Hypomethylation of the c-myc promoter region induced by phenobarbital in rat liver

• Autorzy: Urbanek-Olejnik K. , Liszewska M. , Winczura A. , Hernik A. , Strucinski P. , Ludwicki J.K.
• Języki publikacji: EN

Abstrakty

EN

Background. The changes in DNA methylation are considered as one of the early events in hepatocarcinogenesis. Objective. We evaluated the ability of phenobarbital (PB) – the most widely used anticonvulsant worldwide and classical rodent liver carcinogen – to cause the promoter region of the c-myc protooncogene hypomethylation as well as changes of mRNA level of this gene. Moreover, the expression of Dnmt1 protein in rat treated with this compound was analyzed. Material and Methods. Male Wistar rats received PB in daily oral doses of 92.8 mg kg-1 b.w. day-1 (at 24-h intervals; for one, three and fourteen days). Methylation of the c-myc promoter region was measured by PCR-based methylationsensitive restriction enzyme analysis (MSRA). Levels of mRNA for c-myc and protein Dnmt1 were assayed using Real- Time PCR and Western Blot, respectively. Results. The study showed that phenobarbital stimulated persistent changes in DNA methylation, i.e. loss of methylation in the promoter region of the c-myc gene and up-regulated its mRNA level. In addition, a significant increase in protein level of Dnmt1 in the c-myc over-expressing liver cells was observed. Conclusion. The oppose relationship between Dnmt1 activity and methylation status of c-myc gene was demonstrated. The c-myc over-expression by demethylation might represent an important, early events in the mechanism of action (MOA) of phenobarbital.

PL

Wprowadzenie. Zmiany metylacji DNA są rozważane jako jeden z wczesnych mechanizmów hepatokancerogenezy. Cel pracy. Celem badań była ocena wpływu fenobarbiatlu (PB) - leku przeciwpadaczkowego, modelowego promotora raka wątroby - na poziom metylacji regionu promotorowego i ekspresji na poziomie mRNA protoonkogenu c-myc. Ponadto dokonano analizy poziomu ekspresji białka Dnmt1. Materiał i metody. Samce szczurów szczepu Wistar otrzymywały PB w dawce 92,8 mg/kg m.c. x dzień-1 jednorazowo, 3-krotnie i 14-krotnie. Ocenę poziomu zmian metylacji genu c-myc dokonano metodą MSRA (ang. Methylation-Sensitive Restriction Enzyme Analysis). Analizę względnego poziomu transkryptów genu c-myc i białka Dnmt1 przeprowadzano odpowiednio metodą PCR w czasie rzeczywistymi i techniką Western Blot. Wyniki. W wyniku oddziaływania fenobarbitalu wykazano trwałe zmiany metylacji DNA - obniżenie metylacji w rejonie promotorowym genu c-myc i nadekspresję badanego genu na poziomie mRNA. Jednocześnie obserwowano statystycznie istotny wzrost poziomu białka Dnmt1. Wnioski. Wykazano odwrotną zależność między aktywnością Dnmt1 a poziomem metylacji genu c-myc. Nadekspresja c-myc w wyniku demetylacji może stanowić istotne, wczesne zdarzenie w mechanizmie działania (ang. Mechanism of Action (MOA)) fenobarbitalu.

• Wydawca: -
• Czasopismo: Roczniki Państwowego Zakładu Higieny
• Rocznik: 2018
• Tom: 69
• Numer: 3
• Opis fizyczny: p.307-314,fig.,ref.

Twórcy

autor

Urbanek-Olejnik K.

• Department of Toxicology and Health Risk Assessment, National Institute of Public Health – National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland

autor

Liszewska M.

• Department of Toxicology and Health Risk Assessment, National Institute of Public Health – National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland

autor

Winczura A.

• Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

autor

Hernik A.

• Department of Toxicology and Health Risk Assessment, National Institute of Public Health – National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland

autor

Strucinski P.

• Department of Toxicology and Health Risk Assessment, National Institute of Public Health – National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland

autor

Ludwicki J.K.

• Department of Toxicology and Health Risk Assessment, National Institute of Public Health – National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland

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