DNA Methylation and the Mechanisms of CDKN2A Inactivation in Transitional Cell Carcinoma of the Urinary Bladder

Florl, Andrea R.; Franke, Knut; Niederacher, Dieter; Gerharz, C. D.; Seifert, Hans‐Helge; Schulz, Wolfgang A.

doi:10.1038/labinvest.3780161

Cited by 53 publications

(47 citation statements)

References 30 publications

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“…53 Although P16INK4A deletion has an undisputed role in transitional bladder carcinoma progression, P16INK4A DNA methylation is highly variable between individual bladder carcinoma patients and the contribution of epigenetic factors to bladder carcinoma risk is still controversial. 54,55 UTI-induced CDKN2A methylation could provide a mechanism for the observed correlation between recurrent UTI and bladder carcinoma risk. [56][57][58] After infection, bladder uroepithelial cells undergo apoptosis that results in shedding of infected cells and intracellular pathogens, thereby reducing the overall pathogen load.…”

Section: Discussionmentioning

confidence: 99%

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells

Tölg

Sabha

Cortese

et al. 2011

Laboratory Investigation

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Host cell and bacterial factors determine severity and duration of infections. To allow for bacteria pathogenicity and persistence, bacteria have developed mechanisms that modify expression of host genes involved in cell cycle progression, apoptosis, differentiation and the immune response. Recently, Helicobacter pylori infection of the stomach has been correlated with epigenetic changes in the host genome. To identify epigenetic changes during Escherichia coli induced urinary tract infection (UTI), we developed an in vitro model of persistent infection of human uroepithelial cells with uropathogenic E. coli (UPEC), resulting in intracellular bacteria colonies. Cells inoculated with FimH-negative E. coli (N-UPEC) that are not internalized and non-inoculated cells were used as controls. UPEC infection significantly induced de novo methyltransferase (DNMT) activity (12.5-fold P ¼ 0.002 UPEC vs non-inoculated and 250-fold P ¼ 0.001 UPEC vs N-UPEC inoculated cells) and Dnmt1 RNA expression (6-fold P ¼ 0.04 UPEC vs non-inoculated cells) compared with controls. DNMT1 protein levels were significantly increased in three uroepithelial cell lines (5637, J82, HT-1197) in response to UPEC infection as demonstrated by confocal analysis. Real-time PCR analysis of candidate genes previously associated with bacteria infection and/or innate immunity, revealed UPEC-induced downregulation of the tumor suppressor gene CDKN2A (3.3-fold P ¼ 0.007 UPEC vs non-inoculated and 3.3-fold P ¼ 0.001 UPEC vs N-UPEC) and the DNA repair gene MGMT (9-fold P ¼ 0.03 UPEC vs non-inoculated). Expression of CDH1, MLH1, DAPK1 and TLR4 was not affected. Pyrosequencing of CDKN2A and MGMT CpG islands revealed increased methylation in CDKN2A exon 1 (3.8-fold P ¼ 0.04 UPEC vs N-UPEC and UPEC vs non-inoculated). Methylation of MGMT was not affected. UPEC-induced methylation of CDKN2A exon 1 may increase bladder cancer and presage UTI risk, and be useful as a biological marker for UTI susceptibility or recurrence.

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Section: Discussionmentioning

confidence: 99%

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells

Tölg

Sabha

Cortese

et al. 2011

Laboratory Investigation

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“…22 Point mutations have only rarely been identified in bladder tumors 15,23,24 and promoter methylation, only infrequently. 25 Since the discovery of p14 ARF , 14 much interest has centered on the relative involvement of p16 and p14 ARF as tumor suppressor genes in both mouse models and in human cancer. The tumor suppressor function of p16 and p14 ARF (p19 in the mouse) has been confirmed by many studies including mouse knockout experiments, 26 -29 experiments on cells derived from such knockouts eg, 30 and gene transfer studies in cultured cells eg 31 .…”

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confidence: 99%

Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification

Aveyard¹,

Knowles²

2004

The Journal of Molecular Diagnostics

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Many tumors have large homozygous deletions of the CDKN2A locus (encoding p14 ARF and p16) and of CDKN2B (p15). Our aim was to determine which gene is the major target in bladder cancer. We used quantitative real-time PCR (RTQ-PCR) to determine copy number of p15, of p14 ARF exon 1␤, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex PCR. Titration experiments showed that homozygous deletion could be detected in the presence of up to 30% normal DNA. Results for cell lines were compatible with previous cytogenetic analyses. Ten cell lines and 32 tumors (38.5%) had homozygous deletion of at least one target. Thirteen tumors (15.7%) had deletion of all three targets. Two tumors had deletion of p14 ARF exon 1␤ alone and four of p16 exon 2 alone. RTQ-PCR detected more homozygous deletions than duplex PCR. Finally we used a multiplex ligation-dependent probe amplification kit to provide independent confirmation of results. We conclude that with appropriate controls RTQ-PCR is a sensitive and robust method to detect copy number changes in tumors even in the presence of contaminating normal cell DNA. More than 60% of transitional cell carcinomas (TCC) of the bladder of all stages and grades show loss of heterozygosity (LOH) of chromosome 9. 1-6 Although many tumors have LOH on both arms of the chromosome, a significant number (ϳ10% of tumors overall) have a region of interstitial LOH of 9p. 7 Many such deletions are small and this has allowed the identification of a critical region at 9p21 that contains CDKN2B and CDKN2A/ARF. These two loci encode three tumor suppressor genes, p15, p16, and p14 ARF , all of which are cyclin-dependent kinase inhibitors with key functions in cell cycle regulation. 8,9The CDKN2A locus encodes p16 and p14 ARF , both of which are capable of inducing cell cycle arrest. 10,11 p16 and p14 ARF have different first exons (1␣ and 1␤, respectively) approximately 13 kb apart, 12 giving rise to products in alternate reading frames with no homology at the protein level 13,14 (Figure 1). Exons 1␣, 2, and 3 encode p16, which induces G1 cell cycle arrest via the Rb pathway. Exons 1␤, 2, and 3 encode p14 ARF , which inhibits p53 degradation via binding to mdm2.The mechanism of CDKN2A/ARF inactivation in human cancers is somewhat tumor specific. Homozygous deletions at CDKN2A/9p21 are common in bladder tumors 6,15-17 and have been described in a variety of other sporadic tumors, including melanomas 18 and gliomas. 19 Pancreatic adenocarcinomas show inactivation of CDKN2A by either homozygous deletion or point mutation, 20 whereas esophageal tumors commonly show inactivation by point mutation. 21 A third mechanism of inactivation, transcriptional silencing by promoter hypermethylation, is commonly found in colorectal carcinoma. 22 Point mutations have only rarely been identified in bladder tumors 15,23,24 and promoter methylation, only infrequently. 25 Since the discovery of p14 ARF , 14 much interest has centered on the relative involvement of ...

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“…Investigators have used the methylation status of repeat elements as a biomarker for global methylation status including the Line-1 retrotransposon (LRE1). Various types of cancer, including urothelial bladder carcinoma (25), malignant testicular tumors (26), hepatocellular carcinoma (27), chronic lymphocytic leukemia (28), prostate carcinomas (29), and head and neck squamous cell carcinoma (25), show hypomethylation of LRE1. Head and neck cancers studied to date have been observed to have significantly greater degree of LRE1 hypomethylation compared with their normal tissue counterparts (30), and a lower level of LRE1 methylation in peripheral blood -derived DNA has been associated with risk for head and neck squamous cell carcinoma (31).…”

Section: Introductionmentioning

confidence: 99%

Line Region Hypomethylation Is Associated with Lifestyle and Differs by Human Papillomavirus Status in Head and Neck Squamous Cell Carcinomas

Furniss

Marsit

Houseman

et al. 2008

Cancer Epidemiology, Biomarkers &Amp; Prevention

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Genomic hypomethylation is a hallmark of essentially all cancers, but the degree of this hypomethylation differs among individual tumors. Little work has explored what leads to these differences and or asked whether they are clinically meaningful. In this study of head and neck squamous cell carcinoma, we assessed hypomethylation in tumors using a semiquantitative fragment analysis approach to determine the relative methylation status of the line retroviral element LRE1 (Line-1.2). Because this is an established marker of genomic methylation status, we examined the relationship between the relative methylation, patient demographics, and other risk factors for head and neck squamous cell carcinoma. We determined relative methylation status for 303 patients, 193 of which had complete data for all variables of interest. Using a generalized linear model, we found that patient body mass index was significantly positively associated with tumor LRE1 methylation level. Smoking duration, particularly in tumors lacking human papillomavirus (HPV) DNA, was significantly negatively associated with relative methylation level. Having previously assessed relative methylation in blood-derived DNA, we compared tumor with the blood DNA methylation level and observed these to be independent. Finally, the lower LRE1 methylation in patients whose tumors were HPV DNA negative was associated with poorer patient survival (hazard ratio, 1.6; 95% confidence interval, 1.0-2.6). These findings suggest that HPVassociated tumors differ molecularly from those arising after heavy tobacco use and that this epigenetic alteration may affect survival in HPV-negative patients already exhibiting a more aggressive disease. (Cancer Epidemiol Biomarkers Prev 2008;17(4):966 -71)

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DNA Methylation and the Mechanisms of CDKN2A Inactivation in Transitional Cell Carcinoma of the Urinary Bladder

Cited by 53 publications

References 30 publications

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells

Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification

Line Region Hypomethylation Is Associated with Lifestyle and Differs by Human Papillomavirus Status in Head and Neck Squamous Cell Carcinomas

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