End-labeled DNA Research Articles

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Apoptotic Changes in the Epithelium Germinativum of the Cat (Felis catus s. domestica, L. 1758) at Different Ages and Breeding Seasons

Apoptosis (programmed cell death) could be considered as a physiological process that takes part in a healthy organism, which helps to maintain organism homeostasis. The visible deterioration of semen quality and the number of germ cells is accompanied by a seasonal decrease of the reproductive activity in some species. This post-effect cascade is caused by apoptosis, which is the primary mechanism responsible for the elimination of germ cells during spermatogenesis. The aim of our study was to assess apoptotic changes in the epithelium germinativum in cat testes at different ages. One hundred and two pairs of testes were obtained from domestic cats aged between 4 months and 10 years. The paraffin-embedded tissue sections were labelled using the Oncogene and Calbiochem Research Products DNA Fragmentation Detection Kit (Cat# QIA21; Darmstadt, Germany), which allows the recognition of apoptotic nuclei in tissue sections with Fragment End Labelling (FragEL) of DNA. The activity of apoptotic processes in cat testes collected from the spring-summer period compared with the autumn-winter season revealed that, 59.42% and 51.51%, respectively, males testes were characterized by insignificant changes. The obtained data revealed a distinctive apoptotic changes in the young animal testes before spermatogenesis onset. An intensification of programmed death cells in the epithelium germinativum in the elder cats (between 3-6 and 6-10 years) was not observed. Apoptotic changes slightly intensified in cats aged between 12 and 36 months.

Apoptosis of rat gastric mucosa and of primary cultures of gastric epithelial cells by indomethacin: role of inducible nitric oxide synthase and interleukin-8.

In order to gain insights into indomethacin-induced gastric injury, rats were fed with indomethacin (20 mg/kg), or alternatively, the primary cultures of rat gastric epithelial cells were cultured with different doses of indomethacin (1-1000 microM). Light microscopy, electron microscopy, fluorescence microscopy, TdT-mediated dUTP-biotin nick end labelling staining, ssDNA staining and DNA fragmentation assay were employed to evaluate the levels of gastric injury and apoptosis. Cells expressing inducible nitric oxide synthase (iNOS) and interleukin (IL)-8 were localized at the rat gastric mucosa by immunohistochemistry. Administration of indomethacin to rats caused apoptosis and injury of the gastric mucosal epithelial cells. Indomethacin also induced apoptosis of primary cultures of gastric epithelial cells in a dose-dependent manner. Cells expressing iNOS and IL-8 were detected at and around the sites of gastric injury in the indomethacin-fed rats, but not in the control rats. The induction of apoptosis by indomethacin in the primary cultures of gastric epithelial cells suggests that the direct apoptotic capacity of indomethacin. iNOS and IL-8 may be involved in this process.

Loss of Cyclin G1 Expression in Human Uterine Leiomyoma Cells Induces Apoptosis

Observations from the authors' laboratory suggest a physiological role for increased cyclin G1 protein levels in human uterine leiomyoma. The hypothesis of the present study is that the strategic modulation of cyclin G1 by antisense technology will inhibit the survival of in vitro-grown uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with cyclin G1 ribbon-type antisense oligonucleotide (cyclin G1 RiAS) to effectively reduce cyclin G1 expression. Cell viability, in situ terminal deoxyuridine nick end-labeling (TUNEL) assay, flow cytometry, DNA fragmentation, and expression of cell cycle regulatory-related proteins were evaluated by Western blot. Antisense oligonucleotides compromised uterine leiomyoma cell viability and inducted apoptosis in a caspase-independent mechanism. In situ TUNEL and DNA fragmentation revealed apoptosis induction, and fluorescent-activated cell sorting analysis showed increased sub-G1-phase cells. Furthermore, abrogation of cyclin G1 enhanced p53 accumulation, phosphorylation of p53 at Ser-15 residue, and increased expression of cyclin-dependent kinase inhibitors p21 and p27. These data imply that cyclin G1 expression is associated with growth promotion and the potential utility and novelty of using ribbon-type antisense oligonucleotides as a gene therapy strategy to treat human uterine leiomyoma.

Highly concentrated calcitriol and its analogues induce apoptosis of parathyroid cells and regression of the hyperplastic gland--study in rats

Controlling hyperplasia of the parathyroid gland (PTG) is important in the management of secondary hyperparathyroidism (SHPT). Regression of the hyperplastic PTG requires a decrease in the number of parathyroid cells (PTCs), so the present study investigated cell death caused by toxic agents or by clinically usable vitamin D metabolites. The PTGs of Sprague-Dawley rats, which had been 5/6-nephrectomized and fed a high-phosphate diet for 12 weeks, were treated with two consecutive direct injections (DI) of calcitriol, maxacalcitol, paricalcitol, doxercalciferol or phosphate-buffered saline containing either 0.01% or 90% ethanol (0.01-ET or 90-ET, respectively). Laboratory data, including serum levels of intact parathyroid hormone (intact-PTH), were obtained before and after the treatments. The PTGs were excised 24 h after the final injection and evaluated for PTC apoptosis using light and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) method and DNA electrophoresis. Treatment with any of the vitamin D metabolites and 90-ET significantly decreased the serum intact-PTH level, but only the latter significantly decreased the serum Ca level. Either treatment markedly increased the number of TUNEL-positive PTCs, but not in PTG treated with 0.01-ET. In PTGs treated with DI of any vitamin D metabolites was there ladder formation on DNA electrophoresis, as well as the characteristic morphological features of apoptosis in both the light and electron microscopic studies. DI of vitamin D metabolites may be effective in controlling not only the PTH level, but also PTG hyperplasia, in advanced SHPT by, at least in part, apoptosis-induced cell death. Our study was performed in rats.

Neurotoxicity of MPTP to migrating neuroblasts: Studies in acute and subacute mouse models of Parkinson's disease

The acute or subacute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been widely used in C57BL/6 mice to develop models of Parkinson's disease (PD). The loss of dopaminergic neurons is suggested to be mediated by a mechanism of nonapoptotic cell death or by apoptosis. In recent years, the notion that the neurotoxicity of MPTP is restricted to dopaminergic neurons in the substantia nigra (SN) has been challenged. Here, we provide evidence of rapid cell death in the subventricular zone (SVZ) and rostral migratory stream (RMS) in the adult C57BL/6 mouse brain in response to acute or subacute treatment with MPTP. Significant terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) of fragmented DNA was observed at 24 h (or 1 day) after the last injection in the acute model or after the first injection in the subacute model. Ultrastructural analysis confirmed that dying cells displayed an apoptotic morphology. Using a double labeling method, we demonstrated that the phenotype of the cells undergoing apoptosis is that of migrating neuroblasts. This is further supported by evidence of a subsequent loss of migrating neuroblasts. The results raise the possibility that migrating neuroblasts in the SVZ and RMS may be more vulnerable to MPTP than nigrostriatal dopaminergic neurons in the SN, and the death of migrating neuroblasts may be a primary event in the mouse model of PD. Furthermore, our data suggests that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.

A Diterpenoid Acanthoic Acid from Acanthopanax koreanum Protects against D-Galactosamine/Lipopolysaccharide-Induced Fulminant Hepatic Failure in Mice

The hepatoprotective effects of a diterpenoid acanthoic acid isolated from Acanthopanax koreanum NAKAI were evaluated in a D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure mouse model. Mice were pretreated orally with acanthoic acid 12 and 1 h before intraperitoneal injection of D-galactosamine and lipopolysaccharide. Pretreatment with the compound markedly reduced lethal liver injury in experimental animals. The effects were likely associated with a significant decrease in serum tumor necrosis factor (TNF)-alpha levels, which are correlated not only with those of alanine aminotransferase and aspartate aminotransferase but also with the reduced number of apoptotic hepatocytes in the liver as confirmed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA fragmentation assay. These results suggest that acanthoic acid protects against D-galactosamine/lipopolysaccharide-induced fulminant liver failure at least in part by a mechanism associated with the down-regulation of TNF-alpha secretion.

Inhibition of intestinal ischemia/repurfusion induced apoptosis and necrosis via down-regulation of the NF-kB, c-Jun and caspace-3 expression by epigallocatechin-3-gallate administration

Intestinal ischemia/reperfusion (I/R) produces reactive oxygen species (ROS) activating signal transduction and apoptosis. The aim of this study was to evaluate the effect of (−)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kB, c-Jun and caspace-3 in intestinal I/R. Thirty male wistar rats were used. Group A sham operation, B I/R, C I/R-EGCG 50 mg/kg ip. Intestinal ischemia was induced for 60 min by clamping the superior mesenteric artery. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, Fragment End Labelling of DNA (TUNEL), immunocytochemistry for NF-kB, c-Jun and caspace-3 analysis in intestinal specimens were performed 120 min after reperfusion. Apoptosis as indicated by TUNEL and Caspace-3, NF-kB and c-Jun was widely expressed in I/R group but only slightly expressed in EGCG treated groups. MDA and MPO showed a marked increase in the I/R group and a significant decrease in the EGCG treated group. Light histology showed preservation of architecture in the EGCG treated group. In conclusion, EGCG pre-treatment is likely to inhibit intestinal I/R-induced apoptosis by down-regulating the expression of NF-kB, c-Jun and caspase-3.

Integrated wavelength-selective optical waveguides for microfluidic-based laser-induced fluorescence detection

We demonstrate the fabrication and characterization of a novel, inexpensive microchip capable of laser induced fluorescence (LIF) detection using integrated waveguides with built-in optical filters. Integrated wavelength-selective optical waveguides are fabricated by doping poly(dimethysiloxane) (PDMS) with dye molecules. Liquid-core waveguides are created within dye-doped PDMS microfluidic chips by filling channels with high refractive index liquids. Dye molecules are allowed to diffuse into the liquid core from the surrounding dye-doped PDMS. The amount of diffusion is controlled by choosing either polar (low diffusion) or apolar (high diffusion) liquid waveguide cores. The doping dye is chosen to absorb excitation light and to transmit fluorescence emitted by the sample under test. After 24 h, apolar waveguides demonstrate propagation losses of 120 dB cm(-1) (532 nm) and 4.4 dB cm(-1) (633 nm) while polar waveguides experience losses of 8.2 dB cm(-1) (532 nm) and 1.1 dB cm(-1) (633 nm) where 532 and 633 nm light represent the excitation and fluorescence wavelengths, respectively. We demonstrate the separation and detection of end-labelled DNA fragments using polar waveguides for excitation light delivery and apolar waveguides for fluorescence collection. We demonstrate that the dye-doped waveguides can provide performance comparable to a commercial dielectric filter; however, for the present choice of dye, their ultimate performance is limited by autofluorescence from the dye. Through the detection of a BK virus polymerase chain reaction (PCR) product, we demonstrate that the dye-doped PDMS system is an order of magnitude more sensitive than a similar undoped system (SNR: 138 vs. 9) without the use of any external optical filters at the detector.

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using 32P-5′-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H2O2 generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.

Immunohistochemical analysis of hepatic ganglioside distribution following a partial hepatectomy and exposure to different hyperbaric oxygen treatments

Ganglioside GM3(Neu5Ac) expression is highly increased in liver 54h following 15% partial hepatectomy in pre-operatively oxygenated rats. GM3(Neu5Gc), GM2, GalNAc-GM1b and gangliosides of the neolacto-series are less affected. GM3(Neu5Ac) is a potent inhibitor of epidermal growth factor signaling. Since GM3(Neu5Ac) growth inhibitory effect depends on its cellular localization, the aim of this study was to detect ganglioside cellular localization during liver regeneration. The experiment was performed using the same rat model which previously showed increased ganglioside expression and more efficient liver regeneration. Frozen sections of liver were analyzed using confocal microscopy after labeling for binding of five ganglioside-specific antibodies, with or without hepatocyte membrane permeabilization. Ganglioside precursors, ceramide (Cer), monohexaosylceramide and lactosylceramide (LacCer) were determined by high-performance thin-layer chromatography. Apoptosis was assessed by fluorescein-dUTP end-labeling of fragmented DNA. Liver of pre-operative oxygenated rats showed high perinuclear labeling of GM3(Neu5Ac) which was absent in post-operative oxygenated and control animals. In the same group, Cer content was lower, monohexaosylceramide and LacCer were absent, and content of apoptotic cells was significantly the lowest, compared to other groups examined (F=20.36, p=0.0001). These findings indicate that ganglioside GM3(Neu5Ac) may be involved in mediation of beneficial effects of pre-operatively oxygenation during the liver regeneration.

Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome

Myelodysplastic syndrome (MDS), previously known as preleukemia, comprises a spectrum of heterogeneous, clonal disorders of hematopoiesis. A patient's life expectancy can range from a few months to more than a decade. Recent studies provide some insight into the pathophysiology of MDS. One mechanism contributing to the constellation of hypercellular marrow and peripheral blood cytopenia is a significant increase in programmed cell death (apoptosis) in hematopoietic cells. Tumor necrosis factor (TNF)-alpha, Fas ligand, TNF-related apoptosis-inducing ligand, and other proapoptotic cytokines are upregulated in early-stage/low-risk MDS, and neutralization of these signals can improve hematopoiesis. TNF-related apoptosis inducing ligand induces apoptosis preferentially in clonal cells, which can contribute to containment of the clone. In a proportion of patients, MDS will eventually evolve to acute leukemia. This progression has been correlated with upregulation of nuclear factor kappaB; altered expression of adaptor molecules, such as Flice inhibitory protein; and enhanced activity of antiapoptotic members of the Bcl-2 and inhibitors of apoptosis protein families. Also, the ratio of TNF receptors 1 and 2 changes in favor of receptor 2. The role of the microenvironment in the pathophysiology and progression of MDS has remained controversial, although there is evidence that stroma and matrix components, and their interactions with clonal cells, play an important role. Microarray gene-expression studies are consistent with dysregulation of apoptosis, but not all data are in agreement.

Abstract 2677: Reduction In Tie-2+/flk-1+ Vascular Stem Cells In Thoracic Aortic Aneurysm With Dissections: A Novel Contributor To Acellular Lesion Development

Background. Ascending thoracic aortic aneurysms leading to type A dissections (TAAD) are a life-threatening disease triggered by a complex interactions between environmental and genetic factors. Pathologically, this disease is characterized by a progressive loss of vascular smooth muscle cells (SMCs) and extracellular elastic fibers in the aortic media. Apoptosis, a form of programmed cell death, has been shown to contribute to the loss of medial SMCs. However, little is known about the role of vascular progenitor or stem cells in the pathogenesis of aortic aneurysms. Methods and Results. In this study we examined expression of Tie-2 (receptor for angiopoietin) and Flk-1 (receptor for vascular endothelial cell growth factor), two receptors critical for regulation of vascular stem cell survival and growth in the aortic lesions of patients with TAAD. Immunohistochemistry with specific antibodies against Tie-2 and Flk-1 showed a marked reduction in the numbers of Tie+/Flk+ cells in TAAD when compared to control aortas. There was also a change in the distribution pattern of vascular progenitor cells positive for both Tie-2 and Flk-1 in TAAD patients’ aortic tissues. Compared to normal control aortas, the Tie-2 and Flk-1 immunostained cells were much lower in intensity and scattered. In the regions with paucity of Tie-2+/Flk-1+ cells, there was an increased number of apoptotic cells as determined by in situ DNA end-labeling (TUNEL). Many of the Tie-2 and Flk-1 positive cells in TAAD showed poor morphology with nuclear degeneration and cytoplasmic fragmentation and disarray. Conclusion . These results indicate a paucity of Tie-2+/Flk-1+ vascular stem cells in the aortic wall of patients with TAAD, implying that the stem cell capacity to regenerate SMCs is exhausted. Similar loss of stem cells is observed with Duchenne muscular dystrophy, where the regenerated capacity is significantly compromised. Stem cells loss may similarly impair the capacity of SMCs in aortic tissue in repair and regeneration, and ultimately lead to TAAD formation.

Association of depressed cardiac gp130-mediated antiapoptotic pathways with stimulated cardiomyocyte apoptosis in hypertensive patients with heart failure

To investigate whether the glycoprotein (gp130)-mediated survival pathway, which protects cardiomyocytes from apoptosis, is depressed in left ventricular hypertrophy hypertensive patients with chronic heart failure. Transvenous endomyocardial biopsies were obtained in 52 hypertensive patients with left ventricular hypertrophy: 28 without heart failure and 24 with heart failure. gp130 and gp130-dependent antiapoptotic pathways p42/44 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) as well as gp130 agonist cardiotrophin-1 were analyzed by reverse transcriptase-polymerase chain reaction and western blot. Apoptosis was assessed by DNA end-labeling (TUNEL), caspase-3 immunostaining and caspase substrate poly(ADP-ribose) polymerase cleavage. gp130 protein expression (P < 0.05) and p42/44 MAPK and PI3K/Akt activation (P < 0.01) were decreased in heart-failure hypertensive patients compared with nonheart-failure hypertensive individuals. No changes in gp130 mRNA expression were found between the two groups. Cardiotrophin-1 was increased (P < 0.05) at both the mRNA and protein levels in heart-failure hypertensive individuals compared with nonheart-failure hypertensive individuals. Cardiomyocyte apoptosis was increased (P < 0.01) in heart-failure hypertensive individuals compared with nonheart-failure hypertensive individuals. Inverse correlations (P < 0.01) occurred between cardiomyocyte apoptosis and p42/44 MAPK and PI3K/Akt activation in all hypertensive patients. Cardiotrophin-1 correlated inversely (r = -0.554, P < 0.05) with gp130 in all hypertensive individuals. In cultured HL-1 cardiomyocytes, cardiotrophin-1 decreased (P < 0.05) the gp130:phosphorylated gp130 (at Ser782) ratio and increased (P < 0.05) gp130ubiquitination. An association exists between depression of the gp130 cytoprotective pathway and stimulation of cardiomyocyte apoptosis in hypertensive patients that develop heart failure. Whether the excess of cardiotrophin-1 induces ligand-induced receptor down-regulation in these patients requires further study.

Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue

To evaluate the incidence of apoptosis after vitrification warming of mouse ovaries. Experimental study. University-based research laboratory. Twelve- to 14-day-old National Medical Research Institute female mice. Vitrification of mouse ovaries using ethylene glycol. Follicle viability assessment by trypan blue testing, morphologic examination by hematoxylin-eosin staining and transmission electron microscopy, apoptosis assessment using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method and DNA laddering technique. No statistically significant difference in follicle viability was observed between vitrified and nonvitrified ovaries. On transmission electron microscopy, vitrified ovaries showed a well-preserved ultrastructure. No sign of apoptosis was observed morphologically or by transferase-mediated deoxyuridine triphosphate nick end-labeling technique in either fresh or vitrified-warmed mouse ovaries. Despite the presence of a laddering pattern of DNA in control induced thymic tissue, no similar pattern was observed in fresh or vitrified-warmed ovaries. The data suggest that the vitrification technique does not induce apoptosis in mouse ovarian tissue investigated just after warming.

Microsatellite abnormalities and somatic down‐regulation of mismatch repair characterize nodular‐trabecular muscle‐invasive urothelial carcinoma of the bladder

To correlate histological infiltration patterns with genetic and mismatch repair (MMR) profiles in muscle-invasive bladder urothelial carcinomas (UroC). Infiltration patterns were assessed in the deep compartment of muscle-invasive UroC (nodular-trabecular, 45 cases; infiltrative, 27 cases). Tumour compartment (superficial and deep to muscularis mucosa) analysis included: microsatellite pattern of TP53, RB1, WT1 and NF1 by polymerase chain reaction/denaturing gradient gel electrophoresis; mitotic, Ki67, in situ end labelling (ISEL) indices and DNA ploidy. MMR was assessed by MLH1 and MSH2 sequencing and immunohistochemistry in UroC with two or more abnormal microsatellite loci. Statistical differences were tested using anova and Fisher's exact tests. Infiltrative UroC showed lower Ki67 index 14.94 +/- 4.28, ISEL index 14.1 +/- 10.0 and shorter median survival (20 months) than nodular-trabecular UroC (Ki67 index 20.65 +/- 4.94, ISEL 20.2 +/- 22.7, 37-month survival, respectively). The genetic profile was significantly different for RB1 (P = 0.0003) and NF1 (P = 0.0023) only, being more frequently abnormal in nodular-trabecular UroC. A significant decrease in MLH1 or MSH2 protein expression with no gene mutations was identified in UroC with microsatellite abnormalities and a nodular-trabecular growth pattern. Somatic down-regulation of MMR proteins in nodular-trabecular muscle-invasive UroC results in RB1/NF1 microsatellite abnormalities, correlating with higher cellular turnover and longer survival.

Differential kinetic features by tumour topography in cutaneous small‐cell neuroendocrine (Merkel cell) carcinomas

Merkel cell carcinomas (MCC) reveal epithelial and neuroendocrine differentiation, but its topographic cell kinetics remains unknown. This study analyses proliferation, apoptosis, and DNA ploidy by topography, features that can help planning therapeutic protocols. This study topographically analyses proliferation, apoptosis, and DNA ploidy. We selected 27 small-cell MCCs (expressing one epithelial and two neural markers, with consistent ultrastructural findings) to evaluate mitotic figure counting, Ki-67 index, apoptosis index based on the in situ end labelling of fragmented DNA (using Escherichia coli DNA polymerase I, Klenow fragment), DNA ploidy, and BCL2 and TP53 immuno-expression. At least 50 high-power fields were screened per topographic compartment (superficial or papillary dermis, and deep or reticular dermis), recording average and standard deviation for each variable. Variables were statistically compared in each tumour compartment using analysis of variance and Student's t-test (significant if P < 0.05). MCCs revealed superficial aneuploid DNA content, and no topographic differences for proliferation markers. Apoptosis showed significantly lower values in the deep compartment (average, P = 0.0050, and standard deviation, P = 0.0074), correlating with increased BCL2 and TP53 immuno-expressions. High homogeneously distributed proliferation and superficial aneuploid DNA content defines MCCs. Apoptosis follows proliferation in superficial compartments, being less variable and proliferation independent in deep compartments, where it is inversely correlated with BCL2/TP53 expression.

Formation of archegonium chamber is associated with nucellar-cell programmed cell death in Ginkgo biloba

The archegonium chamber in Ginkgo biloba L. is a pathway for spermatozoids swimming towards the archegonium for fertilization. The objective of this study was to elucidate the mechanism of archegonium chamber formation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and DNA ladder demonstrated that the nucellar cell death, coordinated with the archegonium chamber formation, was a process of programmed cell death. Cytochemical localization of Ca(2+) in these nucellar cells was determined by means of in situ precipitation with potassium pyroantimonate and electron microscopic visualization, in order to study the relation between Ca(2+) and programmed cell death. The results showed an early uptake of the mitochondrial calcium particles in the nucellar cells undergoing programmed cell death. Together with other dynamic changes in Ca(2+) subcellular distribution, this indicates that Ca(2+) may play a role in the regulation of mitochondria-mediated programmed events in the nucellar cells.

Mechanism of UVA-dependent DNA Damage Induced by An Antitumor Drug Dacarbazine in Relation to its Photogenotoxicity

It has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC. We examined DNA damage induced by UVA-irradiated DTIC using 32P-5'-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC. UVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5'-GGT-3' sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC. Photoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.

The Transcription Factor Runx3 Represses the Neurotrophin Receptor TrkB during Lineage Commitment of Dorsal Root Ganglion Neurons

Runx3, a Runt domain transcription factor, determines neurotrophin receptor phenotype in dorsal root ganglion (DRG) neurons. Molecular mechanisms by which Runx3 controls distinct neurotrophin receptors are largely unknown. Here, we show that RUNX3 abolished mRNA induction of TRKB expression, and concomitantly altered the neurotrophin response in a differentiating neuroblastoma cell line. In contrast, RUNX3 did not play a significant role in TRKC regulation even under the relevant BMP signaling pathway. We identified putative regulatory elements of Ntrk2/NTRK2 (a gene that codes for TrkB) using an unbiased computational approach. One of these elements was a highly conserved intronic sequence that contains a cluster of Runx binding sites. In a primary culture of DRG neurons, endogenous Runx3 bound to the consensus cluster, which had repressor activity against the Ntrk2 promoter under the control of NT-3 signaling. Consistent with these findings, Runx3-deficient embryos showed an increased number of trkB+ DRG neurons and failed to maintain trkC expression. Taken together, Runx3 determines TrkC positive sensory neuron identities through the transcriptional repression of TrkB when Trk-BTrkC double positive neurons differentiate into TrkC single positive neurons.