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E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Vass SO, Jarrom D, Wilson WR, Hyde EI, Searle PF - Br. J. Cancer (2009)

Bottom Line: We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB.Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB.NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK.

ABSTRACT
Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

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Sensitisation of Escherichia coli to CB1954 by the expression of NfsA or NfsB. Log phase cultures of E. coli UT5600 stably lysogenised with bacteriophage λ-vectors expressing NfsA or NfsB, or empty vector as control, were diluted and plated on agar containing 0, 50, 100, 200 or 300 μM CB1954. Colonies were counted after 24 h, and expressed as a percentage (%) of the number obtained from the same liquid culture plated in the absence of a prodrug (mean and range of duplicates).
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fig1: Sensitisation of Escherichia coli to CB1954 by the expression of NfsA or NfsB. Log phase cultures of E. coli UT5600 stably lysogenised with bacteriophage λ-vectors expressing NfsA or NfsB, or empty vector as control, were diluted and plated on agar containing 0, 50, 100, 200 or 300 μM CB1954. Colonies were counted after 24 h, and expressed as a percentage (%) of the number obtained from the same liquid culture plated in the absence of a prodrug (mean and range of duplicates).

Mentions: As shown in Figure 1, bacterial lysogens carrying the empty vector (λJG3J1) showed no reduction in plating efficiency at CB1954 concentrations up to 300 μM, indicating that the expression levels of endogenous NfsA or other enzymes do not confer significant sensitisation to the prodrug over this range. Lysogens carrying the vector λJG16C2, which expresses NfsB, showed complete inhibition of colony formation at 300 and 200 μM CB1954, but at 100 μM CB1954 and below the plating efficiency was ⩾80%. Bacteria carrying λSV054, expressing NfsA, showed greater sensitisation to CB1954, with complete inhibition of colony formation at ⩾100 μM CB1954, implying that NfsA catalyses the activation of CB1954 more efficiently than does NfsB.


E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Vass SO, Jarrom D, Wilson WR, Hyde EI, Searle PF - Br. J. Cancer (2009)

Sensitisation of Escherichia coli to CB1954 by the expression of NfsA or NfsB. Log phase cultures of E. coli UT5600 stably lysogenised with bacteriophage λ-vectors expressing NfsA or NfsB, or empty vector as control, were diluted and plated on agar containing 0, 50, 100, 200 or 300 μM CB1954. Colonies were counted after 24 h, and expressed as a percentage (%) of the number obtained from the same liquid culture plated in the absence of a prodrug (mean and range of duplicates).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690450&req=5

fig1: Sensitisation of Escherichia coli to CB1954 by the expression of NfsA or NfsB. Log phase cultures of E. coli UT5600 stably lysogenised with bacteriophage λ-vectors expressing NfsA or NfsB, or empty vector as control, were diluted and plated on agar containing 0, 50, 100, 200 or 300 μM CB1954. Colonies were counted after 24 h, and expressed as a percentage (%) of the number obtained from the same liquid culture plated in the absence of a prodrug (mean and range of duplicates).
Mentions: As shown in Figure 1, bacterial lysogens carrying the empty vector (λJG3J1) showed no reduction in plating efficiency at CB1954 concentrations up to 300 μM, indicating that the expression levels of endogenous NfsA or other enzymes do not confer significant sensitisation to the prodrug over this range. Lysogens carrying the vector λJG16C2, which expresses NfsB, showed complete inhibition of colony formation at 300 and 200 μM CB1954, but at 100 μM CB1954 and below the plating efficiency was ⩾80%. Bacteria carrying λSV054, expressing NfsA, showed greater sensitisation to CB1954, with complete inhibition of colony formation at ⩾100 μM CB1954, implying that NfsA catalyses the activation of CB1954 more efficiently than does NfsB.

Bottom Line: We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB.Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB.NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK.

ABSTRACT
Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Show MeSH
Related in: MedlinePlus