A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase.
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.
Austin
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Wellcome Research Labs, Research Triangle Park, North Carolina 27709.
Huber
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000473:pathology / Q000628:therapy*; D043525:Cytosine Deaminase; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005437:Flucytosine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D015316:Genetic Therapy; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009700:Nucleoside Deaminases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured
D004269:DNA, Bacterial; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil