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N M Wilkie

Publications and source records attributed to N M Wilkie.

At least 19 recordsLinked to original sources
PubMed1999

Identification of amino acids involved in the binding of hMIP-1 alpha to CC-CKR1, a MIP-1 alpha receptor found on neutrophils.

Human macrophage inflammatory protein-1alpha (hMIP-1alpha) and human macrophage inflammatory protein-1beta (hMIP-1beta) are chemokines involved in a diverse range of immunological effects. Both hMIP-1alpha and hMIP-1beta are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1alpha can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1alpha and hMIP-1beta chimeric proteins and mutants show that two amino acids (37K and 43L) are important in the binding and signaling of hMIP-1alpha through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1alpha and hMIP-1beta (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.

Amino Acid Sequence↗
PubMed1996

Identification of a new member of the MNP transcription factor family in differentiated HL60 cells.

MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells, MIP-1 alpha is expressed in transformed B cells and can also be induced during cellular activation of CD4+ T-cells and monocytes. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. In this communication we show evidence identifying a new member of the MNP transcription family, MNP-3, in PMA differentiated HL60 cells.

Base Sequence↗
PubMed1996

HIV-1 tat induces the expression of a new hematopoietic cell-specific transcription factor and downregulates MIP-1 alpha gene expression in activated T-cells.

MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells. The expression of MIP-1 alpha is induced during cellular activation of CD4+ T-cells and monocytes. It is also expressed in transformed B-cells. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation and in transformed B cells. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. Recently, we reported that HIV-1 tat downregulates MIP-1 alpha expression in Jurkat T-cells. In this report we show induction of MNP-1 in Jurkat T-cells expressing HIV-1 tat. Expression of neither HTLV-1 tax in Jurkat T-cells nor EBV in B-cells had any effect on MNP-1 or MNP-2 expression, showing that the effect is specific for HIV-1 tat. We propose that HIV-1 tat may inhibit MIP-1 alpha expression by inducing MNP-1 expression in T-cells, probably by either competing with MNP-2 for binding to the MIP-1 alpha promoter or by sequestering it into inactive forms.

B-Lymphocytes↗
PubMed1995

Regulation of FGF-4 enhancer activity by transcription factor NF-Y.

Regulation of FGF-4 gene expression is controlled both by elements in the promoter and by an enhancer domain located in the untranslated region of the third exon. We have determined that transcription factor NF-Y binds to the FGF-4 promoter. We further show by mutational analysis that binding of NF-Y is essential for FGF-4 enhancer activity but has minimal effect on activity of the FGF-4 promoter alone.

Animals↗
PubMed1995

MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene.

Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.

Animals↗
PubMed1994

The active monomeric form of macrophage inflammatory protein-1 alpha interacts with high- and low-affinity classes of receptors on human hematopoietic cells.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and its human homologue GOS19.1/LD78 are members of the C-C chemokine/intercrine family of secreted proteins. They have proinflammatory properties and also inhibit cell cycle progression of hematopoietic stem cells. Characterization of MIP-1 alpha receptor(s) has been confused because of its reported aggregation to inactive forms. Using a defined monomeric form of MIP-1 alpha that is biologically active for stem cell inhibition and induction of oxidative metabolism in polymorphonuclear cells, we report the detection of high- and low-affinity receptor classes on human leukemic CD34+ blast cells, promyelocytic cells, monocytes, peripheral blood neutrophils, and T cells. Both high- and low-affinity classes are expressed simultaneously in promyelocytes and neutrophils. The calculated kd for high-affinity receptors correlates with the concentrations of MIP-1 alpha required to induce a biologic effect on stem cells and neutrophils. Cross-linking studies show that MIP-1 alpha associates with two cell surface proteins with apparent molecular masses of 92 kD and 52 kD. Direct competition binding studies combined with studies on the inhibition of stem cells show that human and murine MIP-1 alpha have different receptor-binding and biologic properties.

Antigens, CD↗
PubMed1994

The FGF-4 promoter is required for transformation and is active in both embryonal and somatic cells.

We report the independent isolation of a rearranged FGF-4 gene from a patient with chronic myeloid leukaemia. We show that the FGF-4 gene has been truncated 30 nucleotides 3' to the coding sequence and has been fused to the RNA processing signals from a putative unknown gene on chromosome 15. We demonstrate that the promoter region of the FGF-4 gene is active in NIH3T3 cells and is indeed necessary for transformation. Using the luciferase reporter assay we have shown that the FGF-4 5' flanking sequences possess easily detectable promoter activity in both F9 and HeLa cell lines. 5' deletion analysis of the FGF-4 promoter has delineated regions containing cis-acting elements of functional importance. These regulatory regions are common to both embryonal and somatic cell lines. Electrophoretic mobility shift assay, using nuclear extracts from F9 and HeLa cells, has allowed detection of DNA-protein interactions occurring in the functionally significant regions. Subsequent comparison of the human and murine FGF-4 promoters show that the regions of functional significance are highly conserved. We suggest that the FGF-4 gene may be suppressed through a distal suppressor locus and becomes active when separated from this suppressor.

3T3 Cells↗
PubMed1993

Changes in in vivo protein-DNA interactions occur at the c-myc P2 promoter during differentiation.

Down regulation of the c-myc gene is a prerequisite for the differentiation of a number of cell types. Studies have shown that two mechanisms of inactivation are involved in c-myc repression: a block of the elongation of RNA polymerase followed by transcriptional inactivation mediated through promoter sequences. In this study DMS in vivo footprinting was performed on the P2 promoter region of c-myc in differentiated and undifferentiated HL60 cells. A differentiation-specific footprint was observed at G residues immediately upstream of the TATA box. This observation occurred only in cells differentiated for 48 hours or more and hence is likely to be involved in the repression of c-myc by promoter inactivation.

Base Sequence↗
PubMed1993

c-MYC repression of promoter activity through core promoter elements.

c-MYC was found to repress expression of the c-myc promoter in a transient assay system. Deletion constructs showed the cis-acting element(s) were common to both the P1 and P2 promoters. Further analysis involving replacement of the P2 TATA box and initiator elements with the SV40 late promoter initiator localized the cis-acting elements to the core promoter.

Dose-Response Relationship, Drug↗
PubMed1991

Regulation of AP-1/DNA complex formation in vitro.

The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.

Animals↗
PubMed1991

Regulatory domains within the P0 promoter of human c-myc.

Expression of P0 RNA in some Burkitt lymphoma cell lines varies independently of levels of RNA derived from P1 and P2. These data suggest the possibility that expression of P0 RNA may be capable of independent regulation. In order to investigate this possibility we have isolated putative regulatory domains flanking P0 RNA starts within the human c-myc gene and analysed both their ability to direct expression of control reporter genes and their ability to interact with specific transcription factors. Regulatory regions necessary for expression of P0 RNA have been located within 131 bp 5' of the first major P0 RNA start. DNAase 1 footprint analysis and gel retardation assays demonstrate binding of transcription factors Sp1, NF1 and CBP to this region. NF1 binds specifically to two consensus sequences. The more distal site overlaps with the binding site for CBP, and it is likely that concomitant binding of NF1 and CBP within the distal region of the P0 promoter is not possible. Previous work from our laboratory has described a negative regulatory domain within the 5' flanking region of c-myc. The P0 promoter resides within this domain and therefore may contain a negative regulator of c-myc gene expression.

Animals↗
PubMed1990

Identification and characterization of an inhibitor of haemopoietic stem cell proliferation.

The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.

Amino Acid Sequence↗
PubMed1988

Phorbol ester-responsive H-ras1 gene promoter contains multiple TPA-inducible/AP-1-binding consensus sequence elements.

We have constructed recombinant DNA plasmids which carry both the aminoglycoside phosphotransferase (aph) gene and the chloramphenicol acetyl-transferase (CAT) gene linked to the human normal or mutant T24 H-ras1 promoter. We have transfected these plasmids into rat 208F fibroblasts using the calcium phosphate technique and selected for stable transformants by geneticin resistance. These transformants expressed CAT activity at low levels. However, when treated with the phorbol ester TPA, CAT levels increased substantially. Cells transfected with recombinant plasmids carrying a promoterless CAT gene did not respond to TPA. We have noted four motifs in the H-ras1 promoter region which resemble TPA-inducible and AP-1-binding consensus sequences. We suggest that AP-1-like proteins may play a role in control of H-ras1 transcription.

Animals↗
PubMed1988

Transcriptional regulation of the human c-myc gene.

The involvement of c-myc in the genesis of animal neoplasia is now well documented for several systems. In order to define the precise role played by the myc gene in tumorigenesis, a better understanding of the normal regulation of myc expression is necessary. We have begun a study of the cis-acting regulatory sequences within the 5' flanking domain of the human c-myc gene. Regions important for myc promoter function have been identified by linkage to the coding sequences of the bacterial chloramphenicol acetyltransferase (cat) gene. Promoter deletion studies and in vivo competition assays for c-myc/cat recombinant plasmids have allowed the identification of a proximal 'core' promoter region capable of directing high levels of CAT activity. Further upstream a negative regulatory element (NRE2) has been identified which is capable of repressing cat gene expression and which functions by interaction with a transacting factor(s). Preliminary data suggests detection of NRE2 is dependent on both the type and amount of carrier DNA used in transient CAT assays. Initial experiments further indicate the involvement of at least two other distal regulatory domains, a negative regulatory domain (NRE1) and a putative enhancer-type region (E). In vitro footprint analysis has allowed the identification of DNA binding proteins which interact with NRE2 and the 'core' promoter. NRE2 contains binding sites for transcription factors Sp1 and CTF. The 'core' promoter domain appears to be highly complex and possesses several Sp1 binding sites.

Animals↗
PubMed1988

The normal human H-ras1 gene can act as an onco-suppressor.

The altered morphology and tumorigenic phenotypes of rat 208F fibroblasts transformed with the human T24 H-ras1 oncogene is suppressed by transfection with the human normal H-ras1 gene. In the suppressed cells, both the normal and mutant T24 ras gene products are expressed although the normal p21 is expressed at a higher level. Rare transformants or tumours derived from suppressed cells possess reduced expression of normal ras p21. Our findings suggest that transforming ras alleles do not behave in a dominant manner and that elevated expression of the normal allele could cause suppression of the morphologically transformed and tumorigenic phenotypes.

Alleles↗
PubMed1985

Consequences of altered oncogene expression in rodent cells.

By using linkage to transcriptional enhancers to increase gene expression, it was shown that the introduction of the mutant Ha-ras gene from bladder carcinoma cells into primary cultures of Chinese and Syrian hamster cells, and Wistar rat cells, was sufficient to trigger malignant conversion. The normal Ha-ras gene when linked to enhancers did not trigger malignant conversion but gave rise to cell clones with extended growth capacity. We measured the levels of transforming growth factors (TGFs) released from cells containing the ras genes, and the availability of the receptor for epidermal growth factor (EGF) to which alpha-TGF also binds. The level of ras gene expression seemed to correlate with the level of TGF secreted, a reduction in available EGF receptors and the morphology of the transformed cells. However, when an immortalized cell line (rat 208F cells) was used as a recipient for the oncogene vectors a different result was obtained. TGF release was triggered to varying degrees by almost all of the ras and myc oncogene constructions tested, regardless of the level of gene expression. Enhanced expression of the two oncogenes led to increased anchorage-independence. Significantly, both normal ras and normal myc, when linked to transcriptional enhancers, caused the recipient cells to become tumorigeneic without inducing any marked morphological alterations in culture. Enhanced and constitutive expression of the mutant Ha-ras gene resulted in the recipient cells becoming tumorigeneic and the cells displayed a transformed phenotype in culture. The transfected fibroblasts with a markedly transformed phenotype displayed a relative paucity of EGF receptors which correlated with release of alpha-TGF, suggesting a down-regulation of EGF receptors on transformation. While enhanced expression of the normal Ha-ras gene did result in a decrease in EGF receptors on these transfectants, in contrast, the transfectants with enhanced expression of the c-myc and v-myc oncogenes did not display any significant changes in available EGF receptors although the cells were tumorigenic when tested in the nude mouse. These results are discussed in relation to a hypothesis implicating a role for oncogenes at various stages in the multi-stage process of carcinogenesis.

Animals↗
PubMed1984

Transcriptional regulation of a herpes simplex virus immediate early gene is mediated through an enhancer-type sequence.

An enhancer-type sequence has been identified in the promoter region for the herpes simplex virus type 1 (HSV-1) immediate early (IE) mRNA 3. The enhancer-type activity is host cell dependent, being greater in human cells and Syrian hamster cells (the usual host cell for in vitro propagation of HSV) than in Chinese hamster or mouse cells. Enhancer activity is stimulated up to 10-fold after superinfection by tsK, a temperature-sensitive mutant of HSV-1. The induction of enhancer activity is independent of de novo protein synthesis, showing that trans-activation is effected by a component of the virion. We propose that trans-regulation of IE mRNA 3 is mediated through an enhancer-type sequence, and that this provides one explanation for previously described regulation of HSV IE mRNAs by virion components.

Animals↗