Reactive oxygen species mediate a cellular 'memory' of high glucose stress signalling.

AIMS/HYPOTHESIS: A long-term 'memory' of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding. MATERIALS AND METHODS: HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus alpha-lipoic acid in the last week. RESULTS: In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant alpha-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H(2)DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with alpha-lipoic acid added to the last week of normalised glucose. CONCLUSIONS/INTERPRETATION: These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation.

Arsenic alters the function of the glucocorticoid receptor as a transcription factor.

Chronic human exposure to nonovertly toxic doses of arsenic is associated with an increased risk of cancer. Although its carcinogenic mechanism is still unknown, arsenic does not directly cause DNA damage or mutations and is therefore thought to act principally as a co-mutagen, co-carcinogen, and/or tumor promoter. Previous studies in our laboratory demonstrated that effects of low-dose arsenic (III) (arsenite) on expression of the hormone-regulated phosphoenolpyruvate carboxykinase (PEPCK) gene were strongly associated with the glucocorticoid receptor (GR)-mediated regulatory pathway. We therefore examined specifically the effects of arsenite on the biochemical function of GR in hormone-responsive H4IIE rat hepatoma cells. Completely noncytotoxic arsenite treatments (0.3-3.3 microM) significantly decreased dexamethasone-induced expression of transiently transfected luciferase constructs containing either an intact hormone-responsive promoter from the mammalian PEPCK gene or two tandem glucocorticoid response elements (GRE). Western blotting and confocal microscopy of a green fluorescent protein-tagged-GR fusion protein demonstrated that arsenite pretreatment did not block the normal dexamethasone-induced nuclear translocation of GR. These data indicate that nontoxic doses of arsenite can interact directly with GR complexes and selectively inhibit GR-mediated transcription, which is associated with altered nuclear function rather than a decrease in hormone-induced GR activation or nuclear translocation.

Differential effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding.

The toxic metals arsenic(III) and chromium(VI) are considered human carcinogens, although they may act through different mechanisms. We previously showed that when administered at single low, non-overtly toxic doses, chromium, arsenic, and several other chemical carcinogens preferentially alter expression of several model inducible genes in both whole-animal and cell-culture systems. In this study, we assessed whether chromium and arsenic target specific signaling pathways within cells to selectively modulate gene expression. We examined the effects of non-cytotoxic and cytotoxic doses of arsenic(III) and chromium(VI) on nuclear binding of the transcription factors AP-1, NF-kappaB, Sp1, and YB-1 in human MDA-MB-435 breast cancer and rat H4IIE hepatoma cells. These transcription factors were chosen because they may regulate many inducible genes, including those previously shown to be altered by metal treatments. We report that both arsenic and chromium significantly altered nuclear binding levels of these factors to their respective cis-acting elements. However, there were qualitative and quantitative differences in these effects that were dependent on the metal, time, dose, transcription factor, and cell line. These effects may play a significant role in metal-induced alterations in gene expression.

Effects of mitomycin C and carboplatin pretreatment on multidrug resistance-associated P-glycoprotein expression and on subsequent suppression of tumor growth by doxorubicin and paclitaxel in human metastatic breast cancer xenografted nude mice.

Overexpression of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), and several other proteins has been associated with development of multidrug resistance by cancer cells, which represents a significant obstacle to successful treatment by chemotherapy. We had previously demonstrated that a single noncytotoxic dose of mitomycin C (MMC), carboplatin, or one of several other DNA cross-linking agents suppressed mRNA expression of the mdr1 gene coding for Pgp, leading to a subsequent suppression of Pgp protein levels and a concomitant decrease in drug efflux. Pretreatment with MMC led to a 5- to 10-fold decrease in the ED50 for cell killing by a subsequent agent such as the Pgp substrate, doxorubicin, but did not affect killing by the non-Pgp substrate, cisplatin. In this study, we report that MMC and carboplatin each significantly suppressed Pgp protein levels in human MDA-MB-435 cells xenografted as solid tumors into the lateral mammary fat pads of female nude mice, with a similar time course as had previously been observed in cell culture. Pretreatment of mice with MMC or carboplatin 48-72 h prior to receiving either doxorubicin or paclitaxel caused a significantly greater reduction in tumor growth rate compared to either agent alone or the combination given simultaneously. These data suggest that a combination chemotherapy regimen consisting of a DNA cross-linking agent given to modulate the MDR phenotype, followed by a second cytotoxic agent, may be an effective treatment for human patients with de novo or late stage acquired multidrug-resistant malignancies.

Molecular basis for effects of carcinogenic heavy metals on inducible gene expression.

Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure leading to alterations in expression of specific susceptible genes. It is anticipated that such information will provide valuable insight into the mechanistic basis for these effects as well as provide sensitive molecular biomarkers for evaluating human exposure.