• Transcriptional evidence for the "Reverse Warburg Effect" in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and "Neuron-Glia Metabolic Coupling".

      Pavlides, Stephanos; Tsirigos, Aristotelis; Vera, Iset; Flomenberg, Neal; Frank, Philippe G; Casimiro, Mathew C; Wang, Chenguang; Pestell, Richard G; Martinez-Outschoorn, Ubaldo E; Howell, Anthony; et al. (2010-04)
      Caveolin-1 (-/-) null stromal cells are a novel genetic model for cancer-associated fibroblasts and myofibroblasts. Here, we used an unbiased informatics analysis of transcriptional gene profiling to show that Cav-1 (-/-) bone-marrow derived stromal cells bear a striking resemblance to the activated tumor stroma of human breast cancers. More specifically, the transcriptional profiles of Cav-1 (-/-) stromal cells were most closely related to the primary tumor stroma of breast cancer patients that had undergone lymph-node (LN) metastasis. This is consistent with previous morphological data demonstrating that a loss of stromal Cav-1 protein (by immuno-histochemical staining in the fibroblast compartment) is significantly associated with increased LN-metastasis. We also provide evidence that the tumor stroma of human breast cancers shows a transcriptional shift towards oxidative stress, DNA damage/repair, inflammation, hypoxia, and aerobic glycolysis, consistent with the "Reverse Warburg Effect". Finally, the tumor stroma of "metastasis-prone" breast cancer patients was most closely related to the transcriptional profiles derived from the brains of patients with Alzheimer's disease. This suggests that certain fundamental biological processes are common to both an activated tumor stroma and neuro-degenerative stress. These processes may include oxidative stress, NO over-production (peroxynitrite formation), inflammation, hypoxia, and mitochondrial dysfunction, which are thought to occur in Alzheimer?s disease pathology. Thus, a loss of Cav-1 expression in cancer-associated myofibroblasts may be a protein biomarker for oxidative stress, aerobic glycolysis, and inflammation, driving the "Reverse Warburg Effect" in the tumor micro-environment and cancer cell metastasis.
    • Transcriptional regulation of the c-fms proto-oncogene mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) in murine cell lines.

      Helftenbein, G; Krusekopf, K; Just, U; Cross, M; Ostertag, W; Niemann, H; Tamura, T; Institut für Virologie, Justus-Liebig Universität Giessen, Germany. (1996-02-15)
      Differentiation of blood cells is paralleled by a timely ordered expression of cytokine receptor genes. We show here that the expression of the c-fms gene which encodes the lineage-specific receptor for macrophage colony-stimulating factor (M-CSF or CSF-1) is directly linked to ligand-mediated activation of the receptor for the granulocyte/macrophage colony-stimulating factor (GM-CSF). In interleukin-3 (IL-3) dependent multipotent progenitor cells, FDC-Pmix GMV#2 cells, GM-CSF treatment results in the rapid formation of full-length c-fms transcripts. Surprisingly, this upregulation of c-fms transcripts is also observed in mouse NIH3T3 fibroblasts stably transfected with genes coding for the alpha- and beta-subunits of the GM-CSF receptor. These results indicate a direct control by the GM-CSF receptor that takes place regardless of cell differentiation. Furthermore, a 2.1 kb genomic fragment containing the c-fms proximal promoter directs GM-CSF-inducible expression of a reporter gene, suggesting a regulation of c-fms gene expression on the transcriptional level.
    • Transcriptional switch by activating transcription factor 2-derived peptide sensitizes melanoma cells to apoptosis and inhibits their tumorigenicity.

      Bhoumik, Anindita; Jones, Nic; Ronai, Ze'ev; Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029, USA. (2004-03-23)
      The notorious resistance of melanoma cells to drug treatment can be overcome by expression of a 50-aa peptide derived from activating transcription factor 2 (ATF2(50-100)). Here we demonstrate that ATF2(50-100) induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities. Furthermore, ATF2(50-100) binds to c-Jun N-terminal kinase (JNK) and increases its activity. Mutation within ATF2(50-100) that impairs association with JNK and the inhibition of JNK or c-Jun expression by RNA interference (RNAi) reduces the degree of ATF2(50-100)-induced apoptosis. In contrast, TAM67, a dominant negative of the Jun family of transcription factors, or JunD RNAi attenuates sensitization of melanoma cells expressing ATF2(50-100) to apoptosis after treatment with anisomycin, which is used as a model drug. Mutations within the JNK binding region of ATF2(50-100) or expression of TAM67 or JunD RNAi attenuates inhibition of melanoma's tumorigenicity by ATF2(50-100). We conclude that inhibition of ATF2 in concert with increased JNK/Jun and JunD activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity.
    • Transcriptome analysis in a primary human muscle cell differentiation model for myotonic dystrophy type 1

      Todorow, V.; Hintze, S.; Kerr, Alastair R W; Hehr, A.; Schoser, B.; Meinke, P.; Department of Neurology, Friedrich-Baur-Institute, LMU Klinikum, Ludwig-Maximilians-University Munich, 80336 Munich, Germany (2021)
      Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.
    • Transcriptomic rationale for synthetic lethality-targeting ERCC1 and CDKN1A in chronic myelomonocytic leukaemia.

      Hurtado, A; Luengo-Gil, G; Chen-Liang, T; Amaral, Fabio; Batta, Kiran; Palomo, L; Lumbreras, E; Przychodzen, B; Caparros, E; Amigo, M; et al. (2018-05-24)
      Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high-throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA-sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34+ bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down-regulation to be specific to CMML compared with other related disorders. Chromatin-regulator mutated cases showed decreased BAP1 dosage. We validated a significant over-expression of the double strand break-fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.
    • Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.

      Li, Ying; Tew, Simon R; Russell, Amanda M; Gonzalez, Karin R; Hardingham, Timothy E; Hawkins, Robert E; UK Centre for Tissue Engineering, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (2009-10-19)
      Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.
    • Transfection of murine multi-potent haemopoietic stem cells with an E. coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents.

      Jelinek, J; Kleibl, K; Dexter, T Michael; Margison, Geoffrey P; Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1988-01)
      O6-alkylguanine-DNA-alkyltransferase (ATase)-deficient murine haemopoietic stem cells were transfected, following electroporation, with a G418-selectable expression vector containing the protein coding region of the Escherichia coli ATase gene ada. Clones of cells that were resistant to G418 or the chloroethylating agent mitozolomide (Mz) were selected and most were shown to express very high levels of bacterial gene-encoded ATase. In comparison with control cells that were transfected with the parent vector, the ATase-expressing clones were considerably more resistant to the toxic effects of the methylating agents N-methyl-N-nitrosourea and methylmethanesulphonate or the chloroethylating agents Mz or taurine chloroethylnitrosourea, but unchanged in their susceptibility to the bis-chloroethylating agent nitrogen mustard. Thus alkylation damage in DNA that can be repaired by the E. coli ATase constitutes the principal lethal lesion produced by alkylating agents in murine haemopoietic stem cells and the ATase deficiency in these cells can be complemented by electroporation-mediated gene transfection.
    • Transfection of the Escherichia coli nth gene into radiosensitive Chinese hamster cells: effects on sensitivity to radiation, hydrogen peroxide, and bleomycin sulfate.

      Harrison, Lynn; Skorvaga, Milan; Cunningham, R P; Hendry, Jolyon H; Margison, Geoffrey P; Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (1992-10)
      The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.
    • A transfer instrument for neutron dosimetry.

      Greene, D; Miles, J; Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, UK (1976-04)
    • A transferable "resistance factor" from in vitro cultured MDMS-resistant Yoshida sarcoma cells.

      Szende, B; Fox, M; Fox, Brian W; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1973-03)
      Cells of the methylene dimethanesulphonate-(MDMS)-resistant Yoshida sarcoma cell line contain a low molecular weight "resistance factor" which is present in the culture medium of these cells and may be utilized by MDMS-sensitive Yoshida sarcoma cells either by co-culturing the two cell lines or by culturing the MDMS-sensitive Yoshida cells in a medium containing 20% used medium of MDMS-resistant Yoshida cells or in the presence of dialysed medium from resistant cells. The "resistance factor" does not inactivate the drug itself or its metabolites, and it has no influence on the sensitivity of the cells if added after MDMS treatment. Twenty-four hours seems to be enough time for the transfer of the resistance factor, but its effect on whole populations decreases within 24 hours of ceasing the supply. The relationship between these findings and the known phenomena of metabolic co-operation are discussed.
    • Transferrin receptor-mediated gene transfer to the corneal endothelium.

      Tan, Peng H; King, William J; Chen, Daxin; Awad, Hana M; Mackett, Mike; Lechler, Robert I; Larkin, D Frank; George, Andrew J; Department of Immunology, Imperial College, Hammersmith Hospital, London, UK. (2001-02-27)
      BACKGROUND: The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. METHODS: Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. RESULTS: Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eight-fold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. CONCLUSIONS: Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.
    • Transferrin-mediated uptake of plutonium by spermatogenic tubules.

      Hoyes, Katherine P; Bingham, D; Hendry, Jolyon H; Harrison, J D; Sharma, Harbans L; Morris, Ian D; School of Biological Sciences, University of Manchester, UK. (1996-10)
      Using isolated rat seminiferous tubules as an in vitro model, we have found that 238Pu can cross the blood-tubule barrier and accumulate within tubules in a time dependent manner. Furthermore, similar to 59Fe, tubule 238Pu uptake was inhibited by the addition of excess transferrin, suggesting that plutonium may utilize the physiological iron-transferrin pathway to cross the blood-tubule barrier. However unlike 59Fe, 238Pu was only transiently associated with the tubules, suggesting differences in the intracellular processing of these radionuclides. The assumptions made in the estimation of doses to the human testis from incorporated plutonium are considered.
    • Transferrin-mediated uptake of radionuclides by the testis.

      Hoyes, Katherine P; Morris, Ian D; Hendry, Jolyon H; Sharma, Harbans L; Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1996-02)
      In an attempt to explain the deleterious effects of gonadal radionuclide localization, we examined the role of transferrin in testicular radionuclide uptake. METHODS: In vivo testicular uptake and retention of the transferrin binding radionuclides 114mIn-citrate and 59Fe-citrate were compared with that of the nontransferrin binding isotopes 137Cs-citrate and Na125I for 63 days postinjection. Isotope uptake mechanisms were investigated in vitro using isolated seminiferous tubules and Sertoli cell monolayers grown in bicameral culture chambers. RESULTS: Indium-114m, 59Fe and 137Cs were localized in the testis by 24 hr postinjection, but accumulation of 125I was minimal. Although testicular 114mIn remained constant, 59Fe declined slowly over the following 63 days and 137Cs fell very rapidly. When 114mIn- or 59Fe-loaded testes were fractionated, and markedly more 114mIn was associated with the seminiferous tubules than 59Fe, suggesting that 114mIn may be retained. In vitro uptake of 59Fe, 67Ga and 114mIn by isolated seminiferous tubules was inhibited by transferrin, but uptake of 137Cs and 125I was unaffected. Iron-59, 67Ga and 114mIn were retained by isolated tubules in contrast to 137Cs and 125I. Whereas 137Cs, 59Fe and 114mIn all crossed Sertoli cell monolayers, the rate of transcellular transport of 137Cs was faster than that of 59Fe or 114mIn, suggesting differences in the intracellular processing of transferrin binding and nontransferrin binding radionuclides. CONCLUSION: These data suggest that some radionuclides may access the seminiferous epithelium through receptor-mediated endocytosis of transferrin. Such radionuclide localization could lead to continuous irradiation of the testes, resulting in mutagenic damage to spermatogenic cells.
    • Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.

      Arciniegas, E; Sutton, Andrew B; Allen, Terence D; Schor, Ana M; CRC Department of Medical Oncology, Christie Hospital, Manchester, UK. (1992-10)
      Alpha-smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of alpha-smooth muscle actin (alpha-SM actin) may be induced to express the latter when exposed to TGF-beta 1. FVIII was detected by immunofluorescence, alpha-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing alpha-SM actin increased with time of incubation with TGF-beta 1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and alpha-SM actin after 5 days of incubation with TGF-beta 1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed alpha-SM actin. Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-beta 1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-beta 1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and alpha-SM actin-negative.(ABSTRACT TRUNCATED AT 250 WORDS)
    • Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling.

      Booth, Dawn; Haley, John D; Bruskin, Arthur M; Potten, Christopher S; CRC Epithelial Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (2000-04-01)
      Damage to the normal replacing tissues of the body, specifically the gastro-intestinal tract, limits the treatment and hence, cure rate of cancer patients. Here, we investigate the possibility that the sensitivity of the gastro-intestinal tract can be manipulated by transforming growth factor beta3 (TGF-beta3), making it more resistant to radiation in a murine model. The effects of TGF-beta3 were assessed using the crypt microcolony assay, a test of crypt stem-cell functional competence, in animal survival studies examining diarrhoea severity, labelling index and crypt size. Prior treatment with TGF-beta3 can result in a 3- to 4-fold increase (protection factor, PF) in surviving crypts, whilst longer exposure can raise the PF to almost 12. Protection of intestinal clonogenic stem cells results in marked protection of survival with a corresponding reduction in the duration and level of diarrhoea and ultimate restoration of normal histology in surviving mice. Inhibition of proliferation can be demonstrated when sufficient TGF-beta3 exposure is studied. Crypt size is also reduced. In conclusion, TGF-beta3 protects small intestinal clonogenic stem cells from radiation damage, reducing diarrhoea and animal mortality. The mode of action is believed to be specific inhibition of stem-cell proliferation.
    • Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.

      Francis, Julia M; Heyworth, Clare M; Spooncer, Elaine; Pierce, Andrew; Dexter, T Michael; Whetton, Anthony D; Leukaemia Research Fund Cellular Development Unit, Department of Biomolecular Sciences, UMIST, Sackville St., Manchester, M60 1QD, United Kingdom. (2000-12-15)
      Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.
    • Transgenerational effects of preconception paternal contamination with (55)Fe.

      Hoyes, Katherine P; Lord, Brian I; McCann, Christine; Hendry, Jolyon H; Morris, Ian D; Cancer Research Campaign Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 4BX, United Kingdom. (2001-11)
      The conjecture that germline mutations induced by radiation exposure before conception may predispose subsequent offspring to cancer remains contentious. Previous experimental studies have shown that preconception paternal irradiation with (239)Pu induces perturbations in the hemopoietic systems of offspring and influences sensitivity to a secondary carcinogen. In the present study, male DBA2 mice were injected intravenously with the Auger electron emitter (55)Fe (4 kBq g(-1)) 18 or 84 days before mating with normal females. Comet analysis showed an increased incidence of DNA strand breaks in sperm from contaminated animals after 84 days, but not after 18 days, indicating spermatogonial rather than spermatid damage. Offspring were either assayed for changes in bone marrow stem cells and committed progenitors or challenged with the chemical carcinogen methyl nitrosourea (MNU, 50 mg/kg) at 10 weeks of age and monitored for the onset of malignancy. Offspring from irradiated fathers had normal peripheral blood profiles, although the stem cell population was amplified in offspring arising from those exposed to (55)Fe at 84 days before conception. Exposure to MNU significantly increased the incidence of lympho-hemopoietic malignancies in offspring from the 84-day group, but not in those from the 18-day group. These findings support the hypothesis that aberrations that are potentially leukemogenic may be transmitted to offspring after radiation damage to the paternal germline.
    • Transgenerational susceptibility to leukaemia induction resulting from preconception, paternal irradiation.

      Lord, Brian I; CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. blord@picr.man.ac.uk (1999-07)
      The clustered excess of childhood leukaemia and non-Hodgkin's lymphoma at Seascale, close to the nuclear reprocessing plant at Sellafield in the UK is well authenticated and has remained a 'current topic' for over a decade. Its root cause has not been established. Following a study suggesting that parental irradiation exposure prior to conception was a factor, a recent laboratory-based report reopened the debate by indicating the potential for preconception, paternal irradiation (PPI) to result in increased or accelerated induction of lympho-myeloid malignancy in offspring subjected to a recognized leukaemogen. This short commentary presents those new findings in the light of the many and diverse epidemiological investigations of first generation malignancies following parental exposure, the majority of which indicate no real evidence to support the concept that patterns of lympho-myeloid malignancy reflect levels of PPI. Other experimental work supporting PPI are considered against unsuccessful attempts to reproduce them. The alternative, and more popular, hypothesis of infection spread via population mixing, which is more ubiquitous than confinement to nuclear localities, is introduced. Mechanisms of potentiation by PPI are considered, though the danger of applying these current findings to explain the enigma of Seascale, or any other cluster, is recognized.
    • A transient assay for regulatory gene function in haemopoietic progenitor cells.

      McIvor, Zoe J; Heyworth, Clare M; Johnson, Barbra A; Pearson, Stella; Fiegler, Heike; Hampson, Lynne; Dexter, T Michael; Cross, Michael A; Laboratory of Molecular Medicine, IZKF University of Leipzig, Leipzig, Germany. zoem@medizin.uni-leipzig.de (2000-09)
      This work aimed to provide a means of assaying directly the effects of transient expression of introduced genes on the survival, proliferation, lineage commitment and differentiation of haemopoietic progenitor cells. For this purpose, we have developed a system that allows isolation of productively transfected, mulitipotent haemopoietic cells within a few hours of the introduction of test genes. We have shown that FDCP-mix cells productively transfected with expression plasmids encoding green fluorescent protein (GFP) differentiate normally and retain colony-forming potential. We constructed an expression vector consisting of a bicistronic cassette in which a GFP marker gene and a test gene are driven from the same promoter. The vector design has been optimized for co-expression and the test gene was shown to be biologically active. The expression profile from a transiently transfected template under different growth conditions reveals that active expression continues for at least 2 d after transfection. The transient transfection of FDCP-mix cells with the vectors described provides a powerful tool for analysis of the immediate early effects of test gene overexpression during haemopoietic differentiation.
    • Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

      Napolitano, Alessandra; Di Donato, Paola; Prota, Giuseppe; Land, Edward J; The Department of Organic and Biological Chemistry, University of Naples Federico II, Italy. (1999-09)
      Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.