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A 1.5-W single-chip MPEG-2 MP@ML real-time video encoder large scale integrated circuit (LSI) has been developed. To form an MPEG-2 encoder system, we employ two 16-Mb synchronous DRAM's, a microprocessor unit (MPU), and an audio encoder LSI. Owing to a two-step hierarchical search scheme and a novel adaptive search window scheme, the search range of motion estimation is -48/+47 horizontal and -96/+15.5 vertical, and the pseudo search range, which is the size when the location of the search window is adaptively shifted, is -96/+95 horizontal and -32/+31.5 vertical. We have also developed low-power clocking techniques, i.e., demand-clock controller, local-clock controller, and low-power flip-flops, which can eliminate waste of power in clocking. We have successfully fabricated these new designs as a low-power single-chip MPEG-2 encoder LSI. The operating frequency except for a synchronous DRAM interface unit and a video in/out unit is 54 MHz. The supply voltage to the first and second search engines in a motion estimation unit can be successfully lowered to 2.5 V and the others are 3.3 V. Into a 12.45/spl times/12.45 mm/sup 2/ chip with 0.35-/spl mu/m CMOS and triple-metal layer technology are integrated 3.1 M transistors.

Mouse myeloid leukemic M1 cell lines can be induced to differentiate into monocyte/macrophage pathways by various inducers. We have isolated resistant M1 cells that cannot be induced to differentiate into mature cells from the parent M1 cells. The induction of differentiation of M1 cells can be inhibited by protein inhibitors termed differentiation-inhibiting factors (I-factors) in a cell lysate and conditioned medium of the differentiation resistant M1 cells. Production of the I-factor activity in resistant M1 cells is well associated with development of resistance of M1 cells to differentiation inducers. We have purified one of the I-factors from conditioned medium of differentiation-resistant M1 cells. The purified I-factor has a relative molecular mass of approximately 16 000–17 000 Da (16K I-factor) and the amino acid sequences of all fragments of the 16K I-factor are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor is Nm23/nucleoside diphosphate kinase protein.

Recent developments in thermoplastic encapsulation materials, molding techniques, and mold design for encapsulating high performance coils are reviewed. Recently developed encapsulation systems for UL 1446 Class F (155 C) and Class H (180 C) applications are discussed.

Intraperitoneal injection of benzodiazepine receptor agonists (estazolam, zopiclone, triazolam: 0.03–0.24 mmol/kg) induces the head twitch response (HTR). The present study was undertaken to examine the possible participation of the serotonergic system in the mechanism of head twitches induced by benzodiazepine receptor agonists (BZ-RAs). The HTR induced by BZ-RAs was suppressed by pretreatment with ketanserine (1 mg/kg, i.p.), a selective 5-HT2 receptor antagonist. Pretreatment with fluoxetine (10 mg/kg, i.p.), a 5-HT reuptake inhibitor, and 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT1A receptor agonist, also suppressed the HTR induced by BZ-RAs. These results suggest that the HTR induced by BZ-RAs may be the result of an activation of postsynaptic 5-HT2 receptors, probably due to direct action.

Differentiation therapy in patients with acute promyelocytic leukemia (APL) using all-trans-retinoic acid (ATRA) has now been established as an effective strategy for the treatment of leukemia. However, the clinical response of patients with leukemias other than APL to differentiation inducers is limited, often with inconsistent outcome. Recently, numerous new differentiation inducers for various leukemia cells have been developed, some of which have had therapeutic effects on various leukemias in preclinical and clinical trials. Additionally, molecular control mechanisms of differentiation, and apoptosis of various leukemia cells by differentiation inducers have been studied extensively. These studies suggest that problems underlying the current discrepancy between preclinical and clinical therapeutic efficacy of leukemias can be overcome by these basic studies together with multidisciplinary studies on the therapy of leukemias in combination with differentiation therapy and other conventional therapies.

Vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone), a quinolinone derivative, is an orally active inotropic agent used in Japan for the treatment of chronic heart failure. Recently, it has been reported that vesnarinone induces differentiation and apoptosis in certain types of leukaemia and solid tumour cells, and exhibits antitumour effect on several tumours xenografted in nude mice. In the present study, we examined the antitumour effect of vesnarinone in combination with radiation and conventional anticancer agents in nude mice xenografted with human gastric carcinoma, a poorly-differentiated adenocarcinoma, MKN-45 cell line which has a wild-type p53 gene. Vesnarinone treatment combined with radiation resulted in a higher antitumour activity compared with a single treatment with either vesnarinone or radiation alone. Further, vesnarinone treatment together with radiation and conventional anticancer agents including 5-FU and picibanil (an immunopotentiator) produced the highest antitumour effect compared with any other treatment. Additionally, the combination treatment induced marked differentiation and apoptosis of the tumour cells and an increase in the expression of p53 gene in the treated tumour cells. The results suggest that vesnarinone, in combination with radiation and the conventional antitumour agents, may be of clinical interest for treatment of certain types of gastric tumours.

The sites associated with dopamine neurons which produce mouse-killing aggression (muricide) were examined in the rat brain. Muricide appeared in 60-80% of rats after being fed a thiamine-deficient diet for 28 days. Microinjection of dopamine (500 ng/rat) into the olfactory bulb (OB) significantly suppressed muricide, whereas injection into other brain areas failed to do so. The incidence of muricide after dopamine injection was 40% at 5 min and 20% at 15-30 min. When 6-hydroxydopamine (8 micrograms/0.5 microliter), following pretreatment with desmethylimipramine (25 mg/kg i.p.), was injected twice into the ventral tegmental area (VTA) or the olfactory bulb (OB) in nonkiller rats during thiamine-deficient feeding, the occurrence of muricide gradually increased over time. The present results suggest that degeneration of dopamine neurons projecting from the VTA to the OB may be related to mouse-killing aggression in rats.

The relationship between learning and genetic factor on immobility in mice during a forced swimming test (FST) has been studied. The duration of immobility during the FST did not change significantly after the administration of either scopolamine (2.5 mg/kg, ip) or cyclohexamine (150 mg/kg, ip), although both drugs produced impairment in the learning task. This suggests that the increase in immobility observed during the second trial of the FST may not be related to learning which could occur during the first trial. Concerning the strain difference, first, the duration of immobility in C3H mice was shorter than that in ICR, ddY, C57BL and BALB mice. Second, after receiving shock stress in a box, ICR, ddY and C57BL mice, but not C3H mice showed a marked decrease in locomotor activity when placed in the box again without shock. Also during the FST, both C57BL and ddY mice, but not C3H mice showed prolongation of immobility and reduction in swimming after shock stress. The changes in locomotor activity, and immobility and swimming during the FST caused by shock stress in ddY mice recovered to normal levels after treatment with imipramine. From these results, it is suggested that the immobility shown during the FST, which may be independent of learning and dependent on some genetic factor, is a suitable model of depression in animals.

We investigated the effect of 5-aza-2'-deoxycytidine (ADC) (a specific inhibitor of DNA methylation) on differentiation, Lewisy (Ley) antigen expression, and DNA fragmentation (a biochemical marker of apoptosis) in human pancreatic cancer MIA PaCa-2 cells. ADC markedly inhibited the growth of MIA PaCa-2 cells. Flow cytometric analysis showed that ADC suppressed the cell population in the G1 phase, but enhanced it in the G2/M phase. On the other hand, several markers of cell differentiation including morphological and biochemical alterations were distinctly induced in cells treated with ADC. Morphologically, the cells were enlarged and thinner than the untreated control cells, and intracellular alkaline phosphatase (ALP) activity was markedly increased. Additionally, biochemical markers of apoptosis including DNA fragmentation and Ley antigen expression were induced in association with the appearance of morphological and biochemical markers of differentiation (ALP). These results suggest that the hypomethylation of DNA is involved in the molecular mechanism of growth inhibition, induction of apoptosis, and differentiation in human pancreatic cancer MIA PaCa-2 cells.

We examined the effect of vesnarinone, an oral cardiotonic, on the growth and differentiation of human myeloid leukemia cells. Vesnarinone alone markedly induced erythroid differentiation of HEL cells. All-trans-retinoic acid also induced erythroid differentiation of the cells, and the differentiation was greatly enhanced by combined treatment with vesnarinone and retinoic acid. HEL cells are highly resistant to some anticancer drugs, including vincristine, but treatment with vesnarinone greatly increased the sensitivity of HEL cells to vincristine. Enhancement of vincristine sensitivity by vesnarinone was not as significant for other leukemia cells. Expression of P-glycoprotein in HEL cells was effectively inhibited by vesnarinone, suggesting that the restoration of vincristine sensitivity is associated with decrease of P-glycoprotein expression in HEL cells. The plasma level of vesnarinone required to induce differentiation of leukemia cells is 30 micrograms/mL, which could be achieved with oral administration. These results suggest that vesnarinone should be useful in differentiation therapy for some types of myelogenous leukemia.

We recently developed a monoclonal antibody directed to carbohydrate antigen Le(y), BM-1/JIMRO, and found that expression of Le(y) antigen defined by BM-1/JIMRO was associated with the process of apoptosis, but not with cell proliferation or necrosis. In the present experiments, we examined with BM-1/JIMRO the effects of various differentiation inducers on the growth and expression of Le(y) antigen in human lung cancer A549 cells. We found that a specific inhibitor of methylation of DNA, 5-aza-2' deoxycytidine (ADC), could markedly induce expression of Le(y) antigen in association with induction of apoptosis and differentiation in the A549 cells. These results suggest that hypomethylation of DNA is involved in the molecular mechanisms of induction of Le(y) antigen, apoptosis and differentiation in the cells.

The two alpha-methylated tryptamine derivatives, 5- (5-FMT) and 6-fluoro-alpha-methyl- tryptamine (6-FMT), rapidly induced a head-twitch response (HTR) in mice. Two derivatives that lack the methyl group in their chemical structures, 5- (5-FT) and 6-fluorotryptamine (6-FT), did not induce the HTR. The induced HTR was depressed by pretreatment with cycloheptadine, p-chlorophenylalanine or fluoxetine, but was potentiated by 5,7-dihydroxytryptamine. Both 5- and 6-FMT increased brain 5-HT levels in hypothalamus, hippocampus, brainstem, striatum and cortex. 5-FMT decreased the levels of 5-hydroxyindoleacetic acid in those regions, but 6-FMT caused a significant decrease in only the hypothalamus and cortex. The two methylated derivatives inhibited mouse brain MAO-A activity more selectively than non-methylated derivatives. The results suggest that the HTR induced by 5- and 6-FMT may result from increased activity of central 5-HT neurons, probably due to increased 5-HT levels after MAO-A inhibition. This probably results in release of 5-HT with a concomitant increased interaction with postsynaptic 5-HT2 receptors. The present results also indicate the importance of the methyl group to selective MAO-A inhibition by the substrate-analogues tested, and the concomitantly induced animal behavior.

It has been shown recently that various myeloid leukemia cells can be induced by numerous compounds to differentiate terminally into non-growing mature cells. Furthermore, some of these compounds have been found to be clinically useful for the therapy of certain types of myeloid leukemia. To improve this new method of differentiation therapy of leukemia, we have studied on basic issues of such differentiation therapy including 1) development of more effective new inducers of differentiation of leukemia cells, 2) isolation and characterization of cytokines regulating growth and differentiation of leukemia cells, and 3) rationale for chemo-differentiation therapy of leukemia which is a combination chemotherapy of leukemia with differentiation inducers and cytotoxic anticancer drugs. In this article, recent results of our studies on these basic issues on differentiation therapy of leukemia are reviewed.

An animal leukemia model was developed to investigate in vivo induction of differentiation of myeloid leukemia cells. An aneuploid cell line (C15) was isolated from mouse myelomonocytic leukemia WEHI-3B D+ cells. The C15 cells contained twice as much DNA as the parental cells but retained the morphology of myelomonocytic cells and the ability to differentiate into macrophage-like cells in response to all-trans retinoic acid (ATRA) in vitro. When the C15 cells were inoculated into the peritoneal cavity of syngeneic Balb/c mice (10(6) cells/mouse), the mice died of leukemia within 19 days. The DNA content and differentiation antigen (Mac-1) of the cells in the peritoneal cavity were determined by dual-parameter flow cytometry. On day 12 after inoculation, the C15 cells were distinguishable from normal host cells in the peritoneal cavity by their different DNA content. The administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (10 micrograms/day) to mice bearing C15 cells induced the leukemia cells to express Mac-1 antigen and to change morphologically into mature granulocytic cells. Because the C15 cells were not responsive to G-CSF in suspension culture in vitro, this result suggests that the cytokine's actions on the cells in vivo and in vitro are different. This experimental model for analyzing in vivo differentiation of leukemia cells will be useful for studying the therapeutic effects of potential differentiation-inducing agents.

Various alkyl ether lipids were synthesized and their effects on the proliferation of human lung carcinoma cells were examined. The proliferation of Lu-65, a giant cell carcinoma cell line, was significantly decreased with 1 microgram/ml (3-tetradecyloxy-2-methoxy) propyl-2-trimethylammonioethyl phosphate, while the proliferation of Lu-99, another giant cell carcinoma cell line, was unaffected even by treatment with 5 micrograms/ml of the alkyl lysophosphocholine. Adenocarcinoma PC-9 and small cell carcinoma H-69 cells were also fairly resistant to the alkyl ether lipid. Among the alkyl ether lipids tested, 3-nonadecyloxy-2-methoxypropyl 2-trimethylammonioethyl phosphate was the most effective in inhibiting the growth of Lu-65 cells. However, the pyridinioethyl derivative had higher selectivity for the growth of Lu-65 cells than the choline derivative. The sensitivity of Lu-65 cells to the alkyl lysophospholipids was similar to that of human myeloid leukemia cells including HL-60. However, the sensitivities of Lu-65 cells to the other types of alkyl ether lipids were much lower than those of HL-60 cells. These results indicate that Lu-65 cells are selectively sensitive to alkyl lysophospholipids.

Three cDNAs for mouse differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) receptor were isolated from a cDNA library prepared from the liver of a pregnant mouse. A probe for screening was prepared by the RT-PCR method using human cDNA sequences as primers. The mouse D-factor receptor cDNA encoded 1,092 amino acids, which had a marked homology with the human counterpart and consisted of signal sequence, extracellular, transmembrane, and cytoplasmic domains. The WSXWS motif found in members of the cytokine receptor family was also present in the extracellular domain of the mouse D-factor receptor. A second form of cDNA that had a 501 bp insertion was isolated. The insertion introduced a stop codon so that the mRNA encoded the soluble receptor lacking transmembrane and intracellular domains. Because the insertion contained polyadenylation signals, two different sizes of mRNA encoding the soluble receptor were produced, depending on whether or not it utilized these signals. Transcripts utilizing these signals were 2.6-3 kb in size, and were very abundantly expressed in the liver. Transcripts that did not use these signals were longer than 5 kb and of similar size to the mRNA for the cellular receptor.

Interleukin 4 (IL-4) inhibited the differentiation of mouse myeloid leukemia M1 cells induced by leukemia inhibitory factor (LIF), interleukin 6, or dexamethasone and conversely enhanced the induction of M1 cell differentiation by 1 alpha,25-dihydroxyvitamin D3. IL-4 blocked LIF-induced differentiation of M1 cells when it was added to the culture medium within 10 h after LIF, but IL-4 did not block differentiation when it was added 12 h after LIF. These results indicate that IL-4 inhibited a critical intermediate step in myeloid leukemia cell differentiation. LIF markedly stimulated the expression of junB mRNA within 2 h but suppressed the expression of c-myb and c-myc after 2- and 12-h treatment, respectively. IL-4 did not significantly affect LIF-induced junB expression or suppression of c-myb expression. However, it interfered significantly with the LIF-induced suppression of c-myc gene expression. Similar results were obtained when interleukin 6 was used to induce differentiation of M1 cells. Dexamethasone and 1 alpha,25-dihydroxyvitamin D3 did not induce junB gene expression but suppressed the expression of c-myb and c-myc. IL-4 also interfered with dexamethasone-induced suppression of c-myc gene expression. On the other hand, IL-4 enhanced 1 alpha,25-dihydroxyvitamin D3-induced down-regulation of c-myc gene expression, consistent with its enhancement of differentiation. These results indicate that the change in c-myc expression induced by IL-4 in M1 cells is closely associated with the effect of IL-4 on the induction of differentiation of M1 cells.

In severe infection, the host responds to foreign agents and produces cytokines to activate lymphocytes and macrophages. Some of these cytokines can modulate growth and differentiation of myeloid leukemia cells. We examined differentiation-inducing activities in the sera from 9 patients with leukemia or lymphoma. These results indicate that some sera from infected patients, even with acute leukemia, have significant differentiation-inducing activities on both mouse and human leukemia cells, and that cytokines having differentiation-inducing activities varied for different specimens.

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.

The authors examined the effect of a tyrosine kinase inhibitor, erbstatin, and its analogues on abl oncogene functions. Erbstatin and its stable analogue methyl 2,5-dihydroxycinnamate (2,5-MeC) inhibited the growth of v-ablts-NIH3T3 cells at the permissive temperature (33 degrees C) at lower concentrations than at the non-permissive temperature (39 degrees C). 2,5-MeC inhibited the morphological transformation and the activation of v-abl tyrosine kinase by the temperature shift (39 degrees C to 33 degrees C) more effectively than erbstatin. Previously the authors reported that erbstatin induced erythroid differentiation of K562 human chronic myelogenous leukaemia cells, so they examined the effect of erbstatin analogues on the erythroid differentiation. Among eight erbstatin analogues studied, ethyl 2,5-dihydroxycinnamate induced erythroid differentiation of K562 cells most effectively. Ethyl 2,5-dihydroxycinnamate also inhibited bcr-abl tyrosine kinase. These results indicate that the stable analogues of erbstatin suppress oncogene functions of Abl by inhibiting its tyrosine kinase.

1 alpha, 25-Dihydroxyvitamin D3 (D3) (100 nM) and interferon-gamma (IFN-gamma) (100 U/ml) cooperatively inhibited the proliferation of HL-60 cells, and synergistically induced their monocytic differentiation. The growth-promoting effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/ml) was inhibited appreciably by D3 and slightly by IFN-gamma. Despite the clear difference in their effects on growth of HL-60 cells, both IFN-gamma and GM-CSF in combination with D3 induced cell cycle changes, decreasing the number of cells in the S phase and increasing their percentage in the G1/0 phase. GM-CSF alone had no effect on differentiation, but enhanced differentiation induced by D3 distinctly though to a limited extent, and also enhanced monocytic differentiation, including morphological changes of HL-60 cells in the presence of D3 and IFN-gamma. GM-CSF as well as D3 and IFN-gamma induced interleukin-1 beta (IL-1 beta) production by the HL-60 cells, clearly indicating their importance in differentiation of these cells. IFN-gamma and GM-CSF had mutually potentiating effects and induced maximum IL-1 beta production in response to lipopolysaccharide (LPS) in the presence of D3. Thus despite its growth-promoting effect, GM-CSF is a potential inducer of monocytic differentiation of human myeloid leukemia cells, because in cooperation with IFN-gamma it induced monocyte-macrophage differentiation of HL-60 cells in the presence of D3.

Twenty-seven novel nucleobases and nucleosides were synthesized by structural modification of uracil, and their effects on growth and differentiation of human myeloid leukemia HL-60 cells were examined. Some of the compounds inhibited the growth of HL-60 effectively. The nitroblue tetrazolium (NBT)-reducing activities of cells treated with the concentrations of these compounds for 50% inhibition of growth were compared. TI-66 (2,4-dibenzyl-6-fluoro-7,7,8,8-tetramethyl-cis-2,4-diazabicyclo-[4.2.0] octane-3,5-dione) was the most effective inducer of NBT-reducing activity and morphological differentiation of HL-60 cells into cells of the myelomonocytic lineage. TI-66 was also effective for induction of differentiation of another human myelogenous leukemia cell line, ML-1 cells, but not for differentiation of human erythroid leukemia K562 or HEL cells, or monocytic U937 cells. The effect of TI-66 in inducing differentiation of HL-60 cells was additive or more than additive in combination with retinoic acid or vitamin D3. Adenine or hypoxanthine alone induced NBT-reducing activity of the cells, and at suboptimal concentrations these compounds enhanced the effect of TI-66, but the enhanced NBT-reducing activities did not exceed the maximal activity induced by TI-66 alone. Simultaneous treatment of HL-60 cells with hypoxanthine reduced the growth inhibition by TI-66 alone. TI-66 was about 150 times more potent on a molar basis than adenine in inducing differentiation of HL-60 cells. These results suggest that nucleobase analogs such as TI-66 should be useful for differentiation therapy of some types of myelogenous leukemia.

Herbimycin A, a benzoquinonoid ansamycin antibiotic, reduces intracellular phosphorylation by some tyrosine kinases, including v-abl. The mouse megakaryoblastic cell line C1 expresses v-abl protein at high levels. Herbimycin A at about 20 ng/ml caused 50% inhibition of growth of C1 cells but at 100 ng/ml scarcely affected the growth of another mouse leukemia cell line, M1 cells, or of normal bone marrow cells. Injection of 10(6) C1 cells into nude mice resulted in death of all the mice within 30 days. Administration of herbimycin A significantly enhanced the survival of mice inoculated with C1 cells but scarcely affected the survival of mice inoculated with M1 cells. These results suggest that herbimycin A and/or related compounds may be useful for treatment of some types of leukemia in which tyrosine kinase activity is implicated as a determinant of the oncogenic state.

There is increasing evidence 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. The differentiated cells lose their abilities for proliferation and transplantation in animals. Furthermore, survival times of the animals inoculated with various tumors were found to prolong by administration with the differentiation inducers. These basic findings suggest that induction of terminal tumor cell differentiation is another strategy for cancer therapy: differentiation therapy of cancer. Principles, current status and perspectives of the differentiation therapy of leukemia are reviewed in this article.

A patient with acute myelomonocytic leukemia (M4 in FAB classification) refractory to various kinds of intensive chemotherapy was intravenously administered low doses (7 or 14 mg/m2) of aclacinomycin-A (ACM-A) daily. This increased mature neutrophils and monocytes and decreased leukemia cells in the peripheral blood. Pelger Huet-like nuclear anomaly, observed in the neutrophils, suggested the leukemic nature of these cells. The in vivo findings were clearly correlated with the in vitro results in which ACM-A could induce myelomonocytic differentiation of the leukemia cells. During the course of the treatment, the patient achieved and maintained good general condition for more than nine months after the initiation of the treatment. In clinical trials, nine patients, five with acute myeloid leukemia (AML) and four with myelodysplastic syndrome (MDS), were treated with low doses of ACM-A. Five patients, three AML and two MDS, responded to the treatment. The results suggest that low doses of ACM-A may induce in vivo differentiation of leukemia cells, and may be a potential therapeutic strategy in the treatment of AML or MDS that is refractory to conventional chemotherapy.

Herbimycin A, a specific tyrosine kinase inhibitor, induced erythroid differentiation of human myelogenous leukemia K562 cells with a high level of bcr/abl tyrosine kinase. Several derivatives of herbimycin A were synthesized and their effects on cell proliferation and differentiation of K562 cells were examined. Of the compounds tested, 19-allylaminoherbimycin A was the most effective in inducing differentiation of K562 cells. However, the parent compound was the most potent growth inhibitor, suggesting that chemical modification of herbimycin A reduces the growth-inhibiting activity. The sensitivities of K562 cells to herbimycin derivatives were different from those of a rat kidney cell line infected with Rous sarcoma virus (v-src), suggesting that bcr/abl kinase may differ in sensitivity from other tyrosine kinases. These results indicate that a specific inhibitor of bcr/abl kinase could be an effective antitumor agent against chronic myelogenous leukemia.

The sensitivity of human myelogenous leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) during induction of differentiation was examined. Treatment with hemin greatly increased the sensitivity of erythroid leukemia cells to ara-C. The enhancement of ara-C sensitivity by hemin was not as remarkable in nonerythroid leukemia cells. Hemin altered the metabolism of ara-C in human erythroleukemia K562 cells by reducing ara-C deaminase activity, increasing intracellular accumulation of ara-C, and activating the nucleoside kinases. These alterations may be involved in the enhancing effect of hemin on sensitivity of ara-C. These results suggest that some inducers of differentiation potentiate the antileukemic effect of ara-C on human erythroleukemia cells.

Mouse C1 line cells are megakaryoblastic cells established by coinfection of Abelson murine leukemia virus and recombinant simian virus 40. We examined the effects of various compounds on growth and differentiation of these cells. Megakaryocytic differentiation of C1 cells was not induced by cytokines that stimulate megakaryocytic maturation of normal progenitor cells, such as interleukin 3 and 6 and granulocyte-macrophage colony-stimulating factor. However, the cells were induced to differentiate into megakaryocytes by treatment with some protein kinase inhibitors. The inhibition of v-abl tyrosine kinase activity preceded induction of differentiation of the cells treated with tyrosine kinase inhibitors such as genistein, herbimycin A, and erbstatin. Treatment of C1 cells with a v-abl antisense oligomer inhibited their proliferation and induced acetylcholinesterase activity, a typical marker of megakaryocytic differentiation. These results suggest that inhibition of v-abl function is associated with induction of megakaryocytic differentiation of C1 cells. Among the compounds tested, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of cyclic nucleotide-dependent and Ca(2+)-phospholipid-dependent (protein kinase C) protein kinases, was the most potent inducer of differentiation of C1 cells. However, the differentiation-inducing effect of H-7 was unlikely to be mediated through inhibition of protein kinase C or cyclic nucleotide-dependent kinases, because other types of inhibitors of these kinases were not effective, and a protein kinase activator (phorbol ester) induced differentiation of C1 cells. Moreover, neither v-abl mRNA expression nor v-abl kinase activity in C1 cells was affected by treatment with H-7. These findings indicate that induction of megakaryocytic differentiation by H-7 is not related to inhibition of v-abl kinase, but rather to some novel function of H-7.

The K562/VCR cell line, exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (P-glycoprotein), was isolated from human erythroleukemia K562 cells. Various compounds that induced erythroid differentiation of K562 cells were tested for their effects on growth and differentiation of these K562/VCR cells. Sodium butyrate, hemin, 1-beta-D-arabinofuranosylcytosine, and erythroid differentiation factor (EDF) induced erythroid differentiation of K562/VCR cells as well as K562 cells. The MDR of K562/VCR cells was partly overcome by treatment with EDF but not with the other inducers. Expression of P-glycoprotein by K562/VCR cells was measured by radioimmunoassay using MRK16 monoclonal antibody. Results showed that EDF caused down-regulation of P-glycoprotein in K562/VCR cells, whereas the other inducers did not cause its down-regulation. Thus, in addition to inducing erythroid differentiation, EDF enhanced the sensitivity of K562/VCR cells to multidrugs and suppressed expression of P-glycoprotein. These results suggest that differentiation inducers may be useful in chemotherapy of leukemic MDR cells.

We examined the effects of transforming growth factors beta(TGF-beta) alone and in combination with 1 alpha,25-dihydroxyvitamin D3 (VD3) on human leukemic cell lines (HEL/S, HL-60 and K562). TGF-beta 1 alone at higher concentrations induced monocytic differentiation of HEL/S cells. Combinations of TGF-beta 1 and VD3 synergistically inhibited cell proliferation and induced monocytic differentiation of HEL/S and HL-60 cells. TGF-beta 2 (a subtype of TGF-beta) and erythroid differentiation factor (EDF/Activin A, a member of the TGF-beta gene family) also inhibited growth and induced monocytic differentiation of HL-60 cells in synergy with VD3. Thus combinations of VD3 and TGF-beta (TGF-beta 1, -beta 2, or EDF) acted synergistically in inhibiting cell proliferation and inducing monocytic differentiation of human leukemia cells.

The interaction of colony-stimulating factors (CSF) and retinoic acid (RA) in the proliferation and differentiation of HL-60 cells was examined. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the proliferation of HL-60 cells in a dose-dependent manner at concentrations of 0.01-100 ng/ml; however, the proliferation due to GM-CSF was suppressed by 100 nM RA. Granulocyte colony-stimulating factor (G-CSF) slightly stimulated the proliferation of HL-60 cells at concentrations above 10 ng/ml. Neither G-CSF nor GM-CSF alone induced 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)- or N-formyl-methionyl-phenylalanine (FMLP)-stimulated nitro-blue tetrazolium (NBT) reduction at concentrations of 0.01-100 ng/ml. G-CSF induced TPA- and FMLP-stimulated NBT reduction in the presence of 100 nM RA, but GM-CSF induced only TPA-stimulated NBT reduction. RA in addition to G-CSF synergistically increased FMLP binding to HL-60 cells, accompanied by increased NBT reduction in response to FMLP. RA in addition to GM-CSF markedly increased FMLP binding to HL-60 cells more than that induced by RA alone, but the combined treatment with RA and GM-CSF did not increase FMLP-stimulated NBT reduction more than that induced by RA alone. The results suggest that G-CSF stimulates RA-induced morphological and functional differentiation of HL-60 cells, but the differentiation-enhancing effects of GM-CSF are limited, whereas the growth-stimulating effect of GM-CSF on HL-60 cells is greater than that of G-CSF.

We have purified and characterized several protein factors that regulate the growth and differentiation of mouse myeloid leukaemia M1 cells. The differentiation factor (D-factor) from conditioned medium (CM) of Ehrlich ascites tumour cells is a glycoprotein of Mr 40,000-50,000. Its amino acid sequence was found to be almost identical to that of leukaemia inhibitory factor (LIF) from Krebs II ascites cells. The differentiation inhibitory factor (I-factor) from the CM of variant M1 cell clones which were resistant to several differentiation inducers is a basic protein of apparent Mr 68,000. The growth inhibitory factor (GI-factor) that specifically inhibits the partially differentiated and still growing monocytic leukaemia M1 cells was isolated from the CM of a clone of M1 cells resistant to the differentiation inducers. This GI-factor is a basic protein with an Mr of 25,000. Regulation by these protein factors together with other known cytokines of growth and differentiation of M1 cells is reported.

Mouse peritoneal macrophages release a factor(s) that stimulates differentiation of a mouse myeloid leukaemic cell line into mature granulocytes and macrophages. Treatment of the macrophages with the synthetic double-stranded polyribonucleotides poly(I).poly(C) and poly(A).poly(U) resulted in enhanced release of the factor into the culture medium. The effect was maximal after treatment with polyribonucleotides for 1 h, and the optimal dose of poly(I).poly(C) was 50 microgram/ml. The single-stranded polyribonucleotides poly(I) and poly(C) at the same concentration were far less effective. The differentiation-stimulating factor was detected not only in the cultured medium but also in the cell lysate. Exposure of macrophages to poly(I).poly(C) enhanced the total activity of the factor in both the culture medium and the cell lysate. The effect of this compound was blocked by the presence of cycloheximide. These results suggest that double-stranded polyribonucleotides enhance production of the differentiation-stimulating factor by peritoneal macrophages.

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 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.

Mouse myeloid leukemic cells (M1) could be induced to differentiate into macrophage-like and granulocyte-like cells by a lysine-rich, histone H1 fraction (10 to 100 microgram/ml). The differentiated M1 cells expressed phagocytic and lysozyme activity and were macrophage-like and granulocyte-like cells. The differentiation-inducing activity of histone H1 was found in histone H1 fractions isolated from calf thymus, rat liver, and mouse leukemia M1 cells. Histone H2A and H2B fractions did not induce differentiation of M1 cells at concentrations of 10 to 100 microgram/ml but did induce differentiation at a high concentration (200 microgram/ml). The histone H3 fraction, poly-L-lysine and poly-L-arginine, inhibited induction of differentiation of M1 cells.

Mouse myeloid leukemia cells (M1) were induced to differentiate into mature macrophages and granulocytes by glucocorticoids or a protein inducer in ascitic fluid from tumor-bearing rats. Addition of nonsteroidal antiinflammatory agents to M1 cells in suspension cultures inhibited the induction of differentiation by glucocorticoid (dexamethasone) or the protein inducer. The inhibition was unrelated to cytotoxicity and was reversible. The nonsteroidal antiinflammatory agent indomethacin inhibited dexamethasone-induced differentiation only when added before the time of commitment of the cells to differentiation. The indomethacin-mediated inhibition was counteracted by prostaglandins E1 or E2 but not by prostaglandins F1alpha or F2alpha. Prostaglandin E stimulated phagocytosis induced by a suboptimal concentration of dexamethasone, but prostaglandin F did not. Moreover, lysozyme activity, which is a typical biochemical marker of macrophages, was induced in M1 cells by prostaglandin E alone, as well as by inducers of differentiation. These results suggest that prostaglandin E may be important in the induction of differentiation of myelod 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.