nitroreductases

Summary

Summary: Enzymes which reduce nitro groups (NITRO COMPOUNDS) and other nitrogenous compounds.

Top Publications

  1. Aiub C, Mazzei J, Pinto L, Felzenszwalb I. Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity. Chem Biol Interact. 2006;161:146-54 pubmed
    ..b>Nitroreductases from bacteria seem to have a wide spectrum of substrates, as observed by the reduction of several ..
  2. Lovering A, Hyde E, Searle P, White S. The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution. J Mol Biol. 2001;309:203-13 pubmed
    ..Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity. ..
  3. Curado S, Stainier D, Anderson R. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat Protoc. 2008;3:948-54 pubmed publisher
    ..Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h. ..
  4. Grove J, Searle P, Weedon S, Green N, McNeish I, Kerr D. Virus-directed enzyme prodrug therapy using CB1954. Anticancer Drug Des. 1999;14:461-72 pubmed
    ..The efficacy of this enzyme-prodrug approach in model systems compared with other VDEPT approaches demonstrates the feasibility and future promise of this gene therapy strategy. ..
  5. Anlezark G, Melton R, Sherwood R, Coles B, Friedlos F, Knox R. The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol. 1992;44:2289-95 pubmed
    ..The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM). ..
  6. Green N, McNeish I, Doshi R, Searle P, Kerr D, Young L. Immune enhancement of nitroreductase-induced cytotoxicity: studies using a bicistronic adenovirus vector. Int J Cancer. 2003;104:104-12 pubmed
  7. Nakamura J, Kubota Y, Miyaoka M, Saitoh T, Mizuno F, Benno Y. Comparison of four microbial enzymes in Clostridia and Bacteroides isolated from human feces. Microbiol Immunol. 2002;46:487-90 pubmed
    ..05) higher than that in the clostridia isolated from healthy subjects and the subjects given high beef diets. However, the activities of some microorganisms tested showed marked differences in each strain. ..
  8. Race P, Lovering A, White S, Grove J, Searle P, Wrighton C, et al. Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954. J Mol Biol. 2007;368:481-92 pubmed
  9. Cellier M, Fabrega O, Fazackerley E, James A, Orenga S, Perry J, et al. 2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria. Bioorg Med Chem. 2011;19:2903-10 pubmed publisher
    ..The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested. ..

More Information

Publications62

  1. Vass S, Jarrom D, Wilson W, Hyde E, Searle P. E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954. Br J Cancer. 2009;100:1903-11 pubmed publisher
    ..NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs. ..
  2. Felmer R, Clark J. The gene suicide system Ntr/CB1954 causes ablation of differentiated 3T3L1 adipocytes by apoptosis. Biol Res. 2004;37:449-60 pubmed
  3. Ma D, Allen N, Van Bergen Y, Jones C, Baum M, Keverne E, et al. Selective ablation of olfactory receptor neurons without functional impairment of vomeronasal receptor neurons in OMP-ntr transgenic mice. Eur J Neurosci. 2002;16:2317-23 pubmed
    ..This novel technique could potentially be applied to selectively ablate olfactory receptor neurons expressing a particular olfactory receptor by linking its expression to that of the nitroreductase enzyme. ..
  4. Singh R, Manjunatha U, Boshoff H, Ha Y, Niyomrattanakit P, Ledwidge R, et al. PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science. 2008;322:1392-5 pubmed publisher
    ..Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system. ..
  5. Wilkinson S, Kelly J. Trypanocidal drugs: mechanisms, resistance and new targets. Expert Rev Mol Med. 2009;11:e31 pubmed publisher
  6. Purkayastha A, McCue L, McDonough K. Identification of a Mycobacterium tuberculosis putative classical nitroreductase gene whose expression is coregulated with that of the acr aene within macrophages, in standing versus shaking cultures, and under low oxygen conditions. Infect Immun. 2002;70:1518-29 pubmed
    ..tuberculosis genes of previously unknown function that belong to an unusual superfamily of classical nitroreductases, which may have a role for bacteria within the host environment...
  7. Baker N, Alsford S, Horn D. Genome-wide RNAi screens in African trypanosomes identify the nifurtimox activator NTR and the eflornithine transporter AAT6. Mol Biochem Parasitol. 2011;176:55-7 pubmed publisher
    ..Thus, bloodstream-form T. brucei RNAi libraries present a versatile tool for selective genetic screening and for the rapid identification of drug-activation, uptake and potential resistance mechanisms. ..
  8. Atwell G, Yang S, Pruijn F, Pullen S, Hogg A, Patterson A, et al. Synthesis and structure-activity relationships for 2,4-dinitrobenzamide-5-mustards as prodrugs for the Escherichia coli nfsB nitroreductase in gene therapy. J Med Chem. 2007;50:1197-212 pubmed
    ..A bromomesylate mustard showed superior activity against WiDr tumors grown from 1:9 mixtures of NTR+ve and NTR-ve cells, indicating a strong bystander effect in vivo. ..
  9. Hall B, Bot C, Wilkinson S. Nifurtimox activation by trypanosomal type I nitroreductases generates cytotoxic nitrile metabolites. J Biol Chem. 2011;286:13088-95 pubmed publisher
    ..These experiments indicate that the basis for the selectivity of nifurtimox against T. brucei lies in the expression of a parasite-encoded type I nitroreductase. ..
  10. Kwon D, Kato M, el Zaatari F, Osato M, Graham D. Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance. FEMS Microbiol Lett. 2000;188:197-202 pubmed
    ..coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates. ..
  11. Prosser G, Copp J, Syddall S, Williams E, Smaill J, Wilson W, et al. Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954. Biochem Pharmacol. 2010;79:678-87 pubmed publisher
    ..In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation. ..
  12. Palmer D, Mautner V, Mirza D, Oliff S, Gerritsen W, van der Sijp J, et al. Virus-directed enzyme prodrug therapy: intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer. J Clin Oncol. 2004;22:1546-52 pubmed
    ..The high level of nitroreductase expression observed at 1 to 5 x 10(11) virus particles mandates further studies in patients with inoperable tumors who will receive CTL102 and CB1954. ..
  13. Race P, Lovering A, Green R, Ossor A, White S, Searle P, et al. Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme. J Biol Chem. 2005;280:13256-64 pubmed
    ..We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity. ..
  14. Nixon J, Wang A, Field J, Morrison H, McArthur A, Sogin M, et al. Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica. Eukaryot Cell. 2002;1:181-90 pubmed
    ..giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis...
  15. Chung Faye G, Palmer D, Anderson D, Clark J, Downes M, Baddeley J, et al. Virus-directed, enzyme prodrug therapy with nitroimidazole reductase: a phase I and pharmacokinetic study of its prodrug, CB1954. Clin Cancer Res. 2001;7:2662-8 pubmed
    ..We are now conducting a Phase I trial combining adenovirus-mediated NTR and i.v. CB1954 (24 mg/m2) in patients with primary and secondary liver tumors. ..
  16. Montgomery J, Parsons M, Hyde D. A novel model of retinal ablation demonstrates that the extent of rod cell death regulates the origin of the regenerated zebrafish rod photoreceptors. J Comp Neurol. 2010;518:800-14 pubmed publisher
    ..Second, the rod cell death must be acute, rather than chronic, to stimulate regeneration from the Müller glia. This suggests that the zebrafish retina possesses mechanisms to quantify the amount and timing of rod cell death. ..
  17. Cortial S, Chaignon P, Iorga B, Aymerich S, Truan G, Gueguen Chaignon V, et al. NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: insight into its biological role. FEBS Lett. 2010;584:3916-22 pubmed publisher
    ..Finally, we showed that NfrA1 is able to rapidly scavenge H(2)O(2) produced during the oxidative process or added exogenously. ..
  18. Guise C, Grove J, Hyde E, Searle P. Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle. Gene Ther. 2007;14:690-8 pubmed
    ..Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR. ..
  19. Kadiyala V, Nadeau L, Spain J. Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols. Appl Environ Microbiol. 2003;69:6520-6 pubmed
  20. Parkinson G, Skelly J, Neidle S. Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme. J Med Chem. 2000;43:3624-31 pubmed
    ..The structure of the substrate binding pocket has been used to examine substrate specificity and enzyme kinetics for prodrugs used in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT). ..
  21. Bryant C, DeLuca M. Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. J Biol Chem. 1991;266:4119-25 pubmed
    ..The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their ..
  22. Mitchell D, Minchin R. E. coli nitroreductase/CB1954 gene-directed enzyme prodrug therapy: role of arylamine N-acetlytransferase 2. Cancer Gene Ther. 2008;15:758-64 pubmed publisher
    ..Moreover, endogenous NAT2, which is localized predominantly in the liver and gut, may be involved in the dose-limiting hepatic toxicity and gastrointestinal side effects seen in patients treated with the higher doses of CB1954. ..
  23. Pal D, Banerjee S, Cui J, Schwartz A, Ghosh S, Samuelson J. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases). Antimicrob Agents Chemother. 2009;53:458-64 pubmed publisher
    ..Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased ..
  24. Wilson W, Pullen S, Hogg A, Helsby N, Hicks K, Denny W. Quantitation of bystander effects in nitroreductase suicide gene therapy using three-dimensional cell cultures. Cancer Res. 2002;62:1425-32 pubmed
    ..This study demonstrates the utility of the multilayer tissue culture model for quantifying and optimizing bystander effects in tumors and identifies a new lead prodrug for NTR gene-dependent enzyme-prodrug therapy. ..
  25. Kitts C, Green C, Otley R, Alvarez M, Unkefer P. Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). Can J Microbiol. 2000;46:278-82 pubmed
    ..morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive...
  26. Zenno S, Koike H, Kumar A, Jayaraman R, Tanokura M, Saigo K. Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase. J Bacteriol. 1996;178:4508-14 pubmed
    ..Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor ..
  27. Jorgensen M, Trend M, Hazell S, Mendz G. Potential involvement of several nitroreductases in metronidazole resistance in Helicobacter pylori. Arch Biochem Biophys. 2001;392:180-91 pubmed
    ..The results confirmed the presence of rdxA null mutations in resistant strains and suggested that other factors involved in the metabolism of metronidazole contributed to the resistant phenotype. ..
  28. de Oliveira I, Henriques J, Bonatto D. In silico identification of a new group of specific bacterial and fungal nitroreductases-like proteins. Biochem Biophys Res Commun. 2007;355:919-25 pubmed
    ..The nitroreductases are found within bacterial and some eukaryotic species...
  29. Davison J, Akitake C, Goll M, Rhee J, Gosse N, Baier H, et al. Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev Biol. 2007;304:811-24 pubmed
  30. Singleton D, Li D, Bai S, Syddall S, Smaill J, Shen Y, et al. The nitroreductase prodrug SN 28343 enhances the potency of systemically administered armed oncolytic adenovirus ONYX-411(NTR). Cancer Gene Ther. 2007;14:953-67 pubmed
    ..However, subsequent administration of SN 28343 resulted in the majority of mice (62.5%) being tumor-free on day 120. ..
  31. Kutty R, Bennett G. Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from Clostridium acetobutylicum ATCC 824. Arch Microbiol. 2005;184:158-67 pubmed publisher
    The genes that encode oxygen-insensitive nitroreductases from Clostridium acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized...
  32. Green N, Youngs D, Neoptolemos J, Friedlos F, Knox R, Springer C, et al. Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene. Cancer Gene Ther. 1997;4:229-38 pubmed
  33. Zenno S, Kobori T, Tanokura M, Saigo K. Purification and characterization of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase. Biosci Biotechnol Biochem. 1998;62:1978-87 pubmed
  34. Roldán M, Pérez Reinado E, Castillo F, Moreno Vivián C. Reduction of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol Rev. 2008;32:474-500 pubmed publisher
    ..Bacterial nitroreductases are flavoenzymes that catalyze the NAD(P)H-dependent reduction of the nitro groups on nitroaromatic and ..
  35. Mendz G, Megraud F. Is the molecular basis of metronidazole resistance in microaerophilic organisms understood?. Trends Microbiol. 2002;10:370-5 pubmed
    ..This review presents the status of our current knowledge of metronidazole resistance and outlines reasons to explain some of the conflicting evidence and controversy in the interpretation of results in this area. ..
  36. Wilkinson S, Taylor M, Horn D, Kelly J, Cheeseman I. A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes. Proc Natl Acad Sci U S A. 2008;105:5022-7 pubmed publisher
    ..This potential for drug resistance by a simple mechanism has important implications, because nifurtimox is currently undergoing phase III clinical trials against African trypanosomiasis. ..
  37. Kobori T, Sasaki H, Lee W, Zenno S, Saigo K, Murphy M, et al. Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution. J Biol Chem. 2001;276:2816-23 pubmed
    ..The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group. ..
  38. Prosser G, Patterson A, Ackerley D. uvrB gene deletion enhances SOS chromotest sensitivity for nitroreductases that preferentially generate the 4-hydroxylamine metabolite of the anti-cancer prodrug CB1954. J Biotechnol. 2010;150:190-4 pubmed publisher
    ..We describe here two efficient and previously uncharacterized nitroreductases, YfkO from Bacillus subtilis which reduces CB1954 exclusively at the 4-NO(2) position, and NfsA from ..
  39. Gurumurthy M, Mukherjee T, Dowd C, Singh R, Niyomrattanakit P, Tay J, et al. Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles. FEBS J. 2012;279:113-25 pubmed publisher
    ..The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding. ..
  40. Rau J, Stolz A. Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases. Appl Environ Microbiol. 2003;69:3448-55 pubmed
    ..In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase. ..
  41. Whiteway J, Koziarz P, Veall J, Sandhu N, Kumar P, Hoecher B, et al. Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J Bacteriol. 1998;180:5529-39 pubmed
    ..compounds is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the nfsA and nfsB genes of a large number of nitrofuran-resistant mutants of ..
  42. Anlezark G, Melton R, Sherwood R, Wilson W, Denny W, Palmer B, et al. Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase. Biochem Pharmacol. 1995;50:609-18 pubmed
    ..This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system. ..
  43. Michael N, Brehm J, Anlezark G, Minton N. Physical characterisation of the Escherichia coli B gene encoding nitroreductase and its over-expression in Escherichia coli K12. FEMS Microbiol Lett. 1994;124:195-202 pubmed
    ..Substitution of the nfrB ribosome binding site with that of the E. coli lacZ gene reduced production levels of nitroreductase. The sequenced region also contained two incomplete open reading frames of unknown function. ..
  44. Minton N. Clostridia in cancer therapy. Nat Rev Microbiol. 2003;1:237-42 pubmed
  45. Hall B, Wu X, Hu L, Wilkinson S. Exploiting the drug-activating properties of a novel trypanosomal nitroreductase. Antimicrob Agents Chemother. 2010;54:1193-9 pubmed publisher
    ..We conclude that halogenated nitrobenzylphosphoramide mustards represent a novel class of antitrypanosomal agents, and their efficacy validates the strategy of specifically targeting NTR activity to develop new therapeutics. ..
  46. Cellitti S, Shaffer J, Jones D, Mukherjee T, Gurumurthy M, Bursulaya B, et al. Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824. Structure. 2012;20:101-12 pubmed publisher
    ..The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data. ..
  47. Djeha A, Hulme A, Dexter M, Mountain A, Young L, Searle P, et al. Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954. Cancer Gene Ther. 2000;7:721-31 pubmed
    ..We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ. ..
  48. Cui W, Allen N, Skynner M, Gusterson B, Clark A. Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain. Glia. 2001;34:272-82 pubmed
    ..These results provide the first evidence that astrocytes are important for the survival of neurons in the adult brain in vivo. ..
  49. Curado S, Anderson R, Jungblut B, Mumm J, Schroeter E, Stainier D. Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev Dyn. 2007;236:1025-35 pubmed
  50. Olekhnovich I, Goodwin A, Hoffman P. Characterization of the NAD(P)H oxidase and metronidazole reductase activities of the RdxA nitroreductase of Helicobacter pylori. FEBS J. 2009;276:3354-64 pubmed publisher
    ..Unlike other nitroreductases, His6-RdxA exhibited potent NAD(P)H-oxidase activity (k(cat) = 2...
  51. Kim P, Zhang L, Manjunatha U, Singh R, Patel S, Jiricek J, et al. Structure-activity relationships of antitubercular nitroimidazoles. 1. Structural features associated with aerobic and anaerobic activities of 4- and 5-nitroimidazoles. J Med Chem. 2009;52:1317-28 pubmed publisher
    ..Aerobic activity appears to be correlated with efficiency as a substrate for Ddn, suggesting a means of structure-based optimization of improved nitroimidazoles. ..
  52. Buddington R, Williams C, Chen S, Witherly S. Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. Am J Clin Nutr. 1996;63:709-16 pubmed
    ..These findings indicate that 4 g neosugar/d alters the fecal flora in a manner perceived as beneficial by decreasing activities of some reductive enzymes. ..
  53. Helsby N, Ferry D, Patterson A, Pullen S, Wilson W. 2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. Br J Cancer. 2004;90:1084-92 pubmed
    ..These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts. ..