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Yukikiyo Kawakami
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P2.03 - Biology (ID 162)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.03-22 - Chromate Exposure Induces DNA Hypermethylation of the Mismatch Repair Gene MLH1 in Lung Cancer (Now Available) (ID 2156)
10:15 - 18:15 | Author(s): Yukikiyo Kawakami
- Abstract
Background
Hexavalent chromium is recognized as a human carcinogen. To elucidate the role of chromate on carcinogenesis, we have investigated molecular features of LC from chromate workers (chromate LC). Chromate LC frequently had the microsatellite instability (MSI), and that the MSI was associated with repression of MLH1, which is one of the essential DNA mismatch repair (MMR) proteins. In the present study, we investigated methylation status of the promoter region of MLH1 determined quantitatively by bisulfite-pyrosequencing in the paired tumorous/ non-tumorous sample sets of chromate and non-chromate LCs. Moreover, we analyzed three DNA double-strand break (DSB) repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment length polymorphism analysis.
Method
Thirty-two lung tumor samples were obtained from chromate workers with LC during surgery or at autopsy at 5 hospitals between August 1975 and October 1997. Thirty-one tumors were obtained from LC patients without chromate exposure during surgery at Tokushima University Hospital as a control group (non-chromate LC).
DNA was extracted and bisulfite conversion of DNA was conducted using the EpiTect Bisulfite Kit (QIAGEN). PCR primers and sequencing primer for quantification of methylation level in region -209 nucleotides (nt) to -181 nt from the transcription start site in the MLH1. Pyrosequencing of 5 CpG sites in MLH1 was performed with sequencing primers using a PyroMark 24 Pyrosequencing System, version 2.0.6 (QIAGEN). Regions encompassing mononucleotide repeated sequences of genes were amplified using nested-PCR procedure. Fragments were separated by automated capillary electrophoresis in an ABI Prism 3130/3130xl Genetic Analyzer (Applied Biosystems) and electropherograms were analyzed using the GeneMapper software (Applied Biosystems).
Result
The mean methylation level of tumorous tissue was 21.1±15.7% and was significantly higher than that of non-tumorous tissue, 10.9±9.4% (P = 0.004) in chromate LC. In non-chromate LC, there was no significant difference between tumorous and non-tumorous tissues: 3.9±5.1% in tumorous tissue versus 4.9±4.1% in non-tumorous tissue. The mean methylation level of tumorous tissues was significantly higher in chromate LC than in non-chromate LC (P < 0.001). The mean methylation level of non-tumorous tissues tended to be higher in the chromate LC than in non-chromate LC (P = 0.062). There was a significant positive correlation between the methylation level and chromate exposure period in tumorous tissue of chromate LC (r = 0.481, P = 0.017). The methylation level was significantly higher in LCs with reduced expression of MLH1 than in LCs with normal expression of MLH1(P = 0.019). The incidence of mutations at mononucleotide repeats was observed in 50.0% of chromate LC and 28.6% of non-chromate LC in MRE11, and 17.4% of chromate LC and 0.0% of non-chromate LC in RAD50, and in 53.8% of chromate LC and 46.2% of non-chromate LC in DNA-PKcs. The incidence of mutation of mononucleotide tended to be higher in chromate LC than in non-chromate LC in MRE11. In RAD50, the mutation was significantly higher frequency in chromate LC than in non-chromate LC.
Conclusion
These results suggest that chromate exposure might induce MLH1 hypermethylation in LC, which is possible cause of carcinogenesis.
