Genetic background of hepatocyte cell lines: Are in vitro hepatitis C virus research data reliable?

Authors

  • Paul Bensadoun M.D.,

    1. Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France
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  • Christophe Rodriguez M.D.,

    1. Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France
    2. National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital University of Paris-Est, Créteil, France
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  • Alexandre Soulier M.D.,

    1. Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France
    2. National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital University of Paris-Est, Créteil, France
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  • Martin Higgs M.D.,

    1. Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France
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  • Stéphane Chevaliez Pharm.D., Ph.D.,

    1. Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France
    2. National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital University of Paris-Est, Créteil, France
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  • Jean-Michel Pawlotsky M.D., Ph.D.

    1. Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France
    2. National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital University of Paris-Est, Créteil, France
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  • Potential conflict of interest: Nothing to report.

Genetic Background of Hepatocyte Cell Lines: Are In Vitro Hepatitis C Virus Research Data Reliable?

To the Editor:

The results of independent genome-wide association studies have recently been reported, describing the identification of single-nucleotide polymorphisms (SNPs) strongly associated with natural hepatitis C virus (HCV) clearance during acute infection and cure of chronic HCV infection on antiviral therapy with pegylated interferon (IFN)-α and ribavirin.1-4 These SNPs, in chromosomal region 19q13 upstream of the interleukin-28B (IL28B; IFN-λ3) gene, appear to be markers of cell responsiveness to type 1 IFNs. Nevertheless, the underlying mechanisms remain unknown. Several articles have been subsequently published that confirmed the association and provided valuable information on the predictive value of the so-called “IL28B genotype” on the outcomes of acute HCV infection, antiviral treatment of chronic infection, and recurrence of HCV infection after liver transplantation. These findings have been recently reviewed by Afdhal et al.5

To date, most of the current knowledge on HCV biology and new HCV drug development is based on in vitro studies using hepatoma cell lines, principally Huh7 cells and their derivatives. These cells are permissive for HCV entry, and harbor replication of subgenomic and genomic replicons of various genotypes, as well as the full life cycle of the genotype 2a JFH1 (Japanese fulminant hepatitis 1) infectious clone and derivatives. Nevertheless, their IL28B genotype remains uncharacterized, despite its likely effects on numerous intracellular biological processes relevant to HCV infection. We therefore genotyped the IL28B rs12979860 SNP of hepatoma cell lines used in HCV research, including Huh7, Huh7.5, Huh7.5.1, and HepG2 cells, as well as the commonly used non-hepatoma HEK293 (human embryonic kidney 293) and Hela cell lines. We used ultra-deep pyrosequencing to sequence 292 nucleotides flanking the rs12979860 locus, using a GS FLX Titanium Sequencing Kit in conjunction with a Genome Sequencer FLX (Roche Molecular Systems, Pleasanton, CA). Data was analyzed using two original in-house softwares: Pyroclass and Pyromute, with the results shown in Table 1.

Table 1. Subject Characteristics
CharacteristicNormal (n = 85)NPC1L1 +/− (n = 85)P Value
  1. Values are median with interquartile range. Data analyzed by Wilcoxon's two-sample test and chi-square test. ALT, alanine aminotransferase; AST, aspartate aminotransferase; HOMA, homeostasis model assessment of insulin resistance.

Age (years)46 (38-56)47 (39-56)0.885
Body mass index (kg/m2)30 (26-36)30 (26-36)0.957
Total cholesterol (mg/dL)182 (152-207)176 (154-204)0.461
Triglycerides (mg/dL)92 (68-136)111 (74-135)0.134
Glucose (mg/dL)93 (82-104)93 (84-101)0.974
HOMA3.0 (1.8-4.6)3.6 (1.8-5.4)0.614
AST (IU/L)21 (17-27)23 (17-30)0.525
ALT (IU/L)20 (15-29)19 (15-31)0.825
Ethanol intake (g/day)0.3 (0.0-5.6)0.4 (0.0-8.4)0.356
Campesterol:lathosterol ratio2.1 (1.4-3.4)1.3 (0.9-2.1)0.005
Statin use (%)2160.038
Hepatic triglyceride content (%)3.6 (2.2-8.0)3.6 (2.2-5.4)0.412

Huh7 cells and derivatives, and even two Huh7 cell lines originating from different laboratories, demonstrated different allelic frequencies. The “non-Mendelian” distribution of these polymorphisms is likely a consequence of the polyploidal nature of hepatoma cells, but may indicate the presence of multiple clonal populations arising under different environmental pressures.

Data obtained from in vitro cell culture systems may often be related to the genetic background of the cell line(s) used. Our results emphasize the need for the genetic characterization of hepatoma cell lines used for HCV research, especially when these studies involve innate immunity, IFN responsiveness, the assessment of antiviral compound efficacy, or experiments aimed at cell cure. This conclusion may be extended to a number of other in vitro experiments in which the genetic background of the cell line has an influence on the properties studied.

Paul Bensadoun M.D.*, Christophe Rodriguez M.D.* †, Alexandre Soulier M.D.* †, Martin Higgs M.D.*, Stéphane Chevaliez Pharm.D., Ph.D.* †, Jean-Michel Pawlotsky M.D., Ph.D.* †, * Institut National de la Santé et de la Recherche Médicale, Unité U955, Créteil, France, † National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital, University of Paris-Est, Créteil, France.

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