• Formation and loss of O6-methyldeoxyguanosine in human leucocyte DNA following sequential DTIC and fotemustine chemotherapy.

      Lee, Siow Ming; Margison, Geoffrey P; Thatcher, Nick; O'Connor, Peter J; Cooper, Donald P; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1994-05)
      There is increasing evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain anti-tumour alkylating agents and that the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide inter-individual variation in the depletion and subsequent regeneration of ATase in human peripheral blood lymphocytes following sequential DTIC (400 mg m-2) and fotemustine (100 mg m-2) treatment, with the nadir ATase activity occurring approximately 4 h after DTIC administration. We have now measured the formation and loss of O6-methyldeoxyguanosine (O6-MedG) in the DNA of peripheral leucocytes of eight patients receiving this treatment regimen. O6-MedG could be detected within 1 h and maximal levels occurred approximately 3-5 h after DTIC administration. Following the first treatment cycle, considerable inter-individual variation was observed in the peak O6-MedG levels, with values ranging from 0.71 to 14.3 mumol of O6-MedG per mol of dG (6.41 +/- 5.53, mean +/- s.d.). Inter- and intra-individual variation in the extent of O6-MedG formation was also seen in patients receiving additional treatment cycles. This may be a consequence of inter-patient differences in the capacity for metabolism of DTIC to release a methylating intermediate and could be one of the determinants of clinical response. Both the pretreatment ATase levels and the extent of ATase depletion were inversely correlated with the amount of O6-MedG formed in leucocyte DNA when expressed either as peak levels (r = -0.59 and -0.75 respectively) or as the area under the concentration-time curve (r = -0.72 and -0.73 respectively). One complete and one partial clinical response were seen, and these occurred in the two patients with the highest O6-MedG levels in the peripheral leucocyte DNA, although the true significance of this observation has yet to be established.
    • Formation and persistence of isoniazid-DNA adducts in mouse tissues.

      Maru, G B; Bhide, S; Saffhill, Roy; O'Connor, Peter J (1987-03)
      Further confirmation is provided to support the identity of the product formed by the in vitro reaction of isoniazid (INH) with cytosine as 4-deamino-4-isoniazidocytosine (INH-cytosine). Use of INH-treated mice, in which the tissue DNA is prelabelled by the neonatal administration of [3H]-deoxycytidine, has revealed the presence of two other DNA products in addition to INH-cytosine. Tissue differences in the persistence of these three DNA products suggest the presence of repair reactions for certain adducts. These processes and the effects of hepatotoxicity lead to a selective retention of adducts in the DNA of lung which is the target tissue for INH-carcinogenicity in mice. INH-modified DNA templates are weakly promutagenic during in vitro DNA synthesis. The implications of these observations for the role of INH as an initiating agent and for the species differences in its carcinogenicity are discussed.
    • Formation and persistence of N7-methylguanine DNA adducts in the target pyloric tissue following chronic exposure to N-methyl-N'-nitro-N-nitrosoguanidine

      Haque, Kemal; Cooper, Donald P; Povey, Andrew C; Cancer Research Campaign Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, M20 9BX, UK. (1999)
    • Formation and persistence of O(6)-methylguanine in the mouse colon following treatment with 1,2-dimethylhydrazine as measured by an O(6)-alkylguanine-DNA alkyltransferase inactivation assay.

      Jackson, Peta E; Cooper, Donald P; Meyer, T A; Wood, M; Povey, Andrew C; Margison, Geoffrey P; CRC Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (2000-06-05)
      Female SWR mice were treated with 1,2-dimethylhydrazine (DMH: 6.8 mg/kg i.p. injection) once weekly for up to 10 weeks, a dosing regime that produced tumours principally within the distal colon (Jackson et al., 1999. Carcinogenesis 20, 509-513). O(6)-Methylguanine (O(6)-MeG) levels, measured using a simple [3H]-based O(6)-alkylguanine-DNA alkyltransferase (ATase) inactivation assay, ranged from 0.6 to 16.7 fmol/microg DNA with: (i) highest levels in the distal colon; and (ii) higher levels after 68 mg/kg total DMH than 6.8 mg/kg DMH. Basal ATase activity varied between 0.97 and 1.22 fmol/microg DNA within the colon but was not associated with adduct levels or tumour induction. After 6.8 mg/kg DMH, the half life of O(6)-MeG in colonic tissue was 36-42 h whereas after 68 mg/kg DMH, t1/2 was approximately 25, 57 and 96 h in the proximal, mid and distal colon, respectively. Tumour induction was thus associated with the levels and persistence of O(6)-MeG in the distal colon.
    • The formation and stability of methyl phosphotriesters in the DNA of rat tissues after treatment with the carcinogen N,N-dimethylnitrosamine.

      Shooter, K; Slade, T; O'Connor, Peter J (1977-12)
      Following the injection i.p. of N,N-dimethylnitrosamine (DMN) into Chester Beatty (CB) hooded, female rats (2 mg/kg) measurable concentrations of methyl phosphotriesters were found in the DNA of liver, lung and kidney but not in spleen, thymus or brain. In lung and kidney these lesions were stable for at least 14 days but in liver there was a steady loss (t 1/2 9-11 days). Administering the same total dose in 10 weekly injections produced the same concentration of phosphotriesters in lung and kidney DNA as the single injection but in liver only half of the concentration induced by the single injection was found. It was calculated that the half-life of methyl phosphotriesters in the liver DNA of animals given repetitive injections was of the order of 6 weeks.
    • The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases.

      Saffhill, Roy; Abbott, Peter J; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX (1983)
      N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.
    • Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo.

      Ashton, B A; Allen, Terence D; Howlett, C R; Eaglesom, C C; Hattori, A; Owen, M; M.R.C. Bone Research Laboratory, Nuffield Orthopaedic Centre, Oxford OX3 7LD, England (1980-09)
      When freshly isolated rabbit marrow cells were cultured either in vitro or in diffusion chambers in vivo, the hemopoietic cells disappeared and there was a proliferation of the stromal cell population. The colonies formed in vitro were mainly fibroblastic, and this cell type predominated in confluent cultures. Staining for alkaline phosphatase activity and for the Von Kossa reaction was negative in in vitro cultures. However, marrow cell suspensions or fibroblasts harvested from in vitro culture of marrow cells, gave rise to a mixture of bone, cartilage and fibrous tissue in diffusion chambers implanted into the peritoneal cavity. In contrast, only a soft fibrous tissue developed from spleen fibroblasts in diffusion chambers. Differentiation of osteogenic tissue within diffusion chambers fell into two categories: (1) Formation of bone in a fibrous layer surrounding cartilage; (2) intramembranous bone formed directly within fibrous tissue unassociated with cartilage. In both cases alkaline phosphatase activity appeared before the onset of mineralization, and decreased as the first signs of mineral became apparent. The present results suggest that postnatal marrow contains osteogenic precursors with the potential to differentiate via either of the two major paths followed during skeletal development in the embryo. Clonal analysis of the marrow stromal cell population will be required to clarify whether osteo-, chondro-, and fibrogenic cells are the products of one stromal cell line modulated by the microenvironment, or whether there are distinct cell lines for each type.
    • Formation of O2-methylthymine in poly(dA-dT) on methylation with N-methyl-N-nitrosourea and dimethyl sulphate. Evidence that O2-methylthymine does not miscode during DNA synthesis.

      Saffhill, Roy; Abbott, Peter J; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1978-06)
      The alternating co-polymer has been methylated with either N methyl-N-nitrosourea (MNU) or dimethyl sulphate (DMS) and the levels of the various methylated thymidines (O2-methylthymidine, 3-methylthymidine and O4-methylthymidine) measured. MNU produced all three compounds whereas DMS only produced 3-methylthymidine and O2-methylthymidine at detectable levels. These results have been combined with our earlier results concerning the misincorporation of dGMP with E. coli DNA polymerase using MNU-methylated poly(dA-dT). These results indicate that O2-methylthymidine does not miscode during DNA synthesis.
    • Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity.

      Whetton, Anthony D; Needham, L; Dodd, N J; Heyworth, C M; Houslay, M D; Biophysical Chemistry Section, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1983-05-15)
      Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.
    • Fotemustine combined with procarbazine in recurrent malignant gliomas: a phase I study with evaluation of lymphocyte 06-alkylguanine-DNA alkyltransferase activity.

      Boiardi, Amerigo; Silvani, Antonio; Ciusani, Emilio; Watson, Amanda J; Margison, Geoffrey P; Berger, Elisabeth; Lucas, Catherine; Giroux, Bruno; Istituto Carlo Besta, Milan, Italy. (2001-04)
      The aims of this phase I study in patients with recurrent malignant gliomas were to determine the maximum tolerated dose (MTD) and toxicity profile of fotemustine when combined with a fixed dose of procarbazine (PCZ), and to evaluate the extent of O6-alkylguanine-DNA alkyltransferase (ATase) depletion in circulating lymphocytes during treatment. Sixteen patients received an induction cycle consisting of 100 mg/day oral PCZ for 12 consecutive days and a 1-h intravenous infusion of fotemustine given 4 h after PCZ on days 5 and 12 at escalated doses (50, 75, 100 and 125 mg/m2/day). After a 6-week rest period, a maximum of 4 maintenance cycles (PCZ 300 mg/day, 4 days; fotemustine, day 4) was given every 4 weeks. ATase activity was measured on days 1, 5 and 12 over 4 h after PCZ intake. Fifteen patients had previously received at least one nitrosourea-based chemotherapy, associated with PCZ in 12 cases. The MTD of fotemustine was 125 mg/m2 (days 5 and 12) with myelosuppression as the dose limiting toxicity (DLT). At this dose level, half of patients experienced grade 3 anemia, neutropenia or thrombopenia. No extra-hematological DLT was observed. No significant depletion of ATase activity by PCZ was evidenced. One partial response and 7 stable diseases were obtained leading to a disease control rate of 50%. The median times to progression and survival were 2.6 and 9.7 months, respectively. This combined regimen of PCZ and fotemustine was well tolerated with a good disease control rate in heavily pretreated glioma patients and merits further investigation in phase II studies.
    • Four-hourly scheduling of temozolomide improves tumour growth delay but not therapeutic index in A375M melanoma xenografts.

      Middleton, Mark R; Kelly, Jane; Goodger, Sarah J; Thatcher, Nick; Margison, Geoffrey P; Cancer Research Campaign Department of Medical Oncology, Christie Hospital NHS Trust, Manchester, UK. mmiddleton@picr.man.ac.uk (2000)
      PURPOSE: To establish whether temozolomide is more effective against A375M human melanoma xenografts if given every 4 h rather than every 24 h, in order to exploit depletion of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) by prior doses of the drug. METHODS: ATase depletion in A375M human melanoma xenografts was determined over 24 h after a single dose of temozolomide. The effect of different drug schedules (all of total dose 500 mg/kg) in delaying the growth of the xenografts was tested, and ATase depletion and DNA methylation damage assessed in tumour and normal tissue. RESULTS: Maximal depletion of ATase in tumour, to 2.52 +/- 0.23% of pretreatment levels, occurred 4-8 h after a single 100 mg/kg i.p. dose of temozolomide, with 23.0% recovery of protein levels at 24 h. Scheduling of temozolomide every 4 h increased tumour growth delay (33.6 +/- 1.39 days with temozolomide 100 mg/kg 4-hourly x versus 23.2 +/- 1.43 days with temozolomide 100 mg/kg once daily x 5; P < 0.0001) at the expense of increased toxicity (17.4 +/- 1.55% animal weight loss versus 10.6 +/- 1.27%. respectively). Temozolomide every 4 h did not increase ATase depletion compared with the 5-day schedule, but resulted in greater DNA 06-guanine methylation (29.0% more in tumour, 20.8% in liver and 56.0% in brain, comparing areas under the methylation-time curve). CONCLUSIONS: The 4-hourly schedule of temozolomide delayed tumour growth significantly more than the once-daily and 12-hourly schedules, probably as a result of greater DNA damage inflicted, but also increased toxicity. It remains to be seen if this regimen confers a net benefit over the standard schedule.
    • FOXF1 inhibits hematopoietic lineage commitment during early mesoderm specification.

      Fleury, Maud; Eliades, Alexia; Carlsson, P; Lacaud, Georges; Kouskoff, Valerie; Cancer Research UK Stem Cell Hematopoiesis Group, Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, M20 4BX, UK (2015-08-20)
      The molecular mechanisms orchestrating early mesoderm specification are still poorly understood. In particular, how alternate cell fate decisions are regulated in nascent mesoderm remains mostly unknown. In the present study, we investigated both in vitro in differentiating embryonic stem cells and in vivo in gastrulating embryos the lineage specification of early mesodermal precursors expressing or not the Forkhead transcription factor FOXF1. Our data revealed that FOXF1-expressing mesoderm is derived from FLK1(+) progenitors and that in vitro this transcription factor is expressed in smooth muscle and transiently in endothelial lineages but not in hematopoietic cells. In gastrulating embryos, FOXF1 marks most extra-embryonic mesoderm derivatives including the chorion, the allantois, the amnion and a subset of endothelial cells. Similarly to the in vitro situation, FOXF1 expression is excluded from the blood islands and blood cells. Further analysis revealed an inverse correlation between hematopoietic potential and FOXF1 expression in vivo with increase commitment toward primitive erythropoiesis in Foxf1 deficient embryos while FOXF1-enforced expression in vitro was shown to repress hematopoiesis. Altogether our data establish that, during gastrulation, FOXF1 marks all posterior primitive streak extra-embryonic mesoderm derivatives with the remarkable exception of the blood lineage. Our study further suggests that this transcription factor is implicated in actively restraining the specification of mesodermal progenitors to hematopoiesis.
    • FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function.

      Jensen, K; Binderup, T; Jensen, K; Therkelsen, I; Borup, R; Nilsson, E; Multhaupt, H; Bouchard, C; Quistorff, B; Kjaer, A; et al. (2011-11-16)
      Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.
    • Fractionated dose studies with X-rays and various alkylating agents in P388 mouse lymphoma cells.

      Anderson, Diana; Paterson Laboratories, Christie Hospital & Holt Radium Institute, Wilmslow Road, Manchester M20 9BX, UK. (1981)
      The fractionated dose technique has been used in P388F cells to examine the effects of X-rays and four alkylating agents on survival and induction of 5-iodo-2-deoxyuridine (IudR) resistant variants. Fractionation intervals up to 5 1/2 h were used for X-rays and for the alkylating agents up to 192 h. Fractionation of the X-ray dose resulted in a sparing effect for survival and variant induction. A sparing effect was also observed for survival after treatment with alkylating agents. However, variant frequencies were observed as large as or greater than those produced by the full doses of alkylating agents. For such agents this would suggest that survival and variant induction are independent events. Differences in the effects of X-rays and alkylating agents cannot be explained by differences in growth rate or the recovery of viability after treatment.
    • Fractionated high dose rate brachytherapy moulds--a precise treatment for carcinoma of the pinna.

      Allan, Ernest; Stanton, Anthony; Pye, David A; Collins, Conor D; Perry, Lesley A; Filby, Maeve; Wilkinson, John M; Department of Clinical Oncology, Christie Hospital, Withington, Manchester, UK. (1998-09)
      BACKGROUND AND PURPOSE: The aim of this paper is to describe a fractionated high dose rate brachytherapy procedure for the treatment of small superficial cancers of the pinna and to report the outcome in a small series of patients. MATERIALS AND METHODS: Thirteen patients with superficial cancers of the pinna, not invading cartilage, have been treated and in the majority of cases the tumour thickness was determined by a transdermal ultrasound measurement. For the single-plane moulds the prescribed surface dose was 45 Gy in eight fractions over 5 days and the moulds were constructed such that the full thickness of the disease, as determined by the ultrasound measurement, would lie within the 80% isodose surface. One case was treated with a sandwich mould and in this case the dose was reduced to 42.5 Gy. The treatment machine was a high dose rate microselectron, which contains a single stepping iridium source. RESULTS: The radiation reactions were of moderate severity, but were limited to the high dose volume. In all cases there was complete tumour resolution and rapid healing occurred leaving a barely perceptible scar. There were no recurrences over a minimum follow-up time of 18 months and there were no late radiation complications in this period. CONCLUSIONS: The treatment of superficial carcinoma of the pinna by means of HDR moulds is a safe and reliable technique. In this small series of patients there was total tumour control with excellent cosmesis.
    • Fractionation of chromatin by differential solubility in dilute salt.

      Itzhaki, Ruth F; Hell, A; Birnie, G D; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1978-03)
      Chromatin prepared from the livers of rats was fractionated on the basis of solubility in dilute NaCl. Neither of the fractions obtained was enriched in newly synthesized DNA. The salt-soluble fraction had a higher protein content (usually up to 50%) relative to the DNA, and contained 72% or more of the rapidly synthesized RNA. This RNA was found to be complexed with the salt-soluble deoxyribonucleoprotein, not merely co-solubilized with it. Also, polylysine-binding studies showed that about 70% or more of the nucleic acid phosphates were accessible as compared to about 40% in the unfractionated chromatin. These properties suggested that the soluble fraction was enriched in activity transcribed chromatin. In contrast molecular hybridization studies showed that the complexity of the DNA and its homology with cDNA transcribed from rat-liver polysomal mRNA were the same as those of DNA from unfractionated chromatin, or from the salt-insoluble fraction. This suggests that the criteria commonly accepted as distinguishing between euchromatin and heterochromatin in vitro are not invariably valid.
    • Fractionation of mammalian DNA on deae-cellulose.

      Millard, F; Fox, Brian W; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1975-04-09)
      Chromatography on a DEAE-cellulose (DE-52) column of native and denatured DNA from P388F cells has been studied. The main bulk of native DNA is eluted at 0.8M NaC1 and a minor fraction is eluted with 0.5N NaOH. The proportion of the DNA components obtained depends on the type of isotopic labelling used and the method of storing of the DNA preparation following isolation. Heat-denatured DNA elutes in 2 M NaC1 mainly within the pH gradient form 0.1 M NH4OH. When native DNA is chromatographed on DEAE-cellulose, a small fraction of the DNA elutes at 0.5 N NaOH. This fraction is double-stranded, as determined by hydroxyapatite chromatography. A similar component predominates, however, when "newly synthesised" DNA is fracionated. The profiles obtained with "newly synthesised" and "template" labelled DNA differ in their undenatured and heat-denatured configurations. The presence of formaldehyde during the chromatography of undenatured DNA leads to an increased homogeneity of the profile and during heat denaturation considerable modifications to the profile are observed. Some of the changes can be explained in terms of a decrease in the heterogeneity of the charge distribution on the DNA. The technique appears to combine a high degree of reproducibility with sensitivity to charge clustering along the DNA.
    • Fractionation sensitivity and the oxygen effect.

      Hendry, Jolyon H; Thames, H D; Department of Radiobiology, Paterson Institute for Cancer Research, Christie Hospital, Manchester. (1990-01)
    • The fragile X: a scanning electron microscope study.

      Harrison, Christine J; Jack, E M; Allen, Terence D; Harris, R; Department of Cell Biology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1983-08)
      Scanning electron microscopy (SEM) has been used to study the fragile X chromosome. The fragile site appears as an isochromatid gap in the majority of cases, confirming light microscope (LM) observations. SEM has allowed a more precise location of the fragile site to the Xq27 . 3 region.