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Tyrosine phosphorylation of cellular proteins induced by various hematopoietic growth factors such as interleukin 3 (IL3), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin 4 (IL4) was studied in several multi‐factor‐dependent myeloid cell lines. Among the growth factors, IL3 specifically induced rapid tyrosine phosphorylation of a membrane glycoprotein of mol. wt 150 kd (gpp150) in the IL3‐dependent cell lines, IC2 and DA‐1. The IL3‐induced tyrosine phosphorylation of gpp150 was detected within 30 s, reached a maximum at 3 min and decreased thereafter. The concentration of IL3 required for half‐maximum stimulation of gpp150 tyrosine phosphorylation with 2.5 x 10(6)/ml cells was approximately 200 pM, which is the same as the dissociation constant for 125I‐labeled IL3 binding. gpp150 was constitutively phosphorylated on tyrosine residue(s) in growth factor independent variants, IC2Tr and DA‐1Tr, derived from IC2 and DA‐1 respectively. Neither variant synthesized IL3. The present findings suggest that tyrosine phosphorylation of gpp150 is a critical event involved in both IL3‐dependent and ‐independent growth.

p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase- polymerase chain reaction, single strand conformation polymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemias were examined and two with acute lymphoblastic leukemia and one with Waldenstrom's macroglobulinemia were identified to have mutations in the coding region of the p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore, expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias.

A colony-stimulating factor (CSF) that stimulated human and mouse bone marrow cells to proliferate in vitro and form pure granuloid colonies was purified about 4000-fold from normal human urine. Purification procedures included concentration with polyethyleneglycol, ammonium sulfate precipitation, two chromatographic separations on DEAE- cellulose columns, gel filtration, and polyacrylamide gel electrophoresis. The molecular weight of the purified factor was estimated to be about 85,000 daltons by gel filtration, and the specific activity was found to be 10(6) or 6.7 X 10(5) colonies/mg protein using mouse or human bone marrow cells, respectively. A urinary colony-inhibiting factor was separated from the CSF on the first DEAE- cellulose column. This inhibitor suppressed the formation of pure granuloid colonies of human and mouse bone marrow cells when employed in conjunction with the purified urinary CSF.

B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus- induced transient aplastic crisis.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide release and membrane depolarization in parallel in human granulocytes stimulated by the receptor-mediated agonists, N- formyl-methionyl-leucyl-phenylalanine and wheat germ agglutinin, but not by the Ca2+ ionophore ionomycin and phorbol myristate acetate, which bypass the receptors to stimulate the cells. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL (1.3 to 2.6 nmol/L) rhG-CSF for 10 minutes at 37 degrees C. rhG-CSF produced by bacteria and mammalian cells had identical biological effects on a molar basis. rhG-CSF neither affected stimulus-induced increase in cytoplasmic free Ca2+ nor changed the number and affinity of N-formyl- methionyl-leucyl-phenylalanine receptors. The priming effect of rhG-CSF was temperature dependent and did not require new protein synthesis. rhG-CSF increased the expression of C3bi receptors on human granulocytes and enhanced granulocyte adherence to nylon fiber. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL rhG-CSF for 30 minutes at 37 degrees C. rhG-CSF had no effect on human monocytes. These findings demonstrate that rhG-CSF can selectively stimulate mature granulocyte functions.

We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

Changes in the composition and metabolism of glycosphingolipid (GSL), which is one of the cell surface constituents, during cell differentiation of human T-lymphoblastic leukemia cell line MOLT-3 cells were examined with special reference to their alterations in E rosette-forming capacity and expression of surface antigens specific for T-cell lineage. Three molecular species of neutral GSL and greater than or equal to 13 molecular species of acidic sialosyl-GSL (ganglioside) were detectable on high-performance thin-layer chromatography (HPTLC) in untreated MOLT-3 cells. The major components were ceramide monohexoside and gangliosides GM3 and GD1a. When the cells were induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) to differentiate into more mature T cells, the ganglioside composition changed distinctively, and the total ganglioside content increased considerably; mono-, di-, and tri-sialosyl gangliosides concomitantly showed significant increase, but no new molecular species of GSL specific for the differentiation were detected. The activity of one sialyltransferases, CMP-sialic acid:CDH sialyltransferase, which synthesizes ganglioside GM3 and the total sialic acid content of the cell surface, parallelled the extent of cell differentiation. Examination of another human T-lymphoblastic leukemia cell line, HPB- ALL, indicated that TPA could also induce the cells to differentiate along T-cell lineage and that changes in the ganglioside pattern during differentiation are similar to those of MOLT-3 cells. The results indicate that human T-lymphoid cell differentiation intimately involves elongation of neutral oligosaccharide-moieties and the addition of sialic acid residues to gangliosides, resulting in more mature T cells containing higher gangliosides. Both the sialyltransferase activity and the sialic acid content, as well as the ganglioside pattern, might be new biochemical markers specific for human T-lymphoblastic cell differentiation.

In order to better understand the patho-physiologic role of granulocyte colony-stimulating factor (G-CSF), we estimated its serum levels in healthy persons and patients with various disorders, using a newly developed enzyme immunoassay (Motojima et al). In 49 of 56 normal healthy persons (88%), the levels were beneath the sensitivity of the assay (less than 30 pg/mL), while in the remaining seven healthy persons, the levels ranged from 33 to 163 pg/mL. On the other hand, nine of 11 patients (82%) with idiopathic aplastic anemia (AA), one patient with Fanconi's anemia, six of 12 patients (50%) with myelodysplastic syndrome (MDS), five of 12 patients (42%) with acute leukemia without any blast cells in the blood (M4: one, M5: one, L1: one, and L2: two), six of 18 patients (33%) with chronic myeloid leukemia (CML), one of two patients with chronic lymphoid leukemia (CLL), two of four patients with lung cancer, one patient with cyclic neutropenia, two of seven patients with malignant lymphoma, and four patients with acute infection had G-CSF levels ranging from 46 pg/mL to greater than 2,000 pg/mL. Interestingly, a reverse correlation between blood neutrophil count and serum G-CSF level was clearly demonstrated for aplastic anemia (r = -.8169, P less than .01). Moreover, it was found that the G-CSF level rose during the neutropenic phase of cyclic neutropenia and after chemotherapy or bone marrow transplantation (BMT) in three patients with leukemia; also high G-CSF levels were positively correlated to blood neutrophil counts in some cases of infectious disorders and lung cancer. The cellular sources and the mechanisms for production and secretion of circulating G-CSF were not investigated in this study, but the data presented here strongly indicate that G-CSF plays an important role as a circulating neutrophilopoietin.

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10- methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10- methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

Thirty-six patients with aplastic anemia and three with aplastic anemia- PNH syndrome in eight institutes were studied for the presence of peripheral blood mononuclear cells having a suppressive effect on granulocyte-macrophage (G-M) and erythroid colony formation using a uniform protocol. In 11 cases of 29 (38%) and 9 cases of 29 (31%), the presence of mononuclear cells with a suppressive effect on G-M and erythroid colony formation, respectively, was demonstrated. However, the presence of mononuclear cells suppressive both to G-M and erythroid colony formation was demonstrated only in 3 of 19 cases (16%).

Eight adult patients suffering from leukocytopenia have been injected by droplet intravenous infusions with partially purified human urinary colony-stimulating factor (CSFHU), which stimulated human monocytes to produce colony-stimulating activity (CSA) in vitro. Three of eight patients were injected with low-dose CSFHU and five with high-dose CSFHU. The infusion of high-dose CSFHU led to a slightly earlier rise in absolute neutrophil counts and a higher CSA level in the serum, as compared to noninjected leukocytopenic patients. There was little toxicity associated with it. Human urinary colony-stimulating factor used in the clinical studies did not have any CSA in vitro in the presence or absence of the patients' sera. These data may suggest that the intravenous infusion of CSFHU increased the serum CSA level, probably through the stimulation of CSA-producing cells in vivo.

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.

The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor- dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines.

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.

We have isolated essentially full-length cDNA clones for atrial (ALC1) and ventricular (VLC1) myosin alkali light chains from a human fetal heart cDNA library. Comparison of overall nucleotide sequences of ALC1 and VLC1 cDNA clones has revealed that, while these two inserts show significant DNA sequence homology (78.4%) with respect to their coding regions, the 5′- and 3′-untranslated regions are highly divergent. Our statistical analysis suggests that human ALC1 and VLC1 diverged approximately 300 million years ago, during the time of separation of birds and mammals. RNA blot analysis shows that ALC1 mRNA is expressed in fetal ventricular and fetal skeletal muscles as well as fetal and adult atrial muscles and VLC1 mRNA is expressed in adult slow skeletal muscle as well as fetal and adult ventricular muscles. Southern blot analysis indicates that each protein is encoded by a single gene. Finally, we show that VLC1 mRNA is induced in pressure-overloaded human atrium.

By analyzing surface antigens and cytolytic functions of proliferating large granular lymphocytes (LGLs), three types of T cell LGL lymphocytosis were delineated. The first, most commonly encountered type exhibited CD3+4–8+16+, WT31+ phenotype, low or undetectable non- major histocompatibility complex (MHC)-restricted cytotoxicity, and moderate to strong antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). Because these LGLs carried T cell antigen receptor (Ti) recognized by WT31 monoclonal antibody (MoAb), and treatment with anti-Ti, anti-CD3 MoAbs and phytohemagglutinin elicited non-MHC-restricted cytotoxicity, they may have developed from populations of in vivo primed cytotoxic T lymphocytes with unknown antigen specificity. The second, rare type of LGL lymphocytosis exhibited CD3+4–8–16+, WT31 phenotype, and strong non- MHC-restricted, ADCC and LDCC cytotoxicities. These cells were probably derived from the lymphocytes of the same phenotype found in small numbers in normal peripheral blood. Because anti-CD3 MoAb inhibited non- MHC-restricted cytotoxicity of the LGLs, a Ti not detected by WT31 MoAb, but putatively present seemed to serve as a specific receptor for target tumor cell recognition. The third type of LGL lymphocytosis showed CD3+4+8–16+, WT31+ phenotype, and lacked cytolytic activities and parallel tubular arrays. These LGLs probably evolved from cells with the same characteristics selectively located in the germinal centers of lymphoid tissues. Taken together, in patients with LGL lymphocytosis, T cell-associated antigens expressed on LGLs were shown to be involved in the regulation of LGL-mediated cytolytic activities. In addition, studies of surface antigens and the effects of MoAbs and lectins on cytolytic activities may be useful in clarifying the normal counterpart of LGLs from which leukemic or reactively proliferating LGLs originate.

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL- 60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation- induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O- tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.

The regulatory mechanism of differentiation of granulocyte and macrophage precursor cells (G/M CFU-C) in bone marrow and spleen obtained from nude mice bearing colony-stimulating factor (CSF) producing tumor (G-mice), which developed marked granulocytosis, was studied. In these mice, granulopoiesis is enhanced in the spleen, but suppressed in bone marrow. Coculture of G-mouse bone marrow cells with splenic cells of control nude mice (C-mice) and of G-mice resulted in 68% and 62%, respectively, more colonies than expected, while coculture of C-mouse bone marrow cells with these two sources of cell fractions resulted in only 2% and 11% more colonies. In the double-layer agar culture system, bone marrow and splenic cells of C- and G-mice produced a maximal number of colonies containing adequate amounts of human urinary CSF in the upper layer when C-mouse bone marrow cells were added to the lower layer, while these four sources of cells produced a moderate or minimal number of colonies when splenic cells of C- and G- mice or G-mouse bone marrow cells were added to the lower layer. Morphological examination of colonies formed in the upper layer revealed that addition of C-mouse bone marrow cells or irradiated G- mouse bone marrow cells to the lower layer resulted in a massive increment in the number of colonies with pure granulocytes and granulocyte and macrophage mixed (G + G/M) colonies formed by G-mouse bone marrow cells in the upper layer. However, the addition of irradiated C-mouse bone marrow cells or G-mouse bone marrow cells before irradiation to the lower layer did not change G + G/M colony formation by G-mouse bone marrow cells in the upper layer. We could reproduce these findings with conditioned media obtained from 3-day liquid cultures of these cell fractions. This suggests that a diffusible factor may be necessary for inhibition of G + G/M colony formation in G-mouse bone marrow cells. The fine mechanism of such inhibition remains to be clarified.

Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline. This activated c-raf was cloned in a cosmid vector and actively transforming clones were obtained. Comparison of the restriction maps of this activated c-raf and cloned normal rat c-raf revealed a recombination in the 5'-terminal region of the activated form of this gene. The recombined DNA was shown to be actively transcribed and possibly to form a fused mRNA with c-raf, which is slightly smaller than normal c-raf mRNA. Since this recombination was not detected in the original tumor by Southern blot analysis, it presumably occurred during transfection.

Summary A major limitation of studies on the parvovirus B 19, a causative agent of transient aplastic crisis, has been the absence of appropriate cell lines permissive for the virus. In the present study, a human erythroid leukemia cell line (JK-1) was shown to support B 19 virus DNA replication in vitro. Forty-eight hours after virus inoculation of JK-1 liquid cell cultures, the average number of B 19 genome copies was estimated at 3,000 per cell by DNA dot blot analysis. The addition of erythropoietin increased B 19 copy number to 10,000 per cell. The presence of replicative forms of the B 19 virus genome was demonstrated by Southern blot analysis. Although persistent infection of B 19 virus was not observed in JK-1 cells, this culture system will be of value in elucidating the molecular basis of the erythroid specificity of parvovirus B 19.

We report a novel mode of apoptosis induction observed in human leukemic HL-60 cells. These cells spontaneously underwent apoptosis in the course of proliferation when the cell density became higher than 1 x 10(6)/ml. This occurred under ordinary in vitro culture conditions, with or without fetal calf serum. Even the low density cells were committed to undergo apoptosis if they were cultured under artificially concentrated conditions. Replacement of the culture supernatant of the low density cells by that of the high density ones resulted in apoptosis induction in the former cells. This apoptosis-inducing activity of the high density cell culture supernatant was completely eliminated by the action of trypsin but was fully restored following ultrafiltration by 3-kDa pore-sized membrane. A strong apoptosis-inducing activity was recovered from the culture supernatant of the high density HL-60 cells at a specific fraction in reverse-phase column chromatography. Neither an interleukin-beta converting enzyme inhibitor nor CPP-32 inhibitor blocked the induction of cell density-dependent apoptosis in HL-60 cells, although overexpression of Bcl-2 protein markedly attenuated the induction of this mode. Surprisingly, transforming growth factor-beta1 and activin A did not induce but, rather, inhibited the induction of cell density-dependent apoptosis. These data suggest that HL-60 cells release an unknown low molecular weight peptide-containing factor in response to an increase in cell density to induce apoptosis in an autocrine manner and that the interleukin-beta converting enzyme-independent intracellular machinery for this mode of apoptosis is strongly affected by signaling events through the transforming growth factor-beta1 receptor and by the action of Bcl-2 oncoprotein.

Granulocyte colony-stimulating factor(G-CSF), but not granulocyte-macrophage CSF(GM-CSF), induced tyrosine phosphorylation of 95-kDa protein in 13 cases of primary human acute myelogenous leukemic cells. Electrophoretic mobility of 95-kDa phosphoprotein and the protooncogene product p95vav was identical. In addition, p95vav was tyrosine-phosphorylated only in G-CSF-stimulated cells. In contrast to primary leukemic cells, amount of p95vav was under detectable level and G-CSF did not induce tyrosine phosphorylation of 90-100-kDa proteins in human neutrophils. These results indicate specific involvement of vav product in signaling pathway of G-CSF in primary human leukemic cells.Copyright 1995, 1999 Academic Press, Inc.

We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.

It has been documented that streptozotocin-induced diabetes in rats is associated with diminished effects of insulin despite increased insulin binding to its receptor. This paradox led us to examine whether any alterations of insulin receptor-kinase activities occur in this type of insulin resistance. Insulin binding capacity/mg of protein of solubilized, wheat germ agglutinin-purified preparations from livers was increased by 1.8-fold in the streptozotocin (65 mg/kg) diabetic rats. This increase was associated with a parallel increase in receptor protein as measured by an immunoblotting method using anti-insulin receptor antibody. Moreover, no apparent change was observed in the stoichiometry of alpha and beta subunits of the insulin receptor between diabetic and control rats. Insulin-stimulated (10(-7) M) phosphorylation of the beta subunit of the insulin receptor was decreased by 40% in diabetic rats when equal quantities of insulin binding capacity were compared. Phosphorylation of an exogenously added synthetic peptide (similar in sequence to the tyrosine phosphorylation site in pp60src) by the insulin receptor-kinase was also decreased by 25% in diabetic rats. These abnormalities were partially restored by in vivo insulin treatment. These data suggest that diminished insulin receptor autophosphorylation and kinase activity could provide a possible mechanism for the "post-binding insulin resistance" in diabetic rats.

The purified insulin receptor kinase catalyzed the phosphorylation of native tubulin and microtubule-associated proteins (MAPs; MAP2, tau) on tyrosine residues. Insulin (10(-7) M) stimulated the reaction by 4-10-fold by increasing Vmax with little change in Km. alpha-Tubulin was preferred as a substrate for the kinase compared to beta-tubulin. MAP2 was found to be the best substrate among the cytoskeletal proteins tested; in the presence of insulin, the Vmax for MAP2 was 6.3 nmol/min/mg, its Km was 5.1 microM, and 1.7 mol of phosphate were incorporated per mol of MAP2. Under the same conditions used for this phosphorylation of tubulin and MAPs, actin and tropomyosin were very poorly phosphorylated. These data, coupled with previous evidence for potential functional relationships between insulin action and microtubules, raise the possibility that microtubule proteins may be cellular targets for the insulin receptor kinase.

The effects of hematopoietic growth factors on human monocyte superoxide (O2-) release were investigated by using purified human monocytes in suspension. Among growth factors studied, granulocyte- macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), and interleukin-3 (IL-3) primed human monocytes and enhanced O2- release stimulated by the receptor-mediated agonists, N-formyl- methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A), but not by phorbol myristate acetate, which bypasses the receptors to stimulate the cells. The optimal priming was obtained by pretreatment of cells with 1 to 5 ng/mL (0.07 to 0.34 nmol/L) GM-CSF, 50 to 100 ng/mL (0.5 to 1.1 nmol/L) M-CSF, or 10 to 20 ng/mL (0.6 to 1.3 nmol/L) IL-3 for 10 minutes at 37 degrees C. Potency of the maximal priming effects on FMLP- or Con A-induced O2- release was GM-CSF greater than M- CSF = IL-3. The combination of the optimal concentrations of any two CSFs resulted in the effect of more potent priming agent alone. Enhancement of O2- release by GM-CSF was observed over the complete range of effective concentrations of FMLP (10(-8) to 10(-6) mol/L). The pretreatment of monocytes with granulocyte-CSF (50 ng/mL), interferon- gamma (1,000 U/mL), or IL-4 (20 ng/mL) for 10 minutes at 37 degrees C had no effect on O2- release stimulated by FMLP or Con A. These findings show that GM-CSF, M-CSF, and IL-3 selectively enhance O2- release in human monocytes triggered by receptor-mediated agonists after short-term preincubation.

Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time- courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM- CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

Granulocyte colony-stimulating factor (G-CSF) was reported to increase the number of colony-forming units-spleen (CFU-S) and multilineage colonies as well as myeloid-committed cells. We investigated the effects of G-CSF on myeloid progenitors and primitive stem cells in a mouse bone marrow transplantation (BMT) system. Lethally irradiated mice received BM cells from untreated or 5-fluorouracil-treated mice, and then were administered G-CSF or carrier buffer (control) for 5 days from immediately after BMT. A pre-CFU-S assay was performed by the repeated transplantation of BM cells from the first BMT recipients to other mice. By the method of polymerase chain reaction, most of the spleen colonies in the secondary recipients were confirmed to be derived from the first donors. G-CSF did not increase the peripheral white blood cell count significantly, but did increase the number of immature myeloid cells and granulocyte-macrophage colony-forming cells in the BM. The number of erythroid cells in the BM was initially suppressed and then increased by G-CSF treatment. In addition, the pre- CFU-S assay showed an increase in pre-CFU-S cells due to G-CSF administration. The number of spleen colonies of first BMT recipients did not increase, but a higher percentage of them were committed to a certain lineage by G-CSF treatment. These findings suggest that G-CSF has important roles in the early stages of hematopoiesis.

We characterized the molecular species of human macrophage colony- stimulating factor (hM-CSF) found in serum and urine, using immunoblot analysis after partial purification on an antibody-bound affinity column. Although antibodies were prepared using the recombinant product of the large form of hM-CSF with a molecular weight (MW) of 85 Kd as the antigen, this immunoblot system was also capable of detecting the small form of hM-CSF with a MW of 40 to 60 Kd. A single band with a MW of 43 Kd, which reacted with anti-recombinant hM-CSF IgG but not with control IgG, was found when serum and urine from normal adults underwent electrophoresis on reduced sodium dodecyl sulfate- polyacrylamide gel and subsequent immunoblotting. This band represented a subunit of the large form of hM-CSF, because the large form of hM-CSF is a homodimer of a subunit with a MW of 43 Kd and the small form of hM- CSF is a homodimer of a subunit with a MW of 20 to 30 Kd. Analysis of serum and urine from leukemic patients and pregnant women, who had higher serum levels of hM-CSF than normal adults, showed only a single band with a MW of 43 Kd as a hM-CSF-specific molecule. These results suggest that the large form of hM-CSF is the major species in human body fluids.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered (50 to 800 micrograms/m2) once daily as a half-hour intravenous (IV) infusion for 14 days to seven patients with malignant lymphoma. In all patients, administration of rhG-CSF not only ameliorated the decrease in absolute neutrophil count after the cytotoxic chemotherapy but also enhanced superoxide (O2-) release in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The priming effect of rhG-CSF on neutrophil O2- release was rapid (evident within 6.5 hours) and sustained at least for 24 hours after a single IV administration of rhG-CSF. The responsiveness to further in vitro challenge of rhG-CSF was lost or reduced in neutrophils isolated after rhG-CSF treatment, indicating that neutrophils already primed in vivo by rhG-CSF are desensitized to this factor. In contrast to the results obtained with FMLP, when phorbol myristate acetate (PMA) was used as stimulus, no consistent enhancement of O2- release was observed, suggesting that rhG-CSF modulates the signal transduction pathways linked to FMLP receptors rather than increases the components of the O2- producing enzyme complexes. Administration of rhG-CSF also rapidly (evident within 15 minutes) caused an increase in expression of neutrophil C3bi-receptors that was sustained for at least 24 hours after a single IV administration of rhG- CSF. Pharmacokinetic study of rhG-CSF showed a half-life (t1/2) of 114 min. These findings show that rhG-CSF is a potent activator for neutrophil functions both in vivo and in vitro.

We examined the in vitro effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) on neutrophil anomalies in 20 patients with myelodysplastic syndromes (MDS) and eight patients with chronic myelogenous leukemia (CML). Neutrophil alkaline phosphatase (NAP) activity was determined in nine MDS patients and eight CML patients by a scoring method. NAP scores were decreased in six of the nine patients with MDS and in all of the patients with CML. In all patients with these diseases, NAP scores increased by incubating the blood with rhG- CSF. An increase in NAP scores by rhG-CSF was observed even at a concentration of 1 U/mL in patients with MDS but was observed only at higher concentrations (1,000 to 10,000 U/mL) in patients with CML. Significant increases in NAP scores occurred at 12 hours' incubation in patients with MDS, whereas the increase was more gradual in patients with CML. This time course difference was thought to be due mainly to the difference in cell populations of circulating myeloid cells between MDS patients and CML patients. Induction of NAP activity by rhG-CSF in patients with both these diseases was suppressed by the addition of inhibitors of RNA or protein synthesis. Neutrophil superoxide anion (O2- ) production induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was determined in the other 11 patients with MDS. This neutrophil function was decreased in seven of the 11 patients with MDS, normal in two patients, and increased in two patients. Preincubation with rhG-CSF caused a significant increase in fMLP-induced O2- production in nine of the 11 patients with MDS. rhG-CSF enhanced this neutrophil function in a time- and dose-dependent manner, and maximal stimulation was observed at 2,000 to 4,000 U/mL of rhG-CSF and at five to ten minutes' incubation. The present results show that rhG-CSF is able to repair at least in part the neutrophil anomalies in these patients, and our data, especially for patients with MDS, suggest the clinical usefulness of rhG-CSF for this preleukemic disorder.

A case of WT31-, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4–8–16+ and WT31-. By two-color immunofluorescence staining, CD3+4- 8- cells were found to be WT31-, and a small population of WT31+ cells expressed either CD4 or CD8. WT31-, CD3+ cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31-reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-beta and Ti-gamma genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody- and lectin- dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.

High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay.

Summary A multi-institutional randomized study for the evaluation of ubenimex (Bestatin) in the treatment of adult acute nonlymphocytic leukemia was performed. One hundred and ninety-five patients were registered from February 1988 to December 1990. Patients who had reached complete remission by one or two courses of remission induction chemotherapy were divided into the ubenimex group or the control group by randomization. Patients of the ubenimex group started to receive 30 mg ubenimex orally once a day when maintenance therapy began and continued as long as possible. Remission duration and survival were analyzed based on the data as of August 31, 1991. Remission duration of the ubenimex group was superior to that of the control group (generalized Wilcoxon test:-p=0.0338). Fifty percent remission duration was 508 days in the ubenimex group and 386 days in the control group. There has been no statistical difference in survival between the two groups as yet.

Abstract:  Acyclic retinoid (all-trans-3, 7, 11, 15-tetramethyl-2, 4, 6, 10, 14-hexadecapentaenoic acid) binds cellular retinoic acid-binding protein with an affinity similar to that of all-trans retinoic acid and induces differentiation of human hepatoma cell lines and a human acute myelogenous leukemia cell line (HL-60). We investigated the in vitro efficacy of acyclic retinoid to induce the differentiation of acute promyelocytic leukemia (APL) cells using primary cultured cells obtained from 11 APL patients. Five days' incubation with acyclic retinoid effected a dose-dependent induction of differentiation. Cells from eight patients showed maximum differentiation at 10–6 M acyclic retinoid. Cells from one patient required 10–5 M for maximum differentiation, while those from two patients exhibited moderate differentiation at 10–5 M. Five days' incubation with acyclic retinoid (10–7∼10–5 M) did not affect the viability or number of cells from any patient except one, whose cells showed a slight decrease in viability at 10–5 M. Thus, we conclude that acyclic retinoid induced the differentiation of primary cultured APL cells at concentrations of 10–6∼10–5 M, a range at which it is not toxic.

To examine the molecular mechanisms by which mechanical stimuli induce cardiac hypertrophy and specific gene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin. Nuclear run-off transcription assay revealed that this increase in c-fos mRNA level by stretching at least partially reflects changes in the transcriptional status. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene indicated that sequences containing a serum response element were required for efficient transcription by stretching and that sequences containing a cAMP/calcium response element might not be involved in the c-fos response to myocyte stretching. The accumulation of c-fos mRNA by stretching was suppressed by protein kinase C inhibitors at the transcriptional level and inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbol esters stimulated the expression of c-fos and skeletal alpha-actin genes. These findings suggest that mechanical stimuli (myocyte stretching) might directly induce cardiac hypertrophy and specific gene expression possibly via protein kinase C activation.

Leukocyte tyrosine kinase (LTK) is a tyrosine kinase that has been suggested to be specific for hematopoietic cells and neuronal cells and reported as an unusual membrane protein lacking an extracellular domain. Here we report the cloning of a human LTK cDNA clone containing the complete open reading frame of a putative receptor tyrosine kinase protein. The extracellular domain of the receptor protein is larger than previously predicted. Furthermore, we have cloned a set of cDNAs representing differently spliced human LTK mRNAs. These cDNAs predict a truncated receptor protein lacking the tyrosine kinase domain and a soluble receptor protein that has neither a transmembrane nor a tyrosine kinase domain. Our results suggest that the LTK gene produces not only the putative receptor tyrosine kinase for unknown ligand but also multiple protein products that may have different functions.

CD4+ cells comprise approximately 3% to 6% of murine bone marrow (BM) cells. The majority are CD4dull+, but there are two distinct sub populations: CD4 brightly positive Gr-1- cells (CD4hiGr-1-) and CD4+ Gr- 1+ cells (CD4loGr-1lo). CD4hiGr-1- cells are considered to be mature T cells by cell surface antigen expression and morphology. CD4loGr-1lo cells, which comprise approximately 0.6% of the BM cells, express small amount of B220 and Thy1 antigens. Interestingly, colony-forming units (CFU)-spleen and CFU-C are not enriched in this population. However, when injected into lethally irradiated mice, CD4loGr-1lo cells were shown to differentiate into T-cell, B-cell, and myelo-monocyte lineages when assayed 26 weeks after transplantation. Furthermore, donor-derived CD4loGr-1lo cells were present in the recipients' BM at least 16 weeks after transplantation. These observations suggest that murine CD4loGr- 1lo cells in BM have self-renewal capability and retain the ability to differentiate into at least three lineages in long-term hematopoiesis.

The influence of leukaemic cells on the colony formation of human bone marrow cells was studied in vitro as an extension of our previous work (Chiyoda et al., 1975). An extract of leukaemic bone marrow cells significantly suppressed colony forming ability of the normal bone marrow cells, whereas an extract of normal bone marrow cells did not suppress it except in two cases. The suppressive effect of normal bone marrow cells, however, was obviously less intense than that of leukaemic cells. This suppressive effect was dose dependent and was fairly stable to heat treatment. These results suggest that leukaemic bone marrow cells contain factor(s) which suppress normal colony formation.

Monoclonal antibody TFS-4 has previously been shown to react selectively with human small cell lung cancer (SCLC). We evaluated the use of 131I-labelled TFS-4 for the treatment of established human SCLC transplanted in nude mice. The specific accumulation of the antibody in the transplanted tumour was recorded by both scintigraphic and biodistribution studies. Administration of 200 microCi 131I-labelled TFS-4 inhibited tumour growth when compared with the same radiation dose of the control monoclonal antibody. The therapeutic effect was dose-dependent and complete disappearance of the tumour was observed transiently in one out of the three animals following the administration of 500 microCi 131I-labelled TFS-4.

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the β-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the β-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.

We have previously reported that fodrin (beta subunit), tubulin (alpha subunit) and microtubule-associated proteins (MAPs; MAP2 and tau) are good substrates for the purified insulin receptor kinase (Kadowaki, T., Nishida, E., Kasuga, M., Akiyama, T., Takaku, F., Ishikawa, M., Sakai, H., Kathuria, S., and Fujita-Yamaguchi, Y. (1985) Biochem. Biophys. Res. Commun. 127, 493-500 and Kadowaki, T., Fujita-Yamaguchi, Y., Nishida, E., Takaku, F., Akiyama, T., Kathuria, S., Akanuma, Y., and Kasuga, M. (1985) J. Biol. Chem. 260, 4016-4020). In this study, to investigate the substrate specificities of tyrosine kinases, we have examined the actions of the purified epidermal growth factor (EGF) receptor kinase and Rous sarcoma virus src kinase on purified microfilament- and microtubule-related proteins. Among microfilament-related proteins examined, the purified EGF receptor kinase phosphorylated the beta subunit, but not the alpha subunit, of fodrin on tyrosine residues with a Km below the micromolar range. The fodrin phosphorylation by the EGF receptor kinase was markedly inhibited by F-actin. In contrast, the purified src kinase preferentially phosphorylated the alpha subunit of fodrin on tyrosine residues. Fodrin phosphorylation by the src kinase was not inhibited by F-actin. Among microtubule proteins examined, MAP2 was the best substrate for the EGF receptor kinase. By contrast, src kinase favored phosphorylation of tubulin as compared to MAP2. The peptide mapping of MAP2 phosphorylated by the EGF receptor kinase and by the insulin receptor kinase produced very similar patterns of phosphopeptides, while that of MAP2 phosphorylated by the src kinase gave a distinctly different pattern. When the phosphorylation of the tubulin subunits was examined, the EGF receptor kinase preferred beta subunit to alpha subunit, but the src kinase phosphorylated both alpha and beta subunits to a similar extent. These results, together with our previous results, indicate that the substrate specificities of the EGF receptor kinase and the insulin receptor kinase are very similar, but not identical, while that of the src kinase is distinctly different from that of these growth factor receptor kinases.