A filter elution assay for the simultaneous detection of DNA double and single strand breaks.

This paper reports a gravity-flow filter elution assay that permits the simultaneous detection of DNA double strand breaks (DSBs, as neutral elutable DNA) and DNA single strand breaks (SSBs, as alkaline elutable DNA). This assay is based on a modification of a conventional gravity-flow alkaline elution procedure. The elution fraction resulting from cell lysis before the application of the alkaline elution buffer provides an estimate of DNA DSBs, while the fraction eluted by the alkaline buffer (pH 12.3) measures the extent of DNA SSBs. Data show that, in addition to its good detection sensitivity for SSBs, this method provides a sensitive measure of DNA DSBs. As an illustration of its specificity, this method unambiguously distinguished DNA SSBs induced by X-irradiation of mouse leukemia L1210 cells from DNA DSBs occurring in cells undergoing nucleosomal DNA fragmentation and apoptosis. The neutral elution fraction is a good predictor of loss of cell viability induced either by selenite at pharmacological levels or by growth factor depletion. The filter elution method reported is more specific and informative than a nick translation assay for the detection of DNA strand breaks.

Treatment with chemopreventive agents, difluoromethylornithine and retinyl acetate, results in altered mammary extracellular matrix.

The effect of treatment with D-L-2-difluoromethylornithine (DFMO) plus retinyl acetate (RA) on the promotion stage of 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis was evaluated in female Sprague-Dawley rats. Combined treatment was more effective than single agent treatment in decreasing cancer incidence and multiplicity and in prolonging cancer latency. Increased efficacy was associated with reduced morphological complexity of the gland and increased mammary extracellular matrix. Using ovarian hormones to model mitogen stimulation in the mammary gland, DFMO plus RA treatment reduced mammary gland complexity in the absence of an effect on bromodeoxyuridine (BrDU) labeling index measured immunohistochemically. Morphological and biochemical evaluation of these glands revealed increased levels of extracellular matrix in rats treated with chemopreventive agents. Tenascin expression and fibronectin levels were elevated and laminin levels were decreased. The fact that matrix degrading proteinase activity was also increased indicated that tissue remodeling was modulated by these chemopreventive agents. These data provide evidence of alterations in epithelial cell-extracellular matrix interactions that could account in part for the chemopreventive effects of DFMO plus RA.

Alkaline elution analysis of DNA fragmentation induced during apoptosis.

We report that alkaline elution analysis fails to provide a complete profile of DNA fragmentation induced by some genotoxic agents, particularly if these agents induce apoptotic cell death resulting in fragmentation of DNA into oligonucleosomal length multimers. It was questioned whether these oligonucleosome fragments are responsible for the more rapidly eluting component of DNA that is observed as a biphasic elution profile when cells treated with certain genotoxic agents are subjected to alkaline elution. The results of this study indicate that DNA fragmented during apoptotic cell death is eluted in fractions before alkaline denaturation that are normally discarded. Collection of fractions prior to alkaline denaturation is recommended for complete evaluation of the total spectrum of damage induced by genotoxic agents, especially when the compound is suspected of inducing cell death by apoptosis.