Association of p53 and p21 polymorphisms with prostate cancer.

Cell cycle deregulation is common in human cancer. Alterations of the tumor-suppressor gene p53 and its downstream effector p21 have been indicated in the development of numerous human malignancies. Therefore, we hypothesize that the p53 codon 72 polymorphism, either on its own or in combination with p21 (C98A and C70T) polymorphisms, modifies the risk of prostate cancer within the Slovak population, and no previous studies have investigated these gene-gene interactions in the pathogenesis of prostate cancer in the Slovak population. Polymerase chain reaction-restriction fragment length polymorphism was used to determine the p53 and p21 genotypes in subjects comprising 300 prostate cancer patients and 446 healthy individuals. These 3 polymorphisms individually did not correlate with the prostate cancer risk. Conversely, the interaction between the p53 and p21 polymorphisms significantly decreased the risk of prostate cancer, with the odds ratio (OR) being 0.49 [95% confidence interval (CI), 0.27-0.86; P<0.05] for subjects carrying the p53 codon 72 arginine (Arg)/proline (Pro)+Pro/Pro and p21 C98A CA genotypes compared to the combined reference genotypes p53 codon 72 Arg/Arg and p21 C98A CC. Neither the p53 genotypes nor the p21 genotypes showed statistically significant differences in Gleason score or serum prostate-specific antigen levels (P>0.05). A decreased risk of prostate cancer association with the p21 C98A CA genotype (OR=0.58; 95% CI, 0.36-0.93; P<0.05) in non-smokers compared to the non-smokers with the p21 C98A CC genotype was observed. Smokers carrying the p53 codon 72 Pro/Pro genotype were not at any significant risk of prostate cancer (OR=2.97; 95% CI, 0.51-17.15) compared to the non-smokers with the Arg/Arg genotype. Taken together, to the best of our knowledge this is the first study to show that a combination of the variant genotypes of p53 codon 72 and p21 C98A may modify the prostate cancer risk within the Slovak population.

Interfraction variation in prostate cancer - analysis of 11726 cone-beam CT.

PURPOSE: To investigate the setup margins in prostate cancer treatment without using daily online repositioning methods. METHODS: We analysed the data from patients treated with curative-intend radiotherapy. Each patient underwent a series of pretreatment online localizations during daily setup using conebeam CT. The skin-prostate shifts and bone-prostate shifts were recorded in anteroposterior (AP), craniocaudal (CC), and laterolateral (LL)direction. The safety margins based on van Herk equation (2.5Σ+0.7σ) were calculated and the correlations between margins and various patient characteristics and prostate locations were investigated. RESULTS: A total of 307 patients were included, representing 11,726 localisations resulting in 70,356 shifts. The man skin-prostate setup inaccuracy was 0.8 ± 5.4mm in AP, 1.3 ± 4.8mm for CC, and 0.1 ± 5.6mm in LL direction. The man bone-prostate setup inaccuracy was 0.4 ± 3.3mm in AP, 0.1 ± 2.5mm for CC, and 0.1 ± 1.4mm in LL direction. According to van Herk equation, clinical target volume (CTV)-planning target volume (PTV) margins of 11.4, 10.6, and 11.8 mm (AP, CC, and LL, respectively) would be required for setup using skin markers and margins of 7.0, 4.7, and 2.1mm would be necessary for setup using bone structures. The average rectal area < 11cm(2) and volume of bladder > 300 cm(3) were associated with smaller CTV-PTV margins for setup using bone structures. The largest margins (15.8 mm in LL direction) were needed in patients with body mass index (BMI) > 35 using skin markers. CONCLUSIONS: Our results confirm that the commonly used CTV-PTV margins are inadequate.

The effects of atorvastatin treatment on the mean platelet volume and red cell distribution width in patients with dyslipoproteinemia and comparison with plasma atherogenicity indicators--A pilot study.

OBJECTIVES: The mean platelet volume (MPV) and red cell distribution width (RDW) have recently arisen interest because of their association with an increased cardiovascular risk. The aim of our study was, therefore, to determine whether an association exists between MPV, RDW and lipoprotein sub-fractions, and to show the impact of statin therapy on these new possible biomarkers of atherosclerotic risk. DESIGN AND METHODS: A cohort of 40 patients with hypercholesterolaemia (29 females, mean age 62.9±9 years), without previous hypolipidaemic treatment were enrolled. The patients were treated with atorvastatin 40 mg/day for 12 weeks. Total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density cholesterol (HDL-C), triglycerides (TG), LDL-C sub-fractions [large LDL-C 1-2 and small dense (sd)-LDL-C 3-7], apolipoproteins (apoA1, apoB), apoB/apoA1 ratio, atherogenic index of plasma (AIP), haematological parameters (including MPV, RDW) and safety parameters (renal, hepatic) were measured before and after 12 weeks of atorvastatin treatment. RESULTS: At baseline, a strong correlation between HDL-C, TG, sd-LDL-C, apoB, apoB/apoA1, and AIP with MPV (r=-0.55, p<0.001; r=0.57, p<0.001; r=0.73, p<0.001; r=0.41, p<0.05; r=0.52, p<0.001; r=0.61, p<0.001, respectively) and RDW (r=-0.49, p<0.001; r=0.62, p<0.001; r=0.67, p<0.001; r=0.41, p<0.05; r=0.43, p<0.05; r=0.65, p<0.001, respectively) was found. After 12 weeks of treatment with atorvastatin, MPV and RDW values underwent significant modification only in those patients displaying the strongest lipid-lowering effect. CONCLUSIONS: Values of MPV and RDW seem to reflect a pro-atherogenic lipoprotein profile mainly represented by the presence of sd-LDL-C.

Influence of oak wood polyphenols on cysteine, homocysteine and glutathione total levels and PON1 activities in human adult volunteers - a pilot study.

Oxidative stress reflects an imbalance between antioxidants and pro-oxidants. Many diseases like atherosclerosis or heart failure are involved in oxidative stress. Increased oxidative stress is one of the potential contributing factors to aging. The aim of this study was to monitor the total thiol levels as markers of oxidative stress in 20 healthy volunteers after polyphenols intake (extract from the French oak wood Quercus robur - Robuvit® (300 mg/day)). Polyphenols are known as biomodulators with antioxidant activities. Homocysteine, cysteine and glutathione total levels were determined by using HPLC with electrochemical detection. The activity of the antioxidant enzyme paraoxonase-1 toward two substrates was determined by spectrophotometry. The level of thiol compounds and paraoxonase-1 activities were controlled after run-in (week 0), intervention (week 4) and washout (week 6) period. After the intervention period the results showed that Robuvit® had no significant influence on glutathione level (p = 0.382) and paraoxonase activities towards both, arylester and lactone substrates. On the other hand, homocysteine and cysteine levels decreased significantly (p = 0.029; p < 0.001, respectively). The negative correlation between paraoxonase lactonase activity and homocysteine level was noticed. This confirms that paraoxonase might play an important role in homocysteine-thiolactone metabolism.

Variation in N-acetyltransferase 2 (NAT2), smoking and risk of prostate cancer in the Slovak population.

N-acetyltransferase 2 (NAT2) is an enzyme involved in the biotransformation of xenobiotics, mainly aromatic and heterocyclic amines and hydrazines, all of which represent an important class of carcinogens found in tobacco smoke. Polymorphism in NAT2 gene is reported to be associated with susceptibility to various types of cancer. This study investigated the relationship between the NAT2 polymorphism and the risk of prostate cancer with reference to the link between cigarette smoking and the xenobiotic-metabolizing enzyme NAT2. Overall, 281 cases and 395 controls from Slovakia were studied using polymerase chain reaction-restriction fragment length polymorphism assay. We found no statistically significant association between NAT2 genotypes and prostate cancer risk (slow acetylation vs. rapid acetylation: OR 1.13; 95 % CI 0.83-1.55). We report here a statistically significant correlation between the NAT2*5C/NAT2*6A slow acetylator genotype and the risk for developing prostate cancer (OR 2.91; 95 % CI 1.43-5.94; p = 0.003) when compared with the rapid phenotype. Smokers with NAT2 rapid phenotype had a five percent (5 %) reduced risk of prostate cancer compared with non-smokers carrying the rapid acetylator genotype. The association was reversed among smokers and non-smokers with NAT2 slow phenotype. On the basis of the foregoing, we conclude that the NAT2 phenotypes whether alone or in association with smoking do not correlate with susceptibility to prostate cancer within the Slovak population.

Age-related alterations in endothelial function of femoral artery in young SHR and WKY rats.

The present study was designed to evaluate the effects of vascular aging in juvenescence on endothelial function in femoral arteries and to assess differences between normotensive and hypertensive rats. The aim of the study was to determine if age affected nitric oxide- (NO-) mediated relaxations in normotensive and hypertensive rats. Juvenile (7-week-old) and young adult (22-week-old) male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were used in this study. Femoral artery (FA) reactivity was determined by wire myograph and NO synthase activity by conversion of [(3)H]-L-arginine. During juvenescence systolic blood pressure (tail-cuff) increased significantly only in SHR, while NO synthesis decreased significantly in both strains. Endothelium-dependent relaxations to acetylcholine were reduced in the FA of SHR compared to age-matched WKY at both ages, yet these parameters were unchanged in adult rats compared with juvenile animals. The NO-dependent component of vasorelaxation was markedly reduced, whereas the NO-independent component was increased in adult compared to juvenile rats in both strains. The endothelial dysfunction in SHR at both ages was associated with reduction of NO-independent mechanisms. In conclusion, aging in early periods of life was associated with reduction of vascular NO production and bioavailability in both strains investigated. This reduction was however fully compensated by accentuation of NO-independent mechanisms.

Genotype-related effect of crowding stress on blood pressure and vascular function in young female rats.

This study investigated the influence of chronic crowding stress on nitric oxide (NO) production, vascular function and oxidative status in young Wistar-Kyoto (WKY), borderline hypertensive (BHR) and spontaneously hypertensive (SHR) female rats. Five-week old rats were exposed to crowding for two weeks. Crowding elevated plasma corticosterone (P<0.05) and accelerated BP (P<0.01 versus basal) only in BHR. NO production and superoxide concentration were significantly higher in the aortas of control BHR and SHR versus WKY. Total acetylcholine (ACh)-induced relaxation in the femoral artery was reduced in control SHR versus WKY and BHR, and stress did not affect it significantly in any genotype. The attenuation of ACh-induced relaxation in SHR versus WKY was associated with reduction of its NO-independent component. Crowding elevated NO production in all strains investigated but superoxide concentration was increased only in WKY, which resulted in reduced NO-dependent relaxation in WKY. In crowded BHR and SHR, superoxide concentration was either unchanged or reduced, respectively, but NO-dependent relaxation was unchanged in both BHR and SHR versus their respective control group. This study points to genotype-related differences in stress vulnerability in young female rats. The most pronounced negative influence of stress was observed in BHR despite preserved endothelial function.

Long-term social stress induces nitric oxide-independent endothelial dysfunction in normotensive rats.

As chronic stress is a significant risk factor for several cardiovascular disorders, this study investigated the hypothesis that long-term stress produced by crowding may lead to alterations in nitric oxide (NO) production and NO-dependent relaxation in the course of stress, resulting in endothelial dysfunction and hypertension in Wistar-Kyoto (WKY) rats. For this purpose, male WKY rats were divided into control (480 cm2/rat, four rats/cage, n = 8) and crowded (200 cm2/rat, five rats/cage, n = 10) groups for 8 or 12 weeks. Vasorelaxation was evaluated in vitro as a response to acetylcholine (ACh) of femoral arteries pre-contracted by serotonin, before and after NO synthase inhibition (N (G)-nitro-l-arginine methyl ester, 300 µmol/l). Crowding increased plasma corticosterone concentration but failed to affect blood pressure (determined by tail-cuff plethysmography) of rats. NO production was unchanged in the hypothalamus and left ventricle of both stressed groups; however it was significantly elevated in the aorta. Maximal ACh-induced relaxation was elevated significantly after 8-week stress, but reduced after 12 weeks. Stress elevated the NO-dependent component and reduced the NO-independent component of ACh-induced relaxation in both crowded groups. However, a reduction in the NO-independent component was more pronounced after 12-week versus 8-week stress. In conclusion, elevated endothelium-dependent relaxation was observed after 8-week stress, while the extension of stress exposure resulted in a reduction in arterial relaxation associated with a more pronounced decrease of its NO-independent component. Thus, elevation of the NO-dependent component of relaxation can be considered as an adaptation mechanism, and impairment of NO-independent relaxation might be the initial step in chronic stress-induced cardiovascular disorders.

Effect of CYP17 and PSA gene polymorphisms on prostate cancer risk and circulating PSA levels in the Slovak population.

Cytochrome P-450c17α (CYP17) and prostate-specific antigen (PSA) genes, which are involved in the androgen metabolism cascade, have been studied as possible candidates for genetic influences on prostate cancer development. Contradictory results prompted us to evaluate the frequencies of polymorphisms in the CYP17 and PSA genes as well as the association between these genetic variants and serum PSA levels in prostate cancer patients and men routinely screened for prostate cancer with PSA in the Slovak male population. The CYP17 and PSA polymorphisms were determined by the PCR-RFLP analysis in 197 Caucasian prostate cancer patients and 256 Caucasian controls. We did not find any association between the CYP17 and PSA genotypes and prostate cancer risk overall, or by grade. Also the total serum PSA levels in the cases with the AG or AA genotype were not significantly higher than in the men with the GG genotype (P > 0.05). Our study did not provide support for the hypothesized relationship between CYP17 and PSA gene polymorphisms and prostate cancer in the Slovak male population.

Predictive validity of MRI in detecting and following cholesteatoma.

High recurrence rate of the middle ear cholesteatoma requires regular postoperative follow-up. This study evaluated data from the patients investigated with DW MRI to ascertain (1) the strength of the technique in detecting primary, and residual recurrent cholesteatoma, and (2) its accuracy in differentiating cholesteatoma from postoperative tissue changes. The diagnostic accuracy of two different DW imaging (EPI and non-EPI) techniques was evaluated. The data have been collected prospectively from 33 consecutive patients with either primary cholesteatoma, or with suspicious symptoms for potential cholesteatoma recurrence. The findings from non-EPI (HASTE) DW MR and EPI DW MR images were blindly compared with those obtained during a primary or secondary surgery. Preoperative non-EPI (HASTE) DWI pointed to a cholesteatoma in 25 out of 33 patients. In this subgroup, cholesteatoma were confirmed also by the surgery. In five cases, the non-EPI (HASTE) DWI did not show a cholesteatoma in the temporal bone, which agreed with the surgical findings. Three misclassifications were made by non-EPI (HASTE) DWI, all in the subgroup of patients indicated for primary surgery. The resulting pooled sensitivity of non-EPI (HASTE) DW imaging for diagnosing cholesteatoma in our study amounted to 96.15% (95% confidence interval (CI) 80.36-99.9), specificity was 71.43% (95% CI 29.04-96.33). Positive predictive value was 92.59% (95% CI 75.71-99.09) and negative predictive value 83.33% (95% CI 35.88-99.58). In conclusion, we recommend the non-EPI (HASTE) DW MRI as a valid method for diagnosing cholesteatoma and follow-up after cholesteatoma surgery.

One-day session LINAC-based stereotactic radiosurgery of posterior uveal melanoma.

PURPOSE: LINAC-based stereotactic radiosurgery (SRS) of posterior uveal melanoma is a conservative method to treat uveal melanoma. METHODS: This was a retrospective clinic-based study of patients with posterior uveal melanoma in stage T2/T3 who underwent 1-day session SRS at LINAC accelerator or SRS plus combined methods from 2001 to 2008. RESULTS: Thirty-nine patients with posterior uveal melanoma were treated with SRS (age 25-80 years, median 54 years). Median tumor volume at baseline was 0.6 cm3 (range 0.2-1.3 cm3). The therapeutic dose (TD) was 35.0 Gy, median of maximal dose applied was 49.0 Gy (range 37.0-60.0 Gy). Patient data were analyzed in groups: group 1, single SRS irradiation; group 2, SRS with subsequent endoresection or cyclectomy or additional transpupillary thermotherapy (TTT) or brachytherapy by Ru106 plaques; group 3a, enucleation after single SRS; group 3b, enucleation after SRS and endoresection/cyclectomy or TTT or brachytherapy Ru106. In patients with visual acuity of 20/40 or better, the median rate of best-corrected visual acuity (BCVA) decline was higher than that of the total and significantly higher than the rate of decline in the complementary group of patients with BCVA less than 20/40 (p=0.0077; Mann-Whitney U test). CONCLUSIONS: One-step LINAC-based SRS with a single dose 35.0 Gy is a method to treat middle-stage posterior uveal melanoma and to preserve the eye globe or as the first step of combined methods: irradiation before endoresection or cyclectomy.

Modulation of intracellular chloride channels by ATP and Mg2+.

We report the effects of ATP and Mg2+ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n=112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5-2 mmol/l) modulated and/or inhibited the chloride channel activities (n=38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n=5/93) or reversible (n=15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n=6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5-2 mmol/l) without the presence of Mg2+ decreased the chloride channel current (n=12/14), whereas Mg2+ significantly reversed the effect (n=4/4). We suggest that ATP-intracellular chloride channel interactions and Mg2+ modulation of these interactions may regulate different physiological and pathological processes.