Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

Erenpreisa, Jekaterina; Freivalds, Tālivaldis; Roach, Helmtrud I.; Alston, Roger

doi:10.1007/s004180050147

Cited by 35 publications

(30 citation statements)

References 18 publications

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“…Plates were also incubated with acridine orange to serve as an indicator of possible cell death (apoptosis) (Erenpreisa et al 1997;Lucas et al 1998;Whitacre and Berger 1997). Viewed through a red filter ( Figs.…”

Section: Resultsmentioning

confidence: 99%

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Angiostatin Induces Intracellular Acidosis and Anoikis in Endothelial Cells at a Tumor-Like Low pH

2002

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Angiostatin inhibits angiogenesis by binding to endothelial cells (ECs) lining the vasculature of growing tumors. These cells are in a dynamic state during angiogenesis and are thus not firmly attached to the extracellular matrix. This makes them more vulnerable to anoikis, a process resulting in cell death initiated by or promoted by loss of attachment. Another potential source of EC vulnerability during tumor angiogenesis is that tumor extracellular pH is typically lower than in normal tissues. This presents an additional challenge to ECs in terms of maintaining ionic homeostasis. We report here that the lethality of angiostatin is significantly enhanced both by reduced matrix attachment during exposure and lowered extracellular pH (pH(e)). Another effect of angiostatin at reduced pH(e) is a decreased intracellular pH (pH(i)). These effects were observed in three model systems: aortic ring sprouts, ECs during tube formation, and ECs in a scratch/migration assay. In these three dynamic assays, angiostatin-induced cell death and intracellular acidification were clearly seen when pH(e) was reduced to 6.7. The intracellular acidification was far greater than that induced by pH(e) reduction alone. In contrast, the effect of angiostatin on pH(i) and on viability were not observed in a subconfluent monolayer in which the cells were allowed to attach to substrate for 48 h prior to exposure to angiostatin. These data suggest that low pH(e) and reduced adhesion to matrix play a role in the specificity of angiostatin for tumor neovasculature in contrast to wound healing and other normal angiogenic processes. The results also implicate roles for both pH(e) and pH(i) regulation in the mechanism of angiostatin action.

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Section: Resultsmentioning

confidence: 99%

“…For scratch width quantitation, plates were fixed and stained with a Leukostat kit (Biomedical Sciences, Swedesboro, NJ). For apoptosis detection, acridine orange staining was used (Erenpreisa et al 1997;Lucas et al 1998;Whitacre and Berger 1997).…”

Section: Scratch/migration Assaymentioning

confidence: 99%

Angiostatin Induces Intracellular Acidosis and Anoikis in Endothelial Cells at a Tumor-Like Low pH

2002

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“…25,2005 nPHGPx IS A PROTEIN THIOL PEROXIDASE 7641 on May 9, 2018 by guest http://mcb.asm.org/ predominance in mammalian male germ cells and its peculiar reducing substrate specificity (21,28,32). Thus, we applied the AO and TB staining methods to assess chromatin susceptibility to denaturation in spermatozoa isolated from caput or cauda epididymis (11,12). Our results show that while chromatin of the WT spermatozoa entering the epididymis is already resistant to denaturation, chromatin of KO spermatozoa is more labile, reaching stability similar to that of the WT only at a later stage in the cauda (Fig.…”

Section: Discussionmentioning

confidence: 99%

The Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase Is a Protein Thiol Peroxidase Contributing to Sperm Chromatin Stability

Conrad

Moreno

Sinowatz

et al. 2005

Molecular and Cellular Biology

238

184

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The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.Sperm chromatin condensation during the final steps of spermatogenesis in mammals is a multistep process that includes the sequential replacement of the majority of histones by transition proteins and protamines in testis (6, 7). During epididymal transit of spermatozoa, protamine thiol oxidation is completed and intra-and intermolecular cross-links are formed. Hence, a transcriptionally inactive and tightly packed haploid genome is generated rendering sperm nuclei more resistant to mechanical and chemical insults (2). Recently, Cho and colleagues showed that chimeric mice hemizygous for protamine 1 or 2 fail to transmit the targeted allele to the germ line (8).Selenium depletion studies of rodents clearly demonstrated the importance of this trace element in male fertility. Third generation selenium deficiency is associated with structural abnormalities, such as broken midpieces of sperm tails, giant heads, and reversible testicular atrophy (5, 34). Due to its particular high expression in mammalian testis (21) and its resistance to selenium deprivation in testis, the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is thought to account for most of the defects associated with severe selenium deficiency.PHGPx was initially characterized as a lipid peroxidationinhibiting protein (33) and was later shown to be an unusual member of the glutathione peroxidase family, in particular for its scarce specificity for both the oxidizing and reducing substrates (32). Most relevant in this respect was the observation that, in the presence of low glutathione (GSH) concentration, specific protein -SH groups may act as a reductant in the catalytic cycle with a stoichiometry of 2 equivalents of thiol per mole of hydroperoxide (13,22,...

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“…Apoptosis was quantified by measuring the proportion of cells with sub-G1 levels of DNA. Apoptosis was also confirmed by microscopy as described below and by TUNEL staining as described by Erenpreisa et al (1997).…”

Section: Methodsmentioning

confidence: 99%

Nuclear envelope-limited chromatin sheets are part of mitotic death

Erenpreisa

Andrey

Cragg

et al. 2002

Histochem Cell Biol

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Nuclear envelope-limited chromatin sheets (ELCS) are enigmatic membranous structures of uncertain function. This study describes the induction of ELCS in p53 mutated Burkitt's lymphoma cell lines after treatment with irradiation or the microtubule inhibitor, SK&F 96365. Both treatments evoked similar mitotic death, involving metaphase arrest followed by extensive endopolyploidisation and delayed apoptosis, although the kinetics were different. We found that induction of ELCS and nuclear segmentation correlated with the amount and kinetics of M-phase arrest, mitosis restitution and delayed apoptosis of endopolyploid cells. In metaphases undergoing restitution, ELCS are seen participating in the restoration of the nuclear envelope, mediating the attachment of peripheral chromatin to it. In interphase cells, ELCS join nuclear segments, ectopically linking and fusing with heterochromatin regions. In cells with segmented nuclei, continued DNA replication was observed, along with activation and redistribution of Ku70, suggestive of non-homologous DNA end-joining. Induction of ELCS also parallels the induction of cytoplasmic stacked membrane structures, such as confronting cisternae and annulate lamellae, which participate in the turnover and degeneration of ELCS. The results suggest that arrest at a spindle checkpoint and the uncoupling of mitosis from DNA replication lead to the emergence of ELCS in the resulting endopolyploid cells.

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Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

Cited by 35 publications

References 18 publications

Angiostatin Induces Intracellular Acidosis and Anoikis in Endothelial Cells at a Tumor-Like Low pH

Angiostatin Induces Intracellular Acidosis and Anoikis in Endothelial Cells at a Tumor-Like Low pH

The Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase Is a Protein Thiol Peroxidase Contributing to Sperm Chromatin Stability

Nuclear envelope-limited chromatin sheets are part of mitotic death

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