Volume 138, Issue 3, Pages 1155-1165.e2 (March 2010) Induction of p53 Renders ATM-Deficient Mice Refractory to Hepatocarcinogenesis  Narci Teoh, Pawan Pyakurel, Yock Young Dan, Karen Swisshelm, Jing Hou, Claudia Mitchell, Nelson Fausto, Yansong Gu, Geoffrey Farrell  Gastroenterology  Volume 138, Issue 3, Pages 1155-1165.e2 (March 2010) DOI: 10.1053/j.gastro.2009.11.008 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 ATM deficiency confers resistance to DEN-induced hepatocarcinogenesis. Gross liver tumor morphology and corresponding representative H&E-stained tumors in DEN-treated WT ATM+/+ (A and D), ATM+/− (B and E), and liver sections from ATM−/− (C and F) mice at 9 months, respectively (original magnification, 200×). Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 High rate of chromosomal abnormalities in ATM+/− derived hepatocellular carcinomas (HCC). Primary culture of liver tumor cells and hepatocytes were prepared from HCCs arising in ATM mice of all genotypes at 9 months (n = 5 per experimental group). (A) Representative passage 1 primary HCC cell culture derived from ATM+/− mouse at 9 months (original magnification, 100×). (B) Typical passage 1 primary hepatocyte culture derived from ATM−/− mouse at 9 months (original magnification, 100×). (C) Multiple numerical and structural chromosomal abnormalities observed in ATM+/− tumors. Primary HCC cells were harvested at passage 1 and metaphase spreads prepared for karyotype analysis. Representative karyotypes from ATM+/− (Ci and Cii) liver tumors at 9 months. Numerical aberrations (gain or loss of chromosomes shown by arrows) and marker chromosomes (denoted “M”), which indicate profound structural chromosomal aberrations are highlighted in these karyotypes. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 p53 and p19ARF Expression in HCCs and liver tissue at 6 and 9 months after DEN injection in WT, heterozygous, and ATM−/− mice. Expression of p53 protein determined by Western blotting. β-actin was used as loading control. HCC, hepatocellular carcinoma. (A) p53 Is induced in all genotypes at 6 months after DEN injection and appears in WT HCCs and HCCs liver from heterozygous mice at 9 months. By comparison, marked induction in p53 protein expression in ATM−/− liver lysates at 9 months is more striking compared with ATM+/− and WT. (B) DEN-mediated induction of p19ARF protein expression is significantly higher in ATM−/− liver compared with WT and ATM+/− liver and HCCs at 6 and 9 months. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Expression of cell cycle proteins, cyclin A, and cyclin D1 in liver tumors and nontumorous liver. The increase in expression of tumor suppressor proteins in ATM−/− liver was associated with reduced expression of the cell cycle proteins cyclins A and D1. Liver and tumor lysates (20 μg/lane) were analyzed by Western blotting. β-actin was used as a loading control. HCC, hepatocellular carcinoma. (A) Reduced cyclin A levels after DEN treatment in ATM−/− liver compared with WT and ATM+/− at 6 and 9 months. (B) Diminished cyclin D1 expression after DEN treatment in ATM−/− liver compared with WT and ATM+/− at 6 and 9 months. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Active caspase-3 is overexpressed in DEN-injected ATM−/− liver compared with WT at 6 and 9 months. (A) Active caspase-3 as determined by fluorogenic assay. (B) Increased cleaved caspase-3 immunostaining in ATM−/− liver at 6 months. At least 10 high-power fields were examined for n = 4 mice per experimental group. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 ATR and Chk1, but not Chk2 or Brca1, mRNA expression are up-regulated in ATM−/− DEN-treated liver compared with WT ATR (A), Chk1 (B), Brca1 (C), and Chk2 (D) mRNA expression (fold induction) by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. *P < .05 compared with WT (ATM+/+) liver. Increased protein expression of ATR (E) and Chk1 (F) in ATM−/− lysates confirmed by immunoblotting and densitometry. β-actin was used as a loading control. *P < .05 compared with WT (ATM+/+) 9-month HCC. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Mechanisms for refractoriness to DEN-induced HCC in ATM−/− mice may involve not only apoptosis, cell cycle arrest, but also p53-related senescence; the latter appears to be balanced by increased telomerase leading to immortalization. (A) Cxcl-1 mRNA expression by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. *P < .05 compared with DEN naïve (untreated) liver from ATM−/− and WT mice. (B) No significant difference in Csf1 and Mcp1 mRNA by RT-PCR between WT HCCs and ATM−/− DEN-treated liver. (C) ATM−/− liver and WT HCCs display different immunostaining patterns for β-galactosidase. Immunohistochemistry (IHC) for β-galactosidase was performed on liver tumors derived from WT mice (original magnification, 200×). Minimal staining (arrows) seen in representative WT No DEN (control) unlike ATM−/− naïve liver and DEN-treated ATM−/− liver at 12 months. *P < .05 compared with WT HCC. (D) Multiple nuclei staining positive for telomerase (TERT) on IHC in WT HCCs (original magnification, 200×), lesser in ATM−/− liver. Number of positive staining nuclei were counted per high power (original magnification, 400×) field. At least 10 high-power fields were examined for n = 4 mice/experimental group. *P < .01 compared with WT and ATM−/− naïve liver, †P < .05 compared with WT HCC. Positive control, human colon cancer. (E) No nuclear staining for TERT in remnant regenerating WT mouse liver at 30 hours postpartial hepatectomy (original magnification, 200×). (F) Flow diagram to illustrate the several possible mechanisms by which hepatocarcinogenesis is abrogated rather than enhanced in ATM-deficient mice. The present observations are congruent with activation of each of these effector pathways, although have not definitively proven their mechanistic involvement. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 WT and ATM+/− mice develop dysplastic foci at 6 months after DEN injection. No development of proliferative foci in ATM−/− DEN-injected mice at 6 months. Foci per square centimeter of liver tissue were counted, expressed as mean ± SD. *,†P < .01 compared with control (non-DEN injected), wild-type (+/+), and heterozygote (+/−) mice at 6 months. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Liver regeneration in ATM null, heterozygous, and WT mice subjected to partial hepatectomy (HP). Proportion of 5-bromo-2-deoxyuride positive hepatocytes at different timepoints after PH (n = 5 mice/group). All data are expressed as mean ± SE. Gastroenterology 2010 138, 1155-1165.e2DOI: (10.1053/j.gastro.2009.11.008) Copyright © 2010 AGA Institute Terms and Conditions