Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromereadjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5 gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from nonneoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.

Facioscapulohumeral muscular dystrophy (FSHD) is a unique dominant disorder involving shortening of an array of tandem 3.3 kb repeats. This copy-number polymorphic repeat, D4Z4, is present in arrays at both 4q35 and 10q26, but only 4q35 arrays with one to 10 copies of the repeat are linked to FSHD. The most popular model for how the 4q35 array-shortening causes FSHD is that it results in a loss of postulated D4Z4 heterochromatinization, which spreads proximally, leading to overexpression of FSHD genes in cis. This would be similar to a loss of position-effect variegation (PEV) in Drosophila. To test for the putative heterochromatinization, we quantitated chromatin immunoprecipitation with an antibody for acetylated histone H4 that discriminates between constitutive heterochromatin and unexpressed euchromatin. Contrary to the above model, H4 acetylation levels of a non-repeated region adjacent to the 4q35 and 10q26 D4Z4 arrays in normal and FSHD lymphoid cells were like those in unexpressed euchromatin and not constitutive heterochromatin. Also, these control and FSHD cells displayed similar H4 hyperacetylation (like that of expressed genes) at the 5' regions of 4q35 candidate genes FRG1 and ANT1. Contrary to the loss-of-PEV model and a recent report, there was no position-dependent increase in transcript levels from these genes in FSHD skeletal muscle samples compared with controls. Our results favor a new model for the molecular genetic etiology of FSHD, such as, differential long-distance cis looping that depends upon the presence of a 4q35 D4Z4 array with less than a threshold number of copies of the 3.3 kb repeat.

We quantitatively analysed hypermethylation at CpG islands in the 5' ends of 12 genes and one non-CpG island 5' region (MTHFR) in 31 Wilms tumors. We also determined their global genomic 5-methylcytosine content. Compared with various normal postnatal tissues, *40 -90% of these pediatric kidney cancers were hypermethylated in four of the genes, MCJ, RASSF1A, TNFRSF12 and CALCA as determined by a quantitative bisulfite-based assay (MethyLight). Interestingly, the non-CpG island 5' region of MTHFR was less methylated in most tumors relative to the normal tissues. By chromatographic analysis of DNA digested to deoxynucleosides, about 60% of the Wilms tumors were found to be deficient in their overall levels of DNA methylation. We also analysed expression of the three known functional DNA methyltransferase genes. No relationship was observed between global genomic 5-methylcytosine levels and relative amounts of RNA for DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. Importantly, no association was seen between CpG island hypermethylation and global DNA hypomethylation in these cancers. Therefore, the overall genomic hypomethylation frequently observed in cancers is probably not just a response or a prelude to hypermethylation elsewhere in the genome. This suggests that the DNA hypomethylation contributes independently to oncogenesis or tumor progression.

ICF (immunodeficiency, centromeric region instability and facial anomalies) is a recessive disease caused by mutations in the DNA methyltransferase 3B gene (DNMT3B). Patients have immunodeficiency, chromosome 1 (Chr1) and Chr16 pericentromeric anomalies in mitogen-stimulated lymphocytes, a small decrease in overall genomic 5-methylcytosine levels and much hypomethylation of Chr1 and Chr16 juxtacentromeric heterochromatin. Microarray expression analysis was done on B-cell lymphoblastoid cell lines (LCLs) from ICF patients with diverse DNMT3B mutations and on control LCLs using oligonucleotide arrays for approximately 5600 different genes, 510 of which showed a lymphoid lineage-restricted expression pattern among several different lineages tested. A set of 32 genes had consistent and significant ICF-specific changes in RNA levels. Half of these genes play a role in immune function. ICF-specific increases in immunoglobulin (Ig) heavy constant mu and delta RNA and cell surface IgM and IgD and decreases in Ig(gamma) and Ig(alpha) RNA and surface IgG and IgA indicate inhibition of the later steps of lymphocyte maturation. ICF-specific increases were seen in RNA for RGS1, a B-cell specific inhibitor of G-protein signaling implicated in negative regulation of B-cell migration, and in RNA for the pro-apoptotic protein kinase C eta gene. ICF-associated decreases were observed in RNAs encoding proteins involved in activation, migration or survival of lymphoid cells, namely, transcription factor negative regulator ID3, the enhancer-binding MEF2C, the iron regulatory transferrin receptor, integrin beta7, the stress protein heme oxygenase and the lymphocyte-specific tumor necrosis factor receptor family members 7 and 17. No differences in promoter methylation were seen between ICF and normal LCLs for three ICF upregulated genes and one downregulated gene by a quantitative methylation assay [combined bisulfite restriction analysis (COBRA)]. Our data suggest that DNMT3B mutations in the ICF syndrome cause lymphogenesis-associated gene dysregulation by indirect effects on gene expression that interfere with normal lymphocyte signaling, maturation and migration.

T3 and T4 sympathicotomies are both effective for the treatment of PH. T4 sympathicotomy, decreases the side effects but do not compromise the therapeutic effects, and should be the method of choice.

No abstract

Esophageal leiomyoma is the most common benign esophageal tumor. Thoracoscopic enucleation is currently a preferred approach to most of these lesions. We present our experiences of enucleation of these tumors using thoracoscopic approach. A retrospective review of 40 patients who underwent enucleation of esophageal leiomyoma from 1997 to 2007 in our institute was conducted. Presenting symptoms, operative approach, tumor size, tumor shape, outcomes, and indication for this approach were analyzed. Forty patients were identified. Postoperative histopathology confirmed the leiomyoma in all patients. The thoracoscopic enucleation was completed in 34 cases, and the operation was converted to open procedure in six cases. Reasons for conversion included too small tumors to be visualized in two cases, thoracic cavity adhesion in one case, and the too large tumors in three cases. The median operating time was 70 min (50 to 210 min). Mean tumor size was 3.7 cm (0.5-10 cm). There were no major postoperative complications. Symptoms especially dysphasia were relieved postoperatively. Short- and long-term follow-up was satisfactory with none of the patients having tumor recurrences or other problems. Thoracoscopic enucleation of esophageal leiomyoma is technically safe and effective. It is currently the best choice for management of esophageal leiomyoma 1 to 5 cm in diameter. It can also be tried on a tumor larger than 5 cm, although the possibility of conversion to thoracotomy increases along with tumor growing and surrounding the esophagus.

The ras mutation, which is observed in 20-30% of human nonsmall cell lung cancers (NSCLCs), is one of common genetic alterations and has been proposed to be a prognostic factor in lung cancer. Oncogene ras appears to be essential for tumor progression and maintenance. Several therapeutic agents have been developed to inhibit ras, such as FTIs and antisense oligonucleotides. A new tool for blocking oncogenes in cancer cells has emerged with the discovery that RNA interference can specifically silence expression of endogenous human genes. In the current study, we used small interfering RNA (siRNA) directed against mutant K-ras to determine the anti-tumor effects of decreasing the levels of this protein in lung cancer cell lines. Adenovirus-mediated siRNA (AdH1/siK-ras(V12)) against K-ras(V12) markedly decreased K-ras(V12) gene expression and inhibited cellular proliferation of NSCLC H441 cells that express the relevant mutation (K-ras codon 12 GGT --> GTT), but produced minimal growth inhibition on NSCLC H1650 cells that lack the relevant mutation. Pretreatment with AdH1/siK-ras(V12) completely abrogated subcutaneous engraftment of H441 cells, as compared with a 100% tumor take in animals that received control vector-treated tumor cells. The in vivo effect of AdH1/siK-ras(V12) treatment was further examined by intratumoral injections after tumor induction. Pre-existing tumor growth was reduced by 45% by a single intratumoral injection. Three or five repeat injections resulted in complete tumor regression in eight of ten nude mice. Further, 23.12% of AdH1/siK-ras(V12) treated H441 cells underwent apoptosis, as compared with 6.13%, and 8.27% in untreated and control vector-treated cells, respectively. These results indicate that adenovirus-mediated siRNA can specifically and efficiently target factors whose expression is altered in malignancy and may have the potential as a therapeutic modality to treat human lung cancer.