Expression of teneurins is associated with tumor differentiation and patient survival in ovarian cancer.

Teneurins are a family of highly conserved pair-rule proteins involved in morphogenesis and development of the central nervous system. Their function in adult tissues and in disease is largely unknown. Recent evidence suggests a role for dysregulated expression of Teneurins in human tumors, but systematic investigations are missing. Here, we investigated Teneurin-2 and Teneurin-4 expression in various cancer cell lines and in ovarian tumor tissues. Teneurin-2 and Teneurin-4 were expressed in most of the breast cancer cell lines tested. Teneurin-4 was also detected in ovarian cancer cell lines, and throughout ovarian tumors and normal ovary tissue. Ovarian tumors with low Teneurin-4 expression showed less differentiated phenotypes and these patients had shorter mean overall survival. Similarly, Teneurin-2 expression correlated with overall survival as well, especially in patients with serous tumors. In the various cell lines, 5-Aza-cytidine-induced changes in DNA methylation did not alter expression of Teneurin-2 and Teneurin-4, despite the existence of predicted CpG islands in both genes. Interestingly, however, we found evidence for the control of Teneurin-2 expression by the oncogenic growth factor FGF8. Furthermore, we identified multiple transcript splicing variants for Teneurin-2 and Teneurin-4, indicating complex gene expression patterns in malignant cells. Finally, downregulation of Teneurin-4 expression using siRNA caused a cell-type dependent increase in proliferation and resistance to cisplatin. Altogether, our data suggest that low Teneurin-4 expression provides a growth advantage to cancer cells and marks an undifferentiated state characterized by increased drug resistance and clinical aggressiveness. We conclude that Teneurin-2 and Teneurin-4 expression levels could be of prognostic value in ovarian cancer.

DT-diaphorase protects astrocytes from aminochrome-induced toxicity.

Astrocytes are exposed to aminochrome via the oxidation of dopamine that is taken up from the synaptic cleft after its release from dopaminergic neurons. Glutathione transferase M2-2 (GSTM2) has been shown to protect astrocytes from aminochrome-induced toxicity, but astrocytes also express DT-diaphorase, which has been shown to prevent aminochrome-induced neurotoxicity in dopaminergic neurons. Therefore, the question is whether DT-diaphorase also protects astrocytes from aminochrome-induced toxicity. DT-diaphorase is constitutively expressed in U373MG cells, and its inhibition by dicoumarol induced a significant increase of aminochrome-induced cell death. However, the inhibition of DT-diaphorase in U373MGsiGST6 cells, which have 74% of GSTM2 gene expression silenced, resulted in a more than 2-fold increase in cell death, suggesting that DT-diaphorase plays an important role in preventing aminochrome-induced toxicity in astrocytes.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction.

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A 1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A 1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.

Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+)CD8(+) T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473) years after the acute infection. Remarkably, Gn(465-473)-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+)CD27(-)CD28(-)CCR7(-)CD127(-) effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.

Aminochrome as a preclinical experimental model to study degeneration of dopaminergic neurons in Parkinson's disease.

Four decades after L-dopa introduction to PD therapy, the cause of Parkinson's disease (PD) remains unknown despite the intensive research and the discovery of a number of gene mutations and deletions in the pathogenesis of familial PD. Different model neurotoxins have been used as preclinical experimental models to study the neurodegenerative process in PD, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone. The lack of success in identifying the molecular mechanism for the degenerative process in PD opens the question whether the current preclinical experimental models are suitable to understand the degeneration of neuromelanin-containing dopaminergic neurons in PD. We propose aminochrome as a model neurotoxin to study the neurodegenerative processes occurring in neuromelanin-containing dopaminergic neurons in PD. Aminochrome is an endogenous compound formed during dopamine oxidation and it is the precursor of neuromelanin, a substance whose formation is a normal process in mesencephalic dopaminergic neurons. However, aminochrome itself can induce neurotoxicity under certain aberrant conditions such as (i) one-electron reduction of aminochrome catalyzed by flavoenzymes to leukoaminochrome o-semiquinone radical, which is a highly reactive neurotoxin; or (ii) the formation of aminochrome adducts with alpha-synuclein, enhancing and stabilizing the formation of neurotoxic protofibrils. These two neurotoxic pathways of aminochrome are prevented by DT-diaphorase, an enzyme that effectively reduces aminochrome with two-electrons preventing both aminochrome one-electron reduction or formation alpha synuclein protofibrils. We propose to use aminochrome as a preclinical experimental model to study the neurodegenerative process of neuromelanin containing dopaminergic neurons in PD.

Association of GST M1 null polymorphism with Parkinson's disease in a Chilean population with a strong Amerindian genetic component.

We have studied the association of a null mutation of Glutathione Transferase M1 (GST M1*0/0) with Parkinson's disease (MIM 168600) in a Chilean population with a strong Amerindian genetic component. We determined the genotype in 349 patients with idiopathic Parkinson's disease (174 female and 175 male; 66.84+/-10.7 years of age), and compared that to 611 controls (457 female and 254 male; 62+/-13.4 years of age). A significant association of the null mutation in GST M1 with Parkinson's disease was found (p=0.021), and the association was strongest in the earlier age range. An association of GSTM1*0/0 with Parkinson's disease supports the hypothesis that Glutathione Transferase M1 plays a role in protecting astrocytes against toxic dopamine oxidative metabolism, and most likely by preventing toxic one-electron reduction of aminochrome.

Dopamine-dependent iron toxicity in cells derived from rat hypothalamus.

We report a new and specific mechanism for iron-mediated neurotoxicity using RCHT cells, which were derived from rat hypothalamus. RCHT cells exhibit immunofluorescent-positive markers for dopamine beta-hydroxylase and the norepinephrine transporter, NET. In the present study, we observed that iron-induced neurotoxicity in RCHT cells was dependent on (i) formation of an Fe-dopamine complex (100 microM FeCl3:100 microM dopamine); (ii) specific uptake of the Fe-dopamine complex into RCHT cells via NET (79+/-2 pmol 59Fe/mg/min; P<0.05), since the uptake of the 59Fe-dopamine complex by the cells was inhibited by 30 microM reboxetine, a specific NET inhibitor (78% inhibition, P<0.001); and (iii) intracellular oxidation of dopamine present in the Fe-dopamine complex to aminochrome; (iv) inhibition of DT-diaphorase, since incubation of RCHT cells with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase, induced significant cell death (51+/-5%; P<0.001). However, this cell death was reduced by 75% when the cells were incubated in the presence of 30 microM reboxetine (P<0.01). No significant cell death was observed when the cells were incubated with 100 microM dopamine, 100 microM Fe-Dopamine complex, 100 microM dicoumarol, or 100 microM FeCl3 (8.3+/-2, 9+/-4, 8.5+/-3, or 9.7+/-2% of control, respectively). ESR studies using the spin trapping agent DMPO showed no formation of hydroxyl radicals when the cells were incubated with 100 microM FeCl3 alone. However, using the same ESR technique, the formation of hydroxyl radicals and a carbon-centered radical was detected when the cells were incubated with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol. These studies suggest that iron can induce cell toxicity by a mechanism that requires the formation and NET-mediated uptake of an Fe-dopamine complex, ultimately resulting in the intracellular formation of reactive species.

On the neurotoxicity mechanism of leukoaminochrome o-semiquinone radical derived from dopamine oxidation: mitochondria damage, necrosis, and hydroxyl radical formation.

Leukoaminochrome o-semiquinone radical is generated during one-electron reduction of dopamine oxidation product aminochrome when DT-diaphorase is inhibited. Incubation of 100 microM aminochrome with 100 microM dicoumarol, an inhibitor of DT-diaphorase during 2 h, induces 56% cell death (P < 0.001) with concomitant formation of (i) intracellular hydroperoxides (4.2-fold increase compared to control; P < 0.001); (ii) hydroxyl radicals, detected with ESR and spin trapping agents (2.4-fold increase when cells were incubated with aminochrome in the presence of dicoumarol compared to aminochrome alone); (iii) intracellular edema, and cell membrane deterioration determined by transmission electron microscopy; (iv) absence of apoptosis, supported by using anexin-V with flow cytometry; (v) a strong decrease of mitochondrial membrane potential determined by the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanineiodide (P < 0.01); (vi) swelling and disruption of outer and inner mitochondrial membranes determined by transmission electron microscopy. These results support the proposed role of leukoaminochrome o-semiquinone radical as neurotoxin in Parkinson's disease neurodegeneration and DT-diaphorase as neuroprotective enzyme.

Levels of cytokine receptor activator of nuclear factor kappaB ligand in gingival crevicular fluid in untreated chronic periodontitis patients.

BACKGROUND: Receptor activator of nuclear factor kappaB ligand (RANK-L) is a cytokine involved in the regulation of osteoclastogenesis in bone remodeling and inflammatory osteolysis. One of the major causes of tooth loss in humans is bone destruction. The aim of our study was to determine the presence of RANK-L in gingival crevicular fluid (GCF) samples from adult patients with untreated chronic periodontitis and in healthy controls. We also identified the RANK-L present in lesions undergoing episodic attachment loss from GCF. METHODS: GCF samples were collected from two periodontally affected sites (probing depth > or = 5 mm, attachment loss > or = 3 mm) in 20 patients (N = 40). After monitoring for 4 months, seven patients showed active periodontal disease, and GCF samples were collected from one active and one inactive site (N = 14 samples). The comparison with healthy controls was carried out by collecting GCF samples from 12 healthy volunteers (N = 24 samples). GCF was collected using a paper strip, and enzyme-linked immunosorbent assay (ELISA) was performed to determine the total amount of RANK-L. RESULTS: RANK-L was found in a higher proportion (85%) of samples from patients than from controls (46%). The total amount of RANK-L was significantly higher in patients (115.53 +/- 78.18 picograms [pg]) than in healthy subjects (63.08 +/- 55.08 pg) (P = 0.003). Active sites, presumably associated with tissue destruction, had significantly higher levels of RANK-L than their inactive counterparts (125.95 pg versus 91.80 pg, P = 0.007). CONCLUSION: GCF total amount of RANK-L is significantly increased in periodontal disease, supporting its role in the alveolar bone loss developed in this disease.

Oxidation of dopamine to aminochrome as a mechanism for neurodegeneration of dopaminergic systems in Parkinson's disease. Possible neuroprotective role of DT-diaphorase.

Although it is generally accepted that free radicals are involved in the neurodegenerative process occurring in the dopaminergic neurons of the nigro-striatal system in Parkinson's disease, the exact mechanism of neurodegeneration in vivo is still unknown. We propose that the degeneration of dopaminergic nigrostriatal system in this condition may depend on: (a) existence of free dopamine which oxidizes to aminochrome as a consequence of: (i) overproduction of dopamine; (ii) inhibition and/or low expression of synaptic vesicle catecholamine transporter; (iii) inhibition or low expression of monoamine oxidases; (b) one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radical, which induces neurotoxicity, due to inhibition of DT-diaphorase or the existence of a polymorphism with a point mutation (C --> T) in the cDNA 609 expressing an inactive DT-diaphorase. We suggest that DT-diaphorase plays a neuroprotective role in dopaminergic neurons, which is supported by the following observations: (i) Cu-toxicity is dependent on DT-diaphorase inhibition with dicoumarol in RCSN-3 cells derived from the rat substantia nigra; (ii) the cytotoxic effect of monoamine oxidase-A inhibitor amiflamine in RCSN-3 cells is increased by 2.4-fold (p < 0.001) in the presence of the inhibitor of DT-diaphorase, dicoumarol; (iii) concomitant intracerebral administration of manganese (Mn3+) together with the DT-diaphorase inhibitor dicoumarol into the left medial forebrain bundle produced a behavioral pattern characterized by contralateral rotational behavior when the rats were stimulated with apomorphine, in a manner similar to that observed in animals injected unilaterally with 6-hydroxydopamine; (iv) incubation of RCSN-3 cells with salsolinol in the presence of DT-diaphorase inhibitor significantly decreased cell survival by 2.5-fold (p < 0.001).