Volume 132, Issue 4, Pages 1465-1475 (April 2007) Telomerase Deletion Limits Progression of p53-Mutant Hepatocellular Carcinoma With Short Telomeres in Chronic Liver Disease  André Lechel, Henne Holstege, Yvonne Begus, Andrea Schienke, Kenji Kamino, Ulrich Lehmann, Stefan Kubicka, Peter Schirmacher, Jos Jonkers, K. Lenhard Rudolph  Gastroenterology  Volume 132, Issue 4, Pages 1465-1475 (April 2007) DOI: 10.1053/j.gastro.2007.01.045 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Mouse model of crisis-induced hepatocarcinogenesis. Trp53F2–10/+ were crossed with mTERC+/− mice followed by intercrossing Trp53F2–10/+, mTERC−/− mice through 3 generations to produce G3 mTERC−/−, Trp53F2–10/F2–10 mice. In parallel, mTERC+/−, Trp53F2–10/F2–10 were crossed with hepatitis B surface antigen (HBs+) transgenic mice to generate mTERC+/−, Trp53F2–10/F2–10, HBs+ mice. These mice were crossed with G3 mTERC−/−, Trp53F2–10/F2–10 mice to generate the different experimental cohorts. Sixteen-week-old mice were intravenously injected with a nonreplicative adenovirus (8 × 109 PFU) expressing Cre-recombinase. All mice were on C57BL/6J background. Gastroenterology 2007 132, 1465-1475DOI: (10.1053/j.gastro.2007.01.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Telomere shortening and p53 deletion in hepatocellular carcinoma of mTERC+/−, p53−/−, HBs+, and mTERC−/−, p53−/−, HBs+ mice. (A) Real-time PCR analysis showing the percentage of Trp53 gene deletion in liver and liver tumors of mTERC+/−, p53−/−, HBs+ and mTERC−/−, p53−/−, HBs+ mice. Similar rates of all macroscopic liver tumors were Trp53 deleted in both cohorts, specifically: 89.5% (51 of 57) in mTERC+/−, p53−/−, HBs+ compared to 86.7% (13 of 15) in mTERC−/−, p53−/−, HBs+ mice. The numbers below the graph show the number of Trp53 wild-type tumors per total number of tumors analyzed. (B) Histogram showing the percentage of hepatocytes (y-axis) analyzed through cell counting from H&E stained sections and the percentage of Trp53 deletion (x-axis) analyzed by real-time PCR for individual tumors. Note that the percentage of Trp53 deletion correlates well with the percentage of stromal/immune cell content in individual tumors. (C–E) The histograms show the distribution pattern of mean telomere fluorescence intensities (TFI) measured in hepatocyte nuclei of (C) C57BL/6J mice (n = 5 mice), (D) mTERC+/−, p53−/−, HBs+ mice (n = 5 mice), and (E) mTERC−/−, p53−/−, HBs+ mice (n = 5 mice). The mean TFI is shown as a dashed line. Note that hepatocyte telomere length was shortened in chronically damaged liver of both cohorts compared to C57Bl/6J wild-type mice. (F) The histogram shows the percentage of telomeres with very low telomere fluorescence intensity (TFI <200 arbitrary units) among all telomere signals from interphase nuclei of the indicated cohorts of mice (n = 5 mice per group). An increased percentage of short telomeres was present in quiescent liver (HBs−) of mTERC−/−, p53−/−, HBs− compared to mTERC+/−, p53−/−, HBs− mice (P = .004). In contrast, a similarly high frequency of short telomeres was detected in chronically damaged liver and HCC of mTERC−/−, p53−/−, HBs+, and mTERC+/−, p53−/−, HBs+ mice. Gastroenterology 2007 132, 1465-1475DOI: (10.1053/j.gastro.2007.01.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Telomerase deficiency suppresses liver tumor development in p53-mutant mice with short telomeres. (A) Histogram showing the average number of macroscopic tumors per liver in 12- to 15-month-old mice. The tumor incidence is shown in square boxes. Note that the number of tumors was significantly suppressed in mTERC−/−, p53−/−, HBs+ (n = 17 mice, n = 25 tumors) compared to mTERC+/−, p53−/−, HBs+ mice (n = 15 mice, n = 98 tumors, P = .001). (B) The tumors were histologically classified as macroscopic nodules or HCCs (histologic criteria: see Materials and Methods). The histogram shows the average number of macroscopic nodules (left side) and HCCs (right side) per mouse liver in 12 to 15-month-old mTERC+/−, p53−/−, HBs+, and mTERC−/−, p53−/−, HBs+ mice. The incidence of macroscopic nodules and HCCs are shown in square boxes. Gastroenterology 2007 132, 1465-1475DOI: (10.1053/j.gastro.2007.01.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Telomerase deficiency increases aneuploidy in p53-mutant HCCs with short telomeres. (A, B) Representative blot diagram of comparative genomic hybridizations of (A) a mTERC−/−, p53−/−, HBs+ HCC and (B) a mTERC+/−, p53−/−, HBs+ HCC. (C, D) The histograms show the percentage of BACs indicating significant gains or losses (log2 ratio significant different from 0 = P < .01) from all analyzed liver tumors in mTERC−/−, p53−/−, HBs+ mice (red bars) and mTERC+/−, p53−/−, HBs+ mice (blue bars): (C) Analysis of BACs on individual chromosomes; (D) analysis of all analyzed BACs covering the whole genome. Gastroenterology 2007 132, 1465-1475DOI: (10.1053/j.gastro.2007.01.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Telomerase-deficient HCC accumulates DNA-damage and show an induction of p53-independent cell cycle arrest and apoptosis. (A) Representative photograph of a TRAP-gel showing activation of telomerase during hepatocarcinogenesis in mTERC+/−, p53−/−, HBs+ mice. The ladder of 6 base pair extension products indicates telomerase activity. Lanes: 1, negative control DEPC dH2O; 2, heat-inactivated control (HCC cell line from p53−/− HBs− mouse); 3, positive control (HCC cell line from a p53−/− HBs− mouse); 4 and 5, HCCs of mTERC−/−, p53−/−, HBs+ mice; 6–10, liver of mTERC+/−, p53−/−, HBs+ mice; 11–15, HCCs of mTERC+/−, p53−/−, HBs+ mice. (B) The histogram shows the frequency of hepatocyte nuclei staining positive for γH2AX-foci in chronically damaged liver and HCC of mTERC+/−, p53−/−, HBs+ mice (n = 5) and mTERC−/−, p53−/−, HBs+ mice (n = 5). Note the sharp increase in γH2AX-foci in mTERC−/−, p53−/−, HBs+ HCC. (C, D) Representative photographs showing γH2AX-foci in (C) mTERC+/−, p53−/−, HBs+ HCC, and (D) mTERC−/−, p53−/−, HBs+ HCC; arrows point to γH2AX-foci (magnification bar: 100 μm; inlet magnification bar: 20 μm). (E) Representative photographs on telomere/γH2AX immuno-FISH: Upper panel: γH2AX-immunofluorescence; middle panel: telomere-FISH; bottom panel: overlay, arrows point to the colocalization of telomere signals and γH2AX-foci. (F) Histogram on the percentage of apoptotic cells (TUNEL-positive) in HCC of mTERC+/−, p53−/−, HBs+ mice and mTERC−/−, p53−/−, HBs+ mice. (G) Representative photographs show TUNEL-staining of HCCs from mTERC+/−, p53−/−, HBs+ mice (top photograph) and mTERC−/−, p53−/−, HBs+ mice (bottom photograph) (magnification bars: 50 μm). (H) Histogram on the percentage of proliferating cells (PCNA-positive) in HCCs of mTERC+/−, p53−/−, HBs+ mice and mTERC−/−, p53−/−, HBs+ mice. (I) Representative photographs show PCNA-staining of HCCs from mTERC+/−, p53−/−, HBs+ (top photograph) and mTERC−/−, p53−/−, HBs+ mice (bottom photograph) (magnification bars: 50 μm). Gastroenterology 2007 132, 1465-1475DOI: (10.1053/j.gastro.2007.01.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 The role of telomerase for the progression of p53 mutant hepatocellular carcinoma with short telomeres. Telomere shortening and loss of p53 checkpoint function leads to an induction of CIN and cancer initiation. Telomerase negative tumors accumulate excessive CIN and DNA damage inducing p53-independent pathways of tumor suppression. In contrast, telomerase activity limits the accumulation of CIN, thus facilitating tumor progression. Gastroenterology 2007 132, 1465-1475DOI: (10.1053/j.gastro.2007.01.045) Copyright © 2007 AGA Institute Terms and Conditions