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Results for “In Situ Nick-End Labeling”

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At least 19 recordsLinked to original sources
PubMed1996

Preferential expression of Fas/APO1 (CD95) and apoptotic cell death in perinecrotic cells of glioblastoma multiforme.

Fas/APO-1 (CD95)-mediated apoptosis is one of the major mechanisms of programmed cell death. We have previously shown by reverse transcriptase-polymerase chain reaction that Fas is frequently expressed in malignant gliomas [Tachibana et al. (1995) Cancer Res 55: 5528-5530]. In this study, we assessed Fas expression in astrocytomas using a polyclonal anti-Fas antibody. Immunoreactivity to Fas was detected in 1 out of 9 (11%) low-grade astrocytomas (WHO grade II), 2 of 11 (18%) anaplastic astrocytomas (WHO grade III) and in 13 of 15 (87%) glioblastomas (WHO grade IV). In glioblastomas, Fas expression was almost exclusively observed in glioma cells surrounding foci of necrosis. In these perinecrotic areas, there was also an accumulation of glioma cells undergoing apoptosis, as detected by in situ nick-end labeling. This suggests that Fas-mediated apoptosis may play a role in the pathogenesis of necrosis which constitutes a histological hallmark of glioblastoma multiforme.

Adolescent↗
PubMed1995

DNA fragmentation in granular cells of human cerebellum following global ischemia.

Vulnerability of human cerebellum in two autopsy cases following global brain ischemia was examined histologically by using a specific in situ nick-end labeling method for DNA breaks. In both cases, DNA fragmentation was observed in approximately one-third of the granular cells in cerebellar cortex, whereas Purkinje cells were still alive and no DNA fragmentation was recognized in the nuclei. The present study suggests that some granular cells of cerebellar cortex are more vulnerable to transient ischemia than Purkinje cells and death of granular cells is induced by an apoptotic DNA fragmentation following global brain ischemia.

Adult↗
PubMed1996

Apoptosis of cultured microglia by the deprivation of macrophage colony-stimulating factor.

Promotion of microglial proliferation and differentiation by colony-stimulating factors (CSFs) and disappearances of microglia at the late neonatal stage by decreasing of CSFs have been reported. In this study, the effects of the deprivation of macrophage CSF (M-CSF) on enriched microglia in cultures were examined by cytochemical methods including in situ nick-end labeling for DNA fragmentation, and Carrazi's hematoxylin nuclear staining. When M-CSF was deprived from the culture medium: (1) at least 40% of the cells were weakly labeled by nick-end within 3 h and more than 70% of the cells were clearly labeled by 16 h; and (2) nuclear condensation or fragmentation, and formation of apoptotic bodies were observed within 48 h. LeY-positive immunoreactivity, identified as a characteristic of cells undergoing apoptosis, was observed on cells positively labeled by nick-end and condensed nucleus, and ones budding apoptotic bodies. From these results, it is conceivable that microglia undergo apoptosis when M-CSF is deprived from the culture medium and, therefore, require CSFs for their survival. This in vitro phenomenon suggests that one of the mechanisms of microglial disappearance in vivo after synaptogenesis may be due to apoptosis by decreasing level of CSFs.

Animals↗
PubMed1996

Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.

Animals↗
PubMed1996

Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord.

We have evaluated by in situ nick-end labeling the presence of apoptotic cells in the spinal cord of rats with compression injury at the level of Th8-9 of mild, moderate, and severe degrees resulting in no neurologic deficit, reversible paraparesis, and paraplegia, respectively. Rats with compression injury surviving 4 or 9 days showed apoptotic glial cells in the longitudinal tracts of the Th8-9, the cranial Th7, and the caudal Th10 segments. The apoptotic cells were most frequently observed in Th7. They did not express glial fibrillar acidic protein (GFAP) and their morphology was compatible with that of oligodendrocytes. Neurons of the gray matter did not present signs of apoptosis. In addition, we studied the immunohistochemical expression of Bcl-2, an endogenous inhibitor of apoptosis. Compression induced Bcl-2 immunoreactivity in axons of the long tracts, particularly after moderate and severe compression and 1-day survival. Neurons of dorsal root ganglia were immunoreactive but the neurons of the spinal cord were unstained. The accumulation, presumably caused by arrested axonal transport in sensory pathways, was absent in rats surviving 9 days. In conclusion, compression trauma to rat spinal cord induces signs of apoptosis in glial cells, presumably oligodendrocytes of the long tracts. This may induce delayed myelin degeneration after trauma to the spinal cord. Bcl-2 does not seem to be upregulated in oligodendrocytes.

Animals↗
PubMed1995

Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis.

The CA1 pyramidal neurons in the hippocampus are selectively vulnerable to transient ischemic damage. In experimental animals, the CA1 pyramidal neurons undergo cell death several days after brief forebrain ischemia. It remains, however, unknown whether this delayed neuronal death is necrosis or apoptosis. To investigate the degenerating processes of the CA1 pyramidal neurons in gerbil hippocampus after brief ischemia, lysosomal and nuclear alterations in the cells were examined using immunocytochemistry, in situ nick-end labeling, and Southern blotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L, representative lysosomal cysteine proteinases, increased in the CA1 pyramidal neurons 3 d after ischemic insult, which showed cell shrinkage. By morphometric analysis, the volume density of cathepsin B-positive lysosomes markedly increased 3 d after ischemic insult, while that of autophagic vacuole-like structures also increased at this stage, suggesting that cathepsin B-immunopositive lysosomes increasing in the neurons after ischemic insult are mostly autolysosomes. Nuclei of the CA1 neurons were nick-end labeled by biotinylated dUTP mediated by terminal deoxytransferase 3 and 4 d after ischemic insult, but not in the prior stages. Simultaneously, dense chromatin masses appeared in nuclei of the neurons. By Southern blotting, laddering of DNA occurred only in CA1 hippocampal tissues obtained 4 d after ischemic insult. Confocal laser scanning microscopy demonstrated that the fragmented DNA in the CA1 pyramidal layer was phagocytosed by microglial cells. The results suggest that delayed death of the CA1 pyramidal neurons after brief ischemia is not necrotic but apoptotic.

Animals↗
PubMed1995

Enhanced cellular proliferative activity and cell death in chronic thyroiditis and thyroid papillary carcinoma.

For the analysis of cellular proliferative activity and cell death in thyroid diseases, the Ki-67 labeling index, bcl-2 protein expression and cell death of follicular epithelia by immunohistochemistry and in situ DNA nick-end labeling methods were evaluated in normal thyroid tissues as well as in surgical specimens from cases of Hashimoto's disease (16 cases), focal lymphocytic thyroiditis (13 cases), Graves' disease (15 cases), follicular adenoma (20 cases) and papillary carcinoma (43 cases). Cellular proliferative activity and cell death were both enhanced in cases of thyroiditis, including Hashimoto's disease and focal lymphocytic thyroiditis. Thyroids from patients with follicular adenoma and papillary carcinoma also showed increased cellular proliferative activity and cell death. In addition, predominant high cellularity and partial loss of bcl-2 protein expression in papillary carcinoma suggested that the overgrowth and dedifferentiation were associated with malignancy.

Carcinoma, Papillary↗
PubMed1995

Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line.

Cell death via apoptosis is an important event involved in a number of immunological processes. Recently, apoptosis has been associated with oxidative stress in a number of cell systems. Here we assessed the inhibitory capacity of different antioxidants on UV- and drug-induced apoptosis in the human leukemic cell line, HL-60. We found that the oxygen radical scavenger, BHA, the radioprotector cysteamine and the metal chelators, pyrrolidinedithiocarbamate (PDTC), diethyldithiocarbamate (DEDTC), and dimethyldithiocarbamate (DMDTC), were able to significantly inhibit nuclear fragmentation and reduce the formation of apoptotic bodies in UV-irradiated human leukemic cells. Both BHA and PDTC were found to reduce DNA fragmentation as assessed by in situ DNA nick-end labelling and quantification thereof using fluorescence flow cytometry. In addition to inhibiting UV-induced apoptosis, PDTC was also capable of reducing the amount of apoptosis induced by a range of cytotoxic drugs, such as actinomycin-D, camptothecin, etoposide, and melphalan, whereas BHA and cysteamine were not as effective in these cases after more than four hours in culture when compared to PDTC. To further elucidate the working mechanism of PDTC, we have looked at the effect of PDTC on DNA fragmentation in isolated nuclei, under conditions that promote activation of endogenous endonuclease involved in apoptosis. In contrast to ZnCl2, a potent inhibitor of endonuclease activity, PDTC was unable to inhibit DNA-ladder formation in this assay. Taken together, these results indicate that oxygen radicals may have a central role to play in the induction of apoptosis and that dithiocarbamates can serve as potent inhibitors of apoptosis induced by a wide variety of stimuli.

Antioxidants↗
PubMed1994

Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes.

We examined whether apoptosis occurs in cardiomyocytes by hypoxia in vitro. Neonatal rat cardiomyocytes and nonmyocytes were cultured in 95% N2-5% CO2 atmosphere to produce hypoxic conditions. DNA fragmentation into integer multiples of the internucleosomal DNA length was observed in cardiomyocytes as early as 12 hours, whereas nonmyocytes did not show fragmentation of DNA up to 72 hours. DNA fragmentation of cardiomyocytes induced by hypoxia was also confirmed by nick-end labeling in situ. Messenger RNA for Fas antigen, a mediator of apoptotic cell death, was expressed in both cardiomyocytes and nonmyocytes as revealed by Northern blotting and in situ hybridization. In hypoxic condition, Fas messenger RNA levels in cardiomyocytes were upregulated by twofold over controls, whereas those of nonmyocytes were downregulated. These results indicate that cardiomyocyte death by hypoxia can occur via apoptosis and that Fas antigen may be associated with the mechanism of this apoptotic process.

Animals↗
PubMed1996

Regulation of apoptosis induced by transforming growth factor-beta1 in nontumorigenic rat prostatic epithelial cell lines.

Transforming growth factor-beta1 (TGF-beta1), which is induced in the prostate following castration, has been speculated to mediate apoptosis of epithelial cells during prostatic involution. Here, we report the first evidence of a direct effect of TGF-beta on induction of apoptosis in prostatic epithelial cells in vitro, using NRP-152 nontumorigenic and NRP-154 tumorigenic rat prostatic epithelial cell lines. TGF-beta1 induces apoptosis of both cell lines within 24 h, as shown by a decrease in cell viability, in situ DNA nick-end labeling, and internucleosomal DNA fragmentation. Moreover, the ability of TGF-beta to induce apoptosis of NRP-152 is strictly dependent on culture conditions, because dexamethasone enhances while insulin and insulin-like growth factor-I specifically block apoptosis induced by TGF-beta. We suggest that TGF-betas are direct physiological regulators of apoptosis of prostatic epithelial cells.

Animals↗
PubMed1996

[Apoptosis and human viral infections].

Homeostasis of cell numbers in tissues is maintained by a critical balance between cell proliferation and programmed cell death or apoptosis. Many human viruses are able to develop suitable strategies for modifying apoptosis in virus-infected cells and in virus-primed T cells. Apoptosis is characterized by the fragmentation of nuclear DNA into 180-200 bp apoptotic bodies and can be analysed microscopically or by flow cytometry using staining with various dyes. Moreover DNA cleavage can be identified by electrophoresis and by specific labeling using in situ nucleotidyltransferase assay (ISNT), terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling technique (Tunel), or by Elisa. Adenovirus E1A induces expression of protooncogenes c-myc and c-fos which sensitize cells to apoptosis; EBV EBNA-5, and adenovirus E1A, HPV E7, and polyomavirus large T act in the same way by displacing pRB-bound E2F. EBV EBNA-5, HPV E6, Adenovirus E1B 55 kDa inactivate the tumor suppressor protein p53 and engage the cells in the transformation process. EBV LMP-1, HHV6, and HTLV1 tax induce the antiapoptotic bcl-2 protein. EBV BHRF1 encodes proteins with homology to bcl-2 and Adenovirus E1B 19 kDa encodes proteins that have protective functions similar to bcl-2. Activated lymphocytes responding to viral infections express high levels of fas and are susceptible to apoptosis. TNF alpha can down- or up-regulate fas and down-regulates TNF-R. Adenovirus E1B 19 kDa blocks the proapoptotic activity of TNF alpha. Inversly, Cytomegalovirus, hepatitis C virus and Myxoviruses up-regulate fas antigen prior to undergoing apoptosis. In HIV-infected patients, CD4+ T-cell apoptosis is mediated by the cytopathic effect of the virus and the cell surface expression of gp 120-env protein. Moreover, an accelerated T-cell apoptosis in HIV-infected individuals is characterized by (i) HIV gp120-CD4+ cross-linking and subsequent aberrant signaling of T-cells, (ii) involvement of TNF alpha-fas/Apo-1 (TNF-R) binding, (iii) involvement of accessory cells as an apoptosis inducer and as a result of defective antigen presentation, (iv) possible superantigen activity induced by HIV products and cofactors. Many viruses also encode proteins with protease activity which could induce apoptosis. The induction of apoptosis may result in virus clearance, in contrast the inhibition of apoptosis may result in virus cell transformation and viral persistence. Indirectly, the apoptosis of infected cells may be induced by CTLs, NK cells and cytokines. In addition, apoptosis-mediated physiological depletion of T lymphocytes in the course of viral infection can silence the immune response and can induce immunodeficiency.

Apoptosis↗
PubMed1996

The phenotype and fate of the antibody-forming cells of the splenic foci.

The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling.

Animals↗
PubMed1996

Human transplant coronary artery disease: pathological evidence for Fas-mediated apoptotic cytotoxicity in allograft arteriopathy.

Cytotoxicity mediated by cytolytic T cells occurs exclusively through Fas- and perforin-based pathways. Such mechanisms may be important in transplant coronary artery disease (TxCAD). We studied Fas and perforin expression and apoptosis using immunohistochemical methods, in situ terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL), and morphologic analysis of vessels with TxCAD taken from 12 transplant patients, which we compared with vessels of 10 patients with native coronary artery disease (CAD) and vessels from 14 patients with normal coronary arteries. Fas was detected in all TxCAD specimens. Fas-positive cells were mainly endothelial cells (EC); 100% of EC and approximately one-third of T cells and macrophages were positive for Fas. Almost all TUNEL-positive cells were Fas-expressing cells. Of the T cells and macrophages that expressed Fas, 18% appeared apoptotic in the mild and moderate-to-severe TxCAD group as compared with 78% in severe TxCAD patients (p = 0.0124). By contrast, EC damage was less evident in the vessels with greater intimal disease severity: 10% in severe TxCAD versus 75% in the mild and moderate-to-severe TxCAD group (p = 0.0122). Perforin was positive in 5% of the total intimal T cells in 3 of 12 arteriopathy specimens. Fas and perforin were virtually negative in vessels taken from CAD patients. TUNEL was diffusely positive in CD68-positive foam cells in the lipid-rich core, but Fas was negative in all CAD specimens. In normal arteries, 8 of 14 specimens contained a few TUNEL-positive and Fas-positive EC and T cells. Perforin was negative. We conclude that EC damage in TxCAD seems to be brought about through a Fas-based apoptotic pathway and that CD4-positive cytolytic T cells may be the major lytic cells involved in TxCAD.

Adolescent↗
PubMed1995

Ultraviolet B-induced apoptosis of keratinocytes in murine skin is reduced by mild local hyperthermia.

Two components of sunlight, ultraviolet (UV) B (290-320 nm) and infrared (greater than 700 nm), each cause damage to the skin. However, we recently identified a protective response in which heat reduces UVB-induced killing of cultured keratinocytes. Here, this investigation is extended to the living epidermis. The effects of hyperthermic preconditioning upon UVB-induced apoptosis were studied morphologically with hematoxylin and eosin staining, and biochemically with TUNEL (terminal deoxynucleotide transferase nick-end labeling) to measure endonucleolytic cleavage of DNA in situ. Anesthetized SKH-1 hairless mice were exposed to UVB light (0 to 120 mJ/cm2), after which their skin was biopsied at 24 h and paraffin sections were stained with hematoxylin and eosin or with TUNEL. Apoptotic keratinocytes were found to increase after UVB in a dose-related manner. In contrast, if one flank of the mouse was pretreated at 40 degrees C for 1 h and both flanks subsequently were UVB-irradiated at 6 h, the resulting formation of apoptotic cells was reduced twofold or more in the heated flank. Protection appeared by 3 h, reached a maximum at 6 h, and disappeared by 12 h. In summary, heat induces a transient protective effect that reduces UVB-mediated death of keratinocytes in skin at physiologically attainable doses of heat and UVB.

Animals↗
PubMed1995

A cautionary note on the use of the TUNEL stain to determine apoptosis.

Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL assay) is now commonly used to investigate apoptosis. Previous reports suggested that physiologically appropriate death is due to apoptosis and that pathological mechanisms involve necrosis. Strong evidence of apoptosis, following ischaemia and epilepsy, has been recently provided by combining genomic DNA gel electrophoresis, light and electron microscopy and in situ, DNA-break labelling. However, only an observation in light microscopy with high magnification permits the detection of chromatin condensation and apoptotic bodies. We report that a positive TUNEL assay reaction should not be considered as a specific marker of apoptosis but can also indicate necrotic cell death.

Animals↗
PubMed1996

In situ labeling of dying cortical neurons in normal aging and in Alzheimer's disease: correlations with senile plaques and disease progression.

We examined the degeneration of neocortical neurons in normal aging and Alzheimer's disease (AD) using terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL), a method that identifies DNA strand breaks and constitutes a positive marker for dying neurons. TUNEL was positive in neurons, glia, and microglial cells in AD but not in younger or age-matched cognitively characterized controls. Neuronal labeling in AD was most conspicuous in cortical layer III in the early stages of the disease and became more widespread as the disease progressed. In addition, we observed TUNEL of lamina III neurons in a subset of older subjects who had normal cognition but abundant neocortical senile plaques. In concert, the availability of a direct marker of dying neurons allows for specific correlations of cell death with other neuropathological markers as well as clinical variables. Observations from the present study suggest that the death of cortical neurons precedes the symptomatic stage of AD and evolves in parallel with the clinical progression of the disease and that there appears to be an association between the degree of cell death and the severity of senile plaques.

Adult↗
PubMedDate not supplied

Effect of tumor necrosis factor alpha and beta on human oligodendrocytes and neurons in culture.

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human-derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor alpha and beta and compared these with the tumor necrosis factor independent effects mediated by activated CD4+ T-cells. We found that activated CD4+ T-cells, but not tumor necrosis factor alpha or beta, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick-end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor alpha did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor beta effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor alpha was less than that induced by serum deprivation. In-situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor alpha mRNA.

Adult↗
PubMed1996

Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos.

Asbestos causes protracted, dose-dependent increases in steady-state mRNA levels of the proto-oncogenes c-fos and c-jun, and AP-1 DNA-binding activity in normal rat pleural mesothelial (RPM) cells (1). To determine the phenotypic end points of overexpression of these early response genes by asbestos, both cell proliferation and apoptosis were examined in confluent RPM cells exposed to a range of concentrations (1.25 to 10 micrograms/cm2 dish) of crocidolite asbestos for 24 and 48 h. Quantitation of RPM cells pulsed with 5-bromo-2'-deoxyuridine revealed that asbestos caused dose-dependent decreases in cells undergoing DNA synthesis. Decreases in cell proliferation were accompanied by dose-related increases in apoptosis using (1) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (i.e., ApopTag technique), (2) 4',6-diamidino-2-phenylindole cell staining, and (3) fluorescent-activated cell sorter after incorporation of propidium iodide. Less striking but significant dose-related increases in apoptosis were observed in RPM cells exposed to H2O2 (300 microM), and no apoptosis was seen after exposure of cells to high concentrations (10 micrograms/cm2 dish) of glass beads. Our results are unique in that they demonstrate that asbestos induces apoptosis in mesothelial cells at concentrations eliciting increased expression of the proto-oncogenes c-fos and c-jun.

Animals↗