Ku70 Deficiency Accelerates PDGF-C Induced Development Of Hepatocellular Carcinoma In Mice

Objective:To create a new animal model that includes mice with overexpressed Platelet-Derived Growth Factor C and deficient in the Ku70 gene, thus having chromosomal instability.Methods:C57BL/6 male PDGF-C+/-, Ku70+/-heterozygous mice were mated with C57BL/6 female PDGF-C-/-, Ku70+/-heterozygous mice to generate new pups, then PCR genotyping method was applied to identify the genotype of each mouse.Results:We collected six types of mice with the genotype of PDGF-C+/-, Ku70+/+; PDGF-C+/-, Ku70+/-; PDGF-C+/-, Ku70-/-; PDGF-C-/-, Ku70+/+; PDGF-C-/-, Ku70+/-and PDGF-C-/-, Ku70-/-. Of the six types, PDGF-C+/-, Ku70-/-mice are of the expected type -- they can develop hepatocellular carcinoma (HCC), following the steps of steatosis, fibrosis, cirrhosis and finally HCC, caused by overexpressed PDGF-C and are deficient in the Ku70 gene. These mice thus have chromosomal instability.Conclusion:We successfully established PDGF-C+/-, Ku70-/-mice model, which has two significant features:first, overexpression of PDGF-C caused liver steatosis, fibrosis, cirrhosis and finally HCC; second, Ku70 deficiency caused DSB repair problems which creating chromosomal instability. This animal model does not require chemical reagents used in traditional HCC animal models. The HCC of these mice having chromosomal instability can thus been driven by only the PDGF-C. This animal model is the first model of such features in the world.Objective:To determine whether Ku70 deficiency could accelerate the development of PDGF-C induced Hepatocellular carcinoma in mice.Methods:Six month old male C57BL/6 mice were divided into four groups according to their genotypes:WT, PDGF-C transgenic, Ku70 deficiency and PDGF-C+/-/Ku70-/-mice. Mice livers were collected and weighted. Continuous slides were cut and H&E staining were done before histopathologic analyze. The pathologic changes were analyzed with Image J software.Results:Experimental PDGF-C Tg/Ku70 KO mice showed significant macroscopic differences in the livers compared to the other three groups. The livers were enlarged, and misshaped. There were yellow pigments deposited on the liver surface. Cysts and nodules were detected on the livers. Histopathologic changes were detected under the microscope. Dysplastic nodules, dysplastic foci or foci of altered hepatocytes, fatty change, small cell change and lots of hepatic nonparenchymal cell proliferation were detected in experimental PDGF-C Tg/Ku70 KO mice livers. The features of dysplastic foci or foci of altered hepatocytes include nuclear anisocytosis, hyperchromasia, pleomorphism, and increased nuclear to cytoplasmic ratio. The incidence of tumor was increased significantly in experimental PDGF-C Tg/Ku70 KO mice (100%) compared to the other three groups (P<0.05), and the number of foci/cm2 (5.26±3.83) as well as percentage of liver tissue involved by the foci (16.66±9.24%) was significantly higher in experimental PDGF-C Tg/Ku70 KO mice than in WT, PDGF-C transgenic and Ku70 KO mice. Experimental PDGF-C Tg/Ku70 KO mice have tumor burden, determined by liver-to-body weight ratio, that is higher than WT (P<0.05), but is not higher than PDGF-C transgenic mice.Conclusion:Experimental PDGF-C Tg/Ku70 KO mice showed (1) increased incidence of early hepatocarcinogenesis; (2) increased number of dysplastic nodules and dysplastic foci or foci of altered hepatocytes; (3) higher number of foci/cm2; and (4) higher percentage of liver tissue involved by the foci. Therefore, Ku70 deficiency accelerated the development of PDGF-C induced hepatocellular carcinoma in mice.Objective:To explore the molecular mechanisms of Ku70 deficiency on the development of PDGF-C induced HCC in mice.Methods:Six month old male C57BL/6 mice were divided into four groups according to their genotypes:WT, PDGF-C transgenic, Ku70 deficiency and PDGF-C Tg/Ku70 KO mice group. Mouse livers were fixed overnight, processed to paraffin blocks, sectioned, and stained with hematoxylin/eosin and Sirus Red using standard techniques. Immunohistochemistry was performed for microscopy. Cell proliferation, apoptosis, liver fibrosis, steatosis and inflammation were analyzed with all liver sections.Results:1. BrdU labeling showed a complex trend -- there were more BrdU labeled hepatocytes in PDGF-C transgenic and PDGF-C Tg/Ku70 KO mouse livers compared to WT, and more in PDGF-C transgenic mouse livers compared to Ku70 KO. Moreover, there were more BrdU labeled nonparenchymal cells in PDGF-C transgenic mouse livers compared to WT and Ku70 KO. Compared with PDGF-C Tg/Ku70 KO group, there were no significant differences in neither BrdU labeled hepatocytes nor nonparenchymal cells in PDGF-C transgenic mouse livers.2. Ki67 labeling showed a complex trend too-there were more Ki67 labeled hepatocytes in PDGF-C transgenic and PDGF-C Tg/Ku70 KO mouse livers compared to WT. There were more Ki67 labeled hepatocytes in PDGF-C Tg/Ku70 KO than in PDGF-C transgenic mouse livers. Both of them had more Ki67 labeled hepatocytes than Ku70 KO group. Moreover, there were more Ki67 labeled hepatocytes and nonparenchymal cells in the dysplastic nodules and/or dysplastic foci compared to adjacent liver tissues.3. Fibrosis was not detected in WT and Ku70 KO mouse livers. There were more collagen deposited in PDGF-C transgenic and PDGF-C Tg/Ku70 KO mouse livers compared to WT and Ku70 KO groups. Although bridging fibrosis and cirrhosis were detected in one of the PDGF-C Tg/Ku70 KO mouse livers, the overall fibrosis severity was not worse than PDGF-C Tg mouse livers.4. Inflammation was not detected in WT and Ku70 KO mouse livers. More severe inflammation was observed in PDGF-C transgenic mouse livers compared to WT and Ku70 KO groups. There was no significant difference of inflammation severity between PDGF-C Tg and PDGF-C Tg/Ku70 KO mouse livers.5. The severity of steatosis in mouse livers among those four groups was similar, and no significant differences were detected.6. Apoptotic bodies were not detected in WT mouse livers and most of the Ku70 KO mouse livers (4 in 5). Only one of the Ku70 KO mouse livers was determined as having many apoptotic bodies. Compared with them, two PDGF-C transgenic mouse livers were detected as having-many apoptotic bodies, and five had none or rare.3 in 7 PDGF-C Tg/Ku70 KO mouse livers showed many apoptotic bodies while the rest showed none or rare. No significant differences were detected between PDGF-C transgenic and PDGF-C Tg/Ku70 KO group.Conclusion:Ku70 deficiency in PDGF-C overexpressed mice did not change the background of hepatic carcinogenesis, as further development of liver fibrosis, steatosis and inflammation were not detected. Under the similar background, Ku70 deficiency accelerated the development of HCC by increasing the proliferation of hepatocytes in dysplastic nodules and/or dysplastic foci, activating the cell cycle G2/M checkpoint, and decreasing apoptosis of damaged hepatocytes. Damaged hepatocytes arrested in G2/M phase and refused to apoptosis may lead to liver tumorigenesis.