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Summary Hypermethylation of exon 1 of p16 INK4a was examined in tumour and plasma DNA of a series of breast cancer patients. De novo methylation was observed in the tumours of eight patients (23%), and in plasma DNA in five (14%) of these eight patients. Our data show that de novo methylation of exon 1 of p16 INK4a can be demonstrated in plasma DNA of breast cancer patients, a fact that provides additional evidence of the tumour-related origin of free plasma DNA in cancer patients.

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The role of NADPH oxidase (Nox)-derived reactive oxygen species in kidney vascular function has extensively been investigated in the harmful context of oxidative stress in diabetes and obesity-associated kidney disease. Since hydrogen peroxide (H2O2) has recently been involved in the non-nitric oxide (NO) non-prostanoid relaxations of intrarenal arteries, the present study was sought to investigate whether NADPH oxidases may be functional sources of vasodilator H2O2 in the kidney and to assess their role in the endothelium-dependent relaxations of human and rat intrarenal arteries. Renal interlobar arteries isolated from the kidney of renal tumor patients who underwent nephrectomy, and from the kidney of Wistar rats, were mounted in microvascular myographs to assess function. Superoxide (O2.-) and H2O2 production was measured by chemiluminescence and Amplex Red fluorescence, and Nox2 and Nox4 enzymes were detected by Western blotting and by double inmunolabeling along with eNOS. Nox2 and Nox4 proteins were expressed in the endothelium of renal arterioles and glomeruli co-localized with eNOS, levels of expression of both enzymes being higher in the cortex than in isolated arteries. Pharmacological inhibition of Nox with apocynin and of CYP 2C epoxygenases with sulfaphenazol, but not of the NO synthase (NOS), reduced renal NADPH-stimulated O2.- and H2O2 production. Under conditions of cyclooxygenase and NOS blockade, acetylcholine induced endothelium-dependent relaxations that were blunted by the non-selective Nox inhibitor apocynin and by the Nox2 or the Nox1/4 inhibitors gp91ds-tat and GKT136901, respectively. Acetylcholine stimulated H2O2 production that was reduced by gp91ds-tat and by GKT136901. These results suggest the specific involvement of Nox4 and Nox2 subunits as physiologically relevant endothelial sources of H2O2 generation that contribute to the endothelium-dependent vasodilatation of renal arteries and therefore have a protective role in kidney vasculature.

The INK4a/ARF locus encodes two unrelated cell cycleregulatory proteins that both function in tumor suppression, p16INK4a and p14ARF. In human tumors including breast cancer, alterations aecting selectively p14ARF have been poorly analysed. We have performed a comprehensive analysis of the inactivation mechanisms (mutation, homozygous and hemizygous deletion, and promoter hypermethylation) in a large series of 100 primary breast carcinomas. RT ± PCR showed expression variable of the p14ARF transcript, with 17% demonstrating overexpression and 26% demonstrating decreased expression. No detectable alterations were observed in the majority of cases with overexpressed p14ARF mRNA, but 77% of tumors with decreased expression presented at least one of these genetic/ epigenetic alterations. Nevertheless, a statistically signi®cant correlation was observed between decreased p14ARF expression and several poor prognostic parameters. Oncogene (2001) 20, 4586 ± 4590.