Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei

Prosperi, Ennio; Giangarè, Maria Chiara; Supino, Rosanna; Bottiroli, Giovanni

doi:10.1111/j.1365-2818.1990.tb03031.x

Cited by 9 publications

(9 citation statements)

References 23 publications

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“…The increase in PI-f luorescence intensity of nuclei digested with nucleases ranged between 15 and 30%, in agreement with previous results (8,26,27). Changes in DNA stainability were produced by enzymes that do not exhibit sequence specificity, but digest preferentially DNA with particular conformation (141, except that micrococcal nuclease is also sensitive to the base composition (10).…”

Section: Discussionmentioning

confidence: 89%

“…Flow cytometry has been shown to be a useful method for analyzing in situ the DNA sensitivity to nucleolytic cleavage, especially for avoiding the prelabelling of the cells with radioactive precursors (26,271. Monitoring of the enzymatic reaction requires the staining of DNA with fluorochromes, the extent of stainability after the nuclease reaction being the parameter to be determined.…”

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confidence: 99%

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Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

1991

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A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strandspecific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enymzes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nucleaseinduced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios > 0.11. Below this value (2 pg/ml PI), the fluorescence intensity of digested samples was 1530% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation-but not sequence-specific nucleases induce a relaxation of DNA supercoils.

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Section: Discussionmentioning

confidence: 89%

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confidence: 99%

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

1991

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“…DNAse I Assay DNA digestion by DNAse I was performed as described by Prosperi et al (25) and Koti et al (28). The mouse thymocytes were fixed before or after treatment with 0.01% Triton X-100 (pH 7.6), washed thrice, mixed together, resuspended in PBS (at a concentration of 1.0 X 10" cells/ml), supplemented with 0.2% Triton X-100 and 10 …”

Section: Fluorochrome Accessibility Assaymentioning

confidence: 99%

Cell membrane‐dependent chromatin condensation

Vinogradov

1995

Cytometry

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It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non-ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNAspecific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed. 0 1995 wiley-Liss, IIIC.

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“…However, detergent precipitation is unlikely because of the relatively low concentration of divalent cations needed to induce the staining effect. Moreover, the ability of divalent cations to decrease DNA accessibility is supported by observations that cations increase chromatin compactness ( 1 ) and thereby reduce the susceptibility of DNA to radiation-induced strand breaks (20), autodigestion ( 3 4 ) , and micronuclease digestion (28,35).…”

Section: The Chromatin Of Cells Undergoing Apoptosis Typically Exhibimentioning

confidence: 97%

Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

1995

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Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we obAnalysis of the DNA content of thymocyte nuclei prepared by detergent-mediated cytolysis has become a common method for single-parameter flow cytometric detection and quantitation of apoptotic cells (6,8,10, 15,21.24,37). On a DNA histogram, apoptotic nuclei appear as a subdiploid peak clearly distinguishable from the Go&, peak. The mechanism underlying the appearance o f the subdiploid peak has been hypothesized as either decreased fluorochrome binding due to chromatin condensation (6,33) or actual loss of DNA either while the cells are in culture or during sample preparation (2,8,17).In this paper, we report that the addition of a specific RNase A preparation to the lysing buffer resulted in a dramatic increase in the DNA fluorescence of toxicantinduced apoptotic nuclei such that they could not be discriminated from nonapoptotic nuclei. We also were able to mimic this effect by adding 2 mM CaCIJMgCI, to the lysing buffer. Subsequent analysis of the DNA content o f the lysate indicated that the upscale fluorescence shift was the result of a decreased ability of the detergentbased lysing buffer to extract DNA, most likely as a consequence of cation-induced changes in chromatin conformation (9,18,20,3 I ) In addition, we demonstrate that served a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 p M tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 p M dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. Key terms: Apoptosis, stainability, thymocyte, chromatin, ribonuclease during a 16-h exposure, thymocytes induced to undergo apoptosis either spontaneously or by treatment with 1 pM tributyltin methoxide (TBT), 2% ethanol, 2% me...

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Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei

Cited by 9 publications

References 23 publications

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

Cell membrane‐dependent chromatin condensation

Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

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