PubMed · 6618584
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1983. N-Nitroso compounds.. https://pubmed.ncbi.nlm.nih.gov/6618584/
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DNA damage results from a wide variety of external agents such as chemicals and radiation. The consequences of exposure to agents that damage DNA have been traditionally studied from the perspective of cell survival and mutagenesis. Mutations are late endpoints of DNA damage. Cells respond to the earlier stages of DNA damage by inducing the expression of several genes, including those specific of the nature of the lesion. These early transcriptional responses are likely to predetermine the later fate of the damaged cell. Genes activated during this early response include those involved in DNA repair, replication, and growth control. We are interested in the transcriptional mechanisms by which cells respond to DNA damaging agents. To facilitate the measurement of gene induction, we used seven different reporter constructs integrated stably into the RKO cell line derived from a human colon carcinoma. These constructs were derived from promoters and/or response elements isolated from genes associated with DNA damage responses in human cells, and were fused to the bacterial reporter gene, choramphenicol acetyl transferase (CAT). The cell lines generated in this manner contain the promoters and/or response elements representing DNA polymerase beta, p53, gadd (growth arrest and DNA damage) 45 and 153, c-fos, TPA response element, and tissue-type plasminogen activator. These recombinant cell lines were assembled in a 96-well microtiter plate permitting their simultaneous exposure to compounds and subsequent CAT protein measurement. This assembly has been designated the CAT-Tox (D) assay. These cell lines were exposed to different classes of DNA damaging agents including those which covalently join bases to form dimers (e.g., UVC irradiation), generate DNA adducts by alkylation (e.g., methylmethane sulfonate [MMS], ethylmethane sulfonate [EMS], N-methyl-N-nitro-N-nitrosoguanine [MNNG], dimethylnitrosamine [DMN]), cross-link DNA (e.g., mitomycin C), and inhibit DNA replication by intercalative (e.g., actinomycin D) and nonintercalative (e.g., hydroxyurea) mechanisms. The transcriptional responses were measured as a function of the accumulation of CAT protein using antibodies against CAT protein in a standard ELISA. Endogenous cellular responses were evaluated for a number of the genes represented in the assay at both the mRNA and protein levels by Northern and Western blot analysis, respectively. These data corroborate the stress-induced responses measured by CAT ELISA in the CAT-Tox (D) assay, demonstrating the usefulness of this assay as a rapid and sensitive method for detection of DNA damaging agents in human cells.
In the present study, the effects of data base size on predictivity, informational content and structural overlap of derived Structure-Activity Relationship (SAR) models were investigated. It was found that indices of predictivity (i.e., sensitivity, specificity, and concordance between experimental and predicted results (OCP) increased with increasing size of the data base until the range is 300-400 chemicals, at which point they plateau. The greater the size of the data base, the greater the informational content of the model; however, the rate of this increase is no longer optimal when the size of the data base exceeds 400 chemicals.