Aggregate formation of hepatitis B virus X protein affects cell cycle and apoptosis.
OBJECTIVE
To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.


METHODS
Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography. Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 cells. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry.


RESULTS
Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time. EGFP fluorescence in HBx expression cells was significantly decreased.


CONCLUSIONS
Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.
Song
Chang-Zheng
CZ
Laboratory of Immunobiology, College of Life Sciences, Shandong University, Jinan 250100, Shandong Province, China. songcz@life.sdu.edu.cn
Bai
Zeng-Liang
ZL
Song
Chang-Cheng
CC
Wang
Qing-Wei
QW
D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000502:physiology*; D006528:Carcinoma, Hepatocellular; D002453:Cell Cycle / Q000502:physiology; D045744:Cell Line, Tumor / Q000166:cytology / Q000378:metabolism / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism / Q000821:virology; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000821:virology; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000378:metabolism; D008113:Liver Neoplasms; D008164:Luminescent Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D049452:Green Fluorescent Proteins