S U M M A R YThe single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. T HE single-cell gel assay (also termed comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. The technique was developed to visualize the DNA damage induced by radiation in mammalian cells (Ostling and Johanson 1984). This method has many applications in radiation biology, in estimation of oxidative damages and DNA crosslinks, in apoptosis, and in genotoxicity induced by chemical compounds (McKelvey-Martin et al. 1993). A small number of cells are immersed in an agarose gel, lysed, subjected to an electrophoretic field, and then stained with a DNA-binding fluorescent dye. The broken DNA fragments, negatively charged, migrate towards the anode and the cells can be observed under a fluorescent microscope for estimation of the damaged DNA that forms a tail like a "comet." The quantity of DNA separated from the head of the comet is proportional to the dose of irradiation.Several versions of the comet assay are in use in research laboratories, and there are also commercial kits. There are two forms of comet assays: the neutral method for the detection of DNA double-strand breaks, and the alkaline method, which detects DNA single-strand breaks and alkali-labile lesions (Fairbairn et al. 1995). Usually, the comets are visualized and evaluated with fluorescent DNA stains, such as propidium iodide or ethidium bromide (potential carcinogens). These DNA stainings require a high-quality microscope with epifluorescent optics, a 100-W mercury lamp, a sensitive CCD camera light, and sophisticated image analysis software. Unfortunately, the slides can not be stored for long periods of time (the dye is bleached out within a day) so they must be analyzed immediately (an alternative is to store the slides with the gels and re-stain them when reexamination of the samples is needed).We are working with lymphocytes from the peripheral blood of cancer patients to evaluate the effect of cytotoxic drugs. Cells are analyzed before and after chemotherapy. We have developed a silver staining of the comets that allows long-term storage and retrospective comparative evaluation of the cells before and after therapy. We tested a published silver staining method (Trevigen, Inc. 1999) but obtained a very low sensitivity (data not shown). Here we show the modifications introduced to the silver staining metho...
Heat shock proteins (HSPs), also known as molecular chaperones, participate in important cellular processes, such as protein aggregation, disaggregation, folding, and unfolding. HSPs have cytoprotective functions that are commonly explained by their antiapoptotic role. Their involvement in anticancer drug resistance has been the focus of intense research efforts, and the relationship between HSP induction and DNA repair mechanisms has been in the spotlight during the past decades. Because DNA is permanently subject to damage, many DNA repair pathways are involved in the recognition and removal of a diverse array of DNA lesions. Hence, DNA repair mechanisms are key to maintain genome stability. In addition, the interactome network of HSPs with DNA repair proteins has become an exciting research field and so their use as emerging targets for cancer therapy. This article provides a historical overview of the participation of HSPs in DNA repair mechanisms as part of their molecular chaperone capabilities.
Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor β (RARβ) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARβ protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARβ gene expression that was greatest after 72 hrs with a concentration 1 μM. High concentrations of RA increased the expression of RARβ causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration-related proteins [moesin, c-Src and focal adhesion kinase (FAK)]. The treatment with RARα and RARγ agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARβ-agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARβ gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin, c-Src and FAK expressions. RARβ is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration.
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