Terminal Deoxynucleotidyl Transferase Labeling Research Articles

Immature Terminal Deoxynucleotidyl Transferase Positive B Cells are Detected in a Subset of Adult and Pediatric Liver Biopsies.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear enzyme restricted to precursor lymphoid cells and their malignant counterparts; immunohistochemical TdT labeling is helpful in recognition of lymphoblasts, which can resemble mature lymphocytes. The diagnosis of B-lymphoblastic leukemia/lymphoma (B-ALL) is occasionally first encountered on liver core biopsy, but TdT immunostain specificity for B-ALL is not clearly established in this setting, which can be problematic when only a few TdT-positive cells are identified. In this study, we evaluated the incidence and distribution of immature B lymphocytes coexpressing TdT and PAX-5, in pediatric and adult liver biopsies, to determine whether a normal complement of hepatic immature B cells can be detected, which must be recognized in a workup to exclude B-ALL. We selected 41 pediatric and adult liver biopsies with a significant portal and/or sinusoidal hematolymphoid infiltrate and performed immunohistochemical stains for TdT and PAX-5 to identify and categorize distribution of immature B cells. TdT-positive cells were detected in 40% of pediatric liver biopsies with a significant hematolymphoid infiltrate (4/10), which included all biopsies from neonates (and infants under 9 wk of age). In adults, immature B-cell infiltrates were less common (6%, 2/31). Dual immunostaining was performed on 2 cases of neonatal hepatitis, which documented B-cell lineage in at least a subset of TdT-positive cells and there was no colabeling with CD3. Immature B cells can be detected in liver biopsies in a variety of clinical settings, most commonly in children, and presence of a few TdT-positive cells cannot be considered entirely specific for involvement by B-ALL. Further workup for B-ALL can be warranted if there is more extensive multifocal portal and/or sinusoidal involvement by blasts with TdT labeling.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Induction of Pyruvate Dehydrogenase Kinase-3 by Hypoxia-inducible Factor-1 Promotes Metabolic Switch and Drug Resistance

The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and associated with tumor malignancy. However, the mechanism of this metabolic switch remains largely unknown. Herein, we reported that hypoxia-inducible factor-1 (HIF-1) induced pyruvate dehydrogenase kinase-3 (PDK3) expression leading to inhibition of mitochondrial respiration. Promoter activity assay, small interference RNA knockdown assay, and chromatin immunoprecipitation assay demonstrated that hypoxia-induced PDK3 gene activity was regulated by HIF-1 at the transcriptional level. Forced expression of PDK3 in cancer cells resulted in increased lactic acid accumulation and drugs resistance, whereas knocking down PDK3 inhibited hypoxia-induced cytoplasmic glycolysis and cell survival. These data demonstrated that increased PDK3 expression due to elevated HIF-1alpha in cancer cells may play critical roles in metabolic switch during cancer progression and chemoresistance in cancer therapy.

P166, a link between the trypanosome mitochondrial DNA and flagellum, mediates genome segregation

Kinetoplast DNA (kDNA), the trypanosome mitochondrial genome, is a giant network containing several thousand interlocked DNA rings. Within the mitochondrion, kDNA is condensed into a disk-shaped structure positioned near the flagellar basal body. The disk is linked to the basal body by a remarkable transmembrane filament system named the tripartite attachment complex (TAC). Following kDNA replication, the TAC mediates network segregation, pulling the progeny networks into the daughter cells by their linkage to the basal bodies. So far TAC has been characterized only morphologically with no known protein components. By screening an RNAi library, we discovered p166, a protein localizing between the kDNA and basal body in intact cells and in isolated flagellum-kDNA complexes. RNAi of p166 has only small effects on kDNA replication, but it causes profound defects in network segregation. For example, kDNA replication without segregation causes the networks to grow to enormous size. Thus, p166 is the first reported molecular component of the TAC, and its discovery will facilitate study of kDNA segregation machinery at the molecular level.

EFFECTS OF ANTIOXIDANT AND OXYGEN TENSION ON REACTIVE OXYGEN SPECIES-RESPONSIVE GENE EXPRESSION AFTER TRANSPLANTATION USING HYPOXIA INDUCED BOVINE OVARIAN TISSUE

A large proportion of childhood and young cancer patients will lose their fertility after aggressive cancer therapy because of the gonadotoxicities of chemotherapy and radiotherapy. One of the strategies to preserve fertility for those facing premature ovarian failure is ovarian tissue banking. The efficacy of ovarian tissue banking for fertility restoration in humans has not been proved yet. However, there is ample evidence of its efficacy in animal studies. Ovarian tissue banking requires further investigations and should be considered experimental in humans at present. Probably, the most important and urgent issue for successful ovarian transplantation is how to minimize ischemic injuries before and during establishment of angiogenesis in the ovarian grafts. It could be speculated that reduced oxygen tension and antioxidant treatment would protect ovarian grafts from hypoxic injuries, as hypoxia induces the generation of reactive oxygen species (ROS). The aim of this study was to determine the effect of reduced oxygen tension and antioxidants treatment on the protection of apoptotic cell death in transplanted ovarian tissue. Bovine ovarian tissue was sliced into a size of 2 mm3 using aseptic technique, then cultured in 5% or 20% O2 for 0h, 3h, 24h and 48h in a-MEM media with/without antioxidants (ascrobic acid andS1P). mRNA expressions of ROS-responsive genes (Mn-SOD, CuZn-SOD, catalase, GPx, GCS, Bax and GAPDH) in the bovine ovary tissue were determined by RT-PCR. Apoptosis was quantified in situ by terminaldeoxynucleotidyl transferase labeling (TUNEL) assay. There was no significant association with ROS-responsive genes expression and O2 concentration after 3 hours. After 24 and 48 hours, however, culture in 5% O2 significantly increased ROS-responsive genes expression than 20% O2. In the ascrobic acid and S1P treated groups, the mRNA expressions of catalase, CuZn-SOD, Mn-SOD were increased compared with control group after 24 hours, whereas the mRNA expressionsof GPx, GCS, Bax were not different. There was no difference in ROS-responsive genes expression between control and antioxidant groups after48 hours. These results show that it is desirable to perform ovarian tissue transplantation within 3 hours post-thawing to minimize ROS production. Treatments with antioxidants play some role in protecting transplanted cells from oxidative stress-induced cell death. The strategies to reduce reactive oxygen species should be considered as helpful options in ovarian tissue transplantation. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2005-003- E00145). (poster)

Expression and function of TNF-family proteins and receptors in human osteoblasts☆

We studied how tumor necrosis-factor (TNF)-family proteins interact with osteoblasts to resolve several controversial points. We measured expression of TNFs, TNF-receptors, and nonsignaling (decoy) TNF receptors in human osteoblasts derived from mesenchymal stem cells and in MG63 human osteosarcoma cells using unamplified mRNA screening, with secondary Western or PCR analysis where indicated, and studied the effects of TNFs on osteoblasts in cell culture. Expression of TNFs and receptors was similar in MG63 cells and osteoblasts. TNF-R1 (p55), TRAIL receptor 1 and 2 (DR4 and 5), and Fas were expressed; RANK was undetectable. TNF-family ligands RANKL, TRAIL, and TNFα were expressed, but mRNAs were typically at low levels relative to receptors, suggesting that osteoblastic TNF signals, including RANKL, require specific stimuli. Flow cytometry of MG63 cells confirmed TNFα receptors and identified subpopulations with high surface-bound TNFα. Decoy receptors expressed included a novel soluble form of TNFRSF25 (formerly DR3 or Apo3), implicated in rheumatoid-arthritis linkage studies, as well as osteoprotegerin, a well-characterized osteoblast protein that binds TRAIL and RANKL, and DcR2, which binds TRAIL. Osteoblast apoptosis was studied using terminal deoxynucleotidyl transferase labeling and annexin V binding. MG63 cells were resistant to apoptosis by exogenous TNFα except when grown in media promoting osteoblast-like growth or matrix nodules. However, in media supporting osteoblast-like phenotype, apoptosis was induced by anti-Fas or TNF, in contrast to other studies with human osteoblasts. TRAIL caused cell retraction, supporting functional TRAIL response in cell differentiation, but did not cause apoptosis. We conclude that human osteoblasts have functional receptors for FasL, TNFα, TRAIL, but not RANKL, and that osteoblasts are protected by multiple nonsignaling TNF receptors against destruction by TNF-family proteins under conditions favoring cell growth.

Expression and function of TNF-family proteins and receptors in human osteoblasts>

We studied how tumor necrosis-factor (TNF)-family proteins interact with osteoblasts to resolve several controversial points. We measured expression of TNFs, TNF-receptors, and nonsignaling (decoy) TNF receptors in human osteoblasts derived from mesenchymal stem cells and in MG63 human osteosarcoma cells using unamplified mRNA screening, with secondary Western or PCR analysis where indicated, and studied the effects of TNFs on osteoblasts in cell culture. Expression of TNFs and receptors was similar in MG63 cells and osteoblasts. TNF-R1 (p55), TRAIL receptor 1 and 2 (DR4 and 5), and Fas were expressed; RANK was undetectable. TNF-family ligands RANKL, TRAIL, and TNFα were expressed, but mRNAs were typically at low levels relative to receptors, suggesting that osteoblastic TNF signals, including RANKL, require specific stimuli. Flow cytometry of MG63 cells confirmed TNFα receptors and identified subpopulations with high surface-bound TNFα. Decoy receptors expressed included a novel soluble form of TNFRSF25 (formerly DR3 or Apo3), implicated in rheumatoid-arthritis linkage studies, as well as osteoprotegerin, a well-characterized osteoblast protein that binds TRAIL and RANKL, and DcR2, which binds TRAIL. Osteoblast apoptosis was studied using terminal deoxynucleotidyl transferase labeling and annexin V binding. MG63 cells were resistant to apoptosis by exogenous TNFα except when grown in media promoting osteoblast-like growth or matrix nodules. However, in media supporting osteoblast-like phenotype, apoptosis was induced by anti-Fas or TNF, in contrast to other studies with human osteoblasts. TRAIL caused cell retraction, supporting functional TRAIL response in cell differentiation, but did not cause apoptosis. We conclude that human osteoblasts have functional receptors for FasL, TNFα, TRAIL, but not RANKL, and that osteoblasts are protected by multiple nonsignaling TNF receptors against destruction by TNF-family proteins under conditions favoring cell growth.

Inhibition of Dendritic Cell Differentiation by Fumaric Acid Esters

Fumaric acid esters have proved to be effective for the systemic treatment of severe psoriasis vulgaris. These compounds have been shown to induce a Th2-like cytokine secretion pattern in T cells and to reduce keratinocyte proliferation in vitro. Dendritic cells seem to be of major importance as regulatory cells driving the psoriatic tissue reaction. Monocytes or CD34-positive myeloid progenitor cells are precursors of dendritic cells that can be generated in vitro by culture with granulocyte-macrophage colony-stimulating factor and interleukin-4. Using this model the effect of fumaric acid esters on granulocyte-macrophage colony-stimulating factor/interleukin-4-induced differentiation of monocyte-derived dendritic cells was investigated. The results of this study show that dimethylfumarate as well as methylhydrogenfumarate-calcium-salt (0.01-100 microg per ml) concentration-dependently inhibit monocyte-derived dendritic cell differentiation. This was reflected by an inhibition of CD1a, CD40, CD80, CD86, and HLA-DR expression as well as by a reduced capacity of dimethylfumarate-treated monocyte-derived dendritic cells to stimulate lymphocytes in the allogeneic mixed lymphocyte reaction. Other fumaric acid esters showed no effect on monocyte-derived dendritic cell-differentiation. At higher concentrations (30-100 microg per ml) dimethylfumarate, but not methylhydrogenfumarate calcium-salt induced apoptosis in monocyte-derived dendritic cells as measured by expression of Apo 2.7 and DNA fragmentation (TUNEL assay). These data point to a high susceptibility of the monocyte/dendritic cell system to dimethylfumarate and its main metabolite methylhydrogenfumarate. Other fumaric acid esters investigated were without effect. As the effects of fumarates on monocyte-derived dendritic cells observed occur at concentrations 20-fold lower compared with lymphocytes, our data seem to be of relevance in explaining the possible mode of action of these compounds in psoriasis.

Intrastriatal dopamine injection induces apoptosis through oxidation-involved activation of transcription factors AP-1 and NF-kappaB in rats.

ABSTRACT More and more evidence suggests that increases in dopamine (DA) in striata may participate in neurodegenerative processes during acute ischemia, hypoxia, and excitotoxicity. With a rat model of intrastriatal DA injection, we studied the molecular events involved in DA toxicity. Intrastriatal injections of DA in amounts from 1 to 2 micromol result in apoptotic cell death, as indicated by terminal deoxynucleotidyl transferase labeling of DNA strand breaks and Klenow polymerase-catalyzed [(32)P]deoxycytidine triphosphate-labeled DNA laddering. Injections of DA produce a strong and prolonged activated protein 1 (AP-1) activity that contains c-fos, c-jun, and phosphorylated c-jun protein. DA injections also stimulate the activity of nuclear factor-kappaB (NF-kappaB), an oxidative stress-responsive transcription factor. Injection of curcumin at a dose that selectively inhibits AP-1 activation without affecting NF-kappaB activity attenuates DNA laddering induced by DA. Preinjection with SN50, a specific permeable recombinant NF-kappaB translocation inhibitor peptide, reduces DA-induced NF-kappaB activation and apoptosis. Moreover, preinjection of the antioxidant GSH significantly inhibits both DA-induced activation of transcription factors AP-1 and NF-kappaB and subsequent apoptosis. Thus, our data suggest that DA-oxidative stress-induced apoptosis in vivo is mediated by activation of transcription factors AP-1 and NF-kappaB.

Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations.

Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis). We investigated the effects of As2O3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As2O3 can be observed in these cells at clinically achievable concentrations. Eight malignant lymphocytic cell lines and primary cultures of lymphocytic leukemia and lymphoma cells were treated with As2O3, with or without dithiothreitol (DTT) or buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis). Apoptosis was assessed by cell morphology, flow cytometry, annexin V protein level, and terminal deoxynucleotidyl transferase labeling of DNA fragments. Cellular proliferation was determined by 5-bromo-2'-deoxyuridine incorporation into DNA and flow cytometry and by use of a mitotic arrest assay. Mitochondrial transmembrane potential (delta psi(m)) was measured by means of rhodamine 123 staining and flow cytometry. Protein expression was assessed by western blot analysis or immunofluorescence. Therapeutic concentrations of As2O3 (1-2 microM) had dual effects on malignant lymphocytes: 1) inhibition of growth through adenosine triphosphate (ATP) depletion and prolongation of cell cycle time and 2) induction of apoptosis. As2O3-induced apoptosis was preceded by delta psi(m) collapse. DTT antagonized and BSO enhanced As2O3-induced ATP depletion, delta psi(m) collapse, and apoptosis. Caspase-3 activation, usually resulting from delta psi(m) collapse, was not always associated with As2O3-induced apoptosis. As2O3 induced PML (promyelocytic leukemia) protein degradation but did not modulate expression of cell cycle-related proteins, including c-myc, retinoblastoma protein, cyclin-dependent kinase 4, cyclin D1, and p53, or expression of differentiation-related antigens. Substantial growth inhibition and apoptosis without evidence of differentiation were induced in most malignant lymphocytic cells treated with 1-2 microM As2O3. As2O3 may prove useful in the treatment of malignant lymphoproliferative disorders.

Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis.

Infection with Helicobacter pylori induces chronic gastritis in virtually all infected persons, and such gastritis has been associated with an increased risk of developing gastric cancer. This risk is further enhanced with cagA+ (positive for cytotoxin-associated gene A) H. pylori strains and may be a consequence of induced gastric cell proliferation and/or alteration in apoptosis (programmed cell death) in the gastric epithelium. To determine whether the H. pylori cagA genotype and another virulence-related characteristic, the vacA (vacuolating cytotoxin A) s1a genotype, differentially affect epithelial cell proliferation, apoptosis, and the histologic parameters of inflammation and injury, we quantitated these characteristics in infected and uninfected persons. Fifty patients underwent upper gastrointestinal endoscopy, and biopsy specimens were taken. Apoptotic cells in the specimens were quantitated after terminal deoxynucleotidyl transferase labeling of DNA fragments with digoxigenin-deoxyuridine triphosphate; epithelial cell proliferation was scored by immunohistochemical analysis of the proliferation-associated antigen Ki-67. Antibodies directed against H. pylori and CagA protein were measured in the serum of patients by means of enzyme-linked immunosorbent assays. Analysis of H. pylori genomic DNA, by use of the polymerase chain reaction, was performed to determine the cagA and vacA genotypes. Acute and chronic inflammation, epithelial cell degeneration, mucin depletion, intestinal metaplasia, glandular atrophy, and vacuolation were each scored in a blinded manner. Reported P values are two-sided. Persons harboring cagA+ strains (n = 20) had significantly higher gastric epithelial proliferation scores than persons infected with cagA-strains (n = 9) or uninfected persons (n = 21) (P = .025 and P<.001, respectively), but the difference in cell proliferation between the latter two groups was not statistically significant. The number of apoptotic cells per 100 epithelial cells (apoptotic index) in persons infected with cagA+ strains was lower than in persons infected with cagA-strains (P = .05). Apoptotic indices in the cagA+ group were similar to those in the uninfected group (P = .2). Epithelial cell proliferation was significantly correlated with acute gastric inflammation, but only in the cagA+ group (r = .44; P = .006). The cagA+ and vacA s1a genotypes were found to be concordant, confirming the close relationship between these virulence-related genotypes. Gastric mucosal proliferation was significantly correlated with the severity of acute gastritis in persons infected with cagA+ vacA s1a strains of H. pylori. This increased proliferation was not accompanied by a parallel increase in apoptosis. Increased cell proliferation in the absence of a corresponding increase in apoptosis may explain the heightened risk for gastric carcinoma that is associated with infection by cagA+ vacA s1a strains of H. pylori.

DNA damage and apoptosis in Alzheimer's disease: colocalization with c- Jun immunoreactivity, relationship to brain area, and effect of postmortem delay

Many neurons in Alzheimer's disease (AD) exhibit terminal deoxynucleotidyl transferase (TdT) labeling for DNA strand breaks with a distribution suggestive of apoptosis. We have shown previously that immunoreactivity for c-Jun is elevated in AD and found in association with neuronal pathology. In addition, cultured neurons undergoing beta-amyloid-mediated apoptosis exhibit a selective and prolonged induction of c-Jun. Consequently, we conducted double-labeling experiments to examine whether c-Jun is associated with DNA strand breaks in AD tissue; we observed a strong colocalization between these markers. As would be predicted based on the distribution of AD pathology, we also found that TdT labeling was prominent in the entorhinal cortex, but absent or at very low levels in cerebellum. Furthermore, we confirmed that postmortem delay (PMD) does not affect TdT labeling within the limits used for tissue used in this study. However, in contrast to previous studies, we report an increase in TdT labeling with more extended PMDs. Finally, gel electrophoresis of genomic DNA isolated from AD and control cases failed to reveal evidence for either an apoptotic or a necrotic mechanism of cell death in AD, possibly because of a low number of cells actually undergoing cell death at any given time. Our findings support the hypothesis that DNA damage labeled using TdT reflects neuronal vulnerability and cell loss associated with AD pathology, and that at least a portion of the cells labeled with this technique is undergoing apoptosis. Furthermore, in agreement with in vitro findings, these results suggest a relationship between the expression of c-Jun and neuronal risk and/or cell death in AD.

Apoptosis in Target Organs of Hypertension

Apoptosis or programmed cell death frequently parallels abnormalities in cell proliferation and differentiation. As hypertrophy/hyperplasia or remodeling occurs in organs affected by hypertension, we evaluated the degree of apoptosis in the heart, kidney, and brain in situ in genetically hypertensive mice and rats as well as in cultured vascular smooth muscle cells. Apoptosis was characterized by morphological features, DNA fragmentation, and laddering as well as by terminal deoxynucleotidyl transferase labeling of the 3' OH ends of both extracted DNA and tissue sections. The present report provides the first evidence of increased apoptosis in whole organs of genetically hypertensive rat and mouse strains: in the heart of spontaneously hypertensive rats (SHR) and in the heart (ventricular cardiomyocytes), kidney (inner cortex and medulla), and brain (cortex, striatum, hippocampus, and thalamus) of spontaneously hypertensive mice, with a higher effect of apoptotic inducers in cultured aortic smooth muscle cells derived from SHR. Both types of known apoptotic processes, oligonucleosomal cleavage and large DNA fragmentation, were observed in vascular smooth muscle cells, but only the former appeared to be increased in SHR. This study underlines the importance of cell death dysregulation in hypertension, reveals a new route for investigation of the pathogenesis of hypertension, and suggests novel targets of therapeutic intervention.