Significant DNA Fragmentation Research Articles

Apoptotic cell death in human chronic renal allograft rejection.

Chronic renal allograft rejection is characterized by gradual progression suggesting persistent low-grade injury. Apoptotic cell death may be initiated by low-grade injury secondary to external factors, making apoptosis a potential pathway of chronic rejection. In the present investigation, protocol kidney biopsies of 20 pediatric renal allograft recipients (12 with chronic rejection and 8 with normal histology), 9 pediatric liver allograft recipients, and 7 children with minimal change nephrotic syndrome were evaluated. The presence of apoptotic cell death was studied by determining apoptosis-induced oligonucleosomal DNA fragmentation in the biopsy specimens using 3' end labeling with terminal transferase, gel fractionation, and Southern blotting. The specific cell types with increased DNA fragmentation were determined by in situ 3' end labeling performed on sections of the biopsies. Significant DNA fragmentation was found only in the specimens from patients with chronic rejection. In situ investigation revealed increased apoptosis of both proximal and distal tubular epithelial cells, but not in the glomeruli or interstitium. The mean number of apoptotic tubular epithelial cells per 400x magnified field was higher in the renal allografts than in kidneys of liver transplant recipients or patients with minimal change nephrotic syndrome (2.3 vs. 0.8, P<0.05, in both cases). Our data provide biochemical evidence of increased apoptotic cell death of renal tubular epithelial cells in patients undergoing chronic renal allograft rejection.

Therapy with CD7 monoclonal antibody TH-69 is highly effective for xenografted human T-cell ALL.

Human T-cell acute lymphocytic leukaemia (ALL) was established in athymic nude or severe combined immunodeficient (SCID) mice by injecting CEM or MOLT-16 cells. When nude mice bearing approx. 2 g of tumour were treated with a single injection of CD7 antibody TH-69, 82.6% reached complete remission within 10 d whereas 13.0% showed partial remission. Similarly, in SCID mice with advanced disease a significant prolongation of survival was seen. The therapeutic effects were dependent upon dose and affinity of the antibody. TH-69 is a high-affinity antibody (7.6 x 10(9) M-1) that rapidly induced modulation during treatment. The Fc-portion of the antibody was required for effective tumour cell killing. Complement deposition was found on tumour sections after TH-69 treatment and in part may account for tumour destruction. There was no evidence for antibody-dependent cellular cytotoxicity (ADCC). The kinetics of tumour disappearance suggested the initiation of a programmed cell death (PCD), despite the lack of significant DNA fragmentation. Unmodified high-affinity antibodies to the T-cell antigen CD7 have potential for T-cell ALL therapy.

Neurovirulent Simian Immunodeficiency Virus Infection Induces Neuronal, Endothelial, and Glial Apoptosis

Studies of human immunodeficiency virus type 1 (HIV-1) associated dementia have shown neuronal loss in discrete areas. The presence and mechanism of neuronal death, however, has remained quite elusive. One mechanism of cell death, apoptosis, has been clearly demonstrated outside the central nervous system (CNS) in HIV-1 infection but has not been firmly established within the CNS. Therefore, we set out to ascertain whether neuronal cell loss in simian immunodeficiency virus (SIV) encephalitis, an animal model of HIV-1-associated dementia, is a result of apoptosis. With the aid of an in situ technique for identifying the 3'-OH ends of newly fragmented DNA characteristic of apoptosis, in conjunction with specific detected morphological criteria via light microscopy, we have examined encephalitic and nonencephalitic brains of macaques infected with a neurovirulent, neuroendotheliotropic strain of SIV to see if virus is spatially associated with apoptosis of neurons and non-neuronal cell types. We demonstrate the presence of DNA damage, indicative of apoptosis, in neurons, endothelial cells, and glial cells of the CNS of SIV-infected macaques. Furthermore, we observe an association between the localization of cells with significant DNA fragmentation and perivascular inflammatory cell infiltrates containing SIV-infected macrophages and multinucleated giant cells. Quantitative analysis reveals significantly more cells with DNA fragmentation in the CNS of macaques infected with neurovirulent, neuroendotheliotropic SIV strains as compared with strictly lymphocyte-tropic SIV strains and SIV negative controls. Our findings of apoptosis in SIV-infected CNS may potentially lead to a better understanding of the AIDS dementia complex, ultimately providing a basis for better treatments.

Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat

BACKGROUND & AIMS: Mechanisms of cell death in human and experimental pancreatitis remain poorly understood. The aim of this study was to determine the mechanisms of cell death, apoptosis vs. necrosis, in models of pancreatitis induced by pancreatic duct ligation in the opossum and rat. METHODS: Morphological changes were shown by light and electron microscopy, chromatin condensation and DNA breaks were assessed using Hoechst 33258 staining and DNA nickend labeling, and DNA fragmentation was characterized b gel electrophoresis. RESULTS: Cells with morphology of both necrosis and apoptosis were found in pancreata from duct-ligated animals; however, the apoptosis-necrosis ratio was different for the opossum and the rat. Both apoptotic and necrotic cells stained for breaks in DNA, suggesting that both apoptosis and necrosis are associated with DNA fragmentation and that DNA end labeling cannot specifically detect apoptotic cells. No significant oligonucleosomal DNA fragmentation was found in pancreas from duct- ligated animals, although the endonuclease responsible for oligonucleosomal DNA fragmentation was present in pancreatic nuclei. CONCLUSIONS: Duct ligation resulted predominantly in apoptosis in the rat and necrosis in the opossum. The differences in types of cell death in the two models may result from differences in inflammatory cell infiltration. (Gastroenterology 1996 Mar;110(3):875-84)

Bleomycin induces apoptosis in human alveolar macrophages

Bleomycin (BLM) is an effective antineoplastic drug; however, cumulative dosage is often associated with inflammation that can progress to pulmonary fibrosis. The mechanisms by which this occurs are not understood, but they have been proposed to involve the alveolar macrophage (AM). In this study, we examined the in vitro effects of BLM on human AM cytotoxicity and the role of heat shock proteins (HSP or stress proteins) in this process. Although BLM did not cause marked necrosis, it caused significant DNA fragmentation detected by in situ DNA labeling and confirmed by BLM-induced DNA ladder formation after 24 h. The DNA fragmentation was significantly blocked by 10 and 50 microM ZnCl2, suggesting that BLM was inducing apoptosis. BLM did not alter intracellular protooncogene bcl-2 or glutathione levels. However, BLM significantly (50%) blocked HSP-72 expression by 4 h during a mild heat stress (39.8 degrees C). This inhibition occurs without affecting mRNA levels (in situ hybridization) for HSP-72 or overall protein synthesis ([35S]methionine incorporation), suggesting that BLM is blocking the stress response relatively specifically and post-transcriptionally. In summary, these results suggest that BLM causes apoptosis in human AM in vitro that is preceded by the inhibition of HSP-72 induction that appears to be caused by a posttranscriptional mechanism.

Effects of intracellular pH on apoptosis in HL-60 human leukemia cells.

The nature of the endonucleases responsible for DNA fragmentation in apoptosis has not yet been clearly defined. The intracellular acidity has been known to greatly affect apoptosis probably by affecting the activity of the endonucleases. In this study, the implication of pH in the apoptosis was investigated through the use of human HL-60 leukemia cells. The cells were incubated in media with different pH ranging from 3.5 to 7.5 for 4 hrs and the mode of cell death was investigated. The trypan blue exclusion assay showed that close to 25% and 90% of the cells were dead when incubated in pH 6.4 and pH 5.0 media, respectively. The agarose gel electrophoresis of DNA demonstrated that significant DNA fragmentation occurred in the HL-60 cells incubated in the pH 6.2-6.4 media for 4 hr indicating cell death by apoptosis. The electron microscopy study also demonstrated that many of the cells incubated in the pH 6.4 medium were in the process of apoptosis while the cells maintained in the pH 5.0 medium were dying by necrosis. The intracellular pH (pHi) of HL-60 cells was 6.6-6.9 when the extracellular pH (pHe) was 6.2-6.4. These results demonstrated that DNase I which has a maximal endonuclease activity near pH 7.0 may be responsible for the apoptosis accompanied by DNA fragmentation in HL-60 cells in the pH 6.4 medium. This observation is at variance with the previous reports that DNase II mediate the DNA fragmentation in apoptosis. The cell death at extremely low pH (pH 5.0) appeared to be due mainly to necrosis.

Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.

Freshly isolated mouse hepatocytes were essentially insensitive to TNF-alpha cytotoxicity. However, TNF-alpha induced a concentration-dependent cell death in hepatocytes that had been pretreated with the transcriptional inhibitors actinomycin D (ActD), D-galactosamine, or alpha-amanitin. Unlike RNA synthesis inhibition, a translational block in the presence of cycloheximide (CHX) or puromycin did not sensitive hepatocytes to TNF. On the contrary, these agents prevented hepatocytotoxicity induced by ActD/TNF. Pretreatment with peroxides or glutathione depletors had no significant influence on TNF cytotoxicity. In vivo treatment of mice with ActD/TNF caused hepatic failure, which was significantly reduced by co-treatment with CHX. These findings demonstrate that protein synthesis is required for this mechanism of cell death. To test whether TNF may trigger an endogenous suicide program in hepatocytes, we examined whether DNA fragmentation preceded cell death. In the culture system, hepatocellular DNA fragmentation in the presence of ActD/TNF was observed several hours before lactate dehydrogenase release and was inhibited by CHX. Similar results were obtained in vivo. Chromatin condensation and the formation of apoptotic bodies were observed in livers from mice treated with ActD/TNF and significant DNA fragmentation was detected as early as 4 h after challenge. At this time, organ total glutathione content and plasma transaminase levels were not significantly different from those of untreated controls. The findings of this study demonstrate that direct hepatotoxicity of TNF-alpha is associated with an apoptotic mechanism that becomes manifest under the metabolic condition of arrested transcription and functional translation.

Antibody-induced apoptosis in a human leukemia cell line is energy dependent: thermochemical analysis of cellular metabolism

The mouse monoclonal anti-BAL antibody induced apoptosis in a pre-B acute lymphocytic leukemia cell line within 2 days of incubation, after being crosslinked by a secondary antibody. The antibody specifically recognized a 37 kDa membrane protein that was expressed on a wide spectrum of normal and malignant cells, but induced programmed cell death in only very few of these cells. In this study, we have followed the initial kinetics of the antibody-induced cell death in the human acute lymphocytic leukemia cell line KM-3, by microcalorimetric measurements in conjunction with determination of the cellular proliferation rate and DNA fragmentation. An increase in metabolic activity was observed already after incubating the cells for 20 min with crosslinked anti-BAL antibody, which was several hours before significant growth inhibition and DNA fragmentation were detected. These data show for the first time that the initiation phase of antibody-induced apoptosis is an active, energy-dependent process and not merely an effect of receptor blocking.

In vivo induction of apoptosis (programmed cell death) in mouse thymus by administration of lipopolysaccharide

In vivo administration of bacterial lipopolysaccharide to mice induced DNA fragmentation in the thymus. Fragmented DNA was confirmed by agarose gel electrophoresis and laser flow cytometry. DNA fragmentation was predominantly detected in the thymus of young mice, while it was undetectable in the spleen, bone marrow, and lymph nodes. DNA fragmentation in the thymus was roughly dependent on the dose of lipopolysaccharide injected and reached the peak about 18 h after the injection. The addition of lipopolysaccharide to in vitro cultures of thymocytes did not cause DNA fragmentation, suggesting that lipopolysaccharide was unable to induce apoptosis of thymocytes directly. The injection of lipopolysaccharide induced no significant DNA fragmentation in adrenalectomized mice. The injection of anti-tumor necrosis factor alpha antibody together with lipopolysaccharide partially inhibited the appearance of DNA fragmentation in the thymus. On the basis of the fact that DNA fragmentation is one of the characteristics typical in apoptotic cell death, it was suggested that lipopolysaccharide could induce apoptosis in the mouse thymus in vivo. This apoptosis in the thymus might be mediated mainly by the adrenal hormones, but it is likely that tumor necrosis factor alpha might also participate in it.

Analysis of the Survival of Mature Human Eosinophils: Interleukin-5 Prevents Apoptosis in Mature Human Eosinophils

AbstractWe and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5–mediated survival. In the absence of human IL-5 (hlL-5) mature eosinophils succumbed after 4 days, while in the presence of hlL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hlL-5, while no significant DNA fragmentation was observed in the culture with hlL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hlL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hlL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.

Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils

We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5-mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.

Modulation of thymocyte apoptosis by isoproterenol and prostaglandin E 2

Isoproterenol and prostaglandin E 2 increased CAMP levels in mouse thymocytes transiently, but failed to induce significant internucleosomal DNA fragmentation in thymocytes with a 24-hr incubation. However, these substances showed synergistic interaction with forskolin in the accumulation of cAMP and DNA fragmentation. The addition of 12- O-tetradecanoyl 13-acetate, an activator of protein kinase C, with isoproterenol or particularly with prostaglandin E 2 enhanced DNA fragmentation. These results indicate that an increase in cAMP mediated by isoproterenol or prostaglandin receptor is involved in thymocyte apoptosis through internucleosomal DNA fragmentation in concert with a second signal, the activation of protein kinase C.

Dexamethasone‐induced killing of neoplastic cells of lymphoid derivation: Lack of early calcium involvement

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).

Alloreactive cytotoxic T cells induce DNA fragmentation in peritoneal macrophages: evidence for target cell killing by cytotoxic T cells in vivo.

This report addresses the question whether cytotoxic T cells can cause target cell death in vivo by examining target cell DNA fragmentation. The results show that alloreactive cytotoxic T cells induced significant DNA fragmentation in peritoneal macrophages in vivo and that the DNA fragment was a multiple of 180 +/- 30 bp. Furthermore, the effector cells which caused this characteristic DNA fragmentation were CD8+ T cells. These results demonstrate that cytotoxic T cells can cause target cell death in vivo.

Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes.

An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing "signal" that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.