia cells (M1). Cellular retinoic acid-binding protein, but not retinol-binding protein, was detected on HL-60 cells. Neither binding protein could be detected on M1 cells. These results suggest that retinoic acid-binding protein may be necessary for induction by retinoids of functional and morphological differentiation of myeloid leukemia cells.

Human promyelocytic leukemia cells (HL-60) were found to be induced by dimethylsulfoxide and several other compounds to phagocytize, reduce NBT dye, and change into forms that were morphologically similar to granulocytes and macrophages. Arginase also induced these differentiation-associated properties of the cells. The induction of differentiation by arginase was significantly inhibited by addition of excess arginine, but not by lysine or leucine; therefore, the effect of arginase may be due to arginase-mediated arginine depletion.

Ml cells were labeled in vitro with [3H]thymidine and injected into the peritoneal cavity of syngeneic mice. After several days the peritoneal cells were harvested and [3H]labeled cells were determined by autoradiography. The inoculated isotope-labeled Ml cells differentiated in the peritoneal cavity and lipopolysaccharide, an inducer of cell differentiation, significantly stimulated differentiation. These results provide direct evidence that Ml cells can be induced to differentiate in vivo under conditions in which leukemia can develop.

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)

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.

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.

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.

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.