Mouse Myeloid Cell Line Research Articles

A Novel FLT3 Inhibitor FI-700 Selectively Suppresses the Growth of Leukemia Cells with FLT3 Mutations

The aim of this study was to evaluate the antileukemia activity of a novel FLT3 kinase inhibitor, FI-700. The antileukemia activity of FI-700 was evaluated in human leukemia cell lines, mutant or wild-type (Wt)-FLT3-expressing mouse myeloid precursor cell line, 32D and primary acute myeloid leukemia cells, and in xenograft or syngeneic mouse leukemia models. FI-700 showed a potent IC(50) value against FLT3 kinase at 20 nmol/L in an in vitro kinase assay. FI-700 showed selective growth inhibition against mutant FLT3-expressing leukemia cell lines and primary acute myeloid leukemia cells, whereas it did not affect the FLT3 ligand (FL)-driven growth of Wt-FLT3-expressing cells. These antileukemia activities were induced by the significant dephosphorylations of mutant FLT3 and STAT5, which resulted in G(1) arrest of the cell cycle. Oral administration of FI-700 induced the regression of tumors in a s.c. tumor xenograft model and increased the survival of mice in an i.v. transplanted model. Furthermore, FI-700 treatment eradicated FLT3/ITD-expressing leukemia cells, both in the peripheral blood and in the bone marrow. In this experiment, the depletion of FLT3/ITD-expressing cells by FI-700 was more significant than that of Ara-C, whereas bone marrow suppression by FI-700 was lower than that by Ara-C. FI-700 is a novel and potent FLT3 inhibitor with promising antileukemia activity.

Antiproliferative and apoptotic effects of wheatgrass (Triticum aestivum L.) extracts on chronic myeloid leukemia (CML) cell line

Wheatgrass (Triticum aestivum L.) is a rich source of vitamins, antioxidants and minerals and it has higher phenolic and flavonoid content in the ethanol extracts than water extracts. Flavonoids in general have been shown to have anticarcinogenic, antimutagenic, properties. Chronic Myeloid Leukemia(CML) is a malignant hematopoietic stem cell disorder that is characterized by BCR-ABL fusion gene encodes cytoplasmic BCR-ABL oncoprotein with a constitutive tyrosine kinase activity that enhances the proliferation and antiapoptotic capacity of affected cell clone. Antiproliferative and apoptotic effects of wheatgrass has not been studied yet. Here we report the effect of wheatgrass extract on CML cell viability, proliferation and apoptosis. 32Dp210 (BCR-ABL fusion gene (+) mouse CML cell line) and 32D (wild type mouse myeloid cell line) cells were grown in RPMI 1640 medium. Cells were incubated with wheatgrass ethanol extracts at final concentrations of 6.5% (w/v) and 13% (w/v) at 0,24,48,72 hours. Cell viability was detected by MTT and trypan blue assays. Apoptosis was determined morphologically and DNA laddering. Both of the concentrations were found to be statistically different (p<0.001) in respect to their antiproliferative and apoptotic effects than their controls. The results showed that the wheatgrass extract inhibited growth of 32Dp210 cells in a dose dependent manner compared to the control cell line(32D). At 6.5% (w/v) and 13% (w/v) concentrations of wheatgrass extracts induced apoptosis at 72, 24 hours respectively. In this study, it has been calculated that the death risk of 32Dp210 was found 6.2 times higher than 32D. It is concluded that wheatgrass extract inhibits proliferation of 32Dp210 cells through the induction of apoptosis.

Effects of aqueous garlic extract on oxidant/antioxidant status in 32 D and 32 Dp cell lines

It was aimed to investigate possible effects of aqueous garlic extract on oxidant/antioxidant status in 32 D (wild type mouse myeloid cell) and 32 Dp210 (BCR-ABL fusion gene (+) mouse myeloid cell) cell lines. Chronic myeloid leukemia (CML) is a myeloproliferative disorder that is characterized by Philedelphia (Ph) chromosome. This chromosome is caused by resiprocal translocation t(9;22)(q34;q11.2) which results in BCR-ABL fusion gene. We hypothesized that garlic could cause apoptosis in CML cells. Therefore, in this study, it is aimed to investigate possible antiproliferative and apoptotic effects of garlic on 32Dp210 (BCR-ABL fusion gene (+) mouse CML cell line) and 32D (wild type mouse myeloid cell line) cell lines.

The Unique N-Terminus of R-Ras Is Required for Rac Activation and Precise Regulation of Cell Migration

The Ras family GTPase, R-Ras, elicits important integrin-dependent cellular behaviors such as adhesion, spreading and migration. While oncogenic Ras GTPases and R-Ras share extensive sequence homology, R-Ras induces a distinct set of cellular behaviors. To explore the structural basis for these differences, we asked whether the unique N-terminal 26 amino acid extension of R-Ras was responsible for R-Ras-specific signaling events. Using a 32D mouse myeloid cell line, we show that full-length R-Ras activates Rac and induces Rac-dependent cell spreading. In contrast, truncated R-Ras lacking its first 26 amino acids fails to activate Rac, resulting in reduced cell spreading. Truncated R-Ras also stimulates more beta3 integrin-dependent cell migration than full-length R-Ras, suggesting that the N-terminus may negatively regulate cell movement. However, neither the subcellular localization of R-Ras nor its effects on cell adhesion are affected by the presence or absence of the N-terminus. These results indicate that the N-terminus of R-Ras positively regulates specific R-Ras functions such as Rac activation and cell spreading but negatively regulates R-Ras-mediated cell migration.

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation.

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.

DNA microarray analysis of stage progression mechanism in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder of haematopoietic stem cells. Despite the high incidence of MDS in the elderly, effective treatment of individuals in its advanced stages is problematic. DNA microarray analysis is a potentially informative approach to the development of new treatments for MDS. However, a simple comparison of 'transcriptomes' of bone marrow mononuclear cells among individuals at distinct stages of MDS would result in the identification of genes whose expression differences only reflect differences in the proportion of MDS blasts within bone marrow. Such a 'population shift' effect has now been avoided by purification of haematopoietic stem-like cells that are positive for the cell surface marker AC133 from the bone marrow of healthy volunteers and 30 patients at various stages of MDS. Microarray analysis with the AC133+ cells from these individuals resulted in the identification of sets of genes with expression that was specific to either indolent or advanced stages of MDS. The former group of genes included that for PIASy, which catalyses protein modification with the ubiquitin-like molecule SUMO. Induction of PIASy expression in a mouse myeloid cell line induced apoptosis. A loss of PIASy expression may therefore contribute directly to the growth of MDS blasts and stage progression.

Comparative studies of resistin expression and phylogenomics in human and mouse

Resistin is a newly identified adipocytokine that has been proposed to be a link between obesity and type 2 diabetes based on animal studies. However, the role of resistin in the pathogenesis of insulin resistance associated with obesity in humans remains unclear. We comparatively and quantitatively studied the tissue distributions of resistin mRNA between human and mouse. The expression level of resistin mRNA in human adipose tissue is extremely low but detectable by real-time PCR and is about 1/250 of that in the mouse. Remarkably, resistin mRNA is abundant in human primary acute leukemia cells and myeloid cell lines U937 and HL60, but not in the Raw264 mouse myeloid cell line. Resistin expression in U937 cells was not affected by lipopolysaccharide (LPS) or by ciglitazone, a PPARγ ligand. Phylogenomics revealed that the human resistin gene is the ortholog of its murine counterpart and is located in a region of chromosome 19p13.3, which is syntenic to mouse chromosome 8A1. In addition to the resistin-like molecule (RELM) sequences already reported, bioinformatics analysis disclosed another RELM sequence in the vicinity of RELMβ on human chromosome 3q13.1, but this sequence is unlikely to encode an expressed gene. Therefore, only two RELMs, resistin and RELMβ, exist in humans, instead of the three RELMs, resistin, RELMα, and RELMβ, that exist in mice. This finding provides a possible answer to the question of why only two RELMs have been cloned in humans and suggests that the RELM family is not well conserved in evolution and may function differently between species. Therefore, caution should be exercised in interpreting resistin as a link between obesity and insulin resistance in humans. The high expression of resistin in human leukemia cells suggests a hitherto unidentified biological function of resistin in leukocytes.

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Sensitivity of hybrid ovine/rat GH receptors to oGH and rat GH in transfected FDC-P1 mouse myeloid cells in vitro.

We have described previously the properties of two mutant ovine growth hormone receptor extracellular domain (oGHR-ECD) proteins which were created by substituting sequences from the rat GHR at two different locations within the framework of the oGHR-ECD. The first mutation occurred at a region close to the N-terminus of oGHR-ECD between residues Thr 28 and Leu34 and created the protein T28E/N29S/N33K/L34P-oGHR-ECD, where the ovine specific residues T, N, N and L are replaced by their equivalent residues E, S, K and P from the rat protein. This site lies N-terminal to the first element of beta-strand structure in the GHR-ECD and we designated this protein as Site-A mutant. The second mutation was made between residues Serl21 andAsp 124 of oGHR-ECD to produce the protein S121T/E123D/D124E-oGHR-ECD where ovine specific residues are again replaced with the equivalent residues from the rat GHR-ECD. This region lies in a loop structure which joins the two beta-barrel domains of the GHR-ECD. This protein is designated as Site-B mutant. A subsequent analysis confirmed the N-terminal region between residues 28-34 of oGHR-ECD as an important epitope defined by antiserum raised to oGHR-ECD. Also of interest was the finding that mutation at both Sites A and B in oGHR-ECD compromised the affinity of the protein for bovine GH (50-fold for Site-A and 4-fold for Site-B). A comparison of the affinity of wild type oGHR for the highly homologous bovine GH with its affinity for rat GH indicated a 10-fold higher affinity for the ruminant GH than for rat GH. Mutation at Site-A of oGHR-ECD reduced the affinity for rat GH a further 3-fold. However, mutation at Site-B of oGHR-ECD increased the affinity for rat GH 2-3 fold. This indicated that the substitution of rat GHR residues for ovine GHR residues in this part of the protein had a beneficial effect in relation to affinity for rat GH and that this region of the GHR-ECD may contain important specificity determinants. In order to test whether these observed differences in affinity for bovine and rat GH affinities have any biological relevance, we have produced the same ovine --> rat mutations in the context of the full length ovine GHR. Transfection of the cDNAs encoding the wt or mutant GHRs into the mouse myeloid pre-B cell line FDC-P1 to create stably transfected clonal lines, has allowed an examination of the relative activities of ovine and rat GH, using a robust and high throughput bio-assay based on the reduction of a cell permeable tetrazolium salt. In the current manuscript, we report that the decrease in binding affinity previously reported for Site-A and Site-B mutant oGHR extracellular domain proteins is not reflected in compromised biological activities when the same mutations are expressed in the context of the full length oGHR protein. We discuss these findings in the context of the relationships between affinity and activity at the GHR.

DNA microarray analysis of genes involved in p53 mediated apoptosis: activation of Apaf-1.

The transcription regulation activity of p53 controls cellular response to a variety of stress conditions, leading to growth arrest and apoptosis. Despite major progress in the understanding of the global effects of p53 on cellular function the pathways by which p53 activates apoptosis are not well defined. To study genes activated in the p53 induced apoptotic process, we used a mouse myeloid leukemic cell line (LTR6) expressing the temperature-sensitive p53 (val135) that undergoes apoptosis upon shifting the temperature to 32 degrees C. We analysed the gene expression profile at different time points after p53 activation using oligonucleotide microarray capable of detecting approximately 11,000 mRNA species. Cluster analysis of the p53-regulated genes indicate a pattern of early and late induced sets of genes. We show that 91 and 44 genes were substantially up and down regulated, respectively, by p53. Functional classification of these genes reveals that they are involved in many aspects of cell function, in addition to growth arrest and apoptosis. Comparison of p53 regulated gene expression profile in LTR6 cells to that of a human lung cancer cell line (H1299) that undergoes growth arrest but not apoptosis demonstrates that only 15% of the genes are common to both systems. This observation supports the presence of two distinct transcriptional programs in response to p53 signaling, one leading to growth arrest and the other to apoptosis. The proapoptotic genes induced only in LTR6 cells like Apaf-1, Sumo-1 and gelsolin among others may suggest a possible explanation for apoptosis in LTR6 cells.

The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways.

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.

Differential sensitivity of murine myeloid FDC-P1 cells and apoptosis resistant mutant(s) to anticancer drugs

There is growing evidence which suggests that dysregulation of apoptosis may lead to several disease states including cancer. To investigate the mechanism controlling the induction of cell death, apoptosis defective/resistant (Apt-) mutants were isolated and characterized in this study. FDC-P1, a mouse myeloid cell line that depends upon IL-3 for survival and growth but undergoes apoptosis when deprived of growth factor, was mutagenized by treatment with ethyl methane sulfonate. We selected cells that survived the growth factor deprivation but did not grow without the factor. Surviving cells were cloned by limiting dilution and four clones that showed the least morphological characteristics and biochemical changes of apoptosis were chosen. Unlike the parent FDC-P1, these mutants were cross resistant to apoptosis induced by a variety of antitumor drugs such as Adriamycin, Dexamethasone, VP-16, as well as reactive oxygen species (ROS) generated by xanthine/xanthine oxidase (X/XO). We used one of these Apt- mutant to test candidate death genes. Our findings suggest that the preferential increase in Bax/Bcl-2 ratio, p53, c-Myc, Caspase-3 and decrease in AP-1 on treatment with various anticancer drugs may contribute to the preferential apoptotic response in FDC-P1 cells but to varying degrees. Whereas, the higher constitutive level of antioxidant enzymes superoxide dismutase and catalase in the Apt- mutant may contribute at least in part to its resistance.

Differential inhibition of recombinant bovine GH (rbGH) activity in vitro by in vivo enhancing monoclonal antibodies.

We have previously described the effects of complexing recombinant bovine growth hormone (rbGH) with the in vivo enhancing monoclonal antibodies (Mabs) OA11 and OA15 and the non-enhancing Mab OA14 on the subsequent activity of GH in different tissue culture models. We reported that all of these Mabs caused the inhibition of GH-stimulated Jak-2 tyrosine kinase phosphorylation in the GH responsive pre-adipocyte cell line 3T3-F442A. However, using the mouse myeloid cell line FDC-P1 transfected with the full length ovine GH receptor (GHR), we subsequently found that OA11 and OA14 remained inhibitory with respect to the end point measurement of GH stimulated mitogenesis but that OA15 had no inhibitory effect on GH stimulated mitogenesis in this cell line. In order to correlate longer term mitogenic effects of Mab-GH complexes with signalling events in this transfected cell line model, we now report on the effects of complexing with Mab on the subsequent GH stimulated phosphorylation of Stat5b (signal transducer and activator of transcription). In agreement with our data for the mitogenic activity of GH-Mab complexes, we found that OA11 and OA14 inhibit GH activation of Stat5b but that OA15 is not inhibitory. Further to this, the dose-response effect of both OA11 and OA14 on the GH stimulation of Stat5b in the FDC-P1-oGHR transfected cells correlates with the previously described dose-response effects for both Mabs in the context of GH stimulation of mitogenic effects. We conclude that in this oGHR transfected cell line model, Mab effects on short and long term GH signalling events are tightly correlated. The observation that neither of the in vivo enhancing Mabs--OA11 or OA15--amplifies the response to GH in our transfected cell line model, coupled with the differential nature of Mab effects on GH activity (OA11--inhibition; OA15--no effect) may argue for an in vivo mechanism for enhancement of GH activity.

Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1.

The molecular pathways responsible for apoptosis in response to granzyme B have remained unresolved. Here we present data supporting the notion that granzyme B-mediated cell death is largely dependent on a pathway that is inhibitable by Bcl-2 or its viral analog BHRF1. We used a panel of stably transfected FDC-P1 mouse myeloid cell lines to show that overexpression of functional, wild-type Bcl-2 or BHRF1 rescued cells from granzyme B-mediated apoptosis, whereas mutated (Gly145-->Glu) Bcl-2, or wild-type Bcl-2 directed to the plasma membrane conferred no protection. Overexpression of Bcl-2 resulted in inhibition of multiple parameters of apoptosis in response to purified perforin and granzyme B, including DNA fragmentation, changes in light scatter profile indicating cell shrinkage and increased refractivity, loss of mitochondrial membrane potential and inhibited colony formation in clonogenic assays. Nevertheless, when exposed to cytotoxic lymphocytes, FDC-P1 and YAC-1 cells overexpressing Bcl-2 remained susceptible to death imparted by cytolytic granules, irrespective of whether the granules contained granzyme B. Thus, alternative granzyme B-independent pathways can be activated by intact lymphocytes to overcome Bcl-2-like inhibitors of apoptosis, enabling CTLs to overcome potential viral blocks to granzyme B-mediated cell death.

Upregulation of telomerase activity by X-irradiation in mouse leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription.

X-irradiation of two mouse myeloid leukaemia cell lines was found to lead to increased telomerase activities. Maximal increases in activity at 24 h post-irradiation were approximately three times control unirradiated cell levels. These maxima were reached at between 3-5 Gy depending upon cell line. Peak activity was reached at 8h, remained elevated to 24 h and returned to control levels by 48 h. In contrast, X-irradiation did not activate telomerase in a telomerase-negative human fibroblast line, while in cultured normal mouse bone marrow cells irradiation appeared to reduce activities. No simple relationship between radiation-induced increases in telomerase activity in the myeloid leukaemia lines and the proportions of cells in the S or M phases of the cell cycle was apparent. Radiation-induced increases in activity were significantly reduced by inhibitors of transcription (actinomycin D, alpha-amanatin) and protein synthesis (cycloheximide). These data are consistent with two possibilities: (i) X-irradiation leads to increased transcription and/or translation of a component of telomerase, thus increasing activities; or (ii) X-irradiation induces the transcription of a positive regulator of telomerase activity. Northern blot analysis did not indicate that transcription of mTert, the catalytic subunit of telomerase, or mTerc, the RNA component, was elevated after irradiation. Similarly, no significant changes in the expression of Myc or Tnks, the tankyrase gene, two suspected telomerase regulators, were detected. These data are therefore consistent with the induction by X-irradiation of a positive regulator of telomerase activity other than Tnks or Myc or the core protein and RNA components of the enzyme.

Structure and expression pattern of a human MTG8/ETO family gene, MTGR1.

AML1-MTG8 fusion protein, which is produced from the rearranged gene formed between AML1 and MTG8 in myeloid leukemia with t(8;21) chromosomal translocation, plays an important role in the pathogenesis of leukemia. We previously showed that ectopically expressed AML1-MTG8 fusion protein is associated with an MTG8-like protein in the mouse myeloid precursor cell line L-G, and this association seemed to be required for AML1-MTG8 to stimulate proliferation. As a candidate cDNA for this MTG8-like protein, a 6.4 kb MTGR1 cDNA encoding human MTGR1b protein of 604 amino acids was isolated. Since this cDNA was shorter than the main mRNA (about 7.5 kb), the 5'-end of the MTGR1 cDNA was extended using Marathon Ready cDNA. When the newly obtained 5'-sequence was combined with the previous cDNA, the resultant MTGR1 cDNA (6995 bp), including exon 3 that the previous cDNA lacked, could encode MTGR1a protein of 575 amino acids. Transcripts of the MTGR1 gene were expressed ubiquitously in the human tissues and cell lines examined. PCR analyses of the cDNAs from human tissues showed the presence of various splicing variants with regard to the 5'-region including exons 1, 2 and 3. The MTGR1 gene consists of 14 exons and spans about 68 kb. The genomic structure of MTGR1 is highly similar to those of other MTG 8-family genes, MTG8 and MTG16. MTG16 was recently cloned from the translocation breakpoint of myeloid malignancies with t(16;21) chromosomal translocation.

Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems.

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

Dual transforming activities of the FUS (TLS)-ERG leukemia fusion protein conferred by two N-terminal domains of FUS (TLS).

The FUS (TLS)-ERG chimeric protein associated with t(16;21)(p11;q22) acute myeloid leukemia is structurally similar to the Ewing's sarcoma chimeric transcription factor EWS-ERG. We found that both FUS-ERG and EWS-ERG could induce anchorage-independent proliferation of the mouse fibroblast cell line NIH 3T3. However, only FUS-ERG was able to inhibit the differentiation into neutrophils of a mouse myeloid precursor cell line L-G and induce its granulocyte colony-stimulating factor-dependent growth. We constructed several deletion mutants of FUS-ERG lacking a part of the N-terminal FUS region. A deletion mutant lacking the region between amino acids 1 and 173 (exons 1 to 5) lost the NIH 3T3-transforming activity but retained the L-G-transforming activity. On the other hand, a mutant lacking the region between amino acids 174 and 265 (exons 6 and 7) lost the L-G-transforming activity but retained the NIH 3T3-transforming activity. These results indicate that the N-terminal region of FUS contains two independent functional domains required for the NIH 3T3 and L-G transformation, which we named TR1 and TR2, respectively. Although EWS intrinsically possessed the TR2 domain, the EWS-ERG construct employed lacked the EWS sequence containing this domain. Since the TR2 domain is always found in chimeric proteins identified from t(16;21) leukemia patients but not in chimeric proteins from Ewing's sarcoma patients, it seems that the TR2 function is required only for the leukemogenic potential. In addition, we identified three cellular genes whose expression was altered by ectopic expression of FUS-ERG and found that these are regulated in either a TR1-dependent or a TR2-dependent manner. These results suggest that FUS-ERG may activate two independent oncogenic pathways during the leukemogenic process by modulating the expression of two different groups of genes simultaneously.

Leukemia inhibitory factor as an intrapituitary regulator of pro-opiomelanocortin gene expression and corticotrope ontogeny.

Along with the well characterized control of pituitary function by hypothalamic hormones, several cytokines mediate signaling between the neuroendocrine and immune systems. Interleukin-1 (IL-1), IL-6, tumor necrosis factor-a and, recently, leukemia inhibitory factor (LIF) and IL-11 (1, 2) are the most thoroughly characterized. LIF belongs to the IL-6 cytokine family, together with IL-6, IL-11, oncostatin M, cilary neurotropic factor and cardiotropin-1. LIF was originally characterized and so named for its ability to promote the differentiation of a mouse myeloid leukemic cell line (3). LIF is a pleiotropic cytokine and has diverse biological activities, including regulation of hematopoietic cell proliferation and differentiation, stimulation of neuronal growth and development, bone formation, and preparation of the uterus for embryo implantation. Melmed and his colleagues have shown that LIF participates in regulating gene expression of pro-opiomelanocortin (POMC), in addition to corticotropin-releasing hormone (CRH) and glucocorticoids (4‐6).

A novel growth-factor–dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential

Constitutive expression of human colony-stimulating factor-1 receptor (CSF-1R) confers long-lasting CSF-1–dependent proliferation to mouse myeloid cell lines. We developed mice transgenic for human CSF-1R because mouse CSF-1 cannot activate human CSF-1R. Then bone marrow cells from transgenic mice were plated onto MS-5 stromal cells expressing the membrane form of human CSF-1 (2M-1 cells) in order to combine the hematopoietic supporting properties of stromal cells and the proliferative effects of CSF-1. Thus, we were able to derive a hematopoietic cell line, called 47.10, that grew indefinitely under these conditions, whereas no cell line could be developed from nontransgenic mice. Proliferation of 47.10 cells is severely affected by neutralizing anti–CSF-1R monoclonal antibodies. Morphologic and cytofluorometry analysis established that most 47.10 cells are immature myelomonocytic cells. Consistent with this phenotype, the myeloid transcription factor PU.1, but not the erythroid transcription factor GATA-1, is expressed in 47.10 cells. A few 47.10 cells (3–5%) do not express lineage specific markers; they differentiate spontaneously to lineage-positive cells after replating on 2M-1 cells. In agar cultures, 47.10 cells form 7- and 14-day colonies in response to a cocktail of granulocyte/macrophage colony-stimulating factor (2.5 ng/mL), interleukin-3 (1 ng/mL), and mouse CSF-1 (10 ng/mL). Under these conditions, about 0.5% of 47.10 cells formed large 14-day colonies (>1 mm) composed of mature monocytes and granulocytes, reflecting the presence of progenitors endowed with high proliferative potential (HPP-47.10 cells). In conclusion, we have characterized a novel continuous myeloid cell line presenting a hierarchical structure similar to that of the bone marrow progenitor cell compartment.