AIM:To investigate whether the formation of aggregatedHBx has a potential linking with its cellular responses.METHODS:Recombinant HBx was expressed in Escherichiacoliand purified by Ni-NTA metal-affinity chromatography.Anti-HBx monoclonal antibody was developed forimmunocytochemical detection.Bicistronic expression vectorharboring full-length DNA of HBx was employed fortransfection of human HepG2 cells.Immunocytochemicalstaining was used to examine the intracellular HBxaggregates in cells.The effects of HBx aggregation on cellcycle and apoptosis were assessed by flow cytometry.RESULTS:Immunocytochemical staining revealed mostof the HBx was formed intracellular aggregate in cytoplasmand frequently accumulated in large granules.Flowcytometry analysis showed that HepG2 cells transfectedwith vector harboring HBx significantly increased apoptosisand largely accumulated in the G0-G1 phase bymaintenance in serum medium for 36 hours.Control cellswithout HBx aggregates in the presence of serum enteredS phase and proliferated more rapidly at the same time.EGFP fluorescence in HBx expression cells was significantlydecreased.CONCLUSION:Our observations show that cells with HBxaggregate undergo growth arrest and apoptosis,whereascontrol cells without HBx remain in growth and progressioninto S phase.Our data may provide helpful information tounderstand the biological effects of HBx aggregates on cells. A: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses. METHODS: Recombinant HBx was expressed in Escherichiacoliand purified by Ni-NTA metal-affinity chromatography. Anti-HBx monoclonal antibody was developed for immunocytochemistry detection. Biistronic expression vectorharboring full -length DNA of HBx was employed fortransfection of human HepG2 cells. Immunocytochemical stain was used to examine the intracellular HBxaggregates in cells. The effects of HBx aggregation on cellcycle and apoptosis were assessed by flow cytometry. RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular Aggregate in cytoplasmand frequently accumulated in large granules. Flowcytometry analysis showed that HepG2 cells transfected with vector harboring HBx increased increased apoptosis and the largest accumulation in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells with out HBx aggregates in the presence of serum enteredSphase and proliferated more rapidly at the same time. EGFP fluorescence in HBx expression cells was significantly decreased. CONCLUSION: Our observations show that cells with HBxaggregate undergo growth arrest and apoptosis, butcontrol cells without HBx remain in growth and progressioninto S phase. Our data may provide helpful information tounderstand the biological effects of HBx aggregates on cells.