19 research outputs found
Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1C in pancreatic endocrine tumors
<p>Abstract</p> <p>Background</p> <p><it>RASSF1A </it>gene silencing by DNA methylation has been suggested as a major event in pancreatic endocrine tumor (PET) but <it>RASSF1A </it>expression has never been studied. The <it>RASSF1 </it>locus contains two CpG islands (<it>A </it>and <it>C</it>) and generates seven transcripts (<it>RASSF1A</it>-<it>RASSF1G</it>) by differential promoter usage and alternative splicing.</p> <p>Methods</p> <p>We studied 20 primary PETs, their matched normal pancreas and three PET cell lines for the (i) methylation status of the <it>RASSF1 </it>CpG islands using methylation-specific PCR and pyrosequencing and (ii) expression of <it>RASSF1 </it>isoforms by quantitative RT-PCR in 13 cases. CpG island A methylation was evaluated by methylation-specific PCR (MSP) and by quantitative methylation-specific PCR (qMSP); pyrosequencing was applied to quantify the methylation of 51 CpGs also encompassing those explored by MSP and qMSP approaches.</p> <p>Results</p> <p>MSP detected methylation in 16/20 (80%) PETs and 13/20 (65%) normal pancreas. At qMSP, 11/20 PETs (55%) and 9/20 (45%) normals were methylated in at least 20% of <it>RASSF1A </it>alleles.</p> <p>Pyrosequencing showed variable distribution and levels of methylation within and among samples, with PETs having average methylation higher than normals in 15/20 (75%) cases (<it>P </it>= 0.01). The evaluation of mRNA expression of <it>RASSF1 </it>variants showed that: i) <it>RASSF1A </it>was always expressed in PET and normal tissues, but it was, on average, expressed 6.8 times less in PET (<it>P </it>= 0.003); ii) <it>RASSF1A </it>methylation inversely correlated with its expression; iii) <it>RASSF1 </it>isoforms were rarely found, except for <it>RASSF1B </it>that was always expressed and <it>RASSF1C </it>whose expression was 11.4 times higher in PET than in normal tissue (<it>P </it>= 0.001). A correlation between <it>RASSF1A </it>expression and gene methylation was found in two of the three PET cell lines, which also showed a significant increase in <it>RASSF1A </it>expression upon demethylating treatment.</p> <p>Conclusions</p> <p><it>RASSF1A </it>gene methylation in PET is higher than normal pancreas in no more than 75% of cases and as such it cannot be considered a marker for this neoplasm. <it>RASSF1A </it>is always expressed in PET and normal pancreas and its levels are inversely correlated with gene methylation. Isoform <it>RASSF1C </it>is overexpressed in PET and the recent demonstration of its involvement in the regulation of the Wnt pathway points to a potential pathogenetic role in tumor development.</p
Effect of blocking amino group couples on the mechanism of Mg and (Ca + Mg) ATPase associated with erythrocyte membrane fragments.
Kinetic alterations of the divalent cation-dependent ATPase activities of human erythrocyte membranes induced by blocking the membrane amino groups.
3-Methylhistidine urinary excretion as an index of skeletal muscle protein metabolism: reference values.
Sensitivity to radiations of Mg2+ and (Ca2+ + Mg2+) ATPase activities associated with erythrocyte membrane fragments.
8-Hydroxy-2 '-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection
In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV+ and HPV- subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis
Polycyclic aromatic hydrocarbon-DNA adducts in cervical smears of smokers and nonsmokers
The aim of this study was to detect polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in single cervical cells collected during a routine Papanicolaou smear and to relate this carcinogen exposure dose marker to smoking habit. An immunohistochemical assay, using a polyclonal antiserum raised against benzo[a]pyrene diol epoxide-DNA adducts, was performed to evaluate PAH-DNA adducts in cervical cells collected from 16 volunteers who smoked at least 20 cigarettes/day and 16 nonsmokers. The mean adduct level, determined as relative staining intensity by an optical density image analyzer, was significantly higher in smokers compared to nonsmokers (AOD x 1000 +/- SD = 98 +/- 32 and 73 +/- 25, respectively) (P = 0.04). These results demonstrate that this immunohistochemical assay, much simpler than other methodologies used to evaluate PAH-DNA adducts in cervical tissue, is sufficiently sensitive for quantitative adduct evaluation in single epithelial cervical cells, as already verified for other exfoliated material. This work thus confirms that tobacco smoke is a risk factor for genotoxic damage generation in cervical cells and indicates a procedure likely adaptable to a large population screening. Copyright 1999 Academic Press