Dataset Information

Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer.

ABSTRACT:

Background

Large-scale screening methods are widely used to reveal cancer-specific DNA methylation markers. We previously identified non-satellite 3.3-kb repeats associated with facioscapulohumeral muscular dystrophy (FSHD) as hypermethylated in cervical cancer in genome-wide screening. To determine whether hypermethylation of 3.3-kb repeats is a tumor-specific event and to evaluate frequency of this event in tumors, we investigated the 3.3-kb repeat methylation status in human papilloma virus (HPV)-positive cervical tumors, cancer cell lines, and normal cervical tissues. Open reading frames encoding DUX family proteins are contained within some 3.3-kb repeat units. The DUX mRNA expression profile was also studied in these tissues.

Methods

The methylation status of 3.3-kb repeats was evaluated by Southern blot hybridization and bisulfite genomic sequencing. The expression of DUX mRNA was analyzed by RT-PCR and specificity of PCR products was confirmed by sequencing analysis.

Results

Hypermethylation of 3.3-kb repeats relative to normal tissues was revealed for the first time in more than 50% (18/34) of cervical tumors and in 4 HPV-positive cervical cancer cell lines. Hypermethylation of 3.3-kb repeats was observed in tumors concurrently with or independently of hypomethylation of classical satellite 2 sequences (Sat2) that were hypomethylated in 75% (15/20) of cervical tumors. We have revealed the presence of transcripts highly homologous to DUX4 and DUX10 genes in normal tissues and down-regulation of transcripts in 68% of tumors with and without 3.3-kb repeats hypermethylation.

Conclusion

Our results demonstrate that hypermethylation rather than hypomethylation of 3.3-kb repeats is the predominant event in HPV-associated cervical cancer and provide new insight into the epigenetic changes of repetitive DNA elements in carcinogenesis.

SUBMITTER: Katargin AN

PROVIDER: S-EPMC2695481 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Publications

Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer.

Katargin Alexey N AN Pavlova Larissa S LS Kisseljov Fjodor L FL Kisseljova Natalia P NP

BMC medical genomics 20090527

<h4>Background</h4>Large-scale screening methods are widely used to reveal cancer-specific DNA methylation markers. We previously identified non-satellite 3.3-kb repeats associated with facioscapulohumeral muscular dystrophy (FSHD) as hypermethylated in cervical cancer in genome-wide screening. To determine whether hypermethylation of 3.3-kb repeats is a tumor-specific event and to evaluate frequency of this event in tumors, we investigated the 3.3-kb repeat methylation status in human papilloma ...[more]

PMID: 19473516

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Project description:BackgroundHigh risk type human papilloma viruses (HR-HPV) induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25-57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomasMethodsMethylation status of p16 INK4a was analyzed by MSP and by bisulfite-modified DNA sequencing. The expression of p16ink4a was analyzed by RT-PCR and by immunohistochemical technique.ResultsThe extensive methylation within p16 INK4a 5' CpG island was not detected either in 13 primary cervical carcinomas or in 5 cancer cell lines by bisulfite-modified DNA sequencing (including those that were positive by MSP in our hands). The number and distribution of rare partially methylated CpG sites did not differ considerably in tumors and adjacent normal tissues. The levels of the p16 INK4a mRNA were increased in carcinomas compared to the normal tissues independently of the number of partially methylated CpGs within 5'CpG island. The transcriptional activation of p16 INK4a was accompanied by p16ink4a cytoplasmic immunoreactivity in the majority of tumor cells and presence of a varied number of the p16 positive nuclei in different tumors.ConclusionHypermethylaion of the p16INK4a 5' CpG island is not a frequent event in HR-HPV-positive cervical carcinomas and cannot be an effective marker of cancer cells with up-regulated expression of p16ink4a. Our data confirm other previous studies claiming specific p16INK4a up-regulation in the majority of cervical carcinomas at both the protein and mRNA levels. Cytoplasmic accumulation of p16ink4a is a feature of cervical carcinomas.

Project description:ObjectiveThis study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 (SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis.MethodsThe methylation status of SALL3 was detected by methylation-specific PCR, and SALL3 gene expression was assessed by real-time quantitative PCR in the cervical cancer cell lines, SiHa, HeLa and C33A, as well as in cervical cancer tissue samples (n = 23), matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells.ResultsThe SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 ?M and 10 ?M of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 (p<0.05, r = 0.408), and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV-negative tissues (p<0.05).ConclusionThe aberrant hypermethylation of SALL3 together with HPV involvement inactivated its function as a tumor suppressor and contributed to carcinogenesis in cervical cancer.

Dataset Information

Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer.

Background

Methods

Results

Conclusion

Publications

Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer.

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