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Molecular Pharmacology

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Abstract

A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase.

E A Austin and B E Huber
Molecular Pharmacology March 1993, 43 (3) 380-387;
E A Austin
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Abstract

We have developed a new approach involving gene therapy for the treatment of primary and metastatic tumors in the liver. As a first step toward the development of this gene therapy treatment for metastatic colorectal carcinoma, the Escherichia coli gene that encodes cytosine deaminase (CD) (EC 3.5.4.1) has been cloned. By using positive genetic selection, a plasmid carrying a 10.8-kilobase BamHI/EcoRI DNA insert was isolated that had CD enzymatic activity. Genetic screening, followed by enzymatic assays, identified a 3-kilobase DNA fragment that retained CD activity. Deamination of cytosine and 5-fluorocytosine (5-FC) by cloned CD was demonstrated. DNA and protein sequencing identified an open reading frame of 427 amino acids that encodes CD. To demonstrate that expression of CD in eukaryotic cells allows metabolism of the nontoxic prodrug 5-FC to the toxic metabolite 5-fluorouracil, CD was cloned into a eukaryotic expression vector and transfected into a human colorectal carcinoma cell line. Growth inhibition studies showed a shift in the IC50 for 5-FC from 17,000 microM in the parental cell line to 30 microM in cells expressing CD.

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Molecular Pharmacology
Vol. 43, Issue 3
1 Mar 1993
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Abstract

A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase.

E A Austin and B E Huber
Molecular Pharmacology March 1, 1993, 43 (3) 380-387;

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Abstract

A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase.

E A Austin and B E Huber
Molecular Pharmacology March 1, 1993, 43 (3) 380-387;
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