The ability to repair DNA damage is strongly associated with the risk of cancer and other human diseases as it is essential for maintenance of genome stability. Moreover, DNA repair capacity is an important factor contributing to the inter-individual variability in mutagen exposure, cancer development and treatment through an individualized adjusted therapy. In addition to genotypes, functional phenotypic assays which integrate the different pathways provide useful tools to explore the role of DNA repair in cancer susceptibility. This review compares the presently available cellular DNA repair phenotype assays based on their characteristics, and discusses their advantages and limitations. Assays for assessment of DNA repair phenotype should be well characterized in terms of reliability, validity, sensitivity, inter- and intra-individual variability, and cancer predictivity. Our comparison reveals that the G 1 and G 2 challenge assays, although labour-intensive, can be considered as very useful assays to investigate DNA repair phenotype. They have been successfully applied to investigate repair capacity of both cancer patients and environmentally exposed populations, and can detect deficiencies in different repair pathways. Moreover, these assays allow to predict the cancer therapy responses and to investigate the cancer prognosis. Nevertheless, the choice of the assay depends on the scientific question addressed and on the objective of its application and more prospective studies are needed since the phenotype could reflect the pathophysiological alterations in the patient secondary to the disease.

Phenotyping for DNA repair capacity.

Quantitative image analysis for immunocytochemistry and in situ hybridization

The unscheduled DNA synthesis (UDS) assay measures a cell's ability to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique in living cells by creating 6-4 photoproducts and pyrimidine dimers using UVC irradiation, then allowing for their repair. Repair is quantified by the amount of radioactive thymidine incorporated after this insult, and the length of time allowed for this incorporation is specific for repair of particular lesions. Radioactivity is evaluated by grain counting after autoradiography. The results are used to diagnosis repair-deficient disorders clinically and provide a basis for investigation of repair deficiency in human tissues or tumors. At the present time, no other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation, as discussed in Chapter 28, is not an equivalent technique, as it specifically measures transcription-coupled repair at active genes, a subset of total NER.

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Unscheduled DNA Synthesis: A Functional Assay for Global Genomic Nucleotide Excision Repair

Grey matter abnormalities are associated only with severe cognitive decline in early stages of Parkinson's disease.

Pseudopregnancy (PSP) in the female rat is a neuroendocrine condition that is induced by repeated and intermittent vaginocervical stimulation received during mating and involves the expression of bicircadian prolactin surges and cessation of normal estrous cyclicity for 10–12 days postmating. The temporal patterning and number of intromissions received by the female are critical for PSP initiation, and thus, short-term encoding of VCS occurs during transduction of intromissions into PSP. In this experiment, we characterized and compared the mating-induced neural activation patterns within amygdalar and hippocampal regions using expression of the immediate early genes FOS, EGR-1, and ARC. Cycling female rats mated on proestrus received 15 or 5 intromissions under paced or nonpaced mating conditions. High numbers of intromissions during nonpaced mating or low numbers received during paced mating are sufficient to induce PSP, while five nonpaced intromissions and mounts without intromission are insufficient. Here we demonstrate that the CA1 region of the hippocampus was selectively sensitive to PSP-sufficient but not PSP-insufficient mating stimulation by showing significant effects of paced mating for all three IEGs. Paced mating also stimulated the expression of ARC within the basolateral, cortical, and central nuclei of the amygdala. The posterodorsal medial amygdala also showed selective EGR-1 responses to PSP-sufficient mating stimulation. There was no effect of hemisphere on IEG expression. The postmating expression profiles of these IEGs provide evidence that limbic areas involved in encoding and consolidation of memory are involved in initiating the neuroendocrine memory of PSP. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007.

Carol Thouron

Papers

Semiautomatic image analysis for grain counting in in situ hybridization experiments.