Telomerase Deletion Limits Progression of p53-Mutant Hepatocellular Carcinoma With Short Telomeres in Chronic Liver Disease
2007; Elsevier BV; Volume: 132; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2007.01.045
ISSN1528-0012
AutoresAndré Lechel, Henne Holstege, Yvonne Begus, Andrea Schienke, Kenji Kamino, Ulrich Lehmann, Stefan Kubicka, Peter Schirmacher, Jos Jonkers, K. Lenhard Rudolph,
Tópico(s)Mitochondrial Function and Pathology
ResumoBackground & 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 late-generation telomerase knockout mice (mTERC−/−) and littermates, genetically rescued for telomerase gene expression (mTERC+/−). Results: The formation of HCCs was strongly suppressed in mTERC−/− mice compared to mTERC+/− 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, mTERC−/− HCC developed increased levels of DNA damage and aneuploidy compared to mTERC+/− 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. 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 late-generation telomerase knockout mice (mTERC−/−) and littermates, genetically rescued for telomerase gene expression (mTERC+/−). Results: The formation of HCCs was strongly suppressed in mTERC−/− mice compared to mTERC+/− 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, mTERC−/− HCC developed increased levels of DNA damage and aneuploidy compared to mTERC+/− 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. Hepatocellular carcinoma (HCC) is one of the most common solid tumors in humans.1Lotze M.T. Flickinger J.C. Carr B.I. Hepatobiliary neoplasm.in: DeVita V.T. Hellmann S. Rosenberg S.A. Cancer: principles and practice of oncology. 4th ed. Lippincott, Philadelphia, PA1993: 883-887Google Scholar The risk of HCC development is low in healthy liver and early stages of chronic liver disease but sharply increases at the cirrhosis stage.1Lotze M.T. Flickinger J.C. Carr B.I. Hepatobiliary neoplasm.in: DeVita V.T. Hellmann S. Rosenberg S.A. Cancer: principles and practice of oncology. 4th ed. Lippincott, Philadelphia, PA1993: 883-887Google Scholar Frequently found, molecular alterations that are associated with HCC development are telomere shortening,2Ohashi K. Tsutsumi M. Nakajima Y. Kobitsu K. Nakano H. Konishi Y. Telomere changes in human hepatocellular carcinomas and hepatitis virus infected noncancerous livers.Cancer. 1996; 77: 1747-1751Crossref PubMed Google Scholar, 3Plentz R.R. Caselitz M. Bleck J.S. Gebel M. Flemming P. Kubicka S. Manns M.P. Rudolph K.L. Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatocellular carcinoma.Hepatology. 2004; 40: 80-86Crossref PubMed Scopus (108) Google Scholar, 4Plentz R.R. Schlegelberger B. Flemming P. Manns M.P. Rudolph K.L. Wilkens L. Telomere shortening correlates with increasing aneuploidy of chromosome 8 on a cellular level in primary human hepatocellular carcinoma.Hepatology. 2005; 42: 522-526Crossref PubMed Scopus (54) Google Scholar loss of p53 checkpoint function,5Thorgeirsson S.S. Grisham J.W. Molecular pathogenesis of human hepatocellular carcinoma.Nat Genet. 2002; 31: 339-346Crossref PubMed Scopus (1289) Google Scholar, 6Tannapfel A. Busse C. Weinans L. Benicke M. Katalinic A. Geissler F. Hauss J. Wittekind C. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas.Oncogene. 2001; 20: 7104-7109Crossref PubMed Scopus (123) Google Scholar and the evolution of aneuploidy.7Nishida N. Nishimura T. Ito T. Komeda T. Fukuda Y. Nakao K. Chromosomal instability and human hepatocarcinogenesis.Histol Histopathol. 2003; 18: 897-909PubMed Google Scholar, 8Hashimoto K. Mori N. Tamesa T. Okada T. Kawauchi S. Oga A. Furuya T. Tangoku A. Oka M. Sasaki K. Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH.Mod Pathol. 2004; 17: 617-622Crossref PubMed Scopus (89) Google Scholar, 9Wilkens L. Flemming P. Gebel M. Bleck J. Terkamp C. Wingen L. Kreipe H. Schlegelberger B. Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma.Proc Natl Acad Sci U S A. 2004; 101: 1309-1314Crossref PubMed Scopus (82) Google Scholar 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,10Greenberg R.A. Chin L. Femino A. Lee K.H. Gottlieb G.J. Singer R.H. Greider C.W. DePinho R.A. Short dysfunctional telomeres impair tumorigenesis in the INK4a(delta2/3) cancer-prone mouse.Cell. 1999; 97: 515-525Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 11Gonzalez-Suarez E. Samper E. Flores J.M. Blasco M.A. Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis.Nat Genet. 2000; 26: 114-117Crossref PubMed Scopus (300) Google Scholar, 12Rudolph K.L. Millard M. Bosenberg M.W. DePinho R.A. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nat Genet. 2001; 28: 155-159Crossref PubMed Scopus (453) Google Scholar, 13Farazi P.A. Glickmann J. Jiang S. Yu A. Rudolph K.L. DePinho R.A. Differential impact of telomere dysfunction on initiation and progression of hepatocellular carcinoma.Cancer Res. 2003; 63: 5021-5027PubMed Google Scholar, 14Chang S. Khoo C.M. Naylor M.L. Maser R.S. DePinho R.A. Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression.Genes Dev. 2003; 17: 88-100Crossref PubMed Scopus (167) Google Scholar but increased the rate of tumor initiation by induction of chromosomal fusions resulting in anaphase bridges, chromosome breakage, and aneuploidy.12Rudolph K.L. Millard M. Bosenberg M.W. DePinho R.A. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nat Genet. 2001; 28: 155-159Crossref PubMed Scopus (453) Google Scholar, 15Rudolph K.L. Chang S. Lee H.W. Blasco M. Gottlieb G.J. Greider C. DePinho R.A. Longevity, stress response, and cancer in aging telomerase-deficient mice.Cell. 1999; 96: 701-712Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar Impaired tumor progression in telomere dysfunctional mice was associated with an activation of p53-dependent DNA-damage responses.12Rudolph K.L. Millard M. Bosenberg M.W. DePinho R.A. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nat Genet. 2001; 28: 155-159Crossref PubMed Scopus (453) Google Scholar, 14Chang S. Khoo C.M. Naylor M.L. Maser R.S. DePinho R.A. Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression.Genes Dev. 2003; 17: 88-100Crossref PubMed Scopus (167) Google Scholar In contrast, mutation of p53 alleviated tumor suppression and accelerated chromosomal instability (CIN) and cancer initiation in telomere dysfunctional mice.16Artandi S.E. Chang S. Lee S.L. Alson S. Gottlieb G.J. Chin L. DePinho R.A. Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice.Nature. 2000; 406: 641-645Crossref PubMed Scopus (944) Google Scholar, 17Chin L. Artandi S.E. Shen Q. Tam A. Lee S.L. Gottlieb G.J. Greider C.W. DePinho R.A. p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis.Cell. 1999; 97: 527-538Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar, 18Farazi P.A. Glickman J. Horner J. DePinho R.A. Cooperative interactions of p53 mutation, telomere dysfunction and chronic liver damage in hepatocellular carcinoma progression.Cancer Res. 2006; 66: 4766-4773Crossref PubMed Scopus (96) Google Scholar The high incidence of telomere shortening, loss of p53 checkpoint function, and aneuploidy in the vast majority of human HCC2Ohashi K. Tsutsumi M. Nakajima Y. Kobitsu K. Nakano H. Konishi Y. Telomere changes in human hepatocellular carcinomas and hepatitis virus infected noncancerous livers.Cancer. 1996; 77: 1747-1751Crossref PubMed Google Scholar, 3Plentz R.R. Caselitz M. Bleck J.S. Gebel M. Flemming P. Kubicka S. Manns M.P. Rudolph K.L. Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatocellular carcinoma.Hepatology. 2004; 40: 80-86Crossref PubMed Scopus (108) Google Scholar, 4Plentz R.R. Schlegelberger B. Flemming P. Manns M.P. Rudolph K.L. Wilkens L. Telomere shortening correlates with increasing aneuploidy of chromosome 8 on a cellular level in primary human hepatocellular carcinoma.Hepatology. 2005; 42: 522-526Crossref PubMed Scopus (54) Google Scholar, 5Thorgeirsson S.S. Grisham J.W. Molecular pathogenesis of human hepatocellular carcinoma.Nat Genet. 2002; 31: 339-346Crossref PubMed Scopus (1289) Google Scholar, 6Tannapfel A. Busse C. Weinans L. Benicke M. Katalinic A. Geissler F. Hauss J. Wittekind C. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas.Oncogene. 2001; 20: 7104-7109Crossref PubMed Scopus (123) Google Scholar, 7Nishida N. Nishimura T. Ito T. Komeda T. Fukuda Y. Nakao K. Chromosomal instability and human hepatocarcinogenesis.Histol Histopathol. 2003; 18: 897-909PubMed Google Scholar, 8Hashimoto K. Mori N. Tamesa T. Okada T. Kawauchi S. Oga A. Furuya T. Tangoku A. Oka M. Sasaki K. Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH.Mod Pathol. 2004; 17: 617-622Crossref PubMed Scopus (89) Google Scholar, 9Wilkens L. Flemming P. Gebel M. Bleck J. Terkamp C. Wingen L. Kreipe H. Schlegelberger B. Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma.Proc Natl Acad Sci U S A. 2004; 101: 1309-1314Crossref PubMed Scopus (82) Google Scholar 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.5Thorgeirsson S.S. Grisham J.W. Molecular pathogenesis of human hepatocellular carcinoma.Nat Genet. 2002; 31: 339-346Crossref PubMed Scopus (1289) Google Scholar, 19Miura N. Horikawa I. Nishimoto A. Ohmura H. Ito H. Hirohashi S. Shay J.W. Oshimura M. Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis.Cancer Genet Cytogenet. 1997; 93: 56-62Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 20Takahashi S. Kitamoto M. Takaishi H. Aikata H. Kawakami Y. Nakanishi T. Shimamoto F. Tahara E. Tahara H. Ide T. Kajiyama G. Expression of telomerase component genes in hepatocellular carcinomas.Eur J Cancer. 2000; 36: 496-502Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar 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.5Thorgeirsson S.S. Grisham J.W. Molecular pathogenesis of human hepatocellular carcinoma.Nat Genet. 2002; 31: 339-346Crossref PubMed Scopus (1289) Google Scholar, 6Tannapfel A. Busse C. Weinans L. Benicke M. Katalinic A. Geissler F. Hauss J. Wittekind C. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas.Oncogene. 2001; 20: 7104-7109Crossref PubMed Scopus (123) Google Scholar 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,2Ohashi K. Tsutsumi M. Nakajima Y. Kobitsu K. Nakano H. Konishi Y. Telomere changes in human hepatocellular carcinomas and hepatitis virus infected noncancerous livers.Cancer. 1996; 77: 1747-1751Crossref PubMed Google Scholar, 3Plentz R.R. Caselitz M. Bleck J.S. Gebel M. Flemming P. Kubicka S. Manns M.P. Rudolph K.L. Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatocellular carcinoma.Hepatology. 2004; 40: 80-86Crossref PubMed Scopus (108) Google Scholar, 4Plentz R.R. Schlegelberger B. Flemming P. Manns M.P. Rudolph K.L. Wilkens L. Telomere shortening correlates with increasing aneuploidy of chromosome 8 on a cellular level in primary human hepatocellular carcinoma.Hepatology. 2005; 42: 522-526Crossref PubMed Scopus (54) Google Scholar, 21Wright W.E. Shay J.W. The two-stage mechanism controlling cellular senescence and immortalization.Exp Gerontol. 1992; 27: 383-389Crossref PubMed Scopus (350) Google Scholar, 22Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1943) Google Scholar, 23d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (2158) Google Scholar, 24Bond J.A. Wyllie F.S. Wynford-Thomas D. Escape from senescence in human diploid fibroblasts induced directly by mutant p53.Oncogene. 1994; 9: 1885-1889PubMed Google Scholar loss of p53-pathway function,5Thorgeirsson S.S. Grisham J.W. Molecular pathogenesis of human hepatocellular carcinoma.Nat Genet. 2002; 31: 339-346Crossref PubMed Scopus (1289) Google Scholar, 6Tannapfel A. Busse C. Weinans L. Benicke M. Katalinic A. Geissler F. Hauss J. Wittekind C. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas.Oncogene. 2001; 20: 7104-7109Crossref PubMed Scopus (123) Google Scholar, 21Wright W.E. Shay J.W. The two-stage mechanism controlling cellular senescence and immortalization.Exp Gerontol. 1992; 27: 383-389Crossref PubMed Scopus (350) Google Scholar, 22Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1943) Google Scholar, 23d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (2158) Google Scholar, 24Bond J.A. Wyllie F.S. Wynford-Thomas D. Escape from senescence in human diploid fibroblasts induced directly by mutant p53.Oncogene. 1994; 9: 1885-1889PubMed Google Scholar and aneuploidy.7Nishida N. Nishimura T. Ito T. Komeda T. Fukuda Y. Nakao K. Chromosomal instability and human hepatocarcinogenesis.Histol Histopathol. 2003; 18: 897-909PubMed Google Scholar, 8Hashimoto K. Mori N. Tamesa T. Okada T. Kawauchi S. Oga A. Furuya T. Tangoku A. Oka M. Sasaki K. Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH.Mod Pathol. 2004; 17: 617-622Crossref PubMed Scopus (89) Google Scholar, 9Wilkens L. Flemming P. Gebel M. Bleck J. Terkamp C. Wingen L. Kreipe H. Schlegelberger B. Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma.Proc Natl Acad Sci U S A. 2004; 101: 1309-1314Crossref PubMed Scopus (82) Google Scholar, 21Wright W.E. Shay J.W. The two-stage mechanism controlling cellular senescence and immortalization.Exp Gerontol. 1992; 27: 383-389Crossref PubMed Scopus (350) Google Scholar, 22Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1943) Google Scholar, 23d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (2158) Google Scholar, 24Bond J.A. Wyllie F.S. Wynford-Thomas D. Escape from senescence in human diploid fibroblasts induced directly by mutant p53.Oncogene. 1994; 9: 1885-1889PubMed Google Scholar We analyzed the functional role of telomerase in late-generation telomerase knockout mice and littermates that were genetically rescued for telomerase gene expression. The 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. We used the Cre-loxP system to achieve a liver specific deletion of the Trp53 gene (conditional p53 mouse).25Jonkers J. Meuwissen R. van der Gulden H. Peterse H. van der Valk M. Berns A. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.Nat Genet. 2001; 29: 418-425Crossref PubMed Scopus (830) Google Scholar 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.25Jonkers J. Meuwissen R. van der Gulden H. Peterse H. van der Valk M. Berns A. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.Nat Genet. 2001; 29: 418-425Crossref PubMed Scopus (830) Google Scholar Heterozygous conditional p53 mice (Trp53F2–10/+) were crossed with mTERC+/− mice.26Blasco M.A. Lee H.W. Hande M.P. Samper E. Lansdorp P.M. DePinho R.A. Greider C.W. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA.Cell. 1997; 91: 25-34Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar 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+) under the liver-specific albumin promoter (C57BL/6J-Tg(Alb1HBV)44Bri/J; Jackson Laboratories, Bar Harbor, ME)27Chisari F.V. Klopchin K. Moriyama T. Pasquinelli C. Dunsford H.A. Sell S. Pinkert C.A. Brinster R.L. Palmiter R.D. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice.Cell. 1989; 59: 1145-1156Abstract Full Text PDF PubMed Scopus (629) Google Scholar 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).28Hemann M.T. Strong M.A. Hao L.Y. Greider C.W. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability.Cell. 2001; 107: 67-77Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar The mice were infected with an adenovirus expressing Cre-recombinase (Ad-Cre),29Akagi K. Sandig V. Vooijs M. Van der Valk M. Giovannini M. Strauss M. Berns A. Cre-mediated somatic site-specific recombination in mice.Nucleic Acids Res. 1997; 25: 1766-1773Crossref PubMed Scopus (199) Google Scholar 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 (Trp53Δ2–10/Δ2–10). All mice were in a C57Bl/6J background. Ad-Cre virus was propagated in 293T cells and prepared according to previously described cesium chloride gradient purification.29Akagi K. Sandig V. Vooijs M. Van der Valk M. Giovannini M. Strauss M. Berns A. Cre-mediated somatic site-specific recombination in mice.Nucleic Acids Res. 1997; 25: 1766-1773Crossref PubMed Scopus (199) Google Scholar 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. 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 μm) were stained with H&E, and the tumor types were classified using the following criteria. Lesions 0.5–2 mm in diameter, with morphologically clonal appearing growth pattern, sometimes slight marginal compression and basophilic, clear cell or eosinophilic cytomorphology. Nodules >2 mm in size representing macroscopic nodules ("adenomas") not showing characteristic markers of HCCs. 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. The telomere repeat amplification protocol (TRAP) was performed on snap-frozen tissues as reported.30Kim N.W. Wu F. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).Nucleic Acids Res. 1997; 25: 2595-2597Crossref PubMed Scopus (638) Google Scholar Briefly, TRAP reactions were incubated for 30 minutes at 30°C for telomerase extension using 32Chung Y.J. Jonkers J. Kitson H. Fiegler H. Humphray S. Scott C. Hunt S. Yu Y. Nishijima I. Velds A. Holstege H. Carter N. Bradley A. A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.Genome Res. 2004; 14: 188-196Crossref PubMed Scopus (58) Google ScholarP-γATP (Hartmann Diagnostics, Germany) labeled telomerase 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). The DNA copy number of Trp53Δ2–10 and Trp53F2–10 was analyzed in triplicate by real-time PCR according to standard protocols. The following primers were used for detection of the Trp53Δ2–10 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 Trp53Δ2–10 by generating a standard curve using mixtures of genomic DNA from Trp53Δ2–10 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 (ΔCt value of Trp53Δ2–10/ΔCt value of Trp53F2–10) to generate standard curves. Quantitative fluorescence in situ hybridization (Q-FISH) was performed on 5-μm paraffin sections. After unmasking, the slides were incubated in Pepsine solution for 10 minutes at 37°C (100 mg Pepsine; 84μL 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 μg/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 TBS-Tween (0.2%). The relative telomere length was measured by the telomere fluorescence intensity by TFL analysis software program.31Poon S.S. Martens U.M. Ward R.K. Lansdorp P.M. Telomere length measurements using digital fluorescence microscopy.Cytometry. 1999; 36: 267-278Crossref PubMed Scopus (192) Google Scholar Genomic DNA was extracted from mouse tissues by proteinase K digestion and organic extraction, and 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–ULS-labeled reference DNA (isolated from the spleen of the same mouse) and hybridized to a Mouse 3K BAC array as described previously.32Chung Y.J. Jonkers J. Kitson H. Fiegler H. Humphray S. Scott C. Hunt S. Yu Y. Nishijima I. Velds A. Holstege H. Carter N. Bradley A. A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.Genome Res. 2004; 14: 188-196Crossref PubMed Scopus (58) Google Scholar 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.33Hughes T.R. Marton M.J. Jones A.R. Roberts C.J. Stoughton R. Armour C.D. Bennett H.A. Coffey E. Dai H. He Y.D. Kidd M.J. King A.M. Meyer M.R. Slade D. Lum P.Y. Stepaniants S.B. Shoemaker D.D. Gachotte D. Chakraburtty K. Simon J. Bard M. Friend S.H. Functional discovery via a compendium of expression profiles.Cell. 2000; 102: 109-126Abstract Full Text Full Text PDF PubMed Scopus (2138) Google Scholar All fluorescence intensities were converted to log2 values to weight gains and losses equally. 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 γH2AX-staining sections were blocked with M.O.M blocking reagent (Vector Labs, Burlingame, CA) for 1 hour and then incubated with a mouse anti-γH2AX antibody (Upstate, Lake Placid, NY) overnight at 4°C. The subsequent steps to detect γH2AX signal was performed using M.O.M.™ Immunodetection Kit (Vector Laboratories) according to manufacturer's recommendations. Double staining of telomere and γH2AX signals was performed by using the telomere quantitative FISH protocol (see above) followed by γH2AX immunofluorescence (see above) using a Cy3-labeled rabbit antimouse IgG secondary antibody (1:500; Zymed). The Mann–Whitney U test
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