DNA damage sensor γ -H2AX is increased in preneoplastic lesions of hepatocellular carcinoma

Abstract

Background: Phosphorylated histone H2AX ( γ -H2AX) is a potential regulator of DNA repair and is a useful tool for detecting DNA damage. To evaluate the clinical usefulness of γ -H2AX in hepatocellular carcinoma (HCC), we measured the level of γ -H2AX in HCC, dysplastic nodule, and nontumorous liver diseases.

Methods: The level of γ -H2AX was measured by immunohistochemistry in fifty-eight HCC, 18 chronic hepatitis, 22 liver cirrhosis, and 19 dysplastic nodules. Appropriate cases were also examined by fluorescence analysis and western blotting. results: All cases with chronic liver disease showed increased levels of γ -H2AX expression. In 40 (69.9%) of 58 cases with HCC, the labeling index (LI) of γ -H2AX was above 50% and was inversely correlated with the histological grade. Mean γ -H2AX LI was the highest in dysplastic nodule (74.1 ± 22.1%), which was significantly higher than HCC (P < 0.005). Moreover, γ -H2AX was significantly increased in nontumorous tissues of HCC as compared with liver cirrhosis without HCC (62.5 ± 24.7%, from 5.1 to 96.0%, P < 0.005).

Conclusions: γ -H2AX was increased in the preneoplastic lesions of HCC and might be a useful biomarker for predicting the risk of HCC.

Figure 1

γ-H2AX is expressed at different levels in HCC. (a) Immunofluorescence staining shows that phosphorylated histone H2AX (γ-H2AX) is located in the nuclei of HCC cells (green) (original magnification ×100). Case  2: HCC with increased γ-H2AX expression; Case 7: HCC with sparse expression of γ-H2AX. DAPI (blue): nucleus counterstain. (b) Representative data of western blotting for γ-H2AX in liver tissues. H1 and H2: healthy livers; Cases  2 and 7: HCC cases. T: tumor tissues; N: adjacent nontumorous liver tissues. (c) Immunohistochemical staining of γ-H2AX. Cases  1 and 7: HCC cases with negative to low expression of γ-H2AX. Cases  2 and 11: HCCs with high expression of γ-H2AX (original magnification ×40). Arrows indicate positive staining in the nuclei.

Figure 2

γ-H2AX is significantly increased in preneoplastic lesions in the liver. (a) Representative images of immunostaining for γ-H2AX in non-HCC tissues. CH: chronic hepatitis; LC: liver cirrhosis; DN: dysplastic nodule (original magnification ×40). (b) Dot plots showing the γ-H2AX labeling index in normal livers (n = 5), chronic hepatitis (CH; n = 18), liver cirrhosis (LC; n = 22), dysplastic nodule (DN; n = 19), and HCC (n = 58). Horizontal bars depict the mean value, and vertical bars indicate the standard deviation. a**: P value of <0.01 versus normal livers; b**: P value of <0.01 versus dysplastic nodule.

Figure 3

γ-H2AX is increased in the adjacent nontumorous liver tissues of HCC patients. (a) Representative data of western blotting for γ-H2AX. L3, 5, and 12: cases with liver cirrhosis without the coexistence of HCC; H3, 5, and 12: adjacent nontumorous liver tissues obtained from HCC patients. (b) Representative images of γ-H2AX immunostaining in liver tissues with and without the coexistence of HCC (original magnification ×40). (c) Dot plots showing the γ-H2AX labeling index. CH: chronic hepatitis without the coexistence of HCC (n = 18) and with HCC (n = 28); LC: liver cirrhosis without HCC (n = 22) and with HCC (n = 30). NS: not significant; **P < 0.01.