PMC

Proliferative and oncogenic potential of HBx in a selectively repopulating liver. (A) RT-PCR analyses of liver nodules and adjacent normal liver at 139-days PHI expressed HBx and Fah transgenes. (B) Semi-quantitative analysis of RT-PCR from (A) demonstrated significantly higher Afp expression in hyperplastic nodules compared with adjacent normal livers (P = 0.0076, unpaired t test) relative to Actb levels.

NRAS does not cooperate with HBx in liver tumorigenesis during selective repopulation. (A) Statistical analysis using the Mann-Whitney two-tail test indicated a highly significance difference (P < 0.001, black lines) in the number of nodules between indicated cohorts. Significant differences (P < 0.01, grey lines) in the number of nodules were seen between indicated cohorts. Marginal significances (P < 0.05) in the number of nodules were seen between HBx/shp53 vs HBx/NRAS, HBx/shp53 vs Gfp, HBx vs HBx/NRAS/shp53 and empty/shp53 vs HBx/NRAS/shp53 (not indicated). (B) Statistical analysis using the Mann-Whitney two-tail test indicated a highly significance difference (P < 0.001, black lines) in the percentage of liver weight relative to the whole mouse mass from each injected cohort. Statistical analysis using the Mann-Whitney two-tail test indicated a significance difference (P < 0.01, grey lines) in the liver weight percentage between indicated cohorts. Marginal significances (P < 0.05) in the liver weight percentage were seen between HBx/shp53 vs HBx/NRAS, HBx vs HBx/NRAS, empty/shp53 vs NRAS/shp53, NRAS/shp53 vs Gfp and HBx/shp53 vs Gfp (not indicated).

Oncogenic potential of NRAS alone or in combination with shp53 in a selectively repopulating liver. (A) HE staining of hyperplastic nodule sections taken from experimental animals injected with NRAS alone at 82-days PHI (left) or in combination with shp53 at 71-days PHI (right). P, parenchymal; T, hyperplastic nodule; scale bars, 100 μm. (B) IHC staining of hyperplastic nodule serial sections taken from experimental animals injected with NRAS alone at 82-days PHI using antibodies against Fah (left), NRAS (middle) and Ki67 (right). Top panels, sections were incubated with the indicated antibodies. Bottom panels, no primary antibodies were used. Scale bars, 100 μm. (C) IHC staining of hyperplastic nodule serial sections taken from NRAS/shp53 experimental animals at 71-days PHI using antibodies against Fah (left), NRAS (middle left), Ki67 (middle right) and Gfp (right). Top panels, sections were incubated with the indicated antibodies. Bottom panels, no primary antibodies were used. Scale bars, 100 μm.

Oncogenic potential of NRAS is associated with the Pi3k/Akt signaling pathway. IHC staining of serial liver sections taken from experimental animals injected with the indicated transgene(s) using antibody against pAkt. HBx, HBx/shp53, NRAS, NRAS/shp53 and HBx/NRAS/shp53 as described in . Left column, sections were incubated with anti-pAkt. Right column, no primary antibody was used. Magnified inserts in upper-left corner of areas indicated by dashed boxes. Arrows and arrowheads indicate pAkt cytoplasmic and nuclear staining detected in indicated cells, respectively; P, parenchymal; T, hyperplastic nodule; scale bars, 100 μm.

Oncogenic potential of HBx is associated with the Wnt signaling pathway. IHC staining of serial liver sections taken from experimental animals injected with the indicated transgene(s) using antibody against Ctnnb1. HBx, 78-day PHI experimental animal injected with HBx alone. HBx/shp53, 72-day PHI experimental animal injected with HBx and shp53. NRAS, an 82-day PHI experimental animal injected with NRAS alone. NRAS/shp53, a 71-day PHI experimental animal injected with NRAS and shp53. HBx/NRAS, a 71-day PHI experimental animal injected with HBx and NRAS. HBx/NRAS/shp53, a 71-day PHI experimental animal injected with HBx, NRAS and shp53. Left column, sections were incubated with anti-Ctnnb1. Right column, no primary antibody was used. Magnified inserts in upper-left corner of areas indicated by dashed boxes. Arrows and arrowheads indicate Ctnnb1 cytoplasmic and membranous staining detected in indicated cells, respectively; scale bars, 100 μm.

Augmented oncogenic potential of HBx with shp53 in a selectively repopulating liver. (A) Representative demonstration of HBx-positive hepatocytes by IHC in a 71-day PHI HBx/shp53 mouse (see also magnified insert in upper-left corner of area indicated by dashed box). HBx was not detected in normal FVB liver. Scale bar, 100 μm. (B) Representative demonstration of high mitotic index by Ki67 IHC in a 71-day PHI HBx/shp53 mouse. Representative field of view containing several Ki67-positive cells (arrows) in a 78-day PHI HBx mouse. Ki67 was not detected in normal FVB liver. Scale bar, 100 μm. Negative controls for (A) and (B), serial sections incubated without the indicated primary antibodies gave no visible signals above background (data not shown).