Major DNA Fragmentation Is a Late Event in Apoptosis

Collins, Jae A.; Schandl, Cynthia A.; Young, Kristy K.; Veselý, Jozef; Willingham, Mark C.

doi:10.1177/002215549704500702

Cited by 408 publications

(281 citation statements)

References 21 publications

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“…7. The well characterized apoptotic features of nuclear blebbing and DNA fragmentation and condensation are evident (34,35). It is interesting to note that eScTop1p becomes cytoplasmic following camptothecin treatment, which is consistent with the recent report that the enzyme changes its subcellular distribution after drug treatment (32).…”

Section: Expression Of Catalytically Active Esctop1p In Cos Cells Leasupporting

confidence: 84%

“…Twenty-four hours after camptothecin treatment, the coverslips were processed for fluorescent microscopy, using M2 as a primary antibody, followed by DAPI staining. The altered nuclear morphology of an apoptotic cell, as assessed by DAPI staining, has been well defined (34,35). Examples of camptothecin-induced apoptotic cells that are both fluorescent positive due to expression of eScTOP1 and fluorescent negative are shown in Fig.…”

Section: Expression Of Catalytically Active Esctop1p In Cos Cells Leamentioning

confidence: 96%

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Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

Hann¹,

Evans²,

Fertala³

et al. 1998

Journal of Biological Chemistry

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The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabiliza- Eukaryotic DNA topoisomerase I catalyzes the relaxation of supercoiled DNA through the transient breakage and religation of a single DNA strand in a DNA duplex (reviewed in Refs. 1-3). This enzyme plays a role in a number of essential cellular processes, such as replication, recombination, and transcription (1, 3-5). Furthermore, the naturally occurring antitumor drug camptothecin specifically targets this enzyme by stabilizing the covalent enzyme-DNA intermediate (Refs. 6 and 7; reviewed in Ref. 8). During DNA replication (S phase), these stabilized enzyme-DNA adducts are converted into lethal double-stranded DNA breaks due to their interaction with the DNA replication fork (9 -12).Although this enzyme participates in numerous cellular processes, strains of the yeast Saccharomyces cerevisiae deleted for the gene encoding DNA topoisomerase I (top1⌬) are viable because other gene products, such as DNA topoisomerase II, can compensate for the loss of TOP1 (11, 13). These top1⌬ strains are completely resistant to the cytotoxic action of camptothecin (14 -16). However, expression of either S. cerevisiae or human DNA topoisomerase I restores the sensitivity of these cells to camptothecin-induced lethality (4, 14 -16). These results demonstrate the specificity of camptothecin for eukaryotic DNA topoisomerase I and the utility of using yeast as a model system for the analysis of drug-enzyme interactions. In fact, mutations in yeast and human TOP1 that render the enzyme resistant to camptothecin (17,18) or render the enzyme cytotoxic even in the absence of camptothecin (19, 20) 1,2 have been defined using this yeast system.These results indicate a significant conservation of function between the yeast and human enzymes, consistent with extensive similarities in TOP1 sequences. Nevertheless, differences between these proteins do exist. For example, expression of a camptothecin-resistant yeast or human DNA topoisomerase I mutant (top1vac) has different effects on the viability of ...

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Section: Expression Of Catalytically Active Esctop1p In Cos Cells Leasupporting

confidence: 84%

Section: Expression Of Catalytically Active Esctop1p In Cos Cells Leamentioning

confidence: 96%

Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

Hann¹,

Evans²,

Fertala³

et al. 1998

Journal of Biological Chemistry

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“…The TUNEL assay is particularly useful for rapidly screening tissues for evidence of apoptosis. However, single-stranded DNA ends can be detected in necrotic cells, causing false-positive signals (Collins et al, 1997); therefore, ultrastructural surveys are usually employed in conjunction with TUNEL to confirm apoptosis.Although there have been more than 700 fatal cases of human filoviral infection, tissues from only a handful of cases are available for study. Logistical problems including cultural mores and environmental influences have hampered the collection of useable material.…”

mentioning

confidence: 99%

Apoptosis Induced In Vitro and In Vivo During Infection by Ebola and Marburg Viruses

Geisbert

Hensley

Gibb

et al. 2000

Laboratory Investigation

289

254

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SUMMARY:Induction of apoptosis has been documented during infection with a number of different viruses. In this study, we used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling to investigate the effects of Ebola and Marburg viruses on apoptosis of different cell populations during in vitro and in vivo infections. Tissues from 18 filovirus-infected nonhuman primates killed in extremis were evaluated. Apoptotic lymphocytes were seen in all tissues examined. Filoviral replication occurred in cells of the mononuclear phagocyte system and other well-documented cellular targets by TEM and immunohistochemistry, but there was no evidence of replication in lymphocytes. With the exception of intracytoplasmic viral inclusions, filovirus-infected cells were morphologically normal or necrotic, but did not exhibit ultrastructural changes characteristic of apoptosis. In lymph nodes, filoviral antigen was co-localized with apoptotic lymphocytes. Examination of cell populations in lymph nodes showed increased numbers of macrophages and concomitant depletion of CD8 ϩ T cells and plasma cells in filovirus-infected animals. This depletion was particularly striking in animals infected with the Zaire subtype of Ebola virus. In addition, apoptosis was demonstrated in vitro in lymphocytes of filovirus-infected human peripheral blood mononuclear cells by TEM. These findings suggest that lymphopenia and lymphoid depletion associated with filoviral infections result from lymphocyte apoptosis induced by a number of factors that may include release of various chemical mediators from filovirus-infected or activated cells, damage to the fibroblastic reticular cell conduit system, and possibly stimulation by a viral protein. (Lab Invest 2000, 80:171-186).I nfections with filoviruses cause severe and often fatal hemorrhagic disease in humans and nonhuman primates, killing up to 90% of infected individuals. Ebola (EBO) and Marburg (MBG) viruses comprise the family Filoviridae (Murphy et al, 1995). The EBO species consists of the Zaire and Sudan subtypes first isolated during separate outbreaks in 1976, the Reston subtype initially isolated from monkeys imported from the Philippines to the United States in 1989, and the Cô te d'Ivoire subtype discovered in the Tai Forest of the Ivory Coast in 1994. The recent re-emergence of EBO subtype Zaire (EBO-Z) in former Zaire and Gabon, and subtype Reston (EBO-R) in the United States, are but the last of a succession of filoviral outbreaks that have occurred sporadically since the initial zoonotic outbreak of MBG in 1967. Although outbreaks have been self-limiting, the lack of proven effective prophylactic or therapeutic regimens to treat filoviral infections has heightened concerns about the public health threat of these pathogens.The factors contributing to the development of severe lesions in EBO and MBG hemorrhagic fevers are unknown. Filoviral infections of humans and nonhuman primates are characterized by a failure o...

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“…Thus, we were able to demonstrate that NCT, a weak oxidant, has the ability to induce loss of mitochondrial membrane potential and thereby activates the intrinsic mitochondrial apoptosis pathway as described before by Klamt and Shacter. 10 Results of the detection of cellular DNA fragmentation, a rather late characteristic biochemical change in the nuclei of apoptotic cells, 23 showed a significant increase in DNA fragments after 6 h of incubation with 3.3 mM NCT solutions only for HOS and MG-63, but not for SAOS-2. One possible explanation of this finding could be different genetic alterations of the cell lines resulting in a delayed formation of DNA fragments at SAOS-2 cells.…”

Section: Discussionmentioning

confidence: 99%

Taurine chloramine induces apoptosis in human osteosarcoma cell lines

Pilz

Holinka

Vavken

et al. 2012

Journal Orthopaedic Research

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Although combination of surgery with chemotherapy has noticeably improved the survival rate of osteosarcoma patients, the application of anticancer drugs is still associated with significant adverse reactions, for instance acquisition of drug-resistant phenotypes, necessitating the development of new chemotherapeutical agents. Therefore, the aim of this study was to research, if taurine chloramine (NCT) induces apoptosis in the osteosarcoma cell lines HOS, MG-63, and SAOS-2. Proliferation of osteosarcoma cells was detected with the ''EZ4U Cell Proliferation and Cyotoxicity Assay'' showing a time-and dose-dependent cytotoxic effect of NCT on these cell lines. After 3 h of incubation all cell lines showed significantly less cells at 5.5 mM NCT solutions, after 6 h at concentrations of 1.1 and 2.2 mM. Acridine-orange fluorescence nuclear staining showed characteristic features of apoptosis. DNA fragmentation was detected via ELISA, showing significant results for HOS and MG-63 after 6 h at an NCT concentration of 3.3 mM. Results of JC-1 mitochondrial FACS analysis presented a significant increase in apoptotic cells after 6 h at 3.3 mM for the tested cell lines. Summarized, the results of this study indicate that NCT is a promising agent in osteosarcoma therapy. ß

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Major DNA Fragmentation Is a Late Event in Apoptosis

Cited by 408 publications

References 21 publications

Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

Apoptosis Induced In Vitro and In Vivo During Infection by Ebola and Marburg Viruses

Taurine chloramine induces apoptosis in human osteosarcoma cell lines

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