Репозиторий Университета

Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.

© 2018 Ruslania. All rights reserved. Liver cirrhosis and hepatocellular carcinoma are the most common outcomes of chronic hepatitis B. Hepatitis B virus (HBV) induces transformation and cell death in chronic hepatitis B (CHB). DNA double strand breaks (DSBs) represent the most dangerous type of genome damage. It was shown previously that generation of phosphorylated histone H2AX foci is a reliable marker of DSBs. The aim of this study was to analyse generation of yH2AX foci in HBV and hepatitis D virus (HDV) infection in vitro and in liver biopsies of patients with CHB and CHB with delta-agent (CHD). Human hepatoma cell line HepG2-1.1merHBV with activated HBV life cycle was used to perform real-time PCR for analysis of pregenomic RNA, HBV DNA, HBV cccDNA and for immunocytochemical analysis of yH2AX. Liver biopsies from CHB and CHD patients were analyzed to confirm the results. HBV induces multiple discrete yH2AX foci in HepG2-1.1merHBV cells in vitro and in biopsies of CHB and CHB+D patients. The ratio of hepatocytes w/o yH2AX foci is significantly lower (49,9+7-12,3% vs. 85,5+/-0,9%, p<0,05), while the proportion of cells with 1-10 yH2AX foci is higher (49,3+7-12,6% vs. 14,5+/-0,9%, p<0,05) compared to healthy control. There is a significant increase In the mean number of yH2AX foci in biopsies from CHB+D patients (3,5+7-1,1 and 5,5+/-1,5 vs. 0,5+/-0,16 in control hepatocytes, p<0.05). The ratio of hepatocytes w/o yH2AX foci is significantly lower in CHB and CHB+D patients, while percentage of cells with 1-10 yH2AX foci is higher. Rare hepatocytes with multiple (11-30 yH2AX foci per cell) foci appear in CHB and CHB+D patients. In conclusion, yH2AX foci are generated in hepatocytes of CHB and CHB+D patients and can be utilized to assess genome damage, associated with HBV and HDV viral infection.