• Expression of CD11b/CD18 on neutrophils after consolidation chemotherapy for acute myeloid leukemia and after high dose chemotherapy with autologous haematopoietic stem cell transplantation.

      Wang, X; Clowes, C; Duarte, R; Pu, Qingqiu; School of Biological Science, The University of Manchester, UK. (2000-09)
      Quantitative expression of neutrophil CD11b/CD18 following chemotherapy (either conventional dose consolidation chemotherapy or high dose chemotherapy with autologous stem cell transplantation) was investigated during the early recovery phase (neutrophil count 0. 5-1.0x109/l) and at full recovery (neutrophil count 1.0-2.5x109/l) following treatment. CD11b/18 expression was normal in stem cell transplantation supported patients during both early and full neutrophil recovery. By contrast CD11b/CD18 expression was markedly decreased in patients who received chemotherapy without stem cell support. These results suggest that recovery of neutrophil count may not always coincide with recovery of neutrophil function and that G-CSF stimulated peripheral stem cell transplantation enhances neutrophil function post chemotherapy.
    • Expression of cytokeratins in epithelial cells isolated from the crypt and villus of the small intestine.

      Flint, Neil; Evans, Gareth S; Cancer Research Department of Epithelial Biology, Paterson Institute, Christie Hospital, Withington, Manchester. (1992-05)
    • Expression of Epstein-Barr virus latent membrane protein influences self-renewal and differentiation in a multipotential murine haemopoietic 'stem cell' line.

      Fairbairn, Leslie J; Stewart, J Philip; Hampson, Ian N; Arrand, John R; Dexter, T Michael; Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K. (1993-02)
      The product encoded by the latent membrane protein (LMP) gene of Epstein-Barr virus (EBV) has been implicated as a transforming protein by a number of studies. We have examined the effects of LMP expression in FDCP-mix cells, a growth factor-dependent multipotential murine 'stem cell' line. Our studies show that LMP reduces the generation of clonogenic cells and leads to the production of cells expressing a marker (lysozyme M) characteristic of mature monocytes and macrophages. Furthermore, cells expressing LMP are compromised in their ability to produce mature neutrophils. These data suggest that expression of LMP in primitive cells can modulate their self-renewal and differentiation potential and provide evidence in support of the suggestion that EBV may be involved in some of the maturation defects of haemopoiesis.
    • Expression of Epstein-Barr virus membrane antigen gp340/220 in mouse fibroblasts using a bovine papillomavirus vector.

      Conway, Margaret J; Morgan, A; Mackett, Mike; Paterson Institute for Cancer Research, Withington, Manchester, U.K. (1989-03)
      Epstein-Barr virus (EBV) membrane antigen glycoproteins gp340 and gp220 are encoded by a single gene. We have inserted this gene into a bovine papillomavirus (BPV) vector and expressed gp340/220 in mammalian cells under the control of the mouse metallothionein promoter. The proteins produced were of similar Mr, showed similar antigenic specificity and were transported to the same subcellular location as the authentic gp340/220. The inclusion of heavy metal ions in the medium had no effect on the levels of gp340/220, which were approximately the same as those found in standard EBV-transformed lymphoblastoid cell lines, e.g. B95-8. Cells that expressed gp340/220 were selected by several rounds of fluorescence-activated cell sorting, but on passage they rapidly lost the ability to express this glycoprotein. In contrast to this we found that BPV-transformed cells expressing a truncated version of gp340/220 still produced it at significant levels after extended passage.
    • Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954.

      Djeha, A Hakim; Hulme, Alison N; Dexter, T Michael; Mountain, Andrew; Young, Lawrence S; Searle, Peter F; Kerr, David J; Wrighton, Christopher J; Cobra Therapeutics Ltd., The Science Park, University of Keele, United Kingdom. hakimd@cancer.bham.ac.uk (2000-05)
      Expression of the Escherichia coli enzyme nitroreductase (NTR) in mammalian cells enables them to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), leading to interstrand DNA cross-linking and apoptosis in both proliferating and quiescent cells. In the work reported here, we used human hepatocellular carcinoma and squamous carcinoma cell lines constitutively expressing NTR to demonstrate that the ntr/CB1954 system results in potent, long-lasting antitumoral effects in mice. We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ.
    • The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

      Bellantuono, Ilaria; Lashford, Linda S; Rafferty, Joseph A; Fairbairn, Leslie J; Department of Immunology, Imperial College School of Medicine, The Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom. i.bellantuono@ic.ac.uk (2000-05)
      BACKGROUND AND OBJECTIVE: As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. DESIGN AND METHODS: To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). RESULTS: An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. INTERPRETATION AND CONCLUSIONS: A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.
    • Expression of HLA-D sub-region products on human colorectal carcinoma.

      Ghosh, Anna K; Moore, Michael; Street, A J; Howat, J; Schofield, Philip F; Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Institute, Manchester M20 9BX, UK. (1986-10-15)
      The major histocompatibility complex (MHC) status of normal, inflamed, pre-malignant and malignant epithelia of the human gastrointestinal tract was investigated by immunocytochemical methods using monoclonal antibodies (MAbs) directed against heavy (alpha)- and light (beta 2m)-chain Class-I molecules and sub-locus products (DP, DQ, DR) of the HLA-D region. Class-I expression on epithelial cells appeared to vary little with pathological status except in the case of 4/32 (13%) colorectal carcinomas in which the antigens were undetectable or scanty. The pattern of Class-II expression was more complex. The antigens were readily detectable on normal stomach epithelium, in villous adenomas and in inflammatory bowel mucosa. In each of these situations DR was the predominant specificity, followed by DP and DQ. Expression on normal colonic epithelium was usually negative but, in the vicinity of a neoplasm or an area of marked leukocyte infiltration, Class-II molecules (DR greater than DP much greater than DQ) were detectable. A similar pattern of non-coordinate expression was found on 23/32 (72%) colorectal carcinomas, but on the remaining 28% no Class-II products were detectable, under conditions wherein stromal leukocytes were strongly stained. The data suggest that in a significant proportion (nearly 30%) of primary colorectal carcinomas, the capacity for Class-II induction, a constitutive or acquired feature of normal colorectal epithelium, is either diminished or lost. Also, tumor Class-II status is not correlated to Dukes' stage or differentiation.
    • Expression of human CD antigens, including CD1 and CD25, by human x mouse interlineage leukaemia hybrids.

      Taylor, G M; Morten, H; Carr, T; Harrison, C; Ridway, J; Morris Jones, P; Department of Medical Genetics, St Mary's Hospital, Manchester, U.K. (1987-12)
      The expression of human cell-membrane antigens by hybrid cell lines derived by fusing a human B-ALL and mouse BW 5147 T-lymphoma cells has been studied. Using monoclonal antibodies (mAb), the phenotypes of 19 of the 24 hybrids which grew 11-44 days post-fusion have been analysed by indirect membrane immunofluorescence (IF). These uncloned hybrid cells were assayed early after outgrowth, prior to extensive human chromosome and antigen loss. Nonetheless, cytogenetic analysis showed that all hybrids contained variable numbers of human chromosomes. Phenotypic analysis showed that the hybrids could be grouped as follows: a high frequency expressing CD25 (IL-2 receptor), human T200, HLA class I alpha and beta 2microglobulin, and reacting with the mAb H207 and 12E7; an intermediate frequency expressing CD1 and CD2; and a low frequency expressing CD3, CD4, CD5, CD7, CD8 and CD9. This pattern of antigen expression resembled the frequency of these cells in the human B-ALL parent line. Cell sorting was used to immunoselect hybrids expressing CD1 and CD2, but CD1 expression was unstable during subsequent culture.
    • Expression of human CD4 by two human-mouse interlineage hybrids.

      Taylor, G M; Morten, J E; Morten, H; Dodge, A B; Ridway, J C; Jones, P M; Harris, R; Department of Medical Genetics, St Mary's Hospital, Manchester, UK. (1988-08)
      Two hybrid cell lines expressing human CD4 were prepared by fusing human B-lymphoid cells with the mouse T-lymphoma BW5147. Hybrid TF42 was derived from a human B-lymphoblastoid line and TF53.1 from a human B-ALL. Variants of these hybrids expressing or lacking CD4 were isolated by sorting cells stained with the monoclonal antibody (mAb) OKT4 on a fluorescence-activated cell sorter (FACS). Cytogenetic, isoenzyme and DNA analysis confirmed the presence of human chromosome 12 in the CD4+ hybrids, and revealed that CD4 expression by TF42 was associated with multiple copies of this chromosome. Of seventy mAb recognizing human T-cell antigens screened on the CD4+ and CD4- variants of the two hybrids, only mAb recognizing CD4 and Leu 8 reacted with the CD4+ cells. These hybrids should be useful in the preparation, screening and analysis of anti-CD4 monoclonal antibodies, and in studies of CD4 epitopes recognized by HIV.
    • Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.

      Stacey, Simon N; Bartholomew, Jennifer S; Ghosh, Anna K; Stern, Peter L; Mackett, Mike; Arrand, John R; Cancer Research Campaign Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K. (1992-09)
      Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.
    • Expression of human papillomavirus type 16 E7 protein by recombinant baculovirus and use for the detection of E7 antibodies in sera from cervical carcinoma patients.

      Stacey, Simon N; Ghosh, Anna K; Bartholomew, Jennifer S; Tindle, R W; Stern, Peter L; Mackett, Mike; Arrand, John R; Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, England. (1993-05)
      Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins.
    • Expression of hypoxia-inducible factor 1 alpha in thyroid carcinomas.

      Burrows, N; Resch, J; Cowen, R L; Von Wasielewski, R; Hoang-Vu, C; West, Catharine M L; Williams, K J; Brabant, Georg E; School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2010-03)
      Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1 alpha and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1 alpha was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1 alpha staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1 alpha pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O(2), anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1 alpha in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1 alpha and HIF-1 alpha targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1 alpha with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.
    • Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index.

      Wilson, James W; Deed, Richard W; Inoue, Toshiaki; Balzi, Manuela; Becciolini, Aldo; Faraoni, Paola; Potten, Christopher S; Norton, John D; Cancer Research Campaign Epithelial Biology Group, Paterson Institute for Cancer Research, Christie Hospital, National Health Service Trust, Manchester M20 4BX, United Kingdom. (2001-12-15)
      Id helix-loop-helix (HLH) proteins function as regulators of cell growth and differentiation and when overexpressed can induce malignant transformation. In a series of 34 cases of primary human colorectal adenocarcinoma, immunoreactivity for Id1, Id2, and Id3 was found to be significantly elevated in tumor compared with normal mucosa (P = 0.001 for Id1 and Id2; P = 0.002 for Id3). No elevation of Id expression was observed in 17 cases of adenoma. Expression of Id1 and to a lesser extent of Id2 was correlated with mitotic index (P = 0.005 for Id1; P = 0.042 for Id2) in human adenocarcinomas, and expression of all three Id proteins was correlated with p53 immunoreactivity (a marker of mutational 'inactivation' of p53 function; P = 0.002 for Id1; P = 0.006 for Id2; P = 0.016 for Id3). In normal intestinal mucosa of p53-null mice and in spontaneous tumors arising in Min+/- mice, expression of all three Id proteins was also found to be up-regulated. Antisense oligonucleotide blockade of Id protein expression inhibited the proliferation of human adenocarcinoma cells. Enforced, ectopic expression of the E47 basic HLH (bHLH) protein in human adenocarcinoma cell lines efficiently sequestered endogenous Id proteins as Id-bHLH heterodimers, as shown by coimmunoprecipitation and subcellular colocalization studies. This led to growth arrest of the cells. Enforced overexpression of a mutant E47 protein, deficient in transactivation and DNA binding function, also partially inhibited cell growth. Taken together, these data imply that deregulated expression of Id proteins in colorectal adenocarcinoma arises at least in part as a consequence of loss of p53 function and contributes to the uncontrolled proliferation of tumor cells in colorectal cancer.
    • Expression of Id2 and Id3 mRNA in human lymphocytes.

      Ishiguro, A; Spirin, K; Shiohara, M; Tobler, A; Norton, John D; Rigolet, M; Shimbo, T; Koeffler, H P; Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine 90048, USA. (1995-12)
      Helix-loop-helix (HLH) transcription factors are involved in cellular growth and differentiation. The Id (inhibitor of DNA binding and differentiation) HLH proteins, in a dominantly negative fashion, regulate transcriptional activities of basic HLH proteins. We examined by northern hybridization the expression of Id2 and Id3 mRNA in human leukemia/lymphoma lines and patient samples, as well as resting and activated normal human lymphocytes from peripheral blood (PBL). The Id2 mRNA was abundantly expressed in 5/12 T-cell and 3/4 B-cell lines, and Id3 mRNA was detected in 4/12 T-cell and 3/4 B-cell lines. Interestingly, Id2, but not Id3, mRNA was strongly expressed in 4/5 T-cell lines infected with human T-cell leukemia virus type I (HTLV-I) (ATL-1k, MT-2, S-LB1) and type II (Mo). Another unexpected finding was that T-cell leukemias and T-cell lines often expressed either Id2 or Id3 mRNA. In addition, resting PBL constitutively expressed prominent levels of Id2 mRNA, but not Id3 mRNA. Upon PHA-stimulation, Id2 expression decreased and Id3 levels increased with biphasic kinetics. Taken together, our studies revealed three unexpected findings which require further analysis: (1) expression of Id2 mRNA is often associated with lymphocytic transformation by HTLV-I or -II; (2) T-cells usually express either Id2 or Id3 mRNA, but B-cells often express both simultaneously; (3) non-dividing, normal PBL express high levels of Id2 and no Id3 mRNA; and with the onset of cellular proliferation, levels of Id2 mRNA decrease while levels of Id3 mRNA increase, suggesting that regulation of expression of these closely related genes is disparate.
    • Expression of kinase-defective mutants of c-Src in human metastatic colon cancer cells decreases Bcl-xL and increases oxaliplatin- and Fas-induced apoptosis.

      Griffiths, Gareth J; Koh, Mei Yee; Brunton, Valerie G; Cawthorne, Christopher; Reeves, Natalie A; Greaves, Martin J; Tilby, Michael J; Pearson, D Graham; Ottley, Christopher J; Workman, Paul; et al. (2004-10-29)
      Tumor resistance to current drugs prevents curative treatment of human colon cancer. A pressing need for effective, tumor-specific chemotherapies exists. The non-receptor-tyrosine kinase c-Src is overexpressed in >70% of human colon cancers and represents a tractable drug target. KM12L4A human metastatic colon cancer cells were stably transfected with two distinct kinase-defective mutants of c-src. Their response to oxaliplatin, to SN38, the active metabolite of irinotecan (drugs active in colon cancer), and to activation of the death receptor Fas was compared with vector control cells in terms of cell cycle arrest and apoptosis. Both kinase-defective forms of c-Src co-sensitized cells to apoptosis induced by oxaliplatin and Fas activation but not by SN38. Cells harboring kinase-defective forms of c-Src carrying function blocking point mutations in SH3 or SH2 domains were similarly sensitive to oxaliplatin, suggesting that reduction in kinase activity and not a Src SH2-SH3 scaffold function was responsible for the observed altered sensitivity. Oxaliplatin-induced apoptosis, potentiated by kinase-defective c-Src mutants, was dependent on activation of caspase 8 and associated with Bid cleavage. Each of the stable cell lines in which kinase-defective mutants of c-Src were expressed had reduced levels of Bcl-x(L.) However, inhibition of c-Src kinase activity by PP2 in vector control cells did not alter the oxaliplatin response over 72 h nor did it reduce Bcl-x(L) levels. The data suggest that longer term suppression of Src kinase activity may be required to lower Bcl-x(L) levels and sensitize colon cancer cells to oxaliplatin-induced apoptosis.
    • Expression of Ku70 correlates with survival in carcinoma of the cervix.

      Wilson, C R; Davidson, Susan E; Margison, Geoffrey P; Jackson, S P; Hendry, Jolyon H; West, Catharine M L; CRC Experimental Radiation Oncology, Carcinogenesis Groups, Department of Clinical Oncology, Christie Hospital (NHS) Trust, Wilmslow Road, Manchester, M20 4BX. (2000-12)
      Cervical carcinoma affects around 3400 women in the UK each year and advanced disease is routinely treated with radiation. As part of a programme to establish rapid and convenient methods of predicting tumour and patient responses to radiotherapy, we have examined the relationship between the pre-treatment expression of the Ku components of the DNA damage recognition complex DNA-PK and patient survival in cervical carcinoma. Using immunohistochemistry of formalin-fixed sections of tumour biopsies, antibodies to Ku70 and Ku80 stained identical regions of tumour and there was a high degree of correlation between the mean number of cells stained positive for the two components in 77 tumours (r = 0.82, P<0.001). In 53 tumours there was a borderline significant correlation between measurements of tumour radiosensitivity (surviving fraction at 2 gray: SF2) and Ku70 expression (r = 0.26, P = 0.057) and no correlation for Ku80 (r = 0.18, P = 0.19). However, all tumours with a low number of Ku70 or Ku80 positive cells were radiosensitive. Furthermore, using log-rank analysis there was significantly higher survival in the patients whose tumours had a low Ku70 expression (P = 0.046). This difference was also reflected with Ku80, but did not reach statistical significance (P = 0.087). The study suggests that lack of Ku protein leads to radiosensitivity in some tumours and that other factors are responsible for radiosensitive tumours with high Ku expression. It is likely that the most accurate prediction of treatment outcome will lie in assessing the expression of several proteins involved in the recognition and repair of DNA damage, one of which will be Ku.
    • Expression of lineage restricted transcription factors precedes lineage specific differentiation in a multipotent haemopoietic progenitor cell line.

      Cross, Michael A; Heyworth, Clare M; Murrell, A M; Bockamp, E O; Dexter, T Michael; Green, Anthony R; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1994-10)
      Lineage commitment and differentiation are likely to be coordinated by the combined effects of multiple transcription factors acting on numerous different target genes. The mechanisms by which lineage-restricted patterns of transcription factor expression are established are therefore of particular relevance to our understanding of the role of transcription factors both in normal development and in oncogenesis. Here, we report that the genes for the lineage-restricted transcription factors SCL, GATA-1 and GATA-2 are expressed in all multipotent, IL-3-dependent, haemopoietic progenitor cell lines tested. Moreover, a liquid differentiation assay has been used to demonstrate down regulation of SCL, GATA-1, GATA-2 and PU-1 during differentiation into non-expressing lineages. These data support the concept that multiple lineage-restricted transcription factors are expressed prior to lineage commitment.
    • Expression of macrophage inflammatory protein-1 receptors in human CD34(+) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferon-gamma.

      Dürig, Jan; De Wynter, Erika A; Kasper, Christoph; Cross, Michael A; Chang, James; Testa, Nydia G; Heyworth, Clare M; CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1998-11-01)
      Macrophage inflammatory protein-1alpha (MIP-1alpha) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1alpha may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1alpha receptors on CD34(+) cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1alpha receptors on CD34(+) cells was analyzed by two-color flow cytometry using a biotinylated MIP-1alpha molecule. The mean percentage of LP CD34(+) cells expressing the MIP-1alpha receptors was 67.7 +/- 7.2% (mean +/- SEM; n = 22) as compared with 89.9 +/- 2.6% (n = 10) and 74.69 +/- 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1alpha receptor subtypes on LP CD34(+) cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34(+) cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34(+) cells for 24 to 36 hours in the presence of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) resulted in a significant increase in MIP-1alpha receptor expression. TNF-alpha induced MIP-1alpha receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1alpha receptor expression on hematopoietic cells.
    • The expression of matrix metalloproteinases and their inhibitors by pig synovial cells and their regulation by combinations of cytokines and growth factors.

      Legendre, P; Richards, C D; Rafferty, Joseph A; Dew, G W; Reynolds, J J; Cell and Molecular Biology Department, Strangeways Research Laboratory, Cambridge, U.K. (1993-11)
      1. Pig synovial fibroblasts in culture were studied to determine if they were an easily reproducible model system for studying the actions of cytokines and growth factors on human synovial cells. The biochemical analyses were conducted by activity assays, enzymography and Northern blot. 2. Human recombinant interleukin-1 alpha, basic fibroblast growth factor and transforming growth factor-beta 1 were studied in combinations because of their known involvement in controlling tissue remodelling. 3. The response of pig fibroblasts to these agents, in terms of the expression of matrix metalloproteinases (collagenase, gelatinase and stromelysin) and their inhibitors (TIMPs), show that they behave similarly enough to human cells for use when supplies of human primary cells are unavailable.
    • Expression of MHC class II products on human colorectal cancer. An immunohistological and flow cytometric study.

      Moore, Michael; Ghosh, Anna K; Johnston, D; Street, A L; Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK. (1986)
      The MHC status of epithelial cells from 32 primary colorectal neoplasms, villous adenomata (VA; 2) and inflammatory bowel disease (IBD; 3) were evaluated using a panel of monoclonal antibodies (mAbs). Class I antigens and beta 2 microglobulin (beta 2m) were expressed on all normal, benign, inflammatory and malignant epithelia with the exception of two carcinomas. A more complex pattern of reactivity was encountered with anti-class II mAbs. Some expression was detected on normal glandular and luminal epithelium, particularly adjacent to the tumour. Inflammatory tissues, VA and 23/32 carcinomas were also antigen-positive, the proportion of stained epithelial cells ranging from 5% to 90%. Expression was usually non-coordinate, DR being the predominant specificity followed by DP and DQ, which is suggestive of independent D region gene regulation. The hypothesis that class II expression is induced in vivo by locally generated IFN gamma was not confirmed by in vitro treatment with this agent of epithelial colorectal carcinoma-derived cell lines. These provisional data suggest that although IFN gamma may be a necessary stimulus for class II expression it is insufficient and that other factors also influence the responsiveness of tumour cells in this respect.