TdT-mediated dUTP-biotin Nick-end Labeling Staining Research Articles

Overexpression of BCL-2, BCL-X, and BAX in Primary Central Nervous System Lymphomas that Occur in Immunosuppressed Patients

In contrast to primary central nervous system lymphomas (PCNSLs) that occur in immunocompetent patients, most of those that occur in immunosuppressed patients are associated with Epstein-Barr virus (EBV). BCL-2–related proteins either block or promote cell death, forming homo- or heterodimers with each other. LMP-1, EBV latent protein, has been shown to upregulate BCL-2 and BCL-XL. This observation suggests that these proteins may be involved in the transformation process of EBV-infected cells.Twenty-three cases of PCNSLs were studied: 12 of the patients were immunosuppressed, and 11 were immunocompetent. For all cases, we collected clinical information, histologic data, and immunophenotype and tested for the presence of EBV (EBER-1, LMP-1). Apoptosis was assessed by the TdT-mediated dUTP-biotin nick-end labeling method and quantified by image analysis. In three cases, electron microscopy was performed. The BCL-2 family proteins (BCL-2, BCL-X, MCL1, and BAX) and p53 expression were studied by immunohistochemistry on paraffin slides.All cases were classified as diffuse large B-cell lymphomas. PCNSLs in immunosuppressed patients were characterized by EBV association, necrosis, important gliosis, and numerous macrophages. There was no significant difference between the two groups regarding the TdT-mediated dUTP-biotin nick-end labeling staining (P =.08). In contrast, PCNSLs in immunosuppressed patients were shown to express high levels of BCL-2, BCL-X, and BAX in more than 80% of tumor cells in 7, 10, and 11 cases, respectively. In immunocompetent patients, only one case showed a high level of BCL-2 expression in more than 80% of the cells, whereas BCL-X and BAX were overexpressed in two cases. These differences are significant (P <.05). In contrast, there was no significant difference between the two groups in MCL-1 expression. Besides EBV association and necrosis, PCNSLs related to immunosuppression are characterized by an overexpression of BCL-2–related proteins, without dramatically modifying their susceptibility for apoptosis.

Poliovirus 2A protease induces apoptotic cell death.

A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.

Caspase Inhibitor Blocks Human Immunodeficiency Virus 1–Induced T-Cell Death Without Enhancement of HIV-1 Replication and Dimethyl Sulfoxide Increases HIV-1 Replication Without Influencing T-Cell Survival

To determine the relationship, if any, between reagents that modulate survival of T-cells and replication of human immunodeficiency virus 1 (HIV-1) and to determine the effects of the solvent dimethyl sulfoxide (DMSO) and drugs such as cyclosporin A and all-trans retinoic acid on HIV-1 replication. To first establish the direct effects of solvent alone (ie, DMSO) at various concentrations on HIV-1 replication, followed by the ability of various compounds such as the caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethylketone (z-VAD-fmk), cyclosporin A, and all-trans retinoic acid on HIV-1 replication. Next, to determine if HIV-1 induces T-cell apoptosis using TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assays and DNA fragmentation and poly-(ADP-ribose)-polymerase (PARP) cleavage, and then to examine how the various compounds influence T-cell survival after HIV-1 exposure. The human T-cell line, CEM cells, were exposed to HIV(IIIB) and viral replication monitored using reverse transcription assays at 3, 6, and 9 days following infection. Cells were pretreated with various compounds dissolved in DMSO over a wide range of concentrations, and DMSO itself was also examined. T-cell death and apoptosis were assessed using TUNEL staining to detect 3'-OH DNA strand breaks and agarose gel electrophoresis to detect DNA fragmentation (laddering). Furthermore, PARP cleavage implicated in the apoptotic process was also examined. At very low levels, such as 0.002%, DMSO itself appears to enhance HIV-1 replication at 6 and 9 days after infection. At low levels of cyclosporin A, such as 0.01 microgram/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 microgram/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 microgram/mL did not influence HIV-1 replication. In addition, a discrepancy was noted in that HIV-1-infected T-cells were TUNEL positive, indicating DNA strand breaks; however, more complete DNA fragmentation was not detected nor was PARP cleavage identified. The induction of TUNEL positivity was blocked by the caspase inhibitor z-VAD-fmk but not by DMSO or cyclosporin A. Even though z-VAD-fmk blocked the appearance of TUNEL-positive T-cells, there was not a consistently observed increase in HIV-1 replication. Low levels of DMSO and cyclosporin A can enhance HIV-1 replication in CEM cells. At higher levels, cyclosporin A inhibits HIV-1 replication with no significant effects by all-trans retinoic acid. No evidence for classic apoptosis was detected in CEM cells after HIV-1 infection, although DNA strand breaks may be present as revealed by TUNEL positivity. There was no correlation between levels of HIV-1 replication and T-cell survival or death. The mechanism of T-cell death after HIV-1 infection requires further study, and investigators who add compounds dissolved in DMSO must include controls to carefully examine the direct effects of even trace levels of this solvent on HIV-1 replication.

Immunohistochemical Analysis of Apoptosis-Related Proteins in Human Embryonic and Fetal Pancreatic Tissues

The growth of both cancer cells and fetal tissue is rapid; however, cancer cells de-differentiate and proliferate in a disorderly manner, whereas fetal tissues differentiate and proliferate in an orderly manner. Thus, there may be both common and different factors that are involved in the process of the uncontrolled cell growth of pancreatic cancers and the development of the fetal pancreas. The common part of the mechanisms should be in the regulation of the cell cycle, resulting in rapid proliferation via such mechanisms as growth stimulation and avoidance of apoptosis. Therefore, in the current study we investigated the expression of apoptosis-related proteins in fetal pancreatic tissues. Sixteen human embryonic and fetal pancreatic tissues obtained between 6 and 32 wk of gestation were used. We immunohistochemically examined the protein expression of Bcl-2, Bcl-XL, Mcl-1, and Bax. Further, the expression of insulin, glucagon, and proliferting cell nuclear antigen (PCNA), and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining were examined. In embryonic and fetal pancreatic tissues, Bcl-2 was not detected in any type of pancreatic cell (acinar, ductal, or islet). Bcl-XL was expressed in all types of pancreatic cells throughout the gestation. Mcl-1 was expressed in all types of pancreatic components, and strongly expressed in the margin of the islets. Bax, a pro-apoptotic protein, was expressed in all components. PCNA was strongly expressed in the embryonic and fetal pancreas, especially in early stages of gestation; however, TUNEL staining was negative in all samples. At least one antiapoptotic protein was expressed in all types of pancreatic cells. The results of the current study indicate that active proliferation and avoidance of apoptosis take place in embryonic and fetal pancreatic tissues, which may be controlled by particular combinations of apoptosis-related proteins. Among these proteins, Bcl-XL and Mcl-1 may play an important role in the proliferation and differentiation of the embryonic and fetal pancreas.

Retinal Cell Death by Light Damage

Purpose: To determine the relationship between apoptotic photoreceptor cell death and the duration of light exposure. Methods: Ten-week-old male albino rats (Wistar strain) were dark-adapted for 2 days and then exposed to intense light for 12 hours, and 1, 2, 3, 7, 14, 21, and 28 days. The presence of apoptosis was confirmed by electron microscopy and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method. Differences in the apoptotic processes of the photoreceptor cells in the superior, posterior pole, and inferior portions of the retina were determined. Results: Photoreceptor cells showed TUNEL-positive staining, whereas the cells in the inner nuclear layer, ganglion cell layer and retinal pigment epithelia exhibited weak positive or negative TUNEL staining. By electron microscopy, photoreceptor cells showed typical apoptotic nuclear changes and formation of apoptotic bodies. Conclusions: The sensitivity to light damage and style of death differed among retinal cells by location and cell type.

Neuronal stress response and neuronal cell damage after cardiocirculatory arrest in rats.

Cardiocirculatory arrest is the most common clinical cause of global cerebral ischemia. We studied neuronal cell damage and neuronal stress response after cardiocirculatory arrest and subsequent cardiopulmonary resuscitation in rats. The temporospatial cellular reactions were assessed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining of DNA fragments, in situ hybridization (heat shock protein hsp70; immediate early genes c-fos and c-jun), and immunocytochemical (HSP70; and myeloperoxidase, specific marker of polymorphonuclear leukocytes [PMNL]) techniques. Cardiac arrest of 10 minutes' duration was induced in mechanically ventilated male Sprague-Dawley rats anesthetized with nitrous oxide and halothane. After cardiopulmonary resuscitation, animals were allowed to reperfuse spontaneously for 6 hours, 24 hours, 3 days, and 7 days (n = 6 per group). Five sham-operated animals were controls. The TUNEL staining revealed an early onset degeneration in the thalamic reticular nucleus (TRN) at 6 hours that peaked at 3 days. In contrast, degeneration was delayed in the hippocampal CA1 sector, showing an onset at 3 days and a further increase in the number of TUNEL-positive cells at 7 days. A minor portion of TUNEL-positive nuclei in the CA1 sector showed condensed chromatin and apoptotic bodies, whereas all nuclei in the TRN revealed more diffuse staining. After 6 hours of reperfusion, levels of mRNA for hsp70 and c-jun were elevated in circumscribed areas of cortex, in all hippocampal areas, and in most nuclei of thalamus, but not in the TRN. After 24 hours, a strong expression of mRNA for hsp70 and c-jun could be observed in the second layer of the cortex and in hippocampal CA1 sector; hsp70 also was observed in hippocampal CA3 sector. Some animals showed expression of hsp70 and c-jun in the dentate gyrus. After 3 days, hsp70 and c-jun were detected mainly in the CA1 sector of hippocampus. At 7 days, mRNA for both returned to control values. Therefore, delayed cell degeneration in the CA1 sector corresponds to a prolonged expression of hsp70 and c-jun in this area. In situ hybridization studies for c-fos revealed a strong signal in CA3 and dentate gyrus and a less prominent signal in TRN at 6 hours. At 24 hours, CA4 and amygdalae were positive, whereas at 3 and 7 days, the signal reached control levels; no prolonged or secondary expression was observed in the CA1 sector. Immunohistochemical study confirmed translation of HSP70 in various areas corresponding to the detection of mRNA, including the CA1 sector. The number of PMNL increased significantly at 6 hours and 7 days after cardiac arrest; PMNL were distributed disseminately and were not regionally associated with neuronal cell damage. The current data support the view that CA1 neurons might undergo an apoptosis-associated death after cardiac arrest, but PMNL are not directly involved in this process. The marked differences in the time course and the characteristics of TUNEL staining and the neuronal stress response in CA1 sector and TRN point to different mechanisms of neuronal injury in the two selectively vulnerable areas.

Apoptosis in the pathogenesis of cutaneous lupus erythematosus.

Defective regulation of apoptosis may play a role in the development of autoimmune diseases such as systemic lupus erythematosus, in which the skin is a prominent target. To our knowledge, however, the nature of epidermal changes in cutaneous lupus erythematosus (LE) has not previously been investigated. We investigated the involvement of apoptosis in cutaneous LE. A total of 44 lesional skin samples from patients with cutaneous LE, 44 skin samples from patients with scleroderma, five skin specimens from patients suffering from dermatomyositis, and 13 normal skin samples were stained immunohistochemically with monoclonal antibodies to Ki-67, p53 (DO-7), and bcl-2. The lesional skin from cutaneous LE, except LE profundus, showed a marked increase in Ki-67- and p53-positive keratinocytes, which were predominantly located in the basal layer of the epidermis and follicle, and a drastic reduction in the number of bcl-2-positive cells localized in the basal cell compartment. With TdT-mediated dUTP-biotin nick end-labeling staining, we demonstrated that extensive apoptosis occurred in almost the whole epidermis of cutaneous LE, except in cases of LE profundus. This abnormal expression of Ki-67, p53, and bcl-2 and the occurrence of apoptosis in the epidermis was also observed in epidermis from patients with dermatomyositis, but not in that from patients with scleroderma.

Flow cytometric measurement of mitochondrial mass and function: a novel method for assessing chemoresistance.

Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line. A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (deltapsi(m)) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH) fluorescence. Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in deltapsi(m) (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or deltapsi(m). Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis. SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis and induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.

Cell Death Suggestive of Apoptosis After Spinal Cord Ischemia in Rabbits

After spinal cord ischemia, some neurons remain viable after an ischemic insult but may be at risk of dying during reperfusion. We searched for morphological and biochemical features of apoptosis, which is a mechanism of delayed neuronal death, in a rabbit model of spinal cord ischemia. The infrarenal aorta of White New Zealand rabbits (n = 24) was occluded for 40 minutes using a loop tourniquet. Rabbits were killed after 12, 24, or 48 hours (n = 8 per group). The loop was placed but never tightened in sham-operated rabbits (n = 6). The lumbar segment of the spinal cord (L5 to L7) was used for morphological studies, including hematoxylin and eosin staining and a modified terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining method. Electron microscopy was used to examine ultrastructural morphology. In addition, lumbar tissue was used for biochemical investigation of DNA laddering by agarose gel electrophoresis. After ischemia, the affected areas contained neurons with positive TUNEL staining. Positive neurons were located in laminae III to IX, although most were concentrated in the intermediate and ventral areas. Adjacent sections stained with hematoxylin and eosin exhibited ischemic cell changes (red and ghost neurons), while apoptotic bodies were also apparent. In addition, electron microscopy of ischemic tissue samples exhibited ultrastructural characteristics of apoptosis, including nuclear condensation and relatively normal organelle morphology. Finally, isolated DNA revealed a ladder on agarose gel electrophoresis, indicating DNA fragmentation into approximately 180 multiples of base pairs. Spinal cord ischemia in rabbits induces morphological and biochemical changes suggestive of apoptosis. These data raise the possibility that apoptosis contributes to neuronal cell death after spinal cord ischemia.