Survey of primary care physicians' views about breast and ovarian cancer screening for true BRCA1/2 non-carriers.

Despite some controversy, true BRCA1/2 non-carriers are generally considered to be at an average risk for breast and ovarian cancer. Primary care physicians are then expected to encourage their non-carrier patients to adopt cancer screening practices appropriate to women of the same age in the general population. This study aimed to describe breast and ovarian cancer screening recommendations that primary care physicians would consider advisable for young true BRCA1/2 non-carriers. One hundred thirty-four family physicians and 123 gynecologists (response rate 45%) completed a cross-sectional mailed survey administered in the Province of Quebec, Canada. The survey included questions about basic genetic knowledge and screening recommendations for two fictitious cases (< 40 years), one carrier and one non-carrier, from a BRCA1/2 mutation-positive family. Screening exams considered advisable did not differ significantly between family physicians and gynecologists. More than 75% of physicians considered the cancer risks of true non-carriers to be comparable with that of the general population and 14% to be a little higher. Still, 53% would prescribe a biennial and or even an annual (27%) mammography to a non-carrier woman before the recommended starting age. Physician considerations of non-carriers' expectations or requests for screening were associated with more screening prescriptions. More than half of primary care physicians would recommend more mammography screenings than expected for a young true BRCA1/2 non-carrier. Personalized cancer risk assessment may help primary care physicians tailor screening of women from BRCA1/2 mutation-positive families and allow these women to make more informed choices regarding cancer risk management options.

No evidence of excessive cancer screening in female noncarriers from BRCA1/2 mutation-positive families.

BACKGROUND: In families with a proven BRCA1/2 mutation, women not carrying the familial mutation should follow the cancer screening recommendations applying to women in the general population. In the present study, we evaluated the cancer screening practices of unaffected noncarriers from families with a proven BRCA mutation, and we assessed the role of family history in their screening practices. METHODS: Self-report data were provided retrospectively by 220 unaffected female noncarriers for periods of up to 10 years (mean: 4.3 years) since disclosure of their BRCA1/2 genetic test result. A ratio for the annual frequency of breast and ovarian cancer screening exams (mammography, breast ultrasonography, breast magnetic resonance imaging, transvaginal or pelvic ultrasound, cancer antigen 125 testing) was calculated as number of screening exams divided by the number of years in the individual observation period. RESULTS: The annual average for mammography exams was 0.15, 0.4, 0.56, and 0.71 in women 30-39, 40-49, 50-59, and 60-69 years of age respectively. The uptake of other breast and ovarian cancer screening exams was very low. Mammography and breast ultrasonography and magnetic resonance imaging were generally more frequent among participants with at least 1 first-degree relative affected by breast cancer. CONCLUSIONS: In most noncarriers, screening practices are consistent with the guidelines concerning women in the general population. When noncarriers adopt screening behaviours that are different from those that would be expected for average-risk women, those behaviours are influenced by their familial cancer history. IMPACT: Decision tools might help female noncarriers to be involved in their follow-up in accordance with their genetic status and their family history, while taking into account the benefits and disadvantages of cancer screening.

No effects of pantoprazole on the pharmacokinetics of rosuvastatin in healthy subjects.

PURPOSE: Rosuvastatin disposition is modulated by the expression and activity of several membrane transporters including BCRP (ABCG2). The objective of our study was to investigate the effects of pantoprazole, a previously proposed BCRP inhibitor, on the disposition of rosuvastatin. METHODS: The impact of pantoprazole (40 mg ID for 2 days) on rosuvastatin pharmacokinetics was evaluated in healthy volunteers (n = 16) who received a single oral dose of rosuvastatin (10 mg) either alone or with pantoprazole. Rosuvastatin, N-desmethylrosuvastatin, and rosuvastatin lactone levels were quantified in plasma while rosuvastatin and N-desmethylrosuvastatin excretion were measured in urine. RESULTS: Ratios and 90 % standard confidence interval of geometric means for C max (1.03 [0.91-1.16]), AUC0-∞ (1.03 [0.89-1.19]) and renal clearance (0.96 [0.85-1.09]) were all within the pre-specified range of 0.8-1.25, indicating a lack of drug-drug interaction between pantoprazole and rosuvastatin. CONCLUSIONS: Concomitant administration of pantoprazole with rosuvastatin did not affect rosuvastatin plasma concentrations. The use of pantoprazole as a BCRP inhibitor should be revisited when characterizing BCRP-mediated transport in humans.

Tobacco smoking strongly modifies the association of prothrombin G20210A with undetermined stroke: consecutive survivors and population-based controls.

OBJECTIVE: Due to contradictory results of previous studies evaluating the association between ischemic stroke (IS) and thrombophilic polymorphisms, their routine screening in IS patients, particularly those older than 60 years, is not recommended. We evaluated the differences in the distribution of rs6025 and rs1799963 polymorphisms according to IS subtypes and their interaction with smoking. METHODS: We conducted a case-control study of 423 hospital-based consecutive survivors of their first-ever IS and 614 population-based controls. Survivors (18-81 years) with IS documented by brain imagining were examined at a median of 16 months after the index event. The stroke subtype was categorized using the Causative Classification of Stroke System. Controls (50-75 years) were free of a history of stroke/TIA, coronary heart disease, and venous thromboembolism. RESULTS: Age- and gender-adjusted prevalence of individuals carrying at least one copy of the rs1799963A minor allele was 5.3% among stroke survivors (by subtypes: 3.1% in large artery atherosclerosis, 2.0% in cardio-aortic embolism, 2.4% in small artery occlusion, and 10.3% in undetermined stroke) vs. 2.4% among controls. In multinomial multivariate adjusted analysis, rs1799963 was exclusively associated with undetermined stroke (OR: 3.67; 95% CI: 1.52-8.85; p = 0.004). There was strong evidence of rs1799963 × smoking synergistic interaction (OR: 5.14; 95% CI: 1.65-16.01; p = 0.005). There was no association of rs6025 with IS in general, or with any subtype. CONCLUSIONS: In our consecutive IS survivors, carriage of the rs1799963A allele is associated with undetermined stroke. This effect appears to be confined to smokers.

Celastrol protects ischaemic myocardium through a heat shock response with up-regulation of haeme oxygenase-1.

BACKGROUND AND PURPOSE: Celastrol, a triterpene from plants, has been used in traditional oriental medicine to treat various diseases. Here, we investigated the cardioprotective effects of celastrol against ischaemia. EXPERIMENTAL APPROACH: Protective pathways induced by celastrol were investigated in hypoxic cultures of H9c2 rat cardiomyoblasts and in a rat model of myocardial infarction, assessed with echocardiographic and histological analysis. KEY RESULTS: In H9c2 cells, celastrol triggered reactive oxygen species (ROS) formation within minutes, induced nuclear translocation of the transcription factor heat shock factor 1 (HSF1) resulting in a heat shock response (HSR) leading to increased expression of heat shock proteins (HSPs). ROS scavenger N-acetylcysteine reduced expression of HSP70 and HSP32 (haeme oxygenase-1, HO-1). Celastrol improved H9c2 survival under hypoxic stress, and functional analysis revealed HSF1 and HO-1 as key effectors of the HSR, induced by celastrol, in promoting cytoprotection. In the rat ischaemic myocardium, celastrol treatment improved cardiac function and reduced adverse left ventricular remodelling at 14 days. Celastrol triggered expression of cardioprotective HO-1 and inhibited fibrosis and infarct size. In the peri-infarct area, celastrol reduced myofibroblast and macrophage infiltration, while attenuating up-regulation of TGF-ß and collagen genes. CONCLUSIONS AND IMPLICATIONS: Celastrol treatment induced an HSR through activation of HSF1 with up-regulation of HO-1 as the key effector, promoting cardiomyocyte survival, reduction of injury and adverse remodelling with preservation of cardiac function. Celastrol may represent a novel potent pharmacological cardioprotective agent mimicking ischaemic conditioning that could have a valuable impact in the treatment of myocardial infarction.

[The search for a sensor of intracellular sodium involved in pathogenesis of hypertensive disease].

Na+,K+ ATPase plays the key role in regulation of intracellular concentration of monovalent cations and related functions essential for electrogenesis and the maintenance of cell volume. This review is focused on the new data showing that a long-term increase of the intracellular Na+ level induces expression of early response genes and genes involved in regulation of apoptosis. Results of the studies of the Na+ sensor and its role in pathogenesis of the salt-sensitive form of hypertensive disease are summarized.

Investigation of mechanism of p38 MAPK activation in renal epithelial cell from distal tubules triggered by cardiotonic steroids.

Ouabain and other cardiotonic steroids (CTS) kill renal epithelial cells from distal tubules (C7-MDCK) via interaction with Na,K-ATPase but independently of inhibition of Na,K-ATPase-mediated ion fluxes. Recently, we demonstrated that modest intracellular acidification and inhibition of p38 MAPK suppress death of C7-MDCK cells triggered by ouabain. In the present study we investigate the mechanism of p38 MAPK activation in renal epithelial cell from distal tubules evoked by cardiotonic steroids. Using Na+/K+ ionophores (monensin, nigericin) and media with different content of monovalent cations, we revealed that p38 MAPK phosphorylation in ouabain-treated renal epithelial cells is not caused by Na,K-ATPase inhibition and inversion of the [Na+](i)/[K+](i) ratio. We also demonstrated that attenuation of pH from 7.45 to 6.75 did not alter the level of p38 MAPK phosphorylation observed in ouabain-treated cells. Inhibitors of PKA, PKC, and PKG as well as protein phosphatases were unable to abolish p38 MAPK activation triggered by ouabain. Using phosphotyrosine antibodies we did not detect any effect of ouabain on activation of tyrosine kinases. Thus, our results show that activation of p38 MAPK and cytotoxic action of CTS are independent of intracellular Na+, K+, and H+ concentrations. The molecular origin of intermediates of death signaling induced by CTS via conformation changes of Na,K-ATPase with following activation of p38 MAPK should be examined further.

The death of cardiotonic steroid-treated cells: evidence of Na+i,K+i-independent H+i-sensitive signalling.

Na/K-ATPase is the only known target of cardiotonic steroids (CTS) identified in plants, amphibians and later on in several mammalian species, including human. We focus our review on recent data implicating CTS in the tissue-specific regulation of cell survival and death. In vascular smooth muscle cells, CTS inhibited cell death triggered by apoptotic stimuli via a novel Na+i-mediated, Ca2+i-independent mechanism of expression of antiapoptotic genes, including mortalin. In contrast, exposure to CTS in vascular endothelial and renal epithelial cells led to cell death, showing combined markers of apoptosis and necrosis. This mode of cell death, termed oncosis, is caused by CTS interaction with Na/K-ATPase but is independent of the inhibition of Na/K-ATPase-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio. The intermediates of intracellular signalling involved in Na+i, K+i-independent oncosis of CTS-treated cells remain unknown. Recently, we found that this mode of cell death can be protected by modest intracellular acidification via the expression of H+i-sensitive genes. The molecular origin of intracellular Na+ and H+ sensor involvement in the development of apoptosis and oncosis is currently under investigation.

The rapid decline of MTT reduction is not a marker of death signaling in ouabain-treated cells.

Decreased staining with dimethylthiazol diphenyltetrazolium (MTT) is widely used for cell death detection. This study examined MTT assay as a marker of the Na+i,K+i-independent mode of cell death revealed in ouabain-treated C7-MDCK cells derived from distal tubule of the Madin-Darby canine kidney. The action of 3-M ouabain on MTT reduction in C7-MDCK cells exhibited bipartite kinetics with a rapid ~2-fold decline occurring in 30-120 min and a delayed ~8-10-fold decrease after 10 hr of ouabain addition. Treatment with ouabain for 18 hr led to 6-fold activation of caspase-3, 4-fold elevation of chromatin fragmentation, and massive cell detachment. Caspase-3 activation, chromatin fragmentation and cell detachment were completely abolished by acidification of the incubation medium from pH 7.2 to 6.7. In contrast, the 2-fold inhibition of MTT reduction seen in 5 hr of ouabain addition was not affected by medium acidification. Within the 5-hr time window, we did not observe any significant impact of ouabain on the cellular redox state estimated by the autofluorescence ratio of reduced pyridine nucleotides and oxidized flavoproteins. In rat aortic endothelial cells and primary astrocytes, exposure to 5-mM ouabain attenuated MTT reduction but did not affect cell survival. Thus, our results show that diminished staining with MTT in ouabain-treated cells is not sufficient proof of triggering of the cell death machinery. We speculate that altered endo- and exocytoses evoked by cardiotonic steroids contribute to decreased MTT reduction.

Analysis of the putative regulatory region of the gastric inhibitory polypeptide receptor gene in food-dependent Cushing's syndrome.

Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome (CS) results from the ectopic expression of non-mutated GIP receptor (hGIPR) in the adrenal cortex. We evaluated whether mutations or polymorphisms in the regulatory region of the GIPR gene could lead to this aberrant expression. We studied 9.0kb upstream and 1.3kb downstream of the GIPR gene putative promoter (pProm) by sequencing leukocyte DNA from controls and from adrenal tissues of GIP- and non-GIP-dependent CS patients. The putative proximal promoter region (800 bp) and the first exon and intron of the hGIPR gene were sequenced on adrenal DNA from nine GIP-dependent CS, as well as on leukocyte DNA of nine normal controls. Three variations found in this region were found in all patients and controls; at position -4/-5, an insertion of a T was seen in four out of nine patients and in five out of nine controls. Transient transfection studies conducted in rat GC and mouse Y1 cells showed that the TT allele confers loss of 40% in the promoter activity. The analysis of the 8-kb distal pProm region revealed eight distal single nucleotide polymorphisms (SNPs) without probable association with the disease, since frequencies in patients and controls were very similar. In conclusion, mutations or SNPs in the regulatory region of the GIPR gene are unlikely to underlie GIP-dependent CS.

Apoptosis in serum-deprived vascular smooth muscle cells: evidence for cell volume-independent mechanism.

Shrinkage is the earliest hallmark of cells undergoing apoptosis. This study examines the role of this phenomenon in the onset of vascular smooth muscle cell (VSMC) apoptosis triggered by growth factor withdrawal. In hyperosmotic media, VSMC showed the same amplitude of shrinkage but were more resistant to apoptosis than endothelial, epithelial and immune system cells. As with growth factor withdrawal, apoptosis in hyperosmotically-shrunken VSMC was sharply potentiated by transfection with E1A-adenoviral protein and was suppressed by activation of cAMP signaling as well as by the pan-caspase inhibitor z-VAD.fmk. Both cell shrinkage and apoptosis in VSMC-E1A treated with hyperosmotic medium were potentiated under sustained Na+, K+ pump inhibition with ouabain that was in contrast to inhibition of apoptosis documented in ouabain-treated, serum-deprived cells. After 1-hr incubation in serum-deprived medium, VSMC-E1A volume declined by approximately 15%. Transfer from hypotonic to control medium decreased VSMC-E1A volume by approximately 25% without any induction of apoptosis. Neither swelling in hyposmotic medium nor dissipation of the transmembrane gradient of K+ and major organic osmolytes protected serum-deprived VSMC-E1A from apoptosis. Thus, our results show that similarly to immune system, endothelial and epithelial cells, extensive VSMC shrinkage in hyperosmotic medium leads to the development of apoptosis. In contrast to hyperosmotic medium, the modest cell volume decrease occurring in serum-deprived VSMC does not contribute to triggering of the apoptotic machinery.

[Na/K pump and intracellular monovalent cations: novel mechanism of excitation-transcription coupling involved in inhibition of apoptosis]. / Na/K-nasos i vnutrikletochnye monovalentnye kationy: novyi mekhanizm sopriazheniia vozbuzhdeniia s transkriptsiei, uchastvuiushchii v podavlenii apoptoza.

The Na/K pump plays a key role in the regulation of the intracellular concentrations of monovalent cations and related cell function leading to electrogenesis and excitation-contraction coupling. We focus this review on the analysis of recent data showing that (i) inhibition of the Na/K pump triggers a signaling cascade independently of modulation of the intracellular [Na+]i/[K+]i ratio; (ii) elevation of [Na+]i under sustained inhibition of the Na/K pump leads to expression of a set of genes by [Ca2+]i-dependent and independent pathways; (iii) [Na+]i-sensitive genes are involved in the inhibition of programmed cell death (apoptosis) in vascular smooth muscle cells.

[3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein.

[(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.

Suppression of programmed cell death by intracellular cAMP is not mediated by expression of genes encoding an inhibitor of apoptosis.

The elevation of intracellular cAMP content is accompanied by expression of genes whose promoter contains a Ca(2+)-cAMP responsive element. In vascular smooth muscle cells (VSMC), activation of cAMP signaling blocks apoptosis triggered by serum deprivation. In the present study we investigated the role of gene expression in the inhibition of apoptosis by cAMP. In VSMC transfected with E1A adenovirus, incubation in the absence of serum for 6 h led to 20-fold elevation of chromatin fragmentation and 10-fold activation of caspase-3 activity, these being employed as markers of apoptosis. Forskolin-induced activation of cAMP signaling was accompanied by 50% elevation of RNA synthesis and completely abolished the development of apoptosis during the initial 6 h incubation in growth factor-free medium. In 12 h apoptosis in forskolin-treated VSMC was slowly developed and after 24 h the content of chromatin fragments was 2-fold less than in control cells. Addition of actinomycin D and cycloheximide completely blocked RNA synthesis and decreased protein synthesis by 80%, respectively. Neither compound affected baseline apoptosis or its inhibition by forskolin. More than 70 newly phosphorylated proteins were observed by 2D-electrophoresis of VSMC after incubation with forskolin for 3 h; in 24 h the number of phosphoproteins triggered by forskolin was decreased by 2-3-fold. These results show that suppression of VSMC apoptosis under activation of cAMP signaling is mediated via posttranslational modification of pre-existing intermediates of the apoptotic machinery rather than by de novo synthesis of inhibitors of programmed cell death.

Chromosomal mapping of HCaRG, a novel hypertension-related, calcium-regulated gene.

We recently identified a novel gene that is negatively regulated by extracellular calcium concentration with higher levels of transcripts in hypertensive animals (SHR). We named this gene HCaRG (Hypertension-related, Calcium-Regulated Gene). In this work we report the chromosomal localization of the HCaRG gene among different species. We identified a BglII RFLP between BN.lx and SHR rats. We then analysed the strain distribution pattern of this RFLP in 31 RIS, originating from BN.lx and SHR rats, and compared it to the segregation of 475 markers localized in the rat genetic map. Hcarg localizes to the rat chromosome 7 between the markers Mit3 and Mit4. This region is homologous to human chromosome 8q21-24. We identified three clones in Genbank that contain the sequence of HCaRG. It was therefore possible to narrow down the localization of human HCaRG to chromosome 8q24.3. Furthermore, a suggestive localization of mouse Hcarg based on conservation of linkage between human and mouse is on chromosome 15. We previously identified a putative calcium-binding motif (EF-Hand) and a nuclear receptor-binding domain (LxxLL) in the rat sequence of the HCaRG protein. Sequence comparison between five different species showed that these domains are highly conserved. Furthermore, a search of ESTs in Genbank for homologous sequences showed that HCaRG is expressed only in eukaryotes, particularly in mammals.

Aberrant membrane hormone receptors in incidentally discovered bilateral macronodular adrenal hyperplasia with subclinical Cushing's syndrome.

Cortisol secretion in adrenal Cushing's syndrome can be regulated by the aberrant adrenal expression of receptors for gastric inhibitory polypeptide, vasopressin, catecholamines, LH/human CG (LH/hCG), or serotonin. Four patients with incidentally discovered bilateral macronodular adrenal hyperplasia without clinical Cushing's syndrome were evaluated for the possible presence of aberrant adrenocortical hormone receptors. Urinary free cortisol levels were within normal limits, but plasma cortisol levels were slightly elevated at nighttime and suppressed incompletely after dexamethasone administration. Plasma ACTH was partially suppressed basally but increased after administration of ovine CRH. A 51-yr-old woman had ACTH-independent increases of plasma cortisol after 10 IU AVP im (292%), 100 microg GnRH iv (184%), or 10 mg cisapride orally (310%); cortisol also increased after administration of NaCl (3%), hCG, human LH, and metoclopramide. In a 61-yr-old man, cortisol was increased by AVP (349%), GnRH (155%), hCG (252%), and metoclopramide (191%). Another 53-yr-old male increased plasma cortisol after AVP (171%) and cisapride (142%). Cortisol secretion was also stimulated by vasopressin in a 54-yr-old female. This study demonstrates that subclinical secretion of cortisol can be regulated via the aberrant function of at least V1-vasopressin, LH/hCG, or 5-HT4 receptors in incidentally identified bilateral macronodular adrenal hyperplasia.