• Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.

      Turner, Suzanne D; Wijnhoven, S W; Tinwell, H; Lashford, Linda S; Rafferty, Joseph A; Ashby, J; Vrieling, H; Fairbairn, Leslie J; Gene Therapy Group, Christie Hospital (NHS) Trust, Wilmslow Road, Manchester M20 4BX, UK. suzanne.turner@bbsrc.ac.uk (2001-06-27)
      The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.
    • bcl-2 delay of alkylating agent-induced apoptotic death in a murine hemopoietic stem cell line.

      Fairbairn, Leslie J; Cowling, Graham J; Dexter, T Michael; Rafferty, Joseph A; Margison, Geoffrey P; Reipert, Brigit M; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, United Kingdom. (1994-09)
      Many cytotoxic agents kill cells by invoking a specific death pathway termed physiological cell death, or apoptosis. Treatment of a murine hemopoietic stem cell line, FDCP-mix, with methylmethanesulfonate (MMS) or N'-methyl-N'-nitrosourea (MNU) leads to death by apoptosis. Retroviral gene transfer was used to overexpress the bcl-2 oncogene in FDCP-mix cells, and this was associated with a delay in apoptosis in these cells after treatment with MNU and MMS and decreased sensitivity of colony formation to the cytotoxic effects of MMS. These data suggest an explanation for the refractory nature of bcl-2-expressing follicular lymphoma to cytotoxic chemotherapy and furthermore suggest that DNA-damaging antitumor therapy may contribute to the progression of disease.
    • Biological consequences of p160v-abl protein tyrosine kinase activity in a primitive, multipotent haemopoietic cell line.

      Spooncer, Elaine; Fairbairn, Leslie J; Cowling, Graham J; Dexter, T Michael; Whetton, Anthony D; Owen-Lynch, P Jane; Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1994-04)
      A temperature sensitive abl protein tyrosine kinase gene was transferred into a multipotent haemopoietic stem cell line, and the primary biological effects of expression of the gene were examined at the permissive and non-permissive temperatures. Unlike previous studies in factor-dependent cell lines, we found that expression of the functional abl protein tyrosine kinase did not lead to growth autonomy. Furthermore, the cells were still able to undergo terminal myeloid differentiation. However, expression of the functional gene did lead to a delay in maturation with a concomitant increase in cell production, had a modest effect in terms of delayed apoptosis particularly when the cells were maintained at a high cell density, and slightly increased the response to sub-optimal concentrations of IL-3. In many respects, therefore, the effects of abl protein tyrosine kinase in these cells mimics the effect of bcr/abl in primary haemopoietic cells where growth factor independence and an aberrant differentiation profile are relatively late events in clonal evolution and are not intermediate consequences of activation of the abl gene.
    • CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors.

      De Wynter, Erika A; Buck, D; Hart, Claire A; Heywood, R; Coutinho, Lucia H; Clayton, Alison J; Rafferty, Joseph A; Burt, Deborah J; Guenechea, G; Bueren, J A; et al. (1998)
      The AC133 antigen is a novel antigen selectively expressed on a subset of CD34+ cells in human fetal liver, bone marrow, and blood as demonstrated by flow cytometric analyses. In this study, we have further assessed the expression of AC133 on CD34+ cells in hemopoietic samples and found that there was a highly significant difference between normal bone marrow and cord blood versus aphereses (p <0.0001) but not between bone marrow and cord blood. Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133- cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 10(5) CD34+AC133- cells. The CD34+AC133+ population was also enriched (seven-fold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.
    • Chemoprotection of normal tissues by transfer of drug resistance genes.

      Rafferty, Joseph A; Hickson, Ian; Chinnasamy, Nachimuthu; Lashford, Linda S; Margison, Geoffrey P; Dexter, T Michael; Fairbairn, Leslie J; CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS)-Trust, Manchester, UK. (1996-09)
      The effectiveness of many types of antitumour agent is limited by (i) acute dose limiting cytotoxicity, principally myelosuppression but also lung, liver and gastrointestinal tract toxicity, (ii) the risk of therapy related secondary malignancy and (iii) the inherent or acquired drug-resistance of tumour cells. As the management of the acute toxic effects improve, the more insidious effects, and particularly haematological malignancies, are anticipated to increase. Furthermore, attempts to overcome tumour cell resistance to treatment can lead to increased collateral damage in normal tissues. One approach to circumventing both the acute toxic and chronic carcinogenic effects of chemotherapy would be to use gene therapy to achieve high levels of expression of drug resistance proteins in otherwise drug-sensitive tissues. To date the products of the multi-drug resistance (MDR-1) and the human O6-alkylguanine-DNA-alkyltransferase (ATase) gene have been used in preclinical experiments to demonstrate proof of principle, and the former of these is now being tested in a clinical situation. Here we discuss the potential of drug-resistance gene therapy to provide chemoprotection to normal tissues and examine the prospects for a dual approach which combines this with pharmacological sensitisation of tumours to chemotherapeutic agents.
    • Chemoprotective gene transfer I: transduction of human haemopoietic progenitors with O6-benzylguanine-resistant O6-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O6-alkylating agents in vitro.

      Hickson, Ian; Fairbairn, Leslie J; Chinnasamy, Nachimuthu; Lashford, Linda S; Thatcher, Nick; Margison, Geoffrey P; Dexter, T Michael; Rafferty, Joseph A; CRC Section of Haemopoietic Cell, Paterson Institute for Cancer Research, Manchester, UK. (1998-06)
      Retroviral transduction was used to introduce cDNAs encoding two mutants of human O6-alkylguanine-DNA alkyltransferase (hAT), one of which (hATPA) is 16 times more resistant to O6-benzylguanine (O6-beG), and the other (hATPA/GA) which is almost totally refractory to inactivation relative to the wild-type protein, into K562 human erythroleukaemic cells. A colony-forming assay was used to demonstrate significant protection (P < 0.001) against mitozolomide or temozolomide toxicity in K562 clones expressing either hAT mutant, as determined from an in vitro assay of activity. However, protection against these agents was reduced in hATPA expressing cells in the presence of 1 microM O6-beG and was lost in the presence of 20 microM O6-beG while cells expressing hATPA/GA retained protection even in the presence of 20 microM O6-beG (P < 0.001). Using primary human cord blood-derived CD34+ haemopoietic cells in which PCR analysis indicated that up to 70% of progenitors were transduced with retroviral constructs harbouring hATPA/GA, we observed significant protection of the granulocyte-macrophage colony-forming cells against mitozolomide (P < 0.05) and temozolomide (P < 0.001) induced toxicity in the presence of O6-beG. These findings indicate that retrovirus-mediated expression of hATPA/GA in primitive primary human haemopoietic cells is possible and does provide O6-beG-resistant protection for these cells. Using this strategy in patients may simultaneously permit attenuated myelosuppression and increased sensitivity of tumour cells to the effects of O6-alkylating agent chemotherapy. These data, taken together with the study reported by Chinnasamy et al in the accompanying article in this issue showing reduced toxicity and clastogenicity in murine haemopoietic progenitors, make a compelling case to test this strategy clinically.
    • Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase.

      Chinnasamy, Nachimuthu; Rafferty, Joseph A; Hickson, Ian; Lashford, Linda S; Longhurst, S J; Thatcher, Nick; Margison, Geoffrey P; Dexter, T Michael; Fairbairn, Leslie J; CRC Sections of Genome Damage and Repair, Paterson Institute for Cancer Research, Manchester, UK. (1998-06)
      Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte-macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocyto-chemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with O6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). This study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an O6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.
    • Cloning of the murine homolog of the leukemia-associated PML gene.

      Goddard, A D; Yuan, J Q; Fairbairn, Leslie J; Dexter, T Michael; Borrow, J; Kozak, C; Solomon, E; Imperial Cancer Research Fund, London, UK. (1995-10)
      PML, a Ring-finger protein, participates in the disruption of normal myeloid differentiation when fused to the retinoic acid receptor alpha (RAR alpha) by the translocation between chromosomes (Chrs) 15 and 17 in acute promyelocytic leukemia (APL). As an initial step in the characterization of PML in species other than human, a murine cDNA clone of the PML gene was isolated and sequenced, and the intron/exon organization of the murine locus determined. The predicted amino acid sequence of the mouse PML protein shows 80% similarity to that of its human homolog. However, the mouse and human proteins show greater than 90% similarity in the proposed functional domains of the proteins. Despite its role in the etiology of APL, PML expression is not detectably altered during granulocytic differentiation in a murine in vitro system. Chromosomal localization of the Pml locus by somatic cell hybrids and by linkage analysis indicates that the gene maps to a region of mouse Chr 9 with known linkage homology to the region on human Chr 15q to which PML has been localized.
    • Diffusible VP22-E2 protein kills bystander cells and offers a route for cervical cancer gene therapy.

      Green, Katie L; Southgate, Thomas D; Mulryan, Kate; Fairbairn, Leslie J; Stern, Peter L; Gaston, Kevin; Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. (2006-02)
      Human papillomaviruses (HPVs) are a causative agent of cervical cancer and are implicated in several other types of malignant disease including cancer of the vulva, oral cancer, and skin cancer. In HPV-transformed cells, expression of the viral E6 and E7 oncogenes increases cell proliferation and inhibits apoptosis. Expression of the viral E2 protein in HPV-transformed cells represses transcription of E6 and E7 and induces apoptosis and/or growth arrest. We have shown previously that herpes simplex virus type 1 (HSV-1) VP22-HPV E2 fusion proteins can traffic between cells and induce apoptosis. Here we show that replication-defective adenoviruses can be used to deliver VP22-E2 fusion proteins to target cells. We show that the use of adenoviral vectors to deliver VP22-E2 proteins leads to high levels of apoptosis. Interestingly, VP22-E2 proteins produced in adenovirus-infected cells are able to enter uninfected cells and induce apoptosis. Trafficking between cells and the induction of apoptosis in bystander cells are detectable in a three-dimensional tumor model. These results suggest that adenoviral vectors expressing VP22-E2 fusion proteins could be used to treat cervical cancer and other HPV-associated diseases.
    • Dual agent chemoprotection by retroviral co-expression of either MDR1 or MRP1 with the P140K mutant of O6-methylguanine-DNA-methyl transferase.

      Southgate, Thomas D; Garside, Elloise; Margison, Geoffrey P; Fairbairn, Leslie J; Cancer Research UK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. Tsouthgate@picr.man.ac.uk (2006-08)
      Tumour resistance to chemotherapeutic agents results in most chemotherapy being administered in a multi-agent fashion that is often associated with a high level of toxicity in highly proliferative tissues such as the haematopoietic compartment. Thus, whilst many genetic manipulation strategies aim to protect normal tissue against a single component of a multi-agent regime, it is clearly preferable to protect normal cells against all toxicities. In this study we have used retroviral gene transfer to achieve co-expression of either p-glycoprotein (MDR1) or multi-drug resistance-related protein 1 (MRP1) with the P140K mutant form of O6-methylguanine-DNA-methyl transferase (MGMT) which, unlike the wild-type protein, is insensitive to inactivation by tumour sensitisers such as O6-benzylguanine (O6-BeG) or PaTrin2. The combination of certain MDR1/MRP1 substrate drugs with O6-alkylating agents (against which MGMT confers resistance) is particularly myelotoxic. We show here that haematopoietic progenitors co-expressing mutant MGMT with an ABC-transporter exhibit resistance to combination chemotherapy in vitro. This combination of drug transporter and DNA repair function may provide an effective in vivo protection of the haematopoietic compartment during tumour ablation using combination chemotherapy.
    • Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-{alpha} on Fanconi anemia hematopoietic stem and progenitor cells.

      Milsom, Michael D; Schiedlmeier, Bernhard; Bailey, Jeff; Kim, Mi-Ok; Li, Dandan; Jansen, Michael; Ali, Abdullah Mahmood; Kirby, Michelle; Baum, Christopher; Fairbairn, Leslie J; et al. (2009-05-21)
      Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha, we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.
    • The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

      Turner, S D; Rafferty, Joseph A; Fairbairn, Leslie J; Ashby, J; Tinwell, H; Eckert, H G; Baum, C; Lashford, Linda S; Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. (2000-10)
      We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.
    • Engineering drug resistance in human cells.

      Fairbairn, Leslie J; Rafferty, Joseph A; Lashford, Linda S; Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. (2000-05)
      Many of the problems with current anti-tumour therapies stem from a lack of specificity for tumour as opposed to normal tissues. To address the problem of collateral toxicity during anti-tumour chemotherapy we have been developing a gene therapy approach to protect normal tissues from the toxic and potentially mutagenic effects of chemotherapeutic agents. As a paradigm for this we have been examining the potential of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) to confer genetic chemoprotection to the bone marrow. By transfer and expression of a mutant form of this protein, which is resistant to inactivation by the tumour sensitising agent O6-benzylguanine (O6-beG), we have been able to demonstrate protection of murine bone marrow in vitro from the cytotoxic and clastogenic effects of O6-beG in combination with the anti-tumour agent temozolomide. This protection is seen in multiple lineages, including erythroid and granulocyte/macrophage progenitors, as well as more primitive cells. Importantly, significant protection of the platelet lineage is also seen, with faster recovery of platelets. The multi-lineage protection seen has encouraged us to take this approach forward to clinical trial in the near future.
    • Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4.

      Milsom, Michael D; Woolford, Lorna B; Margison, Geoffrey P; Humphries, R Keith; Fairbairn, Leslie J; Gene Therapy, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK. (2004-11)
      To attain therapeutic levels of gene-modified hematopoietic stem cells, it may be necessary in the majority of disorders to provide an in vivo selective advantage that facilitates the expansion of their numbers. A popular strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O6-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified hematopoietic stem cells through the delivery of the homeobox transcription factor HOXB4. By developing a novel tricistronic retroviral vector, we have facilitated the simultaneous coexpression of a mutant version of MGMT and HOXB4 in retrovirally transduced bone marrow. Using an in vivo competitive repopulation assay, we demonstrate that primary bone marrow cells containing this construct show enhanced reconstitution following transplant and improved selection subsequent to chemotherapeutic challenge in comparison to cells expressing either HOXB4 or MGMT alone. This selection advantage was evident even when HOXB4/MGMT-coexpressing cells were infused along with a large excess of unmodified cells. We propose that this selection cassette may facilitate the in vivo expansion of gene-modified hematopoietic stem cells at a level in excess of previous strategies.
    • Erythroid development of the FDCP-Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3.

      Heyworth, Clare M; Alauldin, Mazin; Cross, Michael A; Fairbairn, Leslie J; Dexter, T Michael; Whetton, Anthony D; Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester. (1995-09)
      Conditions are described which promote the erythroid development of the FDCP-Mix A4 (A4) cell line with accompanying proliferation of the cells. The requirements for this development are low concentrations of interleukin 3 (IL-3) plus the presence of erythropoietin (epo) and haemin. When high concentrations of IL-3 are added with erythropoietin and haemin the cells do not differentiate and maintain their blast cell morphology. Addition of haemin, in the absence of erythropoietin, does not promote erythroid development, but the presence of haemin with erythropoietin promotes increased proliferation and maturation. The morphological maturation of A4 cells along the erythroid lineage is accompanied by a gradual loss of clonogenic potential, loss of A4 cell multipotency, increased erythropoietin receptor expression, and an increased expression of the beta-globin gene. An initial increase in mitogenic responsiveness to erythropoietin is followed by a decrease as the cells become refractory to all mitogenic stimuli with the acquisition of a postmitotic, mature erythroid cell phenotype.
    • Expression and downregulation of cytotoxic cell protease 1 or Granzyme 'B' transcripts during myeloid differentiation of interleukin-3-dependent murine stem cell lines.

      Hampson, Ian N; Cross, Michael A; Heyworth, Clare M; Fairbairn, Leslie J; Spooncer, Elaine; Cowling, Graham J; Dexter, T Michael; Cancer Research Campaign Department of Experimental Hematology, Paterson Institute for Cancer Research, Withington, Manchester, UK. (1992-12-15)
      Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme 'B'), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.
    • 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.
    • 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 the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.

      Klungland, A; Fairbairn, Leslie J; Watson, Amanda J; Margison, Geoffrey P; Seeberg, E; Biotechnology Centre of Oslo, Blindern, Norway. (1992-12)
      In order to investigate the importance of 3-methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3-methyladenine DNA glycosylase-1 which specifically removes 3-alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus-packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3-methyladenine DNA glycosylase activity in cell extracts showed an up to 10-fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N-methyl-N-nitrosourea than their parent cell lines, indicating that 3-methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3-methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.
    • Gene transfer to augment the therapeutic index of anticancer chemotherapy.

      Baum, C; Margison, Geoffrey P; Eckert, H G; Fairbairn, Leslie J; Ostertag, W; Rafferty, Joseph A (1996-01)