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) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (G10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (G66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.
Melanoma cells that colonize the brain harbor numerous genetically and epigenetically altered genes. This study presents an integrated genomic and epigenomic analysis that reveals MBM-specific molecular alterations and mutually exclusive molecular subtypes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.