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D Askew

Publications and source records attributed to D Askew.

29 records · Page 2Linked to original sources
PubMed1991

Functional dissection of the paired segmentation gene in Drosophila embryos.

An ectopic expression assay in Drosophila embryos was used to investigate the roles of pair-rule segmentation genes in the spatial regulation of the segment-polarity gene, engrailed (en). It is hypothesized that the regions of overlap in expression of two genes, paired (prd) and even-skipped (eve), define the odd-numbered en expression stripes. Consistent with this combinatorial model, ectopic expression of prd caused these en stripes to be expanded posteriorly. Surprisingly, however, ectopic expression of a prd gene with a deletion of the conserved paired box resulted in loss of these odd-numbered en stripes. This dominant negative effect is a phenocopy of en expression in prd embryos and suggests that the paired box is necessary for normal prd- function. A similar deletion of odd-numbered en stripes was also observed after ectopic expression of a chimeric fushi tarazu (ftz) gene containing a substituted prd gene homeo box; in addition, in these embryos, the even-numbered en stripes were expanded anteriorly, as observed when the unaltered ftz gene is ectopically expressed. These effects suggest that the chimeric protein may have DNA or protein targets of both the normal Ftz and Prd proteins.

Animals↗
PubMed1990

Phenotypes and mechanisms in the transformation of hematopoietic cells.

Interleukin 3 (IL-3) is a growth factor that supports the proliferation of early hematopoietic stem cells, as well as cells that are committed to a variety of the myeloid lineages. The mechanisms by which IL-3 functions have been studied through the use of a series of IL-3-dependent cell lines isolated from myeloid leukemias or long-term bone marrow cultures. A variety of studies have implicated tyrosine phosphorylation in IL-3 signal transduction. One of the substrates of phosphorylation is a 140 kDa, IL-3-binding protein that is speculated to be the biologically relevant IL-3 receptor. IL-3, through tyrosine phosphorylation, supports viability and growth through the regulation of transcription of a series of genes including c-myc and c-pim-1. The c-myc gene contributes to viability, in part, by regulating the transcription of the ornithine decarboxylase gene. The role of growth factors in differentiation is less clear. By studying IL-3-dependent myeloid leukemia cell lines, two genes have been identified whose altered expression is associated with blocking the ability of the cells to differentiate. The c-myb gene is a nuclear DNA binding protein that has been implicated in myeloid transformation in a number of systems. The Evi-1 gene is a novel gene of the zinc finger family of transcriptional activators. Possible mechanisms by which these genes interfere with normal differentiation are discussed.

Animals↗
PubMed1990

Modulation of alloreactivity by Mac-1+, -2+, and -3+ macrophages from normal and tumor-bearing hosts: flow cytofluorometrically separated macrophages.

Macrophages (M phi) are multifunctional cells that regulate humoral and cellular immune responses. Our studies of tumor-induced M phi-mediated dysfunction used M phi subsets which were defined by their Mac-1, Mac-2, and Mac-3 surface markers. To measure the accessory activity of M phi for T cell alloreactivity, thioglycollate-elicited peritoneal M phi from normal and tumor-bearing hosts (TBH) were labeled with anti-Mac-1, -2, or -3 antibodies and separated by flow cytofluorometry. The separated Mac-1+, -2+, and -3+ M phi were called sorted M phi, while unseparated M phi were designated unsorted M phi. Both M phi types were added to mixed lymphocyte reaction (MLR) cultures at concentrations ranging from a low of 2% M phi to a high of 20% M phi. The low concentration of unsorted normal host M phi caused a 31% suppression of alloreactivity. Suppression reached 68% when high concentrations of unsorted normal host M phi were added to the MLR cultures. Unsorted TBH M phi reduced alloreactivity by 64% and 86% at low and high concentrations, respectively. When separated into subpopulations, normal host Mac-1+ M phi reduced alloreactivity by 48% and 81% when added at low and high concentrations, respectively. TBH Mac-1+ reduced alloreactivity by 31% and 59% at low and high concentrations, respectively. There were no differences in the suppression caused by normal or TBH Mac-2+ M phi, and by normal or TBH Mac-3+ M phi. Indomethacin treatment did not effect the suppression caused by Mac-1+ M phi, suggesting that proataglandin E2 was not involved. Indomethacin treatment did reduce suppression mediated by Mac-2+, -3+, and unsorted M phi. Mac-2+ M phi dramatically enhanced alloreactivity at low concentrations with normal host Mac-2+ M phi providing greater enhancement of alloreactivity than TBH Mac-2+ M phi. The division of M phi into subpopulations on the basis of Mac antigens suggested that Mac-1+ and -3+ M phi played a major role in immunosuppression in the normal host, while Mac-3+ M phi were more active in TBH immunosuppression. Because no one population of sorted TBH M phi was more suppressive than sorted normal host M phi, we suggest that tumor-induced immunosuppression may involve a network of suppressor M phi.

Animals↗
PubMed1990

Normal and tumor-bearing host splenic macrophage responses to lipopolysaccharide.

Lipopolysaccharide (LPS) was used to assess the responses of normal and tumor-bearing host (TBH) macrophages (M phi) to activation signals. M phi were incubated with LPS for either 3 hr or 24 hr and then assayed for phenotypic, functional, or cell-cycle changes. A 3-hr LPS treatment had no significant effect on M phi phenotype. In contrast, the 24-hr LPS treatment caused a significant decrease in the percentage of normal host Mac+ and Ia+ M phi. In the TBH, a 24-hr LPS treatment caused an increase in the percentage of Mac-1+ and -2+ M phi and a decrease in the percentage of Mac-3+ and Ia+ M phi. When normal host M phi were plated for 24 hr (control), there was an increase in Mac-1+ and -2+ M phi and a decrease in Ia+ M phi. The 24-hr TBH control showed a decrease in Mac-1+ and Ia+ M phi. To assess functional changes, LPS-treated normal and TBH M phi were added to the allogeneic mixed lymphocyte reaction (MLR) or the autologous MLR (AMLR). There were no significant differences in M phi accessory activity after a 3-hr LPS treatment. A 24-hr LPS treatment of normal host M phi also had no effect. A 24-hr LPS treatment of TBH M phi led to a significant decrease in allogeneic T-cell reactivity, and even the 24-hr TBH control showed significant suppression of T-cell responsiveness. In the AMLR, a measure of autoreactive T-cell responsiveness, a 3-hr LPS treatment had no affect on normal host M phi but led to increased accessory ability in TBH M phi. TBH M phi, however, were still less than 50% as responsive as normal host M phi even after LPS treatment. The 24-hr LPS treatment caused a significant decrease in normal or TBH M phi accessory activity. A 24-hr plating of normal host M phi decreased their accessory ability. In addition to the phenotypic and functional changes after LPS treatment, M phi cell-cycle kinetics were also investigated. The percentages of normal host M phi in G0/G1 were not changed significantly after a 3-hr LPS treatment. In contrast, M phi plated for 24 hr with or without LPS had a decreased percentage of cells in G0/G1. Normal host M phi showed little change in total RNA levels after a 3-hr treatment but had increased RNA levels after a 24-hr treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Origins and properties of hematopoietic growth factor-dependent cell lines.

Studies of the growth regulation, differentiation and transformation of myeloid cells have been greatly facilitated by the availability of a variety of hematopoietic growth factor-dependent cell lines. These cell lines have been isolated from long-term bone marrow cultures and myeloid tumors using interleukin 3 (IL-3) as a growth factor. Using growth factor-dependent cells, it has been shown that growth regulation by IL-3 involves binding to a high-affinity receptor of 140 Kd and activation of tyrosine phosphorylation. IL-3 binding is associated with a number of cellular responses which are required for maintenance of viability, including induction of transcription of the c-myc and ornithine decarboxylase (ODC) genes. In addition, IL-3 regulates the expression of transcription of the gamma T cell receptor locus. The properties of the IL-3-dependent lines are consistent with the hypothesis that they are transformed in their ability to terminally differentiate. In some of the cell lines, this transformation may terminally differentiate. In other of the cell lines, this transformation may be due to the altered expression of the c-myb gene. In other cell lines, transformation is associated with the activation of the expression of a novel gene, termed Evi-1, of the zinc finger family of transcriptional factors. Comparable transformation of erythroid lineage cells is speculated to be due to the activation of the expression of another novel gene termed spi-1. These studies have emphasized the value of well-characterized hematopoietic growth factor-dependent cell lines in advancing our understanding in the basic biology of myeloid cells.

Animals↗
PubMed1989

Tumor-induced variations in a high molecular weight inhibitory monokine.

A soluble inhibitor of T cell proliferation was demonstrated in splenic and peritoneal macrophage (M phi) culture supernatants and was variably glycosylated during tumor development. This factor(s) inhibited mixed lymphocyte reaction- (MLR) and concanavalin A-induced T cell proliferation and was not prostaglandin E2 (PGE2), as demonstrated by size (nondialyzable, found in a 30 kilodalton [kd] ultrafiltration retentate and in fractions corresponding to greater than 67 kd on S-200 Sephacryl chromatography) and time course of activity in the MLR. Concentrated supernatants were PGE2-free and yet inhibited proliferation in the MLR. Isoelectric focusing (IEF) revealed normal and tumor-bearing host (TBH) concentrated M phi supernatants' major peaks of inhibitory activity differed in charge, with a pI of 6.5-7.6 for normal hosts and 4.0-6.0 for TBH. Activity in TBH M phi supernatants was found primarily in fractions eluting from hydroxylapatite at 0.3 M sodium phosphate buffer, pH 7.3, was resistant to proteolytic enzymes, but was sensitive to neuraminidase. In contrast, inhibitory activity in normal host M phi supernatants eluted from hydroxylapatite at 0.6 M sodium phosphate buffer and was not sensitive to neuraminidase. Thus, variable glycosylation (presence and absence of sialic residues) could account for the charge difference seen in IEF and may have a bearing on tumor-induced hyporesponsiveness. Kinetic addition of supernatants to the MLR revealed PGE2 may be required for inhibitory activity to be manifested early (0 and 24 h) but not if the high molecular weight (mw) inhibitor was added late (48 and 72 h post initiation). Both normal and TBH M phi supernatants suppressed the generation of interleukin 2 (IL 2) with a dose- and time-dependent difference. Cell-cycle analysis of mitogen-stimulated cells treated with normal and TBH M phi supernatants revealed that TBH M phi supernatants enhanced cell-cycle progression when measured early, but that both normal and TBH M phi supernatants suppressed the number of cells in S phase when measured late in the assay. The production of a high mw inhibitor by both normal and TBH M phi could suggest a homeostatic mechanism, which was upset by increased PGE2 production and production of an inhibitor containing sialic acid, tipping the balance in favor of immunosuppression.

Animals↗
PubMed1989

Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.

Alleles↗
PubMed1989

Retroviral insertions in the CB-1/Fim-3 common site of integration activate expression of the Evi-1 gene.

A common retroviral integration site (CB-1) was identified in two IL-3-dependent myeloid leukemia cell lines (DA-3, DA-34). The CB-1 locus was mapped to murine chromosome 3 and was shown to be closely linked to another myeloid common site of viral integrations in myeloid leukemias, Evi-1. A comparison of the CB-1 restriction map with published restriction maps for other common integration sites demonstrated that it was nearly identical to the restriction map of a common site of Friend MuLV integration in myeloid tumors termed Fim-3 (Bordereaux et al. 1987). Genomic clones representing approximately 110 kb of the CB-1 locus and 80 kb of the 5' region of the Evi-1 locus demonstrate no physical overlap of these viral integration sites. Previous studies (Morishita et al. 1988) have shown that retroviral insertions in the Evi-1 locus activate the expression of a gene potentially encoding a 120 kd zinc finger protein. Evi-1 expression is also activated in cell lines with viral integrations in the CB-1 locus. These results demonstrate that the CB-1/Fim-3 and Evi-1 loci constitute a large genomic region in which viral integrations activate the transcription of a new potential myeloid transforming gene.

Animals↗
PubMed1987

The v-fms oncogene induces factor-independent growth and transformation of the interleukin-3-dependent myeloid cell line FDC-P1.

The normal cellular counterpart of the v-fms oncogene product is a receptor for the mononuclear phagocyte colony-stimulating factor, CSF-1. An interleukin-3 (IL-3)-dependent mouse myeloid cell line, FDC-P1, was infected with a murine retrovirus vector containing v-fms linked to a gene encoding resistance to neomycin (neo). Infected cells selected for resistance to the aminoglycoside G418 contained few proviral DNA copies per haploid genome, expressed low levels of the v-fms-coded glycoprotein, remained IL-3 dependent for growth, and were nontumorigenic in nude mice. In contrast, infected cells selected for their ability to grow in the absence of IL-3 contained an increased number of proviral insertions, expressed high levels of the v-fms-coded glycoprotein, and were tumorigenic in nude mice. The IL-3-independent cells expressed IL-3 receptors of comparable number and affinity to those detected in uninfected FDC-P1 cells and did not produce a growth factor able to support replication of the parental cells. Thus, the synthesis of high levels of the v-fms gene product in FDC-P1 cells abrogated their requirement for IL-3 and rendered the cells tumorigenic by a nonautocrine mechanism. The data suggest that v-fms encodes a promiscuous tyrosine kinase able to transform cells of the myeloid lineage that do not normally express CSF-1 receptors.

Animals↗
PubMedDate not supplied

Normal and tumor-bearing host macrophage responses: variability in accessory function, surface markers, and cell-cycle kinetics.

Normal and tumor-bearing host (TBH) peritoneal macrophage (M phi) responses to in vitro lipopolysaccharide (LPS) treatment were measured by assessing functional and phenotypic changes. Both normal and TBH untreated M phi suppressed mixed lymphocyte reaction (MLR) reactivity at all concentrations. Normal host M phi treated with LPS for 3 h were suppressive at all concentrations. TBH M phi treated with LPS for 3 h were not suppressive in the MLR until more than 5% were added. Surprisingly, 24 h treatment of normal and TBH M phi with LPS induced cells that significantly enhanced MLR reactivity when added at 2% or 5%. These cells were not suppressive until a 20% M phi concentration was reached. LPS treatment of normal and TBH M phi changed the percentage of cells expressing the surface markers Mac-1, -2, -3, and Ia as determined by flow cytometry. Normal host peritoneal M phi treated with LPS for 3 h had decreased Mac-1 and -3 expression, but there was no change in Mac-2 or Ia. Plating for 24 h did not change the percentage of M phi expressing Mac-1, -3, or Ia but did cause an increase in Mac-2+ M phi. Treatment of normal host M phi with LPS for 24 h led to a decrease in Mac-1+ and Ia+ M phi, no change in Mac-3+ M phi, but an increase in Mac-2+ M phi. LPS treatment of TBH M phi for 3 h decreased the number of Mac-1+ M phi, but Mac-2+, -3+, or Ia+ M phi numbers did not change. Plating TBH M phi for 24 h caused a decrease in the number of Mac-1+ M phi, no change in Mac-3+ or Ia+ M phi, but an increase in Mac-2+ M phi. Treatment with LPS for 24 h led to no change in the number of Mac-1+, -3+, or Ia+ TBH M phi, but Mac-2+ M phi increased. The phenotypic and functional changes after LPS treatment led us to ask if these changes were detectable at the level of DNA and RNA. Flow cytometric analysis of acridine orange-stained M phi was used to measure DNA and RNA levels. This analysis determines M phi cell-cycle kinetics and estimates their RNA synthesis. In normal host M phi, a 3-h LPS treatment caused a decrease of cells in G0/G1 but an insignificant change in RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗