A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice

Abstract

Development of new therapies for human hepatitis B virus infection (HBVHepatitis B virus (HBV) is a major cause of morbidity, that affects as many as 350 million people worldwide, resulting in an estimated 2 million deaths per year. 1,2 Currently available therapies for HBV-related hepatitis have limited success. 3 Therefore, new antiviral therapies that inhibit HBV replication or gene expression are being developed. Severe adverse reactions to nucleoside analogues in recent clinical trials 4,5 underscore the need for a small animal model to screen the safety and efficacy of new therapeutic modalities. Because of the narrow host range of HBV, existing animal models are limited to chimpanzees 6 and the recently reported tupaia, 7-10 which are expensive and in short supply. Related hepadnaviridae, duck HBV, 11 and woodchuck HBV, 12 infect their respective natural hosts; however, these viruses are of limited relevance to human HBV infection because of significant structural divergence among the viruses. 13,14 For these reasons, it would be advantageous to have a small animal model for human HBV.In efforts to produce murine models of HBV viremia, other investigators have transferred HBV to mice as a transgene, or by ectopic transplantation of human tumor cell lines producing HBV. HBV transgenic mice produce virus from HBV DNA, which is integrated into mouse chromosomes of all mouse cells. 15 Viral replication and production of all viral antigens have been shown in this model. Disadvantages of transgenic mice include the production of HBV in mouse hepatocytes but not in human hepatocytes, the absense of covalently closed circular DNA (CCC) viral transcription templates, and vertical rather than horizontal transmission of the virus. The other available murine model is the immunodeficient mouse with ectopically transplanted human tumor cell lines containing or producing HBV. 16,17 BALB/c nude mice injected with PLC/PRF/5 tumor cells subcutaneously were shown 2 decades ago to produce tumors, secrete HBsAg, and contain HBV DNA, however their life span and usefulness are restricted by the tumor growth. HepG2.2.15 tumor cell lines subcutaneously injected in SCID mice 17 replicate HBV and secrete viral markers. This model is limited by the ectopic location of the human tumor cells and by their transformed phenotype, both of which prevent normal interactions of engrafted cells with host hepatocytes. Furthermore, human tumor growth in the host mice limits their survival to only a few weeks. 17,18 We developed an immortalized, but nontumorigenic human hepatocyte clone by stable transfection of the HBV genome, which expresses HBV antigens and replicates virus.

PIN1 overexpression and β-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma

“…To show that PIN1 overexpression may lead directly to increases in b-catenin and cyclin D1 in HCC, a PIN1 cDNA expression construct was transfected into a human liver cell line, MIHA (Brown et al, 2000). Western blot analysis confirmed a significant increase in b-catenin and cyclin D1 when PIN1 was overexpressed in the cell line, by both transient and stable transfection (Figure 3a).…”

“…Empty plasmid pGEM was used as a transfection control. A nontumorigenic, immortalized liver cell line, MIHA (a gift kindly provided by Dr J Roy-Chowdhury, Albert Einstein College of Medicine), was used for transfection experiments (Brown et al, 2000). Cells were transfected with 5.25 mg of pPIN1 or pGEM with FuGENE 6 according to the manufacturer's protocol (Boehringer, Mannhein, GmbH, Germany).…”

Infection of Human Hepatocyte Chimeric Mouse With Genetically Engineered Hepatitis B Virus *

“…15 Viremia of up to 3 ϫ 10 8 copy/mL was still observed in these mice at least 5 months after transplantation. This long-term viremia model should be useful for in vivo HBV studies.…”

Mortalin–p53 interaction in cancer cells is stress dependent and constitutes a selective target for cancer therapy

“…Human HCC cell lines MHCC97H, MHCC97L, H2P, H2M, PLC/PRF/5, HepG2, Hep3B and an immortalized human hepatocyte liver cell line MIHA 38 were cultured in Dulbecco's modified Eagle's medium, a high-glucose medium (Invitrogen, Carlsbad, CA, USA), containing 10% heat-inactivated fetal bovine serum (Invitrogen) in a 5% CO 2 humidified atmosphere at 371C. 23 The p53 shRNA plasmids were obtained from Addgene (Addgene Cambridge, MA 021399-1666, USA) plasmid depository.…”