SVCT2-Dependent plasma and mitochondrial membrane transport of ascorbic acid in differentiating myoblasts.

The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.

The mitochondrial transporter of ascorbic acid functions with high affinity in the presence of low millimolar concentrations of sodium and in the absence of calcium and magnesium.

We recently reported that U937 cell mitochondria express a functional Na+-dependent ascorbic acid (AA) transporter recognised by anti-SVCT2 antibodies. The present study confirms and extends these observations by showing that this transporter is characterised by a Km and a pH-dependence comparable with that reported for the plasma membrane SVCT2. In isolated mitochondria, Na+ increased AA transport rate in a cooperative manner, revealed by a sigmoid curve and a Hill coefficient of 2, as also observed in intact Raw 264.7 cells (uniquely expressing SVCT2). There was however a striking difference on the Na+ concentrations necessary to reach saturation, i.e., 1 or 100 mM for the mitochondrial and plasma membrane transporters, respectively. Furthermore the mitochondrial, unlike the plasma membrane, transporter was fully active also in the absence of added Ca++ and/or Mg++. Taken together, the results presented in this study indicate that the U937 cell mitochondrial transporter of AA, because of its very low requirement for Na+ and independence for Ca++ and Mg++, displays kinetic characteristics surprisingly similar with those of the plasma membrane SVCT2.

Intracellular dehydroascorbic acid inhibits SVCT2-dependent transport of ascorbic acid in mitochondria.

Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA.

Differentiation of Promonocytic U937 Cells to Monocytes Is Associated with Reduced Mitochondrial Transport of Ascorbic Acid.

Growth of promonocytic U937 cells in the presence of DMSO promotes their differentiation to monocytes. After 4 days of culture in differentiating medium, these cells ceased to proliferate, displayed downregulated ryanodine receptor expression, and responded to specific stimuli with enhanced NADPH-oxidase-derived superoxide formation or cytosolic phospholipase A2-dependent arachidonic acid release. We found that the 4-day differentiation process is also associated with downregulated SVCT2 mRNA expression, in the absence of apparent changes in SVCT2 protein expression and transport rate of ascorbic acid (AA). Interestingly, under the same conditions, these cells accumulated lower amounts of the vitamin in their mitochondria, with an ensuing reduced response to external stimuli sensitive to the mitochondrial fraction of AA. Further analyses demonstrated an unexpected increase in mitochondrial SVCT2 protein expression, however, associated with reduced SVCT2-dependent AA uptake in isolated mitochondria. A decrease in the transporter Vmax, with no change in affinity, was found to account for this response. Differentiation of promonocytic cells to monocytes is therefore characterized by decreased SVCT2 mRNA expression that, even prior to the onset of SVCT2 protein downregulation or apparent changes in plasma membrane transport activity, impacts on the mitochondrial accumulation of the vitamin through a decreased Vmax of the transporter.

Arsenite induces DNA damage via mitochondrial ROS and induction of mitochondrial permeability transition.

Arsenite is an established DNA-damaging agent and human carcinogen. We initially selected conditions in which the metalloid causes DNA strand scission in the absence of detectable apoptotic DNA degradation in U937 cells. This response was suppressed by catalase and by treatments (rotenone and ascorbic acid), or manipulations (respiration-deficient phenotype), preventing the mitochondrial formation of O2-. ( mitoO2-.). MitoO2-., and its dismutation product, H2 O2 , are therefore critically involved in the arsenite-dependent DNA-damaging response. We then established a link between mitoO2-./H2 O2 and mitochondrial permeability transition (MPT), and found that this second event also promoted the formation of DNA-damaging species. As a consequence, the DNA damage induced by arsenite, in addition to being abolished by the aforementioned treatments/manipulations, was also significantly reduced by the MPT inhibitor cyclosporin A (CsA). A CsA-sensitive induction of p53 mRNA expression was also detected. Finally, evidence of CsA-sensitive DNA strand scission was also obtained in MCF-7, HT22, and NCTC-2544 cells. MitoO2-./H2 O2 therefore directly mediates DNA damage induced by arsenite and indirectly promotes the formation of additional DNA-damaging species via the induction of MPT. © 2017 BioFactors, 43(5):673-684, 2017.

U937 cell apoptosis induced by arsenite is prevented by low concentrations of mitochondrial ascorbic acid with hardly any effect mediated by the cytosolic fraction of the vitamin.

Arsenite directly triggers cytochrome c and Smac/DIABLO release in mitochondria isolated from U937 cells. These effects were not observed in mitochondria pre-exposed for 15 min to 10 µM L-ascorbic acid (AA). In other experiments, intact cells treated for 24-72 h with arsenite were found to die by apoptosis through a mechanism involving mitochondrial permeability transition. Pre-exposure (15 min) to low micromolar concentrations of AA and dehydroascorbic acid (DHA), resulting in identical cytosolic levels of the vitamin, had a diverse impact on cell survival, as cytoprotection was only observed after treatment with AA. Also the mitochondrial accumulation of the vitamin was restricted to AA exposure. An additional indication linking cytoprotection to the mitochondrial fraction of the vitamin was obtained in experiments measuring susceptibility to arsenite in parallel with loss of mitochondrial and cytosolic AA at different times after vitamin exposure. Finally, we took advantage of our recent findings that DHA potently inhibits AA transport to demonstrate that DHA abolishes all the protective effects of AA, under the same conditions in which the mitochondrial accumulation of the vitamin is prevented without affecting the overall cellular accumulation of the vitamin.

A real time PCR platform for the simultaneous quantification of total and extrachromosomal HIV DNA forms in blood of HIV-1 infected patients.

BACKGROUND: The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed. METHODOLOGY/FINDINGS: Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA. CONCLUSIONS/SIGNIFICANCE: Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 10(4) CD4+, for 12 patients within two working days.

TBX3, the gene mutated in ulnar-mammary syndrome, promotes growth of mammary epithelial cells via repression of p19ARF, independently of p53.

TBX3, the gene mutated in ulnar-mammary syndrome (UMS), is involved in the production of a transcription factor of the T-box family, known to inhibit transcription from the p14ARF (p19ARF in mouse) promoter in fibroblasts and to contribute to cell immortalization. One of the main features of the UMS phenotype is the severe hypoplasia of the breast, associated with haploinsufficiency of the TBX3 gene product. In mice homozygous for the targeted disruption of Tbx3, the mammary glands (MGs) are nearly absent from early stages of embryogenesis, whereas in heterozygous adults, the MGs show reduced ductal branching. All these data strongly suggest a specific role of TBX3 in promoting the growth of mammary epithelial cells (MECs), although direct evidence of this is lacking. Here, we provide data showing the growth-promoting function of Tbx3 in several models of MECs, in association with its ability to repress the ARF promoter. However, no effect of Tbx3 on cell differentiation or apoptosis has been observed. The growth promoting function also entails the down-regulation of p21 ( CIP1/WAF ) and an increase in cyclin D1 but is independent of p53 and Mdm2 cell-cycle regulatory proteins, as p53-null MECs show similar growth responses associated with the up- or down-regulation of Tbx3. This is the first direct evidence that the level of Tbx3 expression positively controls the proliferation of MECs via pathways alternative to Mdm2-p53.