Development and field-test validation of an assay for DNA repair in circulating human lymphocytes

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studie...

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Bibliographic Details
Published in:Cancer research (Chicago, Ill.) Ill.), 1991-11, Vol.51 (21), p.5786-5793
Main Authors: ATHAS, W. F, HEDAYATI, M. A, MATANOSKI, G. M, FARMER, E. R, GROSSMAN, L
Format: Article
Language:English
Subjects:
Adult
B-Lymphocytes - physiology
B-Lymphocytes - radiation effects
Benzo(a)pyrene - pharmacology
Biological and medical sciences
Carcinoma, Basal Cell - genetics
Cell Line
Cell Line, Transformed
Chloramphenicol O-Acetyltransferase - genetics
Chloramphenicol O-Acetyltransferase - metabolism
DNA Damage
DNA Repair - radiation effects
DNA, Recombinant - drug effects
DNA, Recombinant - radiation effects
Dose-Response Relationship, Radiation
Female
Fundamental and applied biological sciences. Psychology
Genetic Vectors
Heterozygote
Humans
Male
Middle Aged
Molecular and cellular biology
Molecular genetics
Mutagenesis. Repair
Plasmids - radiation effects
Reference Values
Skin - radiation effects
Skin Neoplasms - genetics
Transfection
Tumor Cells, Cultured
Ultraviolet Rays
Xeroderma Pigmentosum
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Summary:A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.
ISSN:0008-5472
1538-7445