Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

Methylation of endogenous human retroelements in health and disease.

Retroelements constitute approximately 45% of the human genome. Long interspersed nuclear element (LINE) autonomous retrotransposons are predominantly represented by LINE-1, nonautonomous small interspersed nuclear elements (SINEs) are primarily represented by ALUs, and LTR retrotransposons by several families of human endogenous retroviruses (HERVs). The vast majority of LINE and HERV elements are densely methylated in normal somatic cells and contained in inactive chromatin. Methylation and chromatin structure together ensure a stable equilibrium between retroelements and their host. Hypomethylation and expression in developing germ cells opens a "window of opportunity" for retrotransposition and recombination that contribute to human evolution, but also inherited disease. In somatic cells, the presence of retroelements may be exploited to organize the genome into active and inactive regions, to separate domains and functional regions within one chromatin domain, to suppress transcriptional noise, and to regulate transcript stability. Retroelements, particularly ALUs, may also fulfill physiological roles during responses to stress and infections. Reactivation and hypomethylation of LINEs and HERVs may be important in the pathophysiology of cancer and various autoimmune diseases, contributing to chromosomal instability and chronically aberrant immune responses. The emerging insights into the pathophysiological importance of endogenous retroelements accentuate the gaps in our knowledge of how these elements are controlled in normal developing and mature cells.

Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation.

In prostate carcinoma (PCa) increased DNA methylation ('hypermethylation') occurs at specific genes such as GSTP1. Nevertheless, overall methylation can be decreased ('hypomethylation') because methylation of repetitive sequences like LINE-1 retrotransposons is diminished. We analysed DNA from 113 PCa and 36 noncancerous prostate tissues for LINE-1 hypomethylation by a sensitive Southern technique and for hypermethylation at eight loci by methylation-specific PCR. Hypermethylation frequencies for GSTP1, RARB2, RASSF1A, and APC in carcinoma tissues were each >70%, strongly correlating with each other (P<10(-6)). Hypermethylation at each locus was significantly different between tumour and normal tissues (10(-11)82% of PCas. PCa may fall into three classes, that is, with few DNA methylation changes, with frequent hypermethylation, or with additional LINE-1 hypomethylation.

E-cadherin involved in inactivation of WNT/beta-catenin signalling in urothelial carcinoma and normal urothelial cells.

Constitutive activation of WNT signalling through beta-catenin, which leads to increased transcription of TCF/beta-catenin target genes, is crucial in the development of many human tumour types including colorectal carcinoma and hepatoma. Its role in urothelial cancer (TCC) is unclear, since typical activating mutations are not found. We therefore determined the activity of a beta-catenin/TCF-dependent promoter in proliferating normal uroepithelial cells and seven TCC cell lines, using a hepatoma line with oncogenic beta-catenin as a control. Neither normal urothelial cells nor TCC lines exhibited activity under normal growth conditions. In normal cells and 5/7 TCC lines, even transfection of activated beta-catenin did not restore promoter activity, suggesting repression of beta-catenin/TCF activity. TCF mRNAs and total beta-catenin protein levels did not differ qualitatively between inducible and noninducible cell lines, but E-cadherin expression was lacking or low in inducible TCC lines. In these, cotransfection of E-cadherin diminished activation of the TCF-dependent promoter by beta-catenin. Our results make constitutive WNT/beta-catenin signalling in TCC appear unlikely, thereby explaining the lack of reported mutations. However, decreased E-cadherin expression occurring in many TCC, often as a consequence of promoter hypermethylation, may confer inappropriate responsiveness to WNT factors.

Destabilization of chromosome 9 in transitional cell carcinoma of the urinary bladder.

The most frequent genetic alteration in transitional cell carcinoma of the urinary bladder (TCC) is loss of chromosome 9 which targets CDKN2A on 9p. The targets on 9q are not confirmed. Here, 81 advanced TCC specimens were investigated for loss of heterozygosity (LOH) and homozygous deletions (HD) on chromosome 9q using multiplex analysis of microsatellite markers. 41/81 tumours (51%) showed LOH on 9q, with LOH at all markers in 33 cases. Eight partial losses involved three regions in 9q12, 9q22.3, and 9q33- 9q34. No mutations were identified in the candidate tumour suppressor gene DBCCR1 in three tumours showing restricted LOH at 9q32-33. 22% of the specimens had HD at CDKN2A, but no HD was found on 9q. Two tumours had lost 9p only and five 9q only. 9q LOH was not related to tumour grade or stage and present or absent with equal frequency in recurrent TCC. LOH on 9q correlated with the extent of genome-wide hypomethylation (P < 0.0001) which extended into satellite sequences located in 9q12 juxtacentromeric heterochromatin. While the high frequency of chromosome 9q loss in TCC may reflect destabilization of the chromosome related to hypomethylation of repetitive DNA, the data are compatible with the existence of tumour suppressor genes on this chromosome arm.

Hypermethylation of the tumor necrosis factor receptor superfamily 6 (APT1, Fas, CD95/Apo-1) gene promoter at rel/nuclear factor kappaB sites in prostatic carcinoma.

DNA hypermethylation of CpG-rich promoter sequences is associated with tumor suppressor gene inactivation in many human cancers, notably in carcinoma of the prostate and the urinary bladder. Recently, the mouse homologue of the tumor necrosis factor receptor superfamily 6 (TNFRSF6) gene was reported to be inactivated by DNA methylation in various cell types. The Fas (CD95, Apo-1) protein encoded by the TNFRSF6 gene is an important mediator of apoptosis, which also is downregulated in different types of human carcinoma. We therefore investigated the methylation of the TNFRSF6 promoter in prostatic and bladder carcinomas and cell lines. In a restriction enzyme polymerase chain reaction assay, four of 32 prostatic carcinomas and three of 15 advanced bladder carcinomas showed evidence of hypermethylation at the rel/nuclear factor kappaB (NFkappaB) binding sites essential for promoter activity. The DU145 cell line derived from a metastasis of a prostate carcinoma also displayed hypermethylation in this assay, which was confirmed by bisulfite sequencing. Treatment of DU145 cells with the methylation inhibitor deoxyazacytidine slightly increased Fas protein expression, as detected by flow cytometry analysis. In vitro methylation of the TNFRSF6 promoter at the rel/NFkappaB sites completely abolished its activity. Thus, although the TNFRSF6 gene can be inactivated efficiently by DNA methylation, hypermethylation occurs neither frequently nor extensively in human carcinomas and appears to play a limited role in downregulation of Fas expression.

Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder.

Because polymorphisms in the methyl group metabolism genes methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MS), and cystathione beta-synthetase (CBS) affect plasma homocysteine levels and intracellular concentrations of S-adenosylmethionine (SAM), they modify the susceptibility to cardiovascular diseases and cancer. Specifically, genome-wide decreased DNA methylation ('hypomethylation') in human cancers might be a consequence of decreased SAM levels. Because hypomethylation is particularly prevalent in transitional cell carcinoma of the urinary bladder (TCC), the genotype distributions for the two each most prevalent MTHFR, MS, and CBS alleles were compared between 165 TCC patients and 150 population controls. The distributions of the MTHFR 677A/V and the MS 919G/D alleles were not significantly different between cancer patients and controls, even after stratification according to age, gender, tumor stage or grade. The CBS 844INS68 allele was slightly less frequent in TCC patients than in controls (q=0.07 versus 0.10), but was rarer among males in both groups. Among the TCC patients, this gender difference was highly significant (Mantel-Haenszel and chi(2)-test P=0.007). No significant difference between TCC patients and controls was found for any combined genotype. Likewise, the extent of DNA hypomethylation determined in 62 carcinoma specimens was not related to the respective genotypes. Thus, on their own, the MTHFR, MS and CBS genotypes do not appear to act upon susceptibility to TCC or influence the extent of DNA hypomethylation in this cancer.

DNA methylation and the mechanisms of CDKN2A inactivation in transitional cell carcinoma of the urinary bladder.

Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16INK4A cell cycle regulator and the p14ARF1 p53 activator proteins are frequently found in bladder cancer. Here, we present an analysis of 86 transitional cell carcinomas (TCC) to elucidate the mechanisms responsible for inactivation of this locus. Multiplex quantitative PCR analysis for five microsatellites around the locus showed that 34 tumors (39%) had loss of heterozygosity (LOH) generally encompassing the entire region. Of these, 17 tumors (20%) carried homozygous deletions of at least one CDKN2A exon and of flanking microsatellites, as detected by quantitative PCR. Analysis by restriction enzyme PCR and methylation-specific PCR showed that only three specimens, each with LOH across 9p21, had bona fide hypermethylation of the CDKN2A exon 1alpha CpG-island in the remaining allele. Like most other specimens, these three specimens displayed substantial genome-wide hypomethylation of DNA as reflected in the methylation status of LINE L1 sequences. The extent of DNA hypomethylation was significantly more pronounced in TCC with LOH and/or homozygous deletions at 9p21 than in those without (26% and 28%, respectively, on average, versus 11%, p < 0.0015). No association of LOH or homozygous deletions at 9p21 with tumor stage or grade was found. The data indicate that DNA hypermethylation may be rare in TCC and that deletions are the most important mechanism for inactivation of the CDKN2A locus. The predominance of allelic loss may be explained by its correlation with genome-wide DNA hypomethylation, which is thought to favor chromosomal instability and illegitimate recombination.

Novel mutations of the von hippel-lindau tumor-suppressor gene and rare DNA hypermethylation in renal-cell carcinoma cell lines of the clear-cell type.

Renal-cell carcinoma (RCC) is the most common neoplasm of the kidney, accounting for about 3% of all adult malignancies. Histopathologically, 80% of all cases can be classified as clear-cell RCC. Of these, approximately 55% to 70% are associated with mutations in the von Hippel-Lindau (VHL) tumor-suppressor gene. Here, new mutations of the VHL gene were defined by the use of temperature gradient gel electrophoresis and subsequent sequencing. In addition, DNA hypermethylation, an alternative mechanism of VHL gene silencing, was evaluated by methylation-specific PCR. Twenty-six clear-cell, 3 chromophilic, and 2 chromophobic RCC cell lines were analyzed. Among the clear-cell RCC cell lines tested, 12 (47%) contained 13 mutations overall: 8 (62%) in exon 1, 3 (23%) in exon 2, and 2 (15%) in exon 3. Ten of these mutations have thus far not been described. All single base pair changes were transversions. Six mutations led to alteration of a single amino acid. Seven mutations generated a frameshift or a stop codon. One cell line contained a complex duplication of 36 bp. All cell lines with mutations showed loss of heterozygosity in the VHL gene. No mutations could be detected in the chromophilic or chromophobic RCC samples. Significant hypermethylation was not observed in any of the cell lines. These data provide further evidence that distinct mutations in the VHL gene are a characteristic feature of clear-cell RCC. In contrast, hypermethylation of the gene is probably a rare event. The high frequency of transversion mutations suggests a role for exogenous carcinogens in the etiology of clear-cell RCCs.

DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas.

Since DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome, hypomethylation in human tumours may lead to their reactivation. The methylation status of LINE-1 retroposons was determined in 73 samples of urinary bladder cancers, 34 specimens of renal cell carcinoma and in the corresponding normal tissues by Southern blot analysis. LINE-1 sequences were strongly methylated in normal tissues and were significantly hypomethylated in 69 (95%) urothelial carcinomas, but in none of the renal carcinomas. Hypomethylation in bladder cancers was independent of stage and tended to increase with grade. The methylation status of HERV-K proviral DNA in normal and transformed urothelial cells paralleled that of LINE-1 sequences (r2 = 0.87). It was shown by ligation-mediated polymerase chain reaction that hypomethylation also extended to the LINE-1 promoter sequence located at the 5'-ends of full-length elements which is repressed by methylation in somatic tissues. Accordingly, full-length LINE-1 transcripts were detected by Northern blot analysis in two urothelial carcinoma cell lines. In contrast, transcripts from HERV-K proviruses were restricted to teratocarcinoma cell lines. Our data indicate that genome-wide DNA hypomethylation is an early change in urothelial carcinoma, but is absent from renal cell carcinoma. The coordinate changes of LINE-1 and HERV-K DNA methylation suggest that hypomethylation in urothelial cancer affects a variety of different retroelements to similar extents. We speculate that decreased methylation of LINE-1 retroelements, in particular, may contribute to genomic instability in specific human tumours such as urothelial carcinoma by rendering these normally repressed sequences competent for transcription and recombination.