HepG2 Clones Research Articles

Regulation of cellular invasion and matrix metalloproteinase activity in HepG2 cell by connexin 26 transfection.

We previously reported that connexin (Cx) 26 expression is involved in negative growth control of HepG2 cells established from a human hepatoma. We also found that induction of E-cadherin and subsequent formation of a cell adhesion complex were induced in HepG2 cells by Cx 26 expression. To examine the exact role of Cx 26-induced E-cadherin junctions in regulating appearance of malignant phenotypes of HepG2 cells, we expressed a Cx 26 antisense oligodeoxynucleotide (AS-ODN) in an established HepG2 cell clone that has stable expression of Cx 26 genes. We investigated changes in the expression of E-cadherin, the localization of beta-catenin, and some malignant phenotypes of HepG2 clone after the suppression of Cx 26 expression by AS-ODN treatment. The AS-ODN treatment prevented the expression of Cx 26 and E-cadherin, and the localization of beta-catenin was changed from cytoplasmic membrane to the cytoplasm. In parallel, a morphological change from a monolayer of polygonal cells to multilayered colonies was induced by the treatment, indicating a change of a malignant phenotype of HepG2 cells. The activity of matrix metalloproteinase 9 (MMP-9) was elevated by the AS-ODN treatment. A concomitant increase in invasiveness of the Cx 26-expressing cells by the treatment was also observed in an in vitro assay with Matrigel matrix. These results suggest that the induction of E-cadherin and formation of the cell adhesion complex by Cx 26 expression contribute to the reversal of some malignant phenotypes of HepG2 cells. Furthermore, the Cx 26-dependent expression of E-cadherin leads to reduction of the invasiveness of the cells through suppression of MMP-9 activity.

Transactivation of the human MDR1 gene by hepatitis B virus X gene product

Background/Aims: Persistent hepatitis B virus (HBV) infection may cause hepatocellular carcinoma. Patients with hepatocellular carcinoma are characterized by nonresponsiveness to chemotherapeutic agents. While many studies have been devoted to understanding the hepatocarcinogenesis mechanism of HBV, the possible relationship between HBV and the drug sensitivity phenotype of cancer cells has rarely been addressed. The hepatitis B virus X gene encodes a transcription transactivator which has been suggested to be a potential factor in viral hepatocarcinogenesis. The role of HBV pX in mediating the drug resistance phenotype of hepatoma cell lines was examined in this study. Methods: Standard transfection and chloramphenicol acetyltransferase assay was utilized to examine the effect of HBV pX transactivator on a reporter gene under the control of the human multidrug resistance (MDR) 1 upstream regulatory elements. Selected Hep G2 clones with or without HBV pX expression were tested for their sensitivity towards various anti-cancer agents by utilization of MTT assay. Results: The expression of HBV pX in both Hep G2 (p53+) and Hep 3B (p53−) cells resulted in transactivation of the reporter gene under control of the human MDR1 upstream regulatory elements. Northern blot analysis indicated that expression of the endogenous MDR1 gene was also elevated in Hep G2 clones with HBV pX expression. Decreased drug sensitivity towards adriamycin, vinblastine, and VP-16 was observed in Hep G2 clones with HBV pX expression. Conclusions: HBV pX can transactivate the MDR1 gene. Drug sensitivity was altered in Hep G2 cells with HBV pX expression.

Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.

The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line.