Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure.

The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single- versus double-stranded DNA with acridine orange.

Cytometric assessment of DNA damage induced by DNA topoisomerase inhibitors.

Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is mediated by one of the protein kinases of the phosphoinositide kinase family, namely ATM, ATR, and/or DNA-PK. The presence of Ser-139 phosphorylated H2AX (gammaH2AX) is thus a reporter of DNA damage. This protocol describes quantitative assessment of gammaH2AX detected immunocytochemically in individual cells combined with quantification of cellular DNA content by cytometry. The bivariate analysis of gammaH2AX expression versus DNA content allows one to correlate DNA damage with the cell cycle phase or DNA ploidy. The protocol can also be used to assess activation (Ser-1981 phosphorylation) of ATM; this event also revealing DNA damage induced by topo I or topo II inhibitors. Examples where DNA damage was induced by topotecan (topo I) and etoposide (topo II) inhibitors are provided.

NF-kappaB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: difficulties in identification of dead cells in such cultures.

BACKGROUND: Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI. While studying cytostatic effects of the anti-inflammatory sesquiterpene parthenolide (PRT), we have noticed that, concurrent with apoptosis, the cells were dying by necrosis in the same cultures. Furthermore, because apoptosis was atypical, reflected by rapid loss of plasma membrane integrity, it was difficult to distinguish apoptotic from necrotic cells based on this feature. METHODS: Three methods were used to distinguish apoptosis from necrosis: (a) HL-60 cells treated with PRT were subjected to analysis of caspases activation using antibody that detects activated (cleaved) caspase-3, (b) apoptotic cells were identified by binding of fluorochrome-labeled inhibitor of caspases FAM-VAD-FMK combined with the PI exclusion assay, and (c) cellular DNA and RNA were differentially stained with acridine orange (AO). RESULTS: Apoptotic cells were characterized by (a) caspase-3 activation detected immunocytochemically and (b) binding of FAM-VAD-FMK followed by or concurrent with (c) loss of ability to exclude PI, (d) deficit in DNA content, and (e) relatively little changed RNA content. Necrotic cells showed (a) no evidence of caspase-3 activation, (b) no binding of FAM-VAD-FMK, (c) inability to exclude PI, (d) rapid loss of RNA, and (e) unchanged DNA content. CONCLUSIONS: Identification of apoptotic cells versus necrotic cells was possible either based on the evidence of caspase-3 activation, by labeling with FAM-VAD-FMK combined with PI or by differential staining of cellular DNA and RNA with AO. The data indicate that plasma membrane appears to be one of the targets of PRT, because its integrity is lost very early during cell death, which is reflected by atypical apoptosis and by primary necrosis (lysis of the membrane).

Cell cycle effects and caspase-dependent and independent death of HL-60 and Jurkat cells treated with the inhibitor of NF-kappaB parthenolide.

The sesquiterpene parthenolide (PRT) is an active component of Mexican-Indian medicinal plants and also of the common herb of European origin feverfew. PRT is considered to be a specific inhibitor of NF-kappaB. Human leukemic HL-60, Jurkat, and Jurkat IkappaBalphaM cells, the latter expressing a dominant-negative IkappaBalpha and thus having non-functional NF-kappaB, were treated with PRT and activation of caspases, plasma membrane integrity, DNA fragmentation, chromatin condensation (probed by DNA susceptibility to denaturation), and changes in cell morphology were determined. As a positive control for apoptosis cells were treated with topotecan (TPT) and H2O2. At 2-8 microM concentration PRT induced transient cell arrest in G2 and M followed by apoptosis. A narrow range of PRT concentration (2-10 microM) spanned its cytostatic effect, induction of apoptosis and induction of necrosis. In fact, necrotic cells were often seen concurrently with apoptotic cells at the same PRT concentration. Atypical apoptosis was characterized by loss of plasma membrane integrity very shortly after caspases activation. In contrast, a prolonged phase of caspase activation with preserved integrity of plasma membrane was seen during apoptosis induced by TPT or H2O2. Necrosis induced by PRT was also atypical, characterized by rapid rupture of plasma membrane and no increase in DNA susceptibility to denaturation. Using Jurkat cells with inactive NF-kappaB we demonstrate that cell cycle arrest and the mode of cell death induced by PRT were not caused by inhibition of NF-kappaB. The data suggest that regardless of caspase activation PRT targets plasma membrane causing its destruction. A caution, therefore, should be exercised in interpreting data of the experiments in which PRT is used with the intention to specifically prevent activation of NF-kappaB.