DNA Fragmentation In Apoptotic Cells Research Articles

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Apoptosis inducing potential of Cyclosporine C in human lung cancer cells (A549)

Cyclotides are a large group of plant based cyclic peptides that have attracted attention in the field of peptide drug design. Cyclic peptides exhibit a more potent biological activity when compared to linear peptides due to their stable cyclic structure. Cyclosporine C is a naturally occurring lipophilic cyclic peptide which is used as an immunosuppressant drug. The objective of this study was to establish Cyclosporine C as a viable novel anticancer drug in the treatment of lung cancer and to find the optimal concentration at which Cyclosporine C results in a significant decrease of viable cancer cells to determine the physiological changes caused by Cyclosporine C and also to find the pathway of apoptosis that is induced by Cyclosporine C. The cytotoxic effect of Cyclosporine C was assessed using techniques such as MTT, Hoechst staining, DNA fragmentation, Flowcytometry and Western blot which were carried out on Lung adenocarcinoma cell line A549. The results obtained showed a significant decrease in A549 viable cells at a concentration of 200μg/mL of Cyclosporine C as observed by the MTT assay. The nuclear condensation and DNA fragmentation of apoptotic cells were observed using Hoechst 33258 staining and DNA fragmentation assays respectively. Western blot confirms that Cyclosporine C treated cells expressed a significant level of caspase-9 enzyme. From the results obtained, we can conclude that Cyclosporine C induces apoptosis in Lung adenocarcinoma cell line A549. The apoptosis is induced by caspace-9 enzyme dependent pathway. The antitumor activity of Cyclosporine C suggests that it has potential as a novel anticancer drug for human lung cancer treatment.

DNA binding, cleavage and cytotoxicity studies of three mononuclear Cu(II) chloro-complexes containing N-S donor Schiff base ligands.

We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide). 1-3 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92μM for 1, 2 and 3, respectively, as compared to the IC50 value (13 μM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 1-3. Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.

Detection of DNA Fragmentation in Apoptotic Cells by TUNEL.

Degradation of DNA into oligonucleosomal-sized fragments is a unique event in apoptosis that is orchestrated by caspase-activated DNase. Traditionally, this event is observed by resolving cellular DNA by gel electrophoresis, which results in a characteristic "ladder" pattern. However, this technique is time-consuming and cannot be used to quantitate the number of apoptotic cells in a sample. Terminal dUTP nick-end labeling (TUNEL) of fragmented DNA allows researchers to identify DNA fragmentation at the single-cell level. This method involves the specific addition of fluorescently labeled UTP to the 3'-end of the DNA fragments by terminal deoxynucleotidyl transferase. The TUNEL assay is both fast and sensitive. Here, we describe a protocol in which cells are treated with TUNEL reagent and counterstained with Hoechst 33342. In contrast to TUNEL, which only stains apoptotic cells, Hoechst 33342 stains the DNA of all cells.

Profiles of teleost DNA fragmentation factor alpha and beta from rock bream (Oplegnathus fasciatus): molecular characterization and genomic structure and gene expression in immune stress

Apoptosis serves to protect normal cells during the processes of development, aging, tissue homeostasis, immunity, and inflammation. Molecules such as caspases, the Bcl-2 family proteins, cytochrome c, APAF-1, and apoptotic endonucleases are concerned with and regulate different stages of apoptosis. DNA fragmentation factor (DFF) is a key final molecule in chromosomal DNA fragmentation during the last step of apoptosis, and is a heterodimeric complex consisting of alpha/inhibitor of caspase-activated DNase (DFFA/ICAD) and beta/caspase-activated DNase (DFFB/CAD) subunits. DFFB/CAD is normally combined with the inhibitor, DFFA/ICAD, in healthy cells. However, after cleavage of the DFF complex by effector caspases, DFFB/CAD acts as an apoptotic nuclease and induces DNA fragmentation in apoptotic cells. The study of DFF proteins is focused on the roles of the apoptotic pathway in mammals. However, gene expression and functions under immune stimulation are not well known in other species. In the present study, we first characterized teleost DFFA/ICAD and DFFB/CAD from rock bream at the molecular level and analyzed the transcriptional expression pattern under immune challenges, to understand their role in immunity. These putative proteins have shown conserved domains and interaction sites orthologous to those of other species. High mRNA expression of both genes was found in the gills and blood of tissues involved in the immune response. The increased expression of DFF genes upon bacterial and viral stimulation was confirmed by qPCR analysis. DFFA/ICAD and DFFB/CAD of rock bream may play a pivotal role in apoptosis and may be involved in host immunity against bacterial and viral infection.

Growth inhibition and apoptosis induction of Scutellaria luteo-coerulea Bornm. & Sint. on leukemia cancer cell lines K562 and HL-60.

Scutellaria (Lamiaceae) has been implicated for medicinal purposes both in modern and traditional medicine. Some species of the genus Scutellaria has extensively been studied for anticancer activity. Scutellaria luteo-coerulea (S. luteo-coerulea) is one of the Iranian species of the genus Scutellaria. In the present study, cytotoxic and apoptogenic properties of CH2Cl2, EtOAc, n-BuOH, and H2O fractions of S. luteo-coerulea were investigated on K562. Moreover, HL-60. DNA fragmentation in apoptotic cells were determined by propidium iodide (PI) staining (sub-G1 peak). Scutellaria luteo-coerulea inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. luteo-coerulea, the CH2Cl2 fraction was found to be the most cytotoxic one among others. Sub-G1 peak in flow cytometry histogram of treated cells suggested the induction of apoptosis in S. luteo-coerulea. Scutellaria luteo-coerulea could be a novel candidate for further analytical elucidation in respect to fine major components responsible for the cytotoxic effect of the plant also clinical evaluations.

Abstract B250: Evaluation of anticancer efficacy of 6-propionylpterin characterized from native isolate Bacillus subtilis.

Abstract In this context the present study is aimed at purifying a pteridine derivative and studying its apoptotic effect on a cancer cell line. 6-propionyl pterin have been purified from a native strain of Bacillus subtilis and administered via intra peritoneal injection to DLA induced mice. During the course of the experiment reduction in angiogenesis was visualized. Earlier report (Kim et.al., 2011) clearly suggested that sepiapterin, a precursor of tetrahydrobiopterin inhibits VEGF-A induced HUVEC proliferation and adhesion through down-regulation of VEGFR-2 downstream signaling pathways, independently of NO synthesis and this supported the present finding. Based on our preconceived view that administration of 6-pp enhances the activity of iNOS, thereby enhanced production of nitric oxide (NO), an attempt was made to check the pathway of NO induced apoptosis. The expression of iNOS, p53, caspase 3, and mdm2 gene were checked by RT PCR. But only mdm2 gene expression alone was upregulated. p53 downregulation might be due to Mdm2 which block its transcriptional activity, favours its nuclear export and stimulates its degradation (Chene, 2003). Caspase 3 activation does not participate in Mdm2 induced apoptosis as determined by the protein levels or poly (ADP-ribose) polymerase fragmentation (Dilla et al., 2000). The report of Wolf et al., 1999 strongly suggests that caspase 3 is the primary activator of apoptotic DNA fragmentation. This is inconsistent with our present finding wherein there was absence of DNA fragmentation in apoptotic cells and also expression of caspase 3 was absent. Thus overall mechanism may be initiated by expression of Mdm2 which inturn inhibited p53 expression. Mdm2 might have down regulated Bcl2 followed by a cascade of Caspase 2 and Cyt-c expression. Cyt-c activation might have regulated Caspase -7 and ROCK protein. ROCK is responsible for cell shrinking and membrane blebbing and finally leading to apoptosis. Caspase 3 activation does not participate in Mdm2 induced apoptosis and hence DNA fragmentation is not found. This study was done to evaluate the efficacy of the compound as anticancer agent and whether it induces apoptosis or not. Mechanism of action of drug was predicted from the results obtained. To derive the exact mechanism further study on the signaling proteins has to be performed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B250. Citation Format: S D. Nisshanthini, R. Sukirtha, S. Achiraman, M. Palaniswamy, Angayarkanni Jayaraman. Evaluation of anticancer efficacy of 6-propionylpterin characterized from native isolate Bacillus subtilis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B250.

Longitudinal Bioluminescence Imaging of the Dynamics of Doxorubicin Induced Apoptosis

Objectives: Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene. Methods: Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining. Results: Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and GloTM luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil. Conclusions: BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.

Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 microM DAS and 50 microM DADS but slightly affected with 100 microM DADS. Treatment with 50 and 100 microM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 microM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca(2+)](i), increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFkappaB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.

Release of Hypoacetylated and Trimethylated Histone H4 Is an Epigenetic Marker of Early Apoptosis

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.

In vitroeffects of fungi isolated from equine hooves on primary human keratinocytes

The effects of two dermatophytes (Microsporum gypseum and Trichophyton mentagrophytes) and four moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum and Penicillium spp.) on living keratinocyte cultures were examined in vitro using primary human keratinocytes. Rates of apoptosis of infected cells were determined using a colorimetric TUNEL system which detects the characteristic nuclear DNA fragmentation of apoptotic cells. The cytotoxicity of the individual fungi was tested by quantitatively measuring cytosolic enzyme lactate dehydrogenase, released upon cell lysis, in culture supernatants. Additionally, the cell structures within the infected keratinocytes in cultures were examined by scanning electron microscopy. All of the fungi exhibited high cytotoxicity, whereas the development of only the two dermatophytes and the mould Scopulariopsis brevicaulis resulted in distinctly increased apoptosis. Electron microscopy showed that all fungi studied caused similar alterations in the cell structure, with Microsporum gypseum being the most harmful. Increasing loss of cell adhesion as a consequence of a decreasing number of reticulating cell appendices and a reduced cell plasticity were the most evident alterations.

Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

302 PROTEIN SUPPLEMENTATION TO IVM MEDIUM IN RELATION TO THE INCIDENCE OF APOPTOSIS IN BOVINE OOCYTES MATURED IN VITRO

Mammalian embryos derived from in vitro fertilization display lower developmental competence and quality when compared to their in vivo counterparts. The composition of culture media significantly contributes to this phenomenon. Media supplemented with FBS or the serum derivate BSA are described as biochemically undefined. Those macromolecules were shown to exert a wide range of effects on cultured embryos, dependent on batch-to-batch variability. Therefore, replacement of these protein sources with a synthetic macromolecule such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) provides a possibility to use a chemically defined culture medium (Ali et al. 2002 Biol. Reprod. 66, 901–905). Apoptosis as programmed cell death naturally occurs in mammalian oocytes and embryos; however, its incidence is significantly higher in vitro. The aim of this study was to investigate whether protein supplementation (FBS, fatty acid-free (faf)-BSA, PVP40) of IVM medium affects the incidence of apoptotic oocytes. In the present study, the IVM system previously described by Makarevich et al. (2002 Biol. Reprod. 66, 386–392) was used. Briefly, follicular oocytes aspirated from slaughterhouse ovaries were matured in vitro in one of three maturation media supplemented with FBS (10%), faf-BSA (6 mg mL−1) or PVP40 (4 mg mL−1). The terminal TUNEL assay kit was used to detect the DNA fragmentation in apoptotic cells (DeadEndTM Fluorometric TUNEL system, Promega, Madison, WI, USA). The data were analyzed by chi-square test of independence. Altogether, 630 oocytes collected during 12 IVM experiments were subjected to the Tunel test, and 563 (89.4%) of them were successfully investigated: 426 after maturation in vitro and 137 follicular, non-matured. The remaining 67 cells were lost during manipulation. The rate of Tunel-positive cells differed (P < 0.001) between matured (11.8%) and follicular oocytes (1.5%). Protein supplementation of IVM media did not significantly affect the rate of apoptotic oocyte occurrence, which was 9% in the faf-BSA group, 11.5% in the FBS group, and 15% in the PVP group. No differences were observed in the rate of Tunel-positive cells between oocytes at MII and MI stages. In conclusion, protein supplementation of IVM medium used in the present study did not affect the incidence of apoptotic oocytes after maturation in vitro. This research was supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN-084 from 2003 to 2005 year.

Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin‐like protease

Although several lines of evidence support a role for serine proteases in apoptosis, little is known about the mechanisms involved. In the present study, we have examined the apoptosis-inducing potential and dissected the death-signalling pathways of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK), inhibitors of chymotrypsin- and trypsin-like proteases, respectively. Our results designate two distinct roles for serine proteases. Firstly, we show that both inhibitors induce biochemical and morphological characteristics of apoptosis, including proteolysis of poly(ADP-ribose) polymerase 1 (PARP-1) and inhibitor of caspase-activated DNase (ICAD), as well as mitochondrial dysfunction, and that their action is abrogated by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (z-VAD.fmk). These results suggest that inhibition of anti-apoptotic serine proteases governs the onset of the caspase-dependant apoptotic cascade. Secondly, we also demonstrate the involvement of a serine protease in the terminal stage of apoptosis. We showed that chymotrypsin-like protease activity is required for internucleosomal DNA fragmentation in apoptotic cells. Hence, DNA fragmentation is abrogated in TPCK-pre-treated WEHI 231 cells undergoing apoptosis triggered either by anti-IgM or TLCK. These results indicate that internucleosomal DNA cleavage in apoptotic cells is mediated by a chymotrypsin-like protease.

Detection of Histone H2AX Phosphorylation on Ser‐139 as an Indicator of DNA Damage (DNA Double‐Strand Breaks)

This unit describes immunocytochemical detection of phosphorylated histone H2AX for revealing the presence of DNA double-strand breaks. Double-strand breaks indicate DNA damage induced by ionizing radiation or by treatment with antitumor drugs such as DNA topoisomerase inhibitors. However, double-strand breaks can also be intrinsic, occurring in healthy, nontreated cells for a variety of reasons, and are generated in the course of DNA fragmentation in apoptotic cells. The unit presents strategies to distinguish radiation- or drug-induced breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes the immunocytochemical detection of histone H2AX phosphorylated on Ser-139 combined with measurement of DNA content to identify cells that have DNA double-strand breaks and to concurrently assess their cell cycle phase. The detection is based on indirect immunofluorescence using a FITC-labeled secondary antibody, and DNA is counterstained with propidium iodide (PI). Cellular RNA, which may be stained by PI, is removed with RNase A.

Measurement of Apoptosis and Other Forms of Cell Death

As programmed cell death (PCD) or apoptosis has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. The first basic protocol allows the qualitative and quantitative assessment of apoptosis in lymphocyte cell cultures using light or fluorescent microscopy. Three protocols follow that are designed to detect nuclear DNA fragmentation and support protocols describe methods to radiolabel the DNA and cytoplasm of the cells to be tested. Techniques that quantitate apoptotic cells using flow cytometry are then described and support protocols provide methods for priming T cell clones and freshly isolated lymph node cells, respectively, for T cell receptor (TCR)-induced apoptosis. Quantitative detection of DNA fragmentation in apoptotic cells is also described. TdT-mediated dUTP-biotin nick end-labeling (TUNEL) methods are provided for the detection of apoptotic cells, along with procedures for the flow cytometric quantitation of apoptotic cells using TUNEL, and TUNEL, staining of tissue sections to identify apoptotic cells. Since much remains incompletely understood about the molecular pathways of programmed death, and it is probably best to perform more than one of the basic protocols to confirm an observation of apoptotic cell death.

Identification of two functional nuclear localization signals in DNase gamma and their roles in its apoptotic DNase activity.

Among DNase I family members, only DNase gamma causes DNA fragmentation during apoptosis. However, the molecular basis for this functional feature of DNase gamma is poorly understood. Here we describe the identification of functional NLSs (nuclear localization signals) in DNase gamma and their roles in its apoptotic function. DNase gamma contains two NLSs: a classical bipartite-type NLS (NLS1) located in the N-terminal half, and a short basic domain (NLS2) at the C-terminus. No potential NLSs are found in the primary structures of other DNase I family DNases. Inactivation of either NLS1 or NLS2 causes reduced DNA ladder-producing activity in DNase gamma. Disruption of NLS2 suppresses ladder formation more effectively than disruption of NLS1. DNase gamma doubly mutated in both NLSs is enzymically active, but no longer catalyses apoptotic DNA fragmentation. Although DNase I fails to produce ladder formation during apoptosis, DNase I fused to NLS2 of DNase gamma through its C-terminus is able to catalyse DNA fragmentation in apoptotic cells. These results indicate that the presence of either NLS1 or NLS2 is necessary for the apoptotic function of DNase gamma, and that the most important domain for this function is NLS2. These findings also explain the lack of apoptotic DNase activity in the other DNase I family DNases.

Expression of tumour necrosis factor-α is associated with apoptosis in lungs of pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Expression of tumour necrosis factor (tnf)-α and its association with apoptosis was examined in lungs from pig experimentally inoculated with porcine reproductive and respiratory syndrome virus (prrsv). Thetnf -α nucleic acid was detected in prrsv -infected alveolar macrophages by reverse transcription–polymerase chain reaction (rt - pcr). The tnf -α antigen was detected in alveolar macrophages by immunohistochemisty. dna fragmentation in apoptotic cells was detected by in situ terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labelling (tunel). A double-labelling procedure using immunohistochemisty for the detection of prrsv and the in situ tunel assay for the detection of apoptosis demonstrated that a majority of labelled cells were infected with prrsv or apoptotic, but not both. Apoptotic cells were more abundant than prrsv -infected cells in all lungs examined. A double-labelling procedure using immunohistochemisty for the detection of tnf -α and the in situ tunel assay for the detection of apoptosis demonstrated that a majority of labelled cells were apoptotic or for tnf -α, but not both. These data suggested that tnf -α released from macrophages after infection by prrsv may induce apoptosis in uninfected bystander cells.

Relationship between apoptosis, tumour necrosis factor, and cell proliferation in chronic cholestasis.

Target of the immune response in chronic autoimmune cholestasis, is the bile duct epithelium. Lymphocytic infiltration and apoptosis have both been suggested to mediate the destruction of hepatocytes and biliary epithelium in primary biliary cirrhosis. To further address this issue in two cholestatic liver diseases characterized by an autoimmune pathogenesis and, furthermore, evaluate the relationship between apoptosis and both tumour necrosis factor alpha and cell proliferation. Liver tissue specimens from 16 patients with primary biliary cirrhosis, 15 with primary sclerosing cholangitis, and 16 with chronic hepatitis C (controls) were evaluated. DNA-fragmentation of apoptotic cells was ascertained by the TdT-mediated deoxyuridine triphosphate nick-end labelling method. Tumour necrosis factor alpha expression and cell proliferation (Ki-67 antigen) were assayed by immunohistochemistry. Hepatocytes with DNA fragmentation were observed in 75% of patients with primary biliary cirrhosis, in 66.6% with primary sclerosing cholangitis, and in 43.7% with chronic hepatitis C. Biliocytes showed apoptosis in only 3 cases of primary biliary cirrhosis. Biliocytes showed a strong cytoplasmic expression in 4 cases (1 primary biliary cirrhosis, 2 primary sclerosing cholangitis and 1 chronic hepatitis C). A few intralobular and portal inflammatory mononuclear cells expressing tumour necrosis factor alpha were observed in 62.5% of patients with primary biliary cirrhosis, 46.1% with primary sclerosing cholangitis, and 56.2% with hepatitis C virus chronic hepatitis. The amount of intraportal mononuclear cells expressing Ki-67 antigen was significantly higher in primary biliary cirrhosis specimens than in primary sclerosing cholangitis (p<0.001) or hepatitis C virus-related chronic hepatitis (p<0.03). No correlation was found within the 3 groups of patients between the Ki-67 histological score and the severity of liver disease. Moreover, no relationship was found between TdT-mediated deoxyuridine triphosphate nick-end labelling and either tumour necrosis factor alpha or Ki-67 staining. Apoptosis is a phenomenon which frequently involves hepatocytes in chronic autoimmune cholestasis. This process is apparently parallel, but unrelated to cell proliferation. Cell proliferation mainly involves mononuclear cells in portal tracts of primary biliary cirrhosis specimens. The finding of tumour necrosis factor alpha expression in biliocytes deserves further study to establish whether this cytokine is involved in triggering bile duct lesions.

APOPTOSIS IN CHRONIC REJECTION OF HUMAN CARDIAC ALLOGRAFTS1

We investigated the role of apoptosis (programed cell death) in the pathogenesis of chronic rejection. Epicardial coronary arteries from cardiac allografts with chronic rejection were examined for apoptosis by the TUNEL assay. Double labeling was carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (vWF) monoclonal antibodies. Additional immunostaining was carried using anti-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associated oligonucleosomal DNA degradation was assessed by DNA agarose gel electrophoresis. The transcription level of apoptosis-related caspase genes were determined using microarrays. Apoptotic cells (TUNEL+) were detected within the arterial wall and in perivascular areas. Double labeling demonstrated that apoptotic cells included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothelial cells (VWF+). Numbers and densities of TUNEL+ cells did not correlate with the degree of arterial stenosis. Apoptosis-associated oligonucleosomal DNA degradation was assessed by agarose gel electrophoresis of DNA, which showed DNA fragments of approximately 180 bp and multimers thereof (DNA laddering gel), which are characteristic for DNA fragmentation in apoptotic cells. Microarray analysis demonstrated that the apoptosis related caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, were all transcribed (caspases 8, 9, and 10 were highly up-regulated). These results are consistent with the involvement of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was present at the sites of apoptotic cells. Immunoreactivity for Bcl-2 was present in areas with very few apoptotic cells. Apoptotic cells include T cells, monocyte/macrophages, and endothelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved in the pathogenesis of chronic rejection in cardiac allografts.