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Telomerase Deletion Limits Progression of p53-Mutant Hepatocellular Carcinoma With Short Telomeres in Chronic Liver Disease
GASTROENTEROLOGY 2007;132:1465–1475
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,储 STEFAN KUBICKA,* PETER SCHIRMACHER,¶ JOS JONKERS,‡ and K. LENHARD RUDOLPH*
Background & Aims: During early stages of carcinogenesis most human epithelial cancers including hepatocellular carcinoma (HCC) have been observed to transit through a “crisis” stage characterized by telomere shortening, loss of p53 checkpoint function, and a sharp increase in aneuploidy. The function of telomerase during in vivo hepatocarcinogenesis has not been studied in this genetic context. Methods: Here we generated a mouse model in which HCC was induced by chronic organ damage (HBs-AG transgene) in the presence of telomere shortening and p53 deletion. Tumor development was analyzed in lategeneration telomerase knockout mice (mTERCⴚ/ⴚ) and littermates, genetically rescued for telomerase gene expression (mTERCⴙ/ⴚ). Results: The formation of HCCs was strongly suppressed in mTERCⴚ/ⴚ mice compared to mTERCⴙ/ⴚ siblings correlating with reduced rates of tumor cell proliferation and elevated rates of tumor cell apoptosis. Although the prevalence of short telomeres was similar in chronically damaged liver of both cohorts, mTERCⴚ/ⴚ HCC developed increased levels of DNA damage and aneuploidy compared to mTERCⴙ/ⴚ HCC. Conclusions: This study provides direct evidence that telomerase is a critical component for in vivo progression of p53 mutant HCC with short telomeres in the chronically damaged liver. In this molecular context, telomerase limits the accumulation of telomere dysfunction, the evolution of excessive aneuploidy, and the activation of p53-independent checkpoints suppressing hepatocarcinogenesis.
H
epatocellular carcinoma (HCC) is one of the most common solid tumors in humans.1 The risk of HCC development is low in healthy liver and early stages of chronic liver disease but sharply increases at the cirrhosis stage.1 Frequently found, molecular alterations that are associated with HCC development are telomere shortening,2– 4 loss of p53 checkpoint function,5,6 and the evolution of aneuploidy.7–9 Studies on telomerase deficient (mTERC⫺/⫺) mice have shown that telomere shortening has a dual role in cancer
initiation and progression. These studies have demonstrated that telomere shortening inhibited in vivo progression of tumors,10 –14 but increased the rate of tumor initiation by induction of chromosomal fusions resulting in anaphase bridges, chromosome breakage, and aneuploidy.12,15 Impaired tumor progression in telomere dysfunctional mice was associated with an activation of p53-dependent DNA-damage responses.12,14 In contrast, mutation of p53 alleviated tumor suppression and accelerated chromosomal instability (CIN) and cancer initiation in telomere dysfunctional mice.16 –18 The high incidence of telomere shortening, loss of p53 checkpoint function, and aneuploidy in the vast majority of human HCC2–9 indicates that this molecular context is highly relevant for human hepatocarcinogenesis. In addition to these alterations, most human HCC (⬎90%) reactivate telomerase during cancer progression.5,19,20 The functional role of telomerase for in vivo progression of primary, p53 mutant tumors with dysfunctional telomeres is unknown and needs to be addressed given the high frequency of p53 mutations in human cancer including HCC.5,6 Moreover, this question appears of clinical relevance to test whether telomerase-inhibition represents a promising therapeutic approach for the treatment of p53-mutant HCC. Here we generated a mouse model of hepatocarcinogenesis that recapitulates several molecular characteristics, which are associated with the development of human HCC: telomere shortening,2– 4,21–24 loss of p53pathway function,5,6,21–24 and aneuploidy.7–9,21–24 We analyzed the functional role of telomerase in late-generation telomerase knockout mice and littermates that were genetically rescued for telomerase gene expression. The
Abbreviations used in this paper: Ad-Cre, adenovirus expressing Cre-recombinase; CIN, chromosomal instability; FISH, fluorescence in situ hybridization; HCC, hepatocellular carcinoma; PCNA, proliferating cell nuclear antigen; TFI, telomere fluorescence intensities; TRAP, telomere repeat amplification protocol. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.01.045
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*Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Germany; ‡Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands; §Department of Molecular Pathology, Medical School Hannover, Germany; 储Department of Pathology, Medical School Hannover, Germany; ¶Institute of Pathology, University of Heidelberg, Germany
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study provides experimental evidence that telomerase deletion impairs in vivo progression of primary, p53-mutant HCC with short telomeres. The study indicates that in this genetic context telomerase deficient tumors accumulate DNA damage and aneuploidy, which leads to an activation of p53-independent tumor suppressor checkpoints. These data suggest that telomerase inhibition could show antitumor activity in primary HCCs haboring defects in the p53 signaling pathway.
Materials and Methods Mouse Model of Chronic Liver Damage, Telomere Shortening, and Loss of P53 Checkpoint Function in Liver
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We used the Cre-loxP system to achieve a liver specific deletion of the Trp53 gene (conditional p53 mouse).25 In this mouse loxP sites flank Exon 2–10 of the Trp53 gene. When the enzyme Cre-recombinase is expressed, this region of the p53 gene will be deleted, which leads to loss of p53 function.25 Heterozygous conditional p53 mice (Trp53F2–10/⫹) were crossed with mTERC⫹/⫺ mice.26 Intercrosses of mTERC⫹/⫺, Trp53F2–10/⫹ yielded in G1 mTERC⫺/⫺, Trp53F2–10/F2–10 mice, in which both alleles of the murine p53 gene contained loxP sites. To induce telomere shortening, these mice were crossed through successive generations to produce late generation (G3) mTERC⫺/⫺, Trp53F2–10/F2–10 mice (Figure 1). To induce chronic liver damage, mTERC ⫹/⫺ , Trp53F2–10/F2–10 were crossed with transgenic mice expressing the hepatitis B virus surface antigen (HBs⫹)
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.
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under the liver-specific albumin promoter (C57BL/ 6J-Tg(Alb1HBV)44Bri/J; Jackson Laboratories, Bar Harbor, ME)27 to generate mTERC⫹/⫺, Trp53F2–10/F2–10, HBs⫹ mice. These mice were finally crossed with G3 mTERC⫺/⫺, Trp53F2–10/F2–10 mice to generate the experimental groups of mouse siblings that were in the 4th generation telomerase knockout (mTERC⫺/⫺, Trp53F2–10/F2–10, HBs⫹; n ⫽ 17) or genetically rescued for telomerase gene expression (mTERC⫹/⫺, Trp53F2–10/F2–10, HBs⫹; n ⫽ 15) (Figure 1).28 The mice were infected with an adenovirus expressing Crerecombinase (Ad-Cre),29 which led to recombination of the loxP sites in the Trp53 gene locus (see above) resulting in a liver specific deletion of the Trp53 gene (Trp53⌬2–10/⌬2–10). All mice were in a C57Bl/6J background.
Amplification and Purification of Ad-Cre Ad-Cre virus was propagated in 293T cells and prepared according to previously described cesium chloride gradient purification.29 The titer was determined by the plaque assay and mice were infected with 0.8 ⫻ 1010 PFU of Ad-Cre at 4 months of age.
Dissection of Macroscopic Tumors and Histologic Analysis Mice were humanely sacrificed at an age of 12 to 15 months (mean age in mTERC⫺/⫺ p53⫺/⫺ HBs⫹ mice: 12.8, mean age in mTERC⫹/⫺ p53⫺/⫺ HBs⫹ mice: 13.6 months). Macroscopically tumors (⬎2 mm in diameter) were cut in half; 1/2f was frozen down (for DNA preparation), 1/2 was fixed in 4% neutral-buffered formaldehyde and processed further according to histologic routine protocols and embedded in paraffin. Sections (4 m) were stained with H&E, and the tumor types were classified using the following criteria. Microscopic foci. Lesions 0.5–2 mm in diameter, with morphologically clonal appearing growth pattern, sometimes slight marginal compression and basophilic, clear cell or eosinophilic cytomorphology. Macroscopic nodule. Nodules ⬎2 mm in size representing macroscopic nodules (“adenomas”) not showing characteristic markers of HCCs. HCC. Hepatocellular carcinoma defined by either histologic features (trabecular disorganization, more than 2 cell-thick trabecula, pseudoglandular structures, obvious invasion) or cytologic changes (moderate to severe atypia). Grading of HCC was performed on the basis of cytologic criteria on a G1–3 scale reflecting increasing cellular atypia.
Analysis of Telomerase Activity by the Telomere Repeat Amplification Protocol Assay The telomere repeat amplification protocol (TRAP) was performed on snap-frozen tissues as reported.30 Briefly, TRAP reactions were incubated for 30 minutes at 30°C for telomerase extension using 32P␥ATP (Hartmann Diagnostics, Germany) labeled telo-
merase substrate (TS⫺) primer followed by a PCR reaction (94°C 30 seconds, 60°C 30 seconds, 30 repeats). PCR products were size-fractioned on 12% nondenaturing polyacrylamide gel, and visualized after drying the gel on a phosphor imager (Amersham Biosciences, Arlington Heights, IL).
Detection of Deletion of Exons 2–10 of Trp53 The DNA copy number of Trp53⌬2–10 and was analyzed in triplicate by real-time PCR according to standard protocols. The following primers were used for detection of the Trp53⌬2–10 allele: Trp53A 5=-CAC AAA AAC AGG TTA AAC CCA G-3= and Trp53E 5=-CCA TGA GAC AGG GTC TTG CT-3= and for the Trp53F2–10 allele: Trp53A 5=-CAC AAA AAC AGG TTA AAC CCA G-3=, Trp53B 5=-AGC ACA TAG GAG GCA GAG AC-3=. Amplifications were performed using the Applied Biosystems (Foster City, CA) 7300 Real-Time PCR System under the following conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 40 seconds. We determined the relative quantities of Trp53⌬2–10 by generating a standard curve using mixtures of genomic DNA from Trp53⌬2–10 and Trp53F2–10 mouse embryo fibroblasts in fixed ratios (0:100, 20:80, 40:60, 60:40, 80:20, and 90:10). From the quantitative reverse-transcriptase polymer chain reaction (PCR) results of these samples we calculated the ratio (⌬Ct value of Trp53⌬2–10/⌬Ct value of Trp53F2–10) to generate standard curves. Trp53F2–10
Quantitative Fluorescence In Situ Hybridization for Telomere Length Quantitative fluorescence in situ hybridization (Q-FISH) was performed on 5-m paraffin sections. After unmasking, the slides were incubated in Pepsine solution for 10 minutes at 37°C (100 mg Pepsine; 84L HCl 37% up to 100 mL H2O) and washed in phosphate-buffered saline. The hybridization mix (10 mmol/L Tris pH 7.2; MgCl2 buffer: 7.02 mmol/L Na2HPO3, 2.14 mmol/L MgCl2, 0.77 mmol/L citric acid; 70% deionized formamide; 0.5 g/mL PNA probe 5=-Cy3-CCC TAA CCC TAA CCC TAA-3= Applied Biosystems; 0.25% Roche blocking reagent, Indianapolis, IN) was added to the sections, covered with coverslips, and denatured at 80°C for 3 minutes followed by 2 hours of incubation in the dark. Slides were incubated in 70% formamide, 10 mmol/L Tris (pH 7.2), 0.1% BSA 2 times for 20 minutes, and washed 3 times in TBSTween (0.2%). The relative telomere length was measured by the telomere fluorescence intensity by TFL analysis software program.31
Array Comparative Genomic Hybridization Profiling Using BAC Arrays Genomic DNA was extracted from mouse tissues by proteinase K digestion and organic extraction, and
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digested o/n with HaeIII. Digested genomic DNA was labeled with Cy3 or Cy5 using a ULS aRNA Fluorescent Labeling Kit (Kreatech Biotechnology, Amsterdam, the Netherlands) according to the manufacturer’s protocol. Cy5–ULS-labeled tumor DNA was mixed with Cy3–ULSlabeled reference DNA (isolated from the spleen of the same mouse) and hybridized to a Mouse 3K BAC array as described previously.32 All experiments were performed in fluor-reversed pairs of 2-color hybridizations. Mouse 3K BAC arrays were printed by the NKI Microarray Facility (Amsterdam, The Netherlands). After hybridization, arrays were imaged using an Agilent Scanner and the data were processed using ImaGene software. Each array from the pair of fluor-reversed hybridizations contained duplicate spots, allowing weighted averages, errors, and confidence levels for each data point to be computed from quadruple measurements according to the Rosetta Error Model.33 All fluorescence intensities were converted to log2 values to weight gains and losses equally.
Immunohistochemistry Proliferating cell nuclear antigen (PCNA) staining was performed at real time for 2 hours (PCNA Ab-1, Oncogene Science, Uniondale, NY; diluted 1:150 in phosphate-buffered saline) followed by a 1-hour incubation with a Cy3-labeled rabbit antimouse IgG secondary antibody (1:300 in phosphate-buffered saline; Zymed, San Francisco, CA). The number of PCNA positive cells was counted randomly at low-power fields (200⫻). The rate of apoptosis was determined by TUNEL assay (In situ cell death detection kit, Roche, Mannheim, Germany). The numbers of apoptotic and nonapoptotic cells were counted in 20 low-power fields (200⫻) and the percentage of apoptotic cells was calculated. For ␥H2AX-staining sections were blocked with M.O.M blocking reagent (Vector Labs, Burlingame, CA) for 1 hour and then incubated with a mouse anti␥H2AX antibody (Upstate, Lake Placid, NY) overnight at 4°C. The subsequent steps to detect ␥H2AX signal was performed using M.O.M.™ Immunodetection Kit (Vector Laboratories) according to manufacturer’s recommendations. Double staining of telomere and ␥H2AX signals was performed by using the telomere quantitative FISH protocol (see above) followed by ␥H2AX immunofluorescence (see above) using a Cy3-labeled rabbit antimouse IgG secondary antibody (1:500; Zymed).
Statistical Programs The Mann–Whitney U test and T test were used to calculate the statistical significance and standard deviations were calculated by using Graphpad Instat, Graphpad Prism, SPSS, and Microsoft Excel software.
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Results Mouse Model of Hepatocarcinogenesis in the Context of p53 Mutation, Short Telomeres, and Chronic Organ Damage To analyze the impact of telomerase on hepatocarcinogenesis in the presence of the p53-mutation, short telomeres, and chronic organ damage, we crossed conditional p53 knockout mice (Trp53F2–10/F2–10)25 with mTERC⫺/⫺ mice26 through successive generations followed by an intercross of late-generation (G3) mTERC⫺/⫺, Trp53F2–10/F2–10 mice harboring critically short telomeres with mTERC⫹/⫺, Trp53F2–10/F2–10 mice. This crossproduced littermate offspring that was either genetically rescued for telomerase gene expression (iF1 mTERC⫹/⫺ ⫽ mTERC⫹/⫺) or lacked telomerase gene expression (iF1 G4mTERC⫺/⫺ ⫽ mTERC⫺/⫺) (Figure 1).28 Hepatic deletion of Trp53 (Trp53⌬2–10/⌬2–10 ⫽ p53⫺/⫺) was achieved in both groups by adenovirus mediated Cre-recombinase expression (Supplementary Figure 1; see Supplemental material online at www. gastrojournal.org).29 In addition, all mice expressed the hepatitis B surface protein (HBs⫹) as a transgene under the liver specific albumin promoter, which promotes chronic liver damage and hepatocarcinogenesis in 12- to 15-month-old mice,27 thus closely resembling the pathogenesis of human HCC.1,34 Hepatocarcinogenesis was followed in cohorts of mTERC⫺/⫺, p53⫺/⫺, HBs⫹, and mTERC⫹/⫺, p53⫺/⫺, HBs⫹ siblings. Regarding p53 gene status, we confirmed that virtually all HCCs (48 of 51) from both cohorts showed Cremediated deletion of Trp53 using Southern blot and quantitative PCR analyses (Figure 2A, Supplementary Figure 1; see Supplementary Figure 1 online at www. gastrojournal.org). The proportional loss of wild-type Trp53 sequences determined for individual tumors correlated well with the percentage of stromal and immune cells within the tumors (Figure 2B). Telomere length and numbers of critically short telomeres were determined by quantitative FISH in hepatocytes of nontumorous liver and HCCs. Nontransformed hepatocytes of chronically damaged liver of 12- to 15-
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month-old mTERC⫹/⫺, p53⫺/⫺, HBs⫹, and mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice had shorter telomeres (lower telomere fluorescence intensity ⫽ telomere fluorescence intensities [TFI]) compared to hepatocytes from 12- to 15-monthold C57Bl/6J wild-type mice (Figure 2C–E). Previous studies have established that the in vivo phenotypes of telomere dysfunction depend on the prevalence of critically short telomeres rather than mean telomere length.29 To evaluate the number of critically short telomeres we determined the frequency of telomeres with the lowest detectable fluorescence intensity (telomere fluorescence intensity ⬍200 units) in interphase nuclei. This analysis can not detect telomere-free ends, but represents the best possible method to evaluate the frequency of critical telomere shortening in tissues. In quiescent liver (HBs⫺) a higher incidence of critically short telomeres was detected in mTERC⫺/⫺, p53⫺/⫺, HBs⫺ compared with mTERC⫺/⫺, p53⫺/⫺, HBs⫺ littermates or C57Bl/6J mice (Figure 2F). However, in chronically damaged liver (HBs⫹) a similarly high incidence of critically short telomeres was present in nontumorous liver of 12- to 15month-old mTERC⫹/⫺, p53⫺/⫺, HBs⫹, and mTERC⫺/⫺, p53⫺/⫺, HBs⫹ siblings (Figure 2F). Both groups developed HCC at an age of 12 to 15 month (see below). The incidence of critically short telomeres was similarly high in HCC of both cohorts (Figure 2F).
Telomerase Deletion Inhibits Progression of p53-Mutant HCC With Short Telomeres in Chronically Damaged Liver The number of macroscopically visible liver tumors in 12- to 15-month-old mice was strongly 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, Figure 3A). Histologic analysis revealed that 25.2% of all macroscopic tumors were judged to represent macroscopic nodules (adenomas) and 59.3% of the tumors were HCCs (histologic criteria; see Materials and Methods section). HBs transgenic mice develop HCC induced by chronic liver damage.27 In previous studies, our group has shown that
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
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.
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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.
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the incidence of HCC in 12- to 15-month-old HBs transgenic mice on a C57Bl/6J background was 44%.35 In our current study, the incidence of HCC mTERC⫹/⫺ p53⫺/⫺, HBs⫹ mice was higher (see below) indicating that p53 deletion cooperated with chronic liver damage to induce HCC. The incidence and the average numbers of both macroscopic nodules and HCC were suppressed in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 3B). Specifically, 23.5% of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice displayed HCCs compared with 80% of mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (P ⫽ .0008). In total, 13 HCCs were diagnosed in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared with 60 HCCs in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (P ⫽ .002). Importantly, HCC numbers remained significantly suppressed in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice after sorting out Trp53 wild-type HCCs (Figure 2A) from the total number of HCCs in the mTERC⫹/⫺, p53⫺/⫺, HBs⫹ group (Mann–Whitney test: P ⫽ .006). The differentiation of HCCs was assessed according to previously described standards36 and did not reveal significant differences between both cohorts (Supplementary Figure 2A–D; see supplementary material online at www.gastrojournal.org). Histopathologic analysis of nontumorous liver specimens revealed a similar number of premalignant, microscopic foci in both experimental cohorts (Supplementary Figure 2E, see supplementary material online at www.gastrojournal.org; P ⫽ .255). These data indicated that telomerase deficiency impaired the progression of hepatocarcinogenesis in the context of chronic organ damage, short telomeres, and mutant p53 after the stage of microscopic focus formation.
Telomerase Limits Aneuploidy in p53 Mutant HCC With Short Telomeres In primary human cells and mTERC⫺/⫺ mice, telomere dysfunction and loss of p53 checkpoint function cooperate to induce CIN.16,17,22,23 Comparative genomic hybridization array on macroscopic liver tumors with confirmed Trp53 deletion (Figure 2A) revealed chromosomal gains and losses in all liver tumors examined (n ⫽ 16, Supplementary Table 1; see supplementary material online at www.gastrojournal.org). However, the overall level of aneuploidy was significantly higher in liver tumors of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ (n ⫽ 6 tumors) mice compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (n ⫽ 10 tumors, Figure 4A–C, Supplementary Table 1; see supplementary material online at www.gastrojournal.org). Specifically, the proportion of all arrayed BACs showing chromosomal alteration was 44% in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ tumors compared to 28% in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ tumors (Figure 4D, P ⫽ .02). An interspecies comparison of the most common chromosomal alterations in our mouse model (detected in ⬎68% of the tumors) revealed that syntenic regions for 5 out of 6 of these lesions are commonly affected in human HCC (Supplementary Table 2; see supplementary material online at www.gastrojournal.org) indicating that comparable chromosomal regions are affected through CIN in our mouse model and in human HCCs. The increase in CIN in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice did not affect all chromosomes to the same extent (Figure 4C). A common chromosomal alteration in mTERC⫺/⫺, p53⫺/⫺, HBs⫹, and mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice was the gain
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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.
in copy number of chromosome 15, overlapping the c-myc gene locus, which is also very commonly amplified in human HCC (syntenic region on chromosome 8).5,9,37 To confirm the array-comparative genomic hybridization results, we performed FISH analysis for gain in copy number of mouse chromosomes 15 and 17. These experiments showed a significant increase in copy number of chromosome 17 in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC (Supplementary Figure 3A; see supplementary material online at www. gastrojournal.org; P ⫽ .001), but similar gains of chromosome 15 in HCC of both cohorts (Supplementary Figure 3B and C; see supplementary material online at www.gastrojournal.org, P ⫽ .394). These results suggested that the increase in aneuploidy in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC may predominantly affect random chromosomal alterations rather than HCC-specific chromosomal alteration associated with the development of HCC in our mouse model.
Telomerase Activation Limits DNA Damage Accumulation, Cell Cycle Arrest, and Apoptosis in p53 Mutant Tumor With Short Telomeres To evaluate mechanisms that allow tumor progression in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC we analyzed telomerase activity, DNA damage foci formation at dysfunctional telomeres, tumor cell proliferation, and apoptosis in liver tumors of both cohorts. Measurement of telomerase activity by TRAP-assay revealed an activation of telomerase during hepatocarcinogenesis in the vast majority of mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC (18 out of 21 tumors analyzed; Figure 5A) similar to the situation in human hepatocarcinogenesis.19,20 The formation of DNA damage foci is the earliest detectable sign of DNA damage signal induction in response to telomere dysfunction.23 An analysis of ␥H2AXpositive DNA damage foci did not reveal a significant
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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 ␥H2AX-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 ␥H2AX-foci in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC. (C, D) Representative photographs showing ␥H2AX-foci in (C) mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC, and (D) mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC; arrows point to ␥H2AX-foci (magnification bar: 100 m; inlet magnification bar: 20 m). (E) Representative photographs on telomere/␥H2AX immuno-FISH: Upper panel: ␥H2AX-immunofluorescence; middle panel: telomere-FISH; bottom panel: overlay, arrows point to the colocalization of telomere signals and ␥H2AX-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 m). (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 m).
difference in the prevalence of DNA damage foci in nuclei of chronically damaged, nontransformed liver of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5B–D). A sharp increase in DNA damage foci was detected in HCC of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5B–D). These results indicated that telomerase activity prevented an accumulation of DNA damage in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC. Immuno-FISH revealed a colocalization of ␥H2AX foci with telomeric DNA in 25.8% of the damage foci in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ tumors and 25.4% of the DNA damage foci in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ tumors (Figure 5E, P ⫽ .96) indicating that low levels of telomere dysfunction were also present in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ tumors. The incidence of nontelomere-associated DNA damage foci suggested that secondary events may contribute to an accumulation of DNA damage in HCCs possibly involving fusion– bridge– breakage cycles of telomere dysfunctional tumor cells. Alternatively, the complete loss of telomeric sequences at dysfunctional telomeres may have limited the detection of colocalization in these experiments. TUNEL-staining revealed significantly increased rates of apoptosis in HCCs of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5F and G, P ⫽ .029). In addition, the level of tumor cell proliferation, as determined by IHC for PCNA expression, was significantly suppressed in HCCs of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5H and I, P ⫽ .019). Together, these results indicated that p53-independent tumor suppression in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice involved an activation of apoptosis and cell cycle checkpoints.
Discussion The current study provides experimental evidence that telomerase deletion inhibits progression of p53 mutant tumors with dysfunctional telomeres during in vivo hepatocarcinogenesis in chronically damaged liver. In this genetic context, telomerase negative HCC accumulates aneuploidy and DNA damage resulting in p53independent tumor suppression (see model in Figure 6). The study suggests that telomerase can stabilize telomere function during in vivo hepatocarcinogenesis without rescuing telomere length or the prevalence of critically short telomeres. These data appear to be in line with cell culture-based studies that have shown that telomerase gene delivery allows survival of SV40 large T-antigen transduced fibroblasts from crisis without leading to a net elongation of telomeres.38 It has been proposed that telomerase itself might have a capping function at dysfunctional telomeres.38 The current data on in vivo carcinogenesis extend these cell culture-based findings and indicate that telomerase stabilizes telomeres and
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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.
limits the accumulation of DNA damage and aneuploidy during in vivo formation of primary, p53 mutant tumors. The study indicates that the accumulation of aneuploidy and DNA damage can lead to an activation of p53-independent cell cycle arrest and apoptosis, thereby limiting in vivo progression of telomerase negative tumor with short telomeres. An understanding of these p53independent pathways of tumor suppression appears to be relevant for human cancer. It seems possible that an alteration of these p53-independent pathways could allow limited progression of genetically unstable, p53-mutant carcinoma cells, thus facilitating the consequent reactivation of telomerase. The study results appear to be of clinical relevance for therapeutic approaches aiming to treat cancer by telomerase inhibition or telomere destabilization. There is a debate whether p53 mutant tumors might be resistant to therapeutic approaches targeting telomeres because some checkpoints in response to telomere dysfunction depend on p53-function including the senescence checkpoint.21,22,24,39 In addition, studies in mTERC⫺/⫺ mice have linked tumor suppression in response to telomere dysfunction to p53-pathway activation.12,14,18 The current study provides first evidence that p53-independent tumor suppressor pathways impair the progression of telomerase deficient tumors with dysfunctional telomeres during primary in vivo carcinogenesis. Altogether, this study shows a critical role of telomerase for progression of p53 mutant HCCs in primary mouse model relevant to human hepatocarcinogenesis. These data indicate that telomerase inhibitors could be effective for treatment of primary HCC harboring telomere dysfunction and p53 pathway dysfunction.
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Appendix 17.
Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1053/ j.gastro.2007.01.045. References
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34. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;362:1907–1917. 35. Wiemann SU, Satyanarayana A, Buer J, Kamino K, Manns MP, Rudolph KL. Contrasting effects of telomere shortening on organ homeostasis, tumor suppression, and survival during chronic liver damage. Oncogene 2005;24:1501–1509. 36. Frith CH, Wards JM, Turusoz VS. Tumours of the liver. In: Tursov V, Mohr U, Pathology of tumours in laboratory animals, Vol. 2—tumours of the mouse. Lyon, France: IARC, 1994:223–243. 37. Moinzadeh P, Breuhahn K, Stutzer H, Schirmacher P. Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade--results of an explorative CGH meta-analysis. Br J Cancer 2005;92:935–941. 38. Zhu J, Wang H, Bishop JM, Blackburn EH. Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. Proc Natl Acad Sci U S A 1999;96:3723–3728. 39. Lechel A, Satyanarayana A, Ju Z, Plentz RR, Schaetzlein S, Rudolph C, Wilkens L, Wiemann SU, Saretzki G, Malek NP, Manns
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MP, Buer J, Rudolph KL. The cellular level of telomere dysfunction determines induction of senescence or apoptosis in vivo. EMBO Rep 2005;6:275–281.
Received October 2, 2006. Accepted December 27, 2006. Address requests for reprints to: K. Lenhard Rudolph, MD, Department of Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. e-mail: [email protected]; fax: (49) 511-532-6998. Supported by the Deutsche Forschungsgemeinschaft (Heisenberg Professorship to KLR: Ru 745/8-1 and clinical research group on hepatocarcinogenesis: KFO119), the Deutsche Krebshilfe e.V. (102236-Ru 2), the Roggenbuck-Stiftung, the Wilhelm-Sander-Stiftung, and the Fritz-Thyssen Stiftung. We thank Anton Berns for providing the conditional p53 knockout mice and Nabeel Bardeesy for critical discussion.
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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,储 STEFAN KUBICKA,* PETER SCHIRMACHER,¶ JOS JONKERS,‡ and K. LENHARD RUDOLPH*
Background & Aims: During early stages of carcinogenesis most human epithelial cancers including hepatocellular carcinoma (HCC) have been observed to transit through a “crisis” stage characterized by telomere shortening, loss of p53 checkpoint function, and a sharp increase in aneuploidy. The function of telomerase during in vivo hepatocarcinogenesis has not been studied in this genetic context. Methods: Here we generated a mouse model in which HCC was induced by chronic organ damage (HBs-AG transgene) in the presence of telomere shortening and p53 deletion. Tumor development was analyzed in lategeneration telomerase knockout mice (mTERCⴚ/ⴚ) and littermates, genetically rescued for telomerase gene expression (mTERCⴙ/ⴚ). Results: The formation of HCCs was strongly suppressed in mTERCⴚ/ⴚ mice compared to mTERCⴙ/ⴚ siblings correlating with reduced rates of tumor cell proliferation and elevated rates of tumor cell apoptosis. Although the prevalence of short telomeres was similar in chronically damaged liver of both cohorts, mTERCⴚ/ⴚ HCC developed increased levels of DNA damage and aneuploidy compared to mTERCⴙ/ⴚ HCC. Conclusions: This study provides direct evidence that telomerase is a critical component for in vivo progression of p53 mutant HCC with short telomeres in the chronically damaged liver. In this molecular context, telomerase limits the accumulation of telomere dysfunction, the evolution of excessive aneuploidy, and the activation of p53-independent checkpoints suppressing hepatocarcinogenesis.
H
epatocellular carcinoma (HCC) is one of the most common solid tumors in humans.1 The risk of HCC development is low in healthy liver and early stages of chronic liver disease but sharply increases at the cirrhosis stage.1 Frequently found, molecular alterations that are associated with HCC development are telomere shortening,2– 4 loss of p53 checkpoint function,5,6 and the evolution of aneuploidy.7–9 Studies on telomerase deficient (mTERC⫺/⫺) mice have shown that telomere shortening has a dual role in cancer
initiation and progression. These studies have demonstrated that telomere shortening inhibited in vivo progression of tumors,10 –14 but increased the rate of tumor initiation by induction of chromosomal fusions resulting in anaphase bridges, chromosome breakage, and aneuploidy.12,15 Impaired tumor progression in telomere dysfunctional mice was associated with an activation of p53-dependent DNA-damage responses.12,14 In contrast, mutation of p53 alleviated tumor suppression and accelerated chromosomal instability (CIN) and cancer initiation in telomere dysfunctional mice.16 –18 The high incidence of telomere shortening, loss of p53 checkpoint function, and aneuploidy in the vast majority of human HCC2–9 indicates that this molecular context is highly relevant for human hepatocarcinogenesis. In addition to these alterations, most human HCC (⬎90%) reactivate telomerase during cancer progression.5,19,20 The functional role of telomerase for in vivo progression of primary, p53 mutant tumors with dysfunctional telomeres is unknown and needs to be addressed given the high frequency of p53 mutations in human cancer including HCC.5,6 Moreover, this question appears of clinical relevance to test whether telomerase-inhibition represents a promising therapeutic approach for the treatment of p53-mutant HCC. Here we generated a mouse model of hepatocarcinogenesis that recapitulates several molecular characteristics, which are associated with the development of human HCC: telomere shortening,2– 4,21–24 loss of p53pathway function,5,6,21–24 and aneuploidy.7–9,21–24 We analyzed the functional role of telomerase in late-generation telomerase knockout mice and littermates that were genetically rescued for telomerase gene expression. The
Abbreviations used in this paper: Ad-Cre, adenovirus expressing Cre-recombinase; CIN, chromosomal instability; FISH, fluorescence in situ hybridization; HCC, hepatocellular carcinoma; PCNA, proliferating cell nuclear antigen; TFI, telomere fluorescence intensities; TRAP, telomere repeat amplification protocol. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.01.045
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*Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Germany; ‡Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands; §Department of Molecular Pathology, Medical School Hannover, Germany; 储Department of Pathology, Medical School Hannover, Germany; ¶Institute of Pathology, University of Heidelberg, Germany
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study provides experimental evidence that telomerase deletion impairs in vivo progression of primary, p53-mutant HCC with short telomeres. The study indicates that in this genetic context telomerase deficient tumors accumulate DNA damage and aneuploidy, which leads to an activation of p53-independent tumor suppressor checkpoints. These data suggest that telomerase inhibition could show antitumor activity in primary HCCs haboring defects in the p53 signaling pathway.
Materials and Methods Mouse Model of Chronic Liver Damage, Telomere Shortening, and Loss of P53 Checkpoint Function in Liver
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We used the Cre-loxP system to achieve a liver specific deletion of the Trp53 gene (conditional p53 mouse).25 In this mouse loxP sites flank Exon 2–10 of the Trp53 gene. When the enzyme Cre-recombinase is expressed, this region of the p53 gene will be deleted, which leads to loss of p53 function.25 Heterozygous conditional p53 mice (Trp53F2–10/⫹) were crossed with mTERC⫹/⫺ mice.26 Intercrosses of mTERC⫹/⫺, Trp53F2–10/⫹ yielded in G1 mTERC⫺/⫺, Trp53F2–10/F2–10 mice, in which both alleles of the murine p53 gene contained loxP sites. To induce telomere shortening, these mice were crossed through successive generations to produce late generation (G3) mTERC⫺/⫺, Trp53F2–10/F2–10 mice (Figure 1). To induce chronic liver damage, mTERC ⫹/⫺ , Trp53F2–10/F2–10 were crossed with transgenic mice expressing the hepatitis B virus surface antigen (HBs⫹)
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.
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under the liver-specific albumin promoter (C57BL/ 6J-Tg(Alb1HBV)44Bri/J; Jackson Laboratories, Bar Harbor, ME)27 to generate mTERC⫹/⫺, Trp53F2–10/F2–10, HBs⫹ mice. These mice were finally crossed with G3 mTERC⫺/⫺, Trp53F2–10/F2–10 mice to generate the experimental groups of mouse siblings that were in the 4th generation telomerase knockout (mTERC⫺/⫺, Trp53F2–10/F2–10, HBs⫹; n ⫽ 17) or genetically rescued for telomerase gene expression (mTERC⫹/⫺, Trp53F2–10/F2–10, HBs⫹; n ⫽ 15) (Figure 1).28 The mice were infected with an adenovirus expressing Crerecombinase (Ad-Cre),29 which led to recombination of the loxP sites in the Trp53 gene locus (see above) resulting in a liver specific deletion of the Trp53 gene (Trp53⌬2–10/⌬2–10). All mice were in a C57Bl/6J background.
Amplification and Purification of Ad-Cre Ad-Cre virus was propagated in 293T cells and prepared according to previously described cesium chloride gradient purification.29 The titer was determined by the plaque assay and mice were infected with 0.8 ⫻ 1010 PFU of Ad-Cre at 4 months of age.
Dissection of Macroscopic Tumors and Histologic Analysis Mice were humanely sacrificed at an age of 12 to 15 months (mean age in mTERC⫺/⫺ p53⫺/⫺ HBs⫹ mice: 12.8, mean age in mTERC⫹/⫺ p53⫺/⫺ HBs⫹ mice: 13.6 months). Macroscopically tumors (⬎2 mm in diameter) were cut in half; 1/2f was frozen down (for DNA preparation), 1/2 was fixed in 4% neutral-buffered formaldehyde and processed further according to histologic routine protocols and embedded in paraffin. Sections (4 m) were stained with H&E, and the tumor types were classified using the following criteria. Microscopic foci. Lesions 0.5–2 mm in diameter, with morphologically clonal appearing growth pattern, sometimes slight marginal compression and basophilic, clear cell or eosinophilic cytomorphology. Macroscopic nodule. Nodules ⬎2 mm in size representing macroscopic nodules (“adenomas”) not showing characteristic markers of HCCs. HCC. Hepatocellular carcinoma defined by either histologic features (trabecular disorganization, more than 2 cell-thick trabecula, pseudoglandular structures, obvious invasion) or cytologic changes (moderate to severe atypia). Grading of HCC was performed on the basis of cytologic criteria on a G1–3 scale reflecting increasing cellular atypia.
Analysis of Telomerase Activity by the Telomere Repeat Amplification Protocol Assay The telomere repeat amplification protocol (TRAP) was performed on snap-frozen tissues as reported.30 Briefly, TRAP reactions were incubated for 30 minutes at 30°C for telomerase extension using 32P␥ATP (Hartmann Diagnostics, Germany) labeled telo-
merase substrate (TS⫺) primer followed by a PCR reaction (94°C 30 seconds, 60°C 30 seconds, 30 repeats). PCR products were size-fractioned on 12% nondenaturing polyacrylamide gel, and visualized after drying the gel on a phosphor imager (Amersham Biosciences, Arlington Heights, IL).
Detection of Deletion of Exons 2–10 of Trp53 The DNA copy number of Trp53⌬2–10 and was analyzed in triplicate by real-time PCR according to standard protocols. The following primers were used for detection of the Trp53⌬2–10 allele: Trp53A 5=-CAC AAA AAC AGG TTA AAC CCA G-3= and Trp53E 5=-CCA TGA GAC AGG GTC TTG CT-3= and for the Trp53F2–10 allele: Trp53A 5=-CAC AAA AAC AGG TTA AAC CCA G-3=, Trp53B 5=-AGC ACA TAG GAG GCA GAG AC-3=. Amplifications were performed using the Applied Biosystems (Foster City, CA) 7300 Real-Time PCR System under the following conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 40 seconds. We determined the relative quantities of Trp53⌬2–10 by generating a standard curve using mixtures of genomic DNA from Trp53⌬2–10 and Trp53F2–10 mouse embryo fibroblasts in fixed ratios (0:100, 20:80, 40:60, 60:40, 80:20, and 90:10). From the quantitative reverse-transcriptase polymer chain reaction (PCR) results of these samples we calculated the ratio (⌬Ct value of Trp53⌬2–10/⌬Ct value of Trp53F2–10) to generate standard curves. Trp53F2–10
Quantitative Fluorescence In Situ Hybridization for Telomere Length Quantitative fluorescence in situ hybridization (Q-FISH) was performed on 5-m paraffin sections. After unmasking, the slides were incubated in Pepsine solution for 10 minutes at 37°C (100 mg Pepsine; 84L HCl 37% up to 100 mL H2O) and washed in phosphate-buffered saline. The hybridization mix (10 mmol/L Tris pH 7.2; MgCl2 buffer: 7.02 mmol/L Na2HPO3, 2.14 mmol/L MgCl2, 0.77 mmol/L citric acid; 70% deionized formamide; 0.5 g/mL PNA probe 5=-Cy3-CCC TAA CCC TAA CCC TAA-3= Applied Biosystems; 0.25% Roche blocking reagent, Indianapolis, IN) was added to the sections, covered with coverslips, and denatured at 80°C for 3 minutes followed by 2 hours of incubation in the dark. Slides were incubated in 70% formamide, 10 mmol/L Tris (pH 7.2), 0.1% BSA 2 times for 20 minutes, and washed 3 times in TBSTween (0.2%). The relative telomere length was measured by the telomere fluorescence intensity by TFL analysis software program.31
Array Comparative Genomic Hybridization Profiling Using BAC Arrays Genomic DNA was extracted from mouse tissues by proteinase K digestion and organic extraction, and
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digested o/n with HaeIII. Digested genomic DNA was labeled with Cy3 or Cy5 using a ULS aRNA Fluorescent Labeling Kit (Kreatech Biotechnology, Amsterdam, the Netherlands) according to the manufacturer’s protocol. Cy5–ULS-labeled tumor DNA was mixed with Cy3–ULSlabeled reference DNA (isolated from the spleen of the same mouse) and hybridized to a Mouse 3K BAC array as described previously.32 All experiments were performed in fluor-reversed pairs of 2-color hybridizations. Mouse 3K BAC arrays were printed by the NKI Microarray Facility (Amsterdam, The Netherlands). After hybridization, arrays were imaged using an Agilent Scanner and the data were processed using ImaGene software. Each array from the pair of fluor-reversed hybridizations contained duplicate spots, allowing weighted averages, errors, and confidence levels for each data point to be computed from quadruple measurements according to the Rosetta Error Model.33 All fluorescence intensities were converted to log2 values to weight gains and losses equally.
Immunohistochemistry Proliferating cell nuclear antigen (PCNA) staining was performed at real time for 2 hours (PCNA Ab-1, Oncogene Science, Uniondale, NY; diluted 1:150 in phosphate-buffered saline) followed by a 1-hour incubation with a Cy3-labeled rabbit antimouse IgG secondary antibody (1:300 in phosphate-buffered saline; Zymed, San Francisco, CA). The number of PCNA positive cells was counted randomly at low-power fields (200⫻). The rate of apoptosis was determined by TUNEL assay (In situ cell death detection kit, Roche, Mannheim, Germany). The numbers of apoptotic and nonapoptotic cells were counted in 20 low-power fields (200⫻) and the percentage of apoptotic cells was calculated. For ␥H2AX-staining sections were blocked with M.O.M blocking reagent (Vector Labs, Burlingame, CA) for 1 hour and then incubated with a mouse anti␥H2AX antibody (Upstate, Lake Placid, NY) overnight at 4°C. The subsequent steps to detect ␥H2AX signal was performed using M.O.M.™ Immunodetection Kit (Vector Laboratories) according to manufacturer’s recommendations. Double staining of telomere and ␥H2AX signals was performed by using the telomere quantitative FISH protocol (see above) followed by ␥H2AX immunofluorescence (see above) using a Cy3-labeled rabbit antimouse IgG secondary antibody (1:500; Zymed).
Statistical Programs The Mann–Whitney U test and T test were used to calculate the statistical significance and standard deviations were calculated by using Graphpad Instat, Graphpad Prism, SPSS, and Microsoft Excel software.
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Results Mouse Model of Hepatocarcinogenesis in the Context of p53 Mutation, Short Telomeres, and Chronic Organ Damage To analyze the impact of telomerase on hepatocarcinogenesis in the presence of the p53-mutation, short telomeres, and chronic organ damage, we crossed conditional p53 knockout mice (Trp53F2–10/F2–10)25 with mTERC⫺/⫺ mice26 through successive generations followed by an intercross of late-generation (G3) mTERC⫺/⫺, Trp53F2–10/F2–10 mice harboring critically short telomeres with mTERC⫹/⫺, Trp53F2–10/F2–10 mice. This crossproduced littermate offspring that was either genetically rescued for telomerase gene expression (iF1 mTERC⫹/⫺ ⫽ mTERC⫹/⫺) or lacked telomerase gene expression (iF1 G4mTERC⫺/⫺ ⫽ mTERC⫺/⫺) (Figure 1).28 Hepatic deletion of Trp53 (Trp53⌬2–10/⌬2–10 ⫽ p53⫺/⫺) was achieved in both groups by adenovirus mediated Cre-recombinase expression (Supplementary Figure 1; see Supplemental material online at www. gastrojournal.org).29 In addition, all mice expressed the hepatitis B surface protein (HBs⫹) as a transgene under the liver specific albumin promoter, which promotes chronic liver damage and hepatocarcinogenesis in 12- to 15-month-old mice,27 thus closely resembling the pathogenesis of human HCC.1,34 Hepatocarcinogenesis was followed in cohorts of mTERC⫺/⫺, p53⫺/⫺, HBs⫹, and mTERC⫹/⫺, p53⫺/⫺, HBs⫹ siblings. Regarding p53 gene status, we confirmed that virtually all HCCs (48 of 51) from both cohorts showed Cremediated deletion of Trp53 using Southern blot and quantitative PCR analyses (Figure 2A, Supplementary Figure 1; see Supplementary Figure 1 online at www. gastrojournal.org). The proportional loss of wild-type Trp53 sequences determined for individual tumors correlated well with the percentage of stromal and immune cells within the tumors (Figure 2B). Telomere length and numbers of critically short telomeres were determined by quantitative FISH in hepatocytes of nontumorous liver and HCCs. Nontransformed hepatocytes of chronically damaged liver of 12- to 15-
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month-old mTERC⫹/⫺, p53⫺/⫺, HBs⫹, and mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice had shorter telomeres (lower telomere fluorescence intensity ⫽ telomere fluorescence intensities [TFI]) compared to hepatocytes from 12- to 15-monthold C57Bl/6J wild-type mice (Figure 2C–E). Previous studies have established that the in vivo phenotypes of telomere dysfunction depend on the prevalence of critically short telomeres rather than mean telomere length.29 To evaluate the number of critically short telomeres we determined the frequency of telomeres with the lowest detectable fluorescence intensity (telomere fluorescence intensity ⬍200 units) in interphase nuclei. This analysis can not detect telomere-free ends, but represents the best possible method to evaluate the frequency of critical telomere shortening in tissues. In quiescent liver (HBs⫺) a higher incidence of critically short telomeres was detected in mTERC⫺/⫺, p53⫺/⫺, HBs⫺ compared with mTERC⫺/⫺, p53⫺/⫺, HBs⫺ littermates or C57Bl/6J mice (Figure 2F). However, in chronically damaged liver (HBs⫹) a similarly high incidence of critically short telomeres was present in nontumorous liver of 12- to 15month-old mTERC⫹/⫺, p53⫺/⫺, HBs⫹, and mTERC⫺/⫺, p53⫺/⫺, HBs⫹ siblings (Figure 2F). Both groups developed HCC at an age of 12 to 15 month (see below). The incidence of critically short telomeres was similarly high in HCC of both cohorts (Figure 2F).
Telomerase Deletion Inhibits Progression of p53-Mutant HCC With Short Telomeres in Chronically Damaged Liver The number of macroscopically visible liver tumors in 12- to 15-month-old mice was strongly 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, Figure 3A). Histologic analysis revealed that 25.2% of all macroscopic tumors were judged to represent macroscopic nodules (adenomas) and 59.3% of the tumors were HCCs (histologic criteria; see Materials and Methods section). HBs transgenic mice develop HCC induced by chronic liver damage.27 In previous studies, our group has shown that
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
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.
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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.
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the incidence of HCC in 12- to 15-month-old HBs transgenic mice on a C57Bl/6J background was 44%.35 In our current study, the incidence of HCC mTERC⫹/⫺ p53⫺/⫺, HBs⫹ mice was higher (see below) indicating that p53 deletion cooperated with chronic liver damage to induce HCC. The incidence and the average numbers of both macroscopic nodules and HCC were suppressed in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 3B). Specifically, 23.5% of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice displayed HCCs compared with 80% of mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (P ⫽ .0008). In total, 13 HCCs were diagnosed in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared with 60 HCCs in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (P ⫽ .002). Importantly, HCC numbers remained significantly suppressed in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice after sorting out Trp53 wild-type HCCs (Figure 2A) from the total number of HCCs in the mTERC⫹/⫺, p53⫺/⫺, HBs⫹ group (Mann–Whitney test: P ⫽ .006). The differentiation of HCCs was assessed according to previously described standards36 and did not reveal significant differences between both cohorts (Supplementary Figure 2A–D; see supplementary material online at www.gastrojournal.org). Histopathologic analysis of nontumorous liver specimens revealed a similar number of premalignant, microscopic foci in both experimental cohorts (Supplementary Figure 2E, see supplementary material online at www.gastrojournal.org; P ⫽ .255). These data indicated that telomerase deficiency impaired the progression of hepatocarcinogenesis in the context of chronic organ damage, short telomeres, and mutant p53 after the stage of microscopic focus formation.
Telomerase Limits Aneuploidy in p53 Mutant HCC With Short Telomeres In primary human cells and mTERC⫺/⫺ mice, telomere dysfunction and loss of p53 checkpoint function cooperate to induce CIN.16,17,22,23 Comparative genomic hybridization array on macroscopic liver tumors with confirmed Trp53 deletion (Figure 2A) revealed chromosomal gains and losses in all liver tumors examined (n ⫽ 16, Supplementary Table 1; see supplementary material online at www.gastrojournal.org). However, the overall level of aneuploidy was significantly higher in liver tumors of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ (n ⫽ 6 tumors) mice compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (n ⫽ 10 tumors, Figure 4A–C, Supplementary Table 1; see supplementary material online at www.gastrojournal.org). Specifically, the proportion of all arrayed BACs showing chromosomal alteration was 44% in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ tumors compared to 28% in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ tumors (Figure 4D, P ⫽ .02). An interspecies comparison of the most common chromosomal alterations in our mouse model (detected in ⬎68% of the tumors) revealed that syntenic regions for 5 out of 6 of these lesions are commonly affected in human HCC (Supplementary Table 2; see supplementary material online at www.gastrojournal.org) indicating that comparable chromosomal regions are affected through CIN in our mouse model and in human HCCs. The increase in CIN in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice did not affect all chromosomes to the same extent (Figure 4C). A common chromosomal alteration in mTERC⫺/⫺, p53⫺/⫺, HBs⫹, and mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice was the gain
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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.
in copy number of chromosome 15, overlapping the c-myc gene locus, which is also very commonly amplified in human HCC (syntenic region on chromosome 8).5,9,37 To confirm the array-comparative genomic hybridization results, we performed FISH analysis for gain in copy number of mouse chromosomes 15 and 17. These experiments showed a significant increase in copy number of chromosome 17 in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC (Supplementary Figure 3A; see supplementary material online at www. gastrojournal.org; P ⫽ .001), but similar gains of chromosome 15 in HCC of both cohorts (Supplementary Figure 3B and C; see supplementary material online at www.gastrojournal.org, P ⫽ .394). These results suggested that the increase in aneuploidy in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC may predominantly affect random chromosomal alterations rather than HCC-specific chromosomal alteration associated with the development of HCC in our mouse model.
Telomerase Activation Limits DNA Damage Accumulation, Cell Cycle Arrest, and Apoptosis in p53 Mutant Tumor With Short Telomeres To evaluate mechanisms that allow tumor progression in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC we analyzed telomerase activity, DNA damage foci formation at dysfunctional telomeres, tumor cell proliferation, and apoptosis in liver tumors of both cohorts. Measurement of telomerase activity by TRAP-assay revealed an activation of telomerase during hepatocarcinogenesis in the vast majority of mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC (18 out of 21 tumors analyzed; Figure 5A) similar to the situation in human hepatocarcinogenesis.19,20 The formation of DNA damage foci is the earliest detectable sign of DNA damage signal induction in response to telomere dysfunction.23 An analysis of ␥H2AXpositive DNA damage foci did not reveal a significant
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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 ␥H2AX-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 ␥H2AX-foci in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC. (C, D) Representative photographs showing ␥H2AX-foci in (C) mTERC⫹/⫺, p53⫺/⫺, HBs⫹ HCC, and (D) mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC; arrows point to ␥H2AX-foci (magnification bar: 100 m; inlet magnification bar: 20 m). (E) Representative photographs on telomere/␥H2AX immuno-FISH: Upper panel: ␥H2AX-immunofluorescence; middle panel: telomere-FISH; bottom panel: overlay, arrows point to the colocalization of telomere signals and ␥H2AX-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 m). (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 m).
difference in the prevalence of DNA damage foci in nuclei of chronically damaged, nontransformed liver of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5B–D). A sharp increase in DNA damage foci was detected in HCC of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5B–D). These results indicated that telomerase activity prevented an accumulation of DNA damage in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ compared with mTERC⫺/⫺, p53⫺/⫺, HBs⫹ HCC. Immuno-FISH revealed a colocalization of ␥H2AX foci with telomeric DNA in 25.8% of the damage foci in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ tumors and 25.4% of the DNA damage foci in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ tumors (Figure 5E, P ⫽ .96) indicating that low levels of telomere dysfunction were also present in mTERC⫹/⫺, p53⫺/⫺, HBs⫹ tumors. The incidence of nontelomere-associated DNA damage foci suggested that secondary events may contribute to an accumulation of DNA damage in HCCs possibly involving fusion– bridge– breakage cycles of telomere dysfunctional tumor cells. Alternatively, the complete loss of telomeric sequences at dysfunctional telomeres may have limited the detection of colocalization in these experiments. TUNEL-staining revealed significantly increased rates of apoptosis in HCCs of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared with mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5F and G, P ⫽ .029). In addition, the level of tumor cell proliferation, as determined by IHC for PCNA expression, was significantly suppressed in HCCs of mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice compared to mTERC⫹/⫺, p53⫺/⫺, HBs⫹ mice (Figure 5H and I, P ⫽ .019). Together, these results indicated that p53-independent tumor suppression in mTERC⫺/⫺, p53⫺/⫺, HBs⫹ mice involved an activation of apoptosis and cell cycle checkpoints.
Discussion The current study provides experimental evidence that telomerase deletion inhibits progression of p53 mutant tumors with dysfunctional telomeres during in vivo hepatocarcinogenesis in chronically damaged liver. In this genetic context, telomerase negative HCC accumulates aneuploidy and DNA damage resulting in p53independent tumor suppression (see model in Figure 6). The study suggests that telomerase can stabilize telomere function during in vivo hepatocarcinogenesis without rescuing telomere length or the prevalence of critically short telomeres. These data appear to be in line with cell culture-based studies that have shown that telomerase gene delivery allows survival of SV40 large T-antigen transduced fibroblasts from crisis without leading to a net elongation of telomeres.38 It has been proposed that telomerase itself might have a capping function at dysfunctional telomeres.38 The current data on in vivo carcinogenesis extend these cell culture-based findings and indicate that telomerase stabilizes telomeres and
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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.
limits the accumulation of DNA damage and aneuploidy during in vivo formation of primary, p53 mutant tumors. The study indicates that the accumulation of aneuploidy and DNA damage can lead to an activation of p53-independent cell cycle arrest and apoptosis, thereby limiting in vivo progression of telomerase negative tumor with short telomeres. An understanding of these p53independent pathways of tumor suppression appears to be relevant for human cancer. It seems possible that an alteration of these p53-independent pathways could allow limited progression of genetically unstable, p53-mutant carcinoma cells, thus facilitating the consequent reactivation of telomerase. The study results appear to be of clinical relevance for therapeutic approaches aiming to treat cancer by telomerase inhibition or telomere destabilization. There is a debate whether p53 mutant tumors might be resistant to therapeutic approaches targeting telomeres because some checkpoints in response to telomere dysfunction depend on p53-function including the senescence checkpoint.21,22,24,39 In addition, studies in mTERC⫺/⫺ mice have linked tumor suppression in response to telomere dysfunction to p53-pathway activation.12,14,18 The current study provides first evidence that p53-independent tumor suppressor pathways impair the progression of telomerase deficient tumors with dysfunctional telomeres during primary in vivo carcinogenesis. Altogether, this study shows a critical role of telomerase for progression of p53 mutant HCCs in primary mouse model relevant to human hepatocarcinogenesis. These data indicate that telomerase inhibitors could be effective for treatment of primary HCC harboring telomere dysfunction and p53 pathway dysfunction.
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Appendix 17.
Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1053/ j.gastro.2007.01.045. References
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Received October 2, 2006. Accepted December 27, 2006. Address requests for reprints to: K. Lenhard Rudolph, MD, Department of Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. e-mail: [email protected]; fax: (49) 511-532-6998. Supported by the Deutsche Forschungsgemeinschaft (Heisenberg Professorship to KLR: Ru 745/8-1 and clinical research group on hepatocarcinogenesis: KFO119), the Deutsche Krebshilfe e.V. (102236-Ru 2), the Roggenbuck-Stiftung, the Wilhelm-Sander-Stiftung, and the Fritz-Thyssen Stiftung. We thank Anton Berns for providing the conditional p53 knockout mice and Nabeel Bardeesy for critical discussion.
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