Separate origins of hepatitis B virus surface antigen-negative foci and hepatocellular carcinomas in transgenic HBsAg (alb/psx) mice

Abstract

We have examined the development and transgene expression in liver lesions of transgenic mice bearing the hepatitis B surface antigen (HBsAg) gene of hepatitis B virus under the control of the albumin promoter (alb/psx) to study liver regeneration and hepatocellular carcinoma (HCC) associated with hepatitis B virus infection. Storage of the HBsAg in the endoplasmic reticulum precedes loss of liver cells and regenerative hyperplastic nodules that do not express HBsAg. Histological analysis indicated that HBsAg-negative foci and nodules arose from liver progenitor cells in the portal zone and lacked mRNA expression. Genomic DNA from eight of nine HBsAg-negative laser capture-excised liver foci showed loss of part of the alb/psx gene, whereas no loss of the actin gene was observed. The alb/psx DNA was intact in adjacent HBsAg-positive tissue. Sequencing of polymerase chain reaction products suggested that alterations in the HBsAg transgene in HBsAg-negative foci occurred via large-scale deletions as opposed to single-site mutations. Southern blot analysis of HCC from 2-year-old transgenic HBsAg mice, however, revealed an intact alb/psx gene. Thus, HBsAg-negative progenitor cells with deletions in the transgene appear to be responsible for compensatory regeneration of the liver, whereas HCCs arise from clonal expansion of hepatocytes with intact alb/psx transgenes.

Figure 1

A–F: HBsAg staining. A, normal liver; B–F, transgenic male mice (brown color). G–I, Pan-CK staining (black color). A, normal mouse liver, 15 months; original magnification, ×400; B–F, transgenic HBsAg mice. B, 5 months; original magnification, ×400; C, 7 months; D; original magnification, ×200; E; original magnification, ×400; F, 15 months; original magnification, ×20. B: There is uniform accumulation of HBsAg and dysplasia in each hepatocyte at 5 months. C–E: By 7 months, foci of small HBsAg-negative hepatocytes next to ductules (arrow) appear. An HBsAg-negative microcarcinoma is shown in F next to an HBsAg nodule at 15 months. G, Normal male mouse, 9 months; original magnification, ×200; H, transgenic male, 2.5 months; original magnification, ×200; I, transgenic male, 9 months. Arrows in I point to Pan-CK-positive small ductules extending into hepatic plates. p, portal vein.

Figure 2

Sequential sections of HBsAg-negative foci. Top panel: HBsAg staining, 8-month-old transgenic male mouse (original magnification, ×200). Middle panel: Double staining for HBsAg (red) and Pan-CK (blue), 8-month-old transgenic male mouse. In the top panel, an HBsAg-negative focus can be followed from the liver lobule to the portal zone and then back to the lobule. In the middle panel are representative sections from 20 serial sections. The arrows point to small bile ductules associated with an HBsAg-negative focus. p, portal vein; n, HBsAg-negative focus. Bottom panels: mRNA expression in the liver foci of transgenic mice. Liver sections from 7-month-old HBV transgenic mice analyzed for HBsAg mRNA using in situ hybridization. A, mRNA (Green); B, nuclei (red, propidium iodide); C, double stained. Lack of HBsAg mRNA is observed in foci compared with surrounding (nonfocal) tissue.

Figure 3

PCR analysis. A: PCR primers used for amplification of the HBV transgene. Location of three PCR primer combinations with internal nested primer sites in the alb/psx transgene. For clarity, two panels are shown. Top panel: Overlapping PCR primers only. Bottom panel: Same as top panel but with internal nested PCR primer sites indicated. B: Specificity of PCR primers. Genomic DNA from positive control transgenic liver (+) and six nontransgenic mouse livers (S1 to S6) was extracted and PCR amplified using the primers described in the top panel of A (listed to the left of the figure), followed by re-amplification with the nested primers shown in the bottom panel of A. Lane 1, size markers. Lane 2, positive control (+). Lane 3, PCR performed in the absence of DNA template negative control (−). Lanes S1 to S6, nontransgenic liver samples. C: HBsAg-negative foci. Genomic DNA from positive control transgenic liver (+) and nine immunohistochemically HBsAg-negative individual nodules (lanes F1 to F9, F1 to F3, F4 to F6, and F7 to F9 are from three different mice) was extracted and PCR amplified using the primers described in the top panels of A and then with the nested primers shown in the bottom panel of A (listed on the left). Lane 1, size markers. Lane 2, positive control (+). Lane 3, negative control (−). Lanes F1 to F9, HBsAg-negative foci samples. D: HBsAg-positive tissue surrounding the HBsAg-negative foci. Genomic DNA from positive control transgenic liver (+) and six samples of immunohistochemically HBsAg-positive cells surrounding the foci (lanes P1 to P6) was extracted and PCR amplified using the primers described above (listed on the left). Lane 1, size markers. Lane 2, positive control (+). Lane 3, negative control (−). Lanes P1 to P6, HBsAg-positive tissue samples. P1 and P2 are from the same mouse as F1 to F3 in C; P3 and P4 are from the same mouse as F4 to F6; and P5 and P6 are from the same mouse as F7 to F9.

Figure 4

Southern blot analysis of 2-year-old HBV transgenic mice liver tumors. T1 to T7, samples from seven different liver tumors from seven different mice; L1 to L3, liver tissue surrounding the tumors: +, HBV transgenic mouse kidney positive control; and −, nontransgenic liver negative control. Approximately 20 μg of extracted genomic DNA was digested with EcoRI, electrophoresed, transferred to Gene Screen, and probed with labeled PCR product (primer set II) to the HBV gene.