Mechanism of the Cytotoxic Action of Alkylating Agents in Mammalian Cells and Evidence for the Removal of Alkylated Groups from Deoxyribonucleic Acid

Nature ◽  
1966 ◽  
Vol 211 (5045) ◽  
pp. 150-153 ◽  
Author(s):  
A. R. CRATHORN ◽  
J. J. ROBERTS
Keyword(s):  
Mammalian Cells ◽  
Deoxyribonucleic Acid ◽  
Alkylating Agents ◽  
Cytotoxic Action

The influence of radiomimetic substances on deoxyribonucleic acid synthesis and function studied in Escherichia coli / phage systems V. A comparison of the inactivation of phages T 2, T 7 and two strains of T 4 by alkylating agents

1959 ◽  
Vol 151 (942) ◽  
pp. 148-155 ◽  
Keyword(s):  
Deoxyribonucleic Acid ◽  
Osmotic Shock ◽  
Alkylating Agents ◽  
Acid Synthesis ◽  
Acid Moiety ◽  
Rate Of Penetration ◽  
Injection Process ◽  
Deoxyribonucleic Acid Synthesis ◽  
And Function ◽  
Escherichia Coli Phage

The sensitivity of phage T 7 to epoxides and freshly prepared solutions of di(2-chloroethyl) methylamine ( HN 2) was identical with that of T 2. T 7, however, proved considerably the more sensitive to ethylenimine and to aged solutions of HN 2. It was considered that this was due to the cationic nature of these latter agents affecting the rate of penetration into the phage heads, and that the susceptibility of T 2 and resistance of T 7 to osmotic shock was a parallel phenomenon. Confirmation was afforded by the fact that a strain of T 4 sensitive to osmotic shock behaved like T 2, and a resistant strain of T 4 like T 7. These results, together with others previously reported, are believed to offer very strong evidence that inactivation of bacteriophage by alkylating agents derives from reaction with the deoxyribonucleic acid moiety, probably leading to a failure of the injection process.

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

Recovery from Sub-lethal X-ray Damage of Mammalian Cells during Inhibition of Synthesis of Deoxyribonucleic Acid

Nature ◽  
1964 ◽  
Vol 204 (4958) ◽  
pp. 598-599 ◽  
Author(s):  
J. H. KIM ◽  
M. L. EIDINOFF ◽  
J. S. LAUGHLIN
Keyword(s):  
Mammalian Cells ◽  
Deoxyribonucleic Acid ◽  
X Ray

scholarly journals Alterations of Gene Structure in Ethyl Methane Sulfonate-Induced Mutants of Mammalian Cells

1982 ◽  
Vol 2 (11) ◽  
pp. 1459-1462 ◽  
Author(s):  
Mark Meuth ◽  
Janet E. Arrand
Keyword(s):  
Gene Structure ◽  
Restriction Enzyme ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Induced Mutants ◽  
Enzyme Cleavage ◽  
Internal Gene

To determine the types of alterations in gene structure induced by DNA-alkylating agents, we analyzed the restriction enzyme cleavage patterns of adenine phosphoribosyltransferase gene sequences in mutant strains of Chinese hamster ovary cells deficient in this enzyme. Base pair changes as detected by loss of restriction enzyme sites were found, but no major internal gene rearrangements could be detected.

scholarly journals Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3′-5′)thymidine

1978 ◽  
Vol 171 (3) ◽  
pp. 575-587 ◽  
Author(s):  
D H Swenson ◽  
P D Lawley
Keyword(s):  
Alkyl Group ◽  
Deoxyribonucleic Acid ◽  
Dimethyl Sulphate ◽  
Alkylating Agents ◽  
Electrophilic Reagent ◽  
Relative Reactivity ◽  
Ethyl Methanesulphonate ◽  
A Value ◽  
Phosphodiester Group ◽  
Steric Accessibility

1. The ethyl phosphotriester of thymidylyl(3′-5′)thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates.

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