Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Stakeholder perspectives on transitions of nursing home residents to hospital emergency departments and back in two Canadian provinces.

Major gaps exist in our understanding of transitions in care for older persons living in nursing homes. The purpose of the study was to identify key elements, from multiple stakeholder perspectives, that influence the success of transitions experienced by nursing home residents when they required transfer to a hospital emergency department. This interpretive descriptive study was conducted in two cities in the Canadian provinces of British Columbia and Alberta. Data were collected from 71 participants via focus groups and individual interviews with nursing home residents, family members, and professional healthcare providers working in nursing homes, emergency departments, and emergency medical services. Transcripts were analyzed using constant comparison. The elements contributing to the success of transitions reflected a patient- and family-centered approach to care. Transitions were influenced by the complex interplay of multiple elements that included: knowing the resident; critical geriatric knowledge and skilled assessment; positive relationships; effective communication; and timeliness. When one or more of the elements was absent or compromised, the success of the transition was also compromised. There was consistency about the importance of all the identified elements across all stakeholder groups whether they are residents, family members, or health professionals in nursing homes, emergency departments or emergency medical services. Aspects of many of these elements are modifiable and suggest viable targets for interventions aimed at improving the success of transitions for this vulnerable population.

Negative regulation of Pim-1 protein kinase levels by the B56beta subunit of PP2A.

The Pim protein kinases are serine threonine protein kinases that regulate important cellular signaling pathway molecules, and enhance the ability of c-Myc to induce lymphomas. We demonstrate that a cascade of events controls the cellular levels of Pim. We find that overexpression of the protein phosphatase (PP) 2A catalytic subunit decreases the activity and protein levels of Pim-1. This effect is reversed by the application of okadaic acid, an inhibitor of PP2A, and is blocked by SV40 small T antigen that is known to disrupt B subunit binding to PP2A A and C subunits. Pim-1 can coimmunoprecipitate with the PP2A regulatory B subunit, B56beta, but not B56alpha, gamma, delta, epsilon or B55alpha. Using short hairpin RNA targeted at B56beta, we demonstrate that decreasing the level of B56beta increases the half-life of Pim-1 from 0.7 to 2.8 h, and decreases the ubiquitinylation level of Pim-1. We also find that Pin1, a prolyl-isomerase, is capable of binding Pim-1 and leads to a decrease in the protein level of Pim-1. On the basis of these observations, we hypothesize that phosphorylated Pim-1 binds Pin1 allowing the interaction of PP2A through B56beta. Dephosphorylation of Pim-1 then allows for ubiquitinylation and protein degradation of Pim-1.

Caspase cleavage of the nuclear autoantigen LEDGF/p75 abrogates its pro-survival function: implications for autoimmunity in atopic disorders.

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a nuclear autoantigen in atopic disorders implicated in cellular protection against stress-induced apoptosis. We observed that LEDGF/p75 was cleaved during apoptosis into fragments of 65 and 58 kD generated by caspases-3 and -7 cleaving at three sites: DEVPD30/G, DAQD486/G and WEID85/N. Sequence analysis revealed that the DEVPD30/G and WEID85/N sites lie within the highly conserved HATH (homologous to amino terminus of hepatoma-derived growth factor) region, also known as PWWP domain. Alignment of proteins containing this domain failed to reveal conservation of the DEVPD30/G and WEID85/N sites, suggesting that the HATH/PWWP domain of LEDGF/p75 may be specifically targeted by caspases. Overexpression of LEDGF/p75 protected HepG2 cells from serum starvation-induced cell death, whereas expression of the 65 kD fragment failed to protect. The apoptotic cleavage of LEDGF/p75 may contribute to the pathogenesis of atopic disorders by abrogating its pro-survival function and enhancing its immunogenicity.

Distinct domains of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit mediate activation of Jak/Stat signaling and differentiation.

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor has several isoforms that result from alternative splicing events. Two forms, alpha-1 and alpha-2, have intracytoplasmic sequences that are identical within a membrane-proximal domain but differ completely distally. Variant and mutated GM-CSF receptor alpha subunits, along with the beta subunit (beta(c) protein) were expressed in M1 murine leukemia cells. and the ability of the receptors to signal for differentiation events and to activate Jak/Stat signaling pathways was examined. All cell lines expressing both alpha and beta(c) proteins exhibited high-affinity binding of radiolabeled human GM-CSF. Receptor alpha subunits with intact membrane-proximal intracellular domains could induce expression of the macrophage antigen F4/80 and down-regulate the expression of CD11b. Addition of recombinant human GM-CSF to cells expressing alpha-1 subunits induced the expression of CD86 and tyrosine phosphorylation of Jak-2 and its putative substrates SHPTP-2, Stat-5, and the GM-CSF receptor beta(c) subunit. Cells containing alpha subunits that lacked a distal domain (term-3) or had the alternatively spliced alpha-2 distal domain showed markedly decreased ability to support tyrosine phosphorylation of Jak-2 and its substrates or to up-regulate CD86. Ligand binding induced stable association of the alpha-1 subunit and beta(c) protein. In contrast, the alpha-2 subunit did not stably associate with the beta(c) subunit. These data identify potential molecular mechanisms for differential signaling of the alpha-1 and alpha-2 proteins. The association of unique signaling events with the 2 active GM-CSF alpha subunit isoforms offers a model for variable response phenotypes to the same ligand.

Cytoplasmic domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor beta chain (hbetac) responsible for human GM-CSF-induced myeloid cell differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of myeloid progenitor cells. The biologic actions of GM-CSF are mediated by its binding to the alpha and beta subunits of the GM-CSF receptor (GM-CSFRalpha and betac, respectively). To determine whether identical regions of the betac protein mediate both cell growth and differentiation, we expressed cDNA constructs encoding the human wild-type (897 amino acids) and truncated betac (hbetac) subunits along with the wild-type human GM-CSFRalpha subunit in the murine WT19 cell line, an FDC-P1-derived cell line that differentiates toward the monocytic lineage in response to murine GM-CSF. Whereas the WT19 cell line carrying the C-terminal deleted hbetac subunit of 627 amino acids was still able to grow in human GM-CSF (hGM-CSF), 681 amino acids of the hbetac were necessary for cell differentiation. The addition of hGM-CSF to WT19 cell lines containing the hbetac627 subunit stimulated the phosphorylation of ERK (extracellular signal-regulated kinase) and induced the tyrosine-phosphorylation of SHP-2 and STAT5, suggesting that the activation of these molecules is insufficient to mediate the induction of differentiation. A point mutation of tyrosine 628 to phenylalanine (Y628F) within hbetac681 abolished the ability of hGM-CSF to induce differentiation. Our results indicate that the signals required for hGM-CSF-induced differentiation and cell growth are mediated by different regions of the hbetac subunit.

The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.

Pentoxifylline inhibits tumor necrosis factor-alpha-mediated cytotoxicity and cytostasis in L929 murine fibrosarcoma cells.

Tumor necrosis factor-alpha (TNF alpha) is recognized as a principal mediator of a variety of inflammatory conditions. In animal models, pentoxifylline attenuates the morbidity and mortality of bacterial sepsis, an effect which has been attributed to its ability to suppress the induction of TNF alpha. To determine whether pentoxifylline also directly inhibits the effects of TNF alpha, the ability to inhibit cytotoxicity on the TNF alpha-sensitive murine fibrosarcoma cell line, L929, was examined. Cell viability was assessed by crystal violet staining and cell proliferation was assessed by [3H]-thymidine uptake assay. TNF alpha induced dose-dependent cytotoxicity. At concentrations of TNF alpha of 1000 U/ml, viability at 3 days was approximately 35% of control. When L929 cells were co-incubated with TNF alpha (1000 U/ml) and pentoxifylline (1 mM), cell viability increased to approximately 75% of control (P = 0.001). At concentrations of TNF alpha of 10,000 U/ml, cell viability which was 11% of control with TNF alpha alone increased to 53% in the presence of pentoxifylline (P = 0.002). TNF alpha at 1000 and 10,000 U/ml concentrations decreased [3H]-thymidine uptake to approximately 5% of control values. Co-incubation with pentoxifylline significantly increased uptake to 13% of control at both TNF alpha concentrations (P = 0.002). Pentoxifylline did not affect the level of type I TNF alpha receptor--ligand cross-link product. However, in TNF alpha receptor binding assays, incubation with pentoxifylline 1 mM for 4 h was associated with an increase in the receptor affinity (control: KD = 0.42 nM vs pentoxifylline-treated: KD = 0.21 nM, P = 0.006), without significant change in number of type I TNF alpha receptors, suggesting that pentoxifylline affects post-receptor signalling events. We have observed that pentoxifylline prevents the TNF alpha-mediated activation of sn-2 arachidonic acid-specific cytosolic phospholipase A2, an important component of the signal transduction pathway of TNF alpha cytotoxicity. Because pentoxifylline does not inhibit all activities mediated by the type I TNF alpha receptor, its selective inhibition of post-receptor signalling may facilitate further study into the mechanisms underlying the diverse effects of TNF alpha.

Mapping the intracytoplasmic regions of the alpha granulocyte-macrophage colony-stimulating factor receptor necessary for cell growth regulation.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor is composed of an alpha subunit which binds GM-CSF and a beta subunit, which together form the high affinity receptor. By transfecting the human alpha subunit into murine Ba/F3 cells, we have been able to investigate the role of the short 54-amino acid intracytoplasmic portion (amino acid 346-400) of this subunit in mediating cell growth. We have shown that the intracytoplasmic amino acids 346-382 are necessary for GM-CSF-mediated cell growth. In contrast, amino acids 382-400 can be removed without effect. The stable transfection of the human beta subunit into the cell lines containing the mutant alpha subunits did not affect the growth characteristics of these cells. The ability of GM-CSF to stimulate cell growth of the Ba/F3 cells alpha subunit transfectants was correlated with the ability of this hormone to translocate protein kinase C to the particulate fraction. In contrast, the ability of GM-CSF addition to increase phosphorylation of the human beta subunit did not correlate with cell growth and required the entire intracytoplasmic domain of the alpha subunit. These results demonstrate an important role for the intracytoplasmic portion of the alpha subunit in mediating both signal transduction and cell cycle commitment stimulated by GM-CSF.

Effect of granulocyte-macrophage colony-stimulating factor and interleukin-3 on interleukin-8 production by human neutrophils and monocytes.

Interleukin-8 (IL-8) is a major neutrophil chemoattractant and functional stimulant that is induced by IL-1, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS). We report that recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 are also potent inducers of IL-8 messenger RNA (mRNA) accumulation and protein secretion by normal peripheral blood monocytes. Neutrophils produce IL-8 in response to GM-CSF but not to IL-3. In contrast, recombinant human granulocyte-CSF (rhG-CSF), at concentrations as high as 100 ng/mL, does not induce IL-8 in either cell type. rhGM-CSF also induces IL-8 mRNA expression and IL-8 protein in the promonocytic cell line, U-937, whereas rhG-CSF does not. IL-8 secretion by monocytes was stimulated within 2 hours after incubation with rhGM-CSF or rhIL-3. Stimulation of neutrophils with rhGM-CSF resulted in an increase in cell-associated IL-8 at 4 hours. At 24 hours, cell-associated IL-8 levels declined, whereas secreted IL-8 levels increased. In contrast, virtually all IL-8 induced in monocytes appeared as secreted protein. Neither rhGM-CSF nor rhIL-3 induced detectable secretion of IL-1, TNF alpha, or IL-6 protein by monocytes. rhGM-CSF, and to a lesser degree rhIL-3, potently stimulated IL-8 secretion in cultures of heparinized whole blood, whereas rhG-CSF had no significant effect on IL-8 secretion. Induction of IL-8 by GM-CSF may be physiologically important in enhancing the acute inflammatory response.

Sodium vanadate, a tyrosine phosphatase inhibitor, affects expression of hematopoietic growth factors and extracellular matrix RNAs in SV40-transformed human marrow stromal cells.

Protein tyrosine kinases represent a subset of proteins that mediate signal transduction between the extracellular environment and the nucleus. We have previously described a coordinated upregulation between RNA transcripts of a tyrosine kinase, c-abl, and those of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) in human marrow stromal cells (SVMSC). Moreover, an inverse relationship exists between expression of c-abl transcripts and those of extracellular matrix proteins such as type collagen I transcripts. In the present study, these inverse relationships were again seen in SVMSC when tyrosine kinase effects were enhanced by treatment of the cells with the tyrosine phosphatase inhibitor sodium orthovanadate. This suggests that tyrosine kinases are involved in the coordinate regulation of these genes.

Hyperthermia of pet animal tumours with self-regulating ferromagnetic thermoseeds.

Investigations with thermally self-regulating ferromagnetic implants (thermoseeds) were done on healthy rats and pet animals with spontaneous and transmissible venereal tumours (TVT). The thermoseeds were produced from a nickel-copper alloy and electroplated with a gold-silver layer. Manufacturing conditions were varied to produce thermoseeds with various operating temperatures, the critical temperature above which heating power production sharply declines. To test for toxicity, thermoseeds were implanted into the liver of rats and left in place for up to 14 months. While atomic absorption spectroscopy showed increased nickel and copper levels in tissues near the implants, no clinical evidence of ill-effects was noted. For hyperthermia treatment, thermoseeds were implanted into tumours of pet animals, and these were placed into an induction coil which produced an 89 kHz frequency, 4000 A/m amplitude field. The highest recorded tumour temperature correlated with the nominal operating point of the thermoseeds, demonstrating their ability to regulate the temperature. Of the 15 evaluable animals with spontaneous tumours treated, 12 received concomitant 60Co radiation (two of them only after tumour recurrence following an initial treatment course of hyperthermia alone). Five of those treated with both modalities experienced complete response, five responded partially and two had no change. The treatment course of hyperthermia alone resulted in one animal achieving a complete response, and in three partial responders. Animals bearing TVT had a complete local response with hyperthermia alone. Massive tissue necrosis and seed migration caused the major treatment-related toxicity. Our findings suggest that self-regulating thermoseeds offer the possibility of predictable heat delivery to defined tissue volumes, and may be useful in the treatment of human tumours which are amenable to implantation. Until migration can be controlled, clinical trials should be limited to removable implants.

Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression.

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration-dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage-independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL-6 may have indirect as well as direct influences on hematopoietic cell proliferation.

Interleukin-6 and granulocyte-macrophage colony-stimulating factor are candidate growth factors for chronic myelomonocytic leukemia cells.

Previous studies suggest that malignant cells from some patients with myeloid leukemias produce colony-stimulating factors (CSFs) that can function as autocrine growth factors in vitro. We have examined the roles of interleukin-6 (IL-6) and granulocyte-macrophage CSF (GM-CSF) in the proliferation of myeloid leukemia cells. IL-6 activity was assessed in conditioned medium (CM) from myeloid leukemia cell cultures or cell lysates using IL-6-dependent KD83 and 7TD1 murine cell lines. Media conditioned by cells from patients with chronic myelomonocytic leukemia (CMMoL), but not by normal monocytes, chronic myelogenous leukemia (CML), or acute myelogenous leukemia (AML) cells, contained substantial levels (50 to 1,000 U/10(6) cells) of IL-6. The IL-6 content of CM correlated directly with donor peripheral blood WBC count. CM from two of five CMMoL samples also contained greater than 350 pg/mL GM-CSF. Moreover, CMMoL cells spontaneously formed colonies in semisolid medium. CMMoL colony formation could be partially inhibited by antibodies to IL-6 or GM-CSF, whereas combination of these antibodies gave additive, and nearly complete (greater than 93%), inhibition of spontaneous colony formation. Cell lysates from uncultured CMMoL cells from one patient contained abundant GM-CSF protein but no detectable IL-6. These data suggest that IL-6 and GM-CSF act in vitro as autocrine growth factors for CMMoL cells, and that CMMoL cells in vivo may represent a GM-CSF-dependent autocrine growth system.

Varied differentiation responses of human leukemias to bryostatin 1.

Bryostatin 1, a macrocyclic lactone isolated from a marine bryozoan, has significant antineoplastic activity against the murine cell line P388. Like phorbol esters, bryostatin 1 is capable of binding to and activating protein kinase C, but these two compounds differ in the ability of bryostatin 1 to act as a tumor promoter. We have investigated whether bryostatin 1 can modulate the differentiated phenotype of fresh samples of human myeloid leukemia. We find that six of seven samples responded to bryostatin treatment with changes associated with a more differentiated phenotype including increases in macrophage-like morphology and an increase in adherence and OKM1 and alpha-naphthyl acetate esterase activity positivity. The percentage of cells within each sample evidencing these changes varied markedly among the seven patients' cells examined. Because of the effects of bryostatin on fresh samples we examined the ability of bryostatin to differentiate four HL-60 cell sublines obtained from different laboratories. We found that two of the cell lines did not respond either with an inhibition of growth or morphological change, while one was inhibited, and one showed both growth inhibition and some induction of macrophage-like morphology when treated with bryostatin. To test whether other differentiating agents would enhance the effects of bryostatin 1, we added tumor necrosis factor alpha and bryostatin to these four cell lines. The addition of both agents effected an additive inhibition of growth. These data suggest that bryostatin 1 alone or in combination with other biological response modifiers may have a role in the treatment of human leukemia.

Differential staining of neutrophils and monocytes: surface and cytoplasmic iron-binding proteins.

Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and alpha-naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.

Leukemia-differentiating activity expressed by the human melanoma cell line LD-1.

We have identified a leukemia-differentiating activity (LDA) in medium conditioned by the LD-1 melanoma, a G-CSF secreting human tumor line. Partially-purified LDA induces HL-60 cells to produce superoxide, become phagocytic, and to develop macrophage-like morphology and surface markers. The LDA markedly suppresses clonal growth in agar of HL-60 cells, and cells of the human myeloid leukemia lines PBL 985 and K562, but does not suppress clonal growth of the B-lymphoblast lines Raji and Daudi. The molecular weight of this material is approx. 40,000 daltons. It can be separated from the bulk of the colony stimulating activity on phenyl sepharose chromatography. The LDA is not neutralized by antibodies to G-CSF, GM-CSF, IFN alpha, IFN gamma, TNF, urokinase, and tissue plasminogen activator, and is not inhibited by preincubation with aprotinin. The LDA in conditioned medium may be different from previously described differentiating factors, and may represent an additional class of human growth regulators.

Production of granulocyte colony-stimulating factor by a human melanoma cell line.

We have isolated a human melanoma line (LD-1) from a patient with melanoma and unexplained leukocytosis. The LD-1 cells produced a colony-stimulating factor (CSF) which stimulated primarily granulocytic colonies in human and murine bone marrow cultures. Erythroid burst and mixed colony-stimulating activity was not detected. A single CSF species with a molecular weight of 21,000 was detected in LD-1-conditioned media by G-200 chromatography. Nude mice transplanted with LD-1 tumors developed granulocytosis and had increased blood CSF levels. Messenger RNA from LD-1 cells directed the synthesis of CSF by Xenopus oocytes. Northern blots of LD-1 RNA hybridized strongly with oligonucleotide probes based on the published sequences for human G-CSF, but not with a probe based on the human GM-CSF sequence. Northern blots hybridized with an oligonucleotide probe based on the CSF-1 sequence showed a high-molecular weight band; however, low-molecular weight CSF-1 mRNAs, which are present in the CSF-1-producing cell line MIA-PaCa-2, were not detected in the LD-1 mRNA. The CSF activity of LD-1 cells is best described as human granulocyte CSF.

Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity.

A new human diploid cell line, designated PLB-985, has been established from the peripheral blood of a patient with acute nonlymphocytic leukemia (ANLL). Cells of this line are capable of granulocytic and monocytic maturation in the presence of inducing agents. By morphology, the analysis of surface antigens, and cytochemical staining PLB-985 cells are myelomonoblasts. Transmission electron microscopy reveals them to be devoid of neutrophilic primary or secondary granules and to have an open chromatin pattern with frequent nucleoli. The modal karyotype of the line is 46,XX, with no consistent marker chromosomes or recognizable translocations. Myelomonoblasts of this line form colonies in soft agar and induce tumors (chloromas) in nude mice. Growth of the cells in the presence of dimethyl sulfoxide, cis-retinoic acid, or dibutyryl cyclic adenosine monophosphate results in granulocytic maturation as determined by morphology, histochemical staining characteristics, and incorporation of 35S-methionine into the neutrophil primary granule proteinases elastase and cathepsin G. The tumor-promoting phorbol ester phorbol myristate acetate induces PLB-985 cells to differentiate as monocytes. Cells grown in the presence of this agent rapidly become adherent to plastic, display markedly increased phagocytosis of latex particles, stain positively for alpha-naphthyl acetate esterase, and lose the ability to synthesize the neutrophilic proteinases. Induction of differentiation along either pathway is accompanied by a marked decrease in myc oncogene transcription.

Expression of granulocyte colony-stimulating factor by human cell lines.

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.