Annals of Oncology Advance Access originally published online on July 17, 2008
Annals of Oncology 2008 19(11):1875-1881; doi:10.1093/annonc/mdn405
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gastrointestinal tumors |
Telomere shortening is correlated with the DNA damage response and telomeric protein down-regulation in colorectal preneoplastic lesions
1 Laboratoire de radiobiologie et oncologie, CEA, Fontenay-aux-Roses, France
2 Department of Pathology, Assan Medical Center, Uslan University Medical College, Seoul, South Korea
3 FIRC Institute of Molecular Oncology Foundation, Milan, Italy
4 Department of Pathology CHU A Michallon and INSERM U 823 A Bonniot Institut Grenoble, France
5 Department of Onco-Hematology, Hospital l'Archet, CHU de Nice, Nice, France
6 Division of Cancer Medicine, IGR, Villejuif, France
* Correspondence to: Dr J.-C. Soria, Department of Medecine, Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805 Villejuif, France. Tel: +33-1-42-11-42-91; Fax: +33-1-42-11-52-17; E-mail: soria{at}igr.fr
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Abstract |
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Background: A relation between telomere attrition in early carcinogenesis and activation of DNA damage response (DDR) has been proposed. We explored telomere length and its link with DDR in colorectal multistep carcinogenesis.
Patients and methods: We studied normal mucosa, low-grade dysplasia (LGD) and high-grade dysplasia (HGD) and invasive carcinoma (IC) in matched human colon specimens by evaluating p-ataxia telangiectasia mutated (ATM), p-checkpoint kinase 2 (Chk2), c-H2AX, TRF1 and TRF2 expressions by immunohistochemistry. FISH was used to assess telomere length.
Results: Telomeres shortened significantly from normal (N) to LGD and HGD (P < 0.0001; P = 0.012), then increased in length in IC (P = 0.006). TRF1 and TRF2 expressions were diminished from N to LGD and HGD (P = 0.004, P < 0.0001, ns) and were reexpressed at the invasive stage (P = 0.053 and P = 0.046). Phosphorylated ATM, Chk2 and H2AX appeared already in LGD (respectively, P = 0.001, P = 0.002 and P = 0.02). Their expression decreased from HGD to IC (respectively, P = 0.03, P = 0.02 and P = 0.37). These activating phosphorylations were inversely correlated with telomere length and TRF1/2 expression.
Conclusion: In a model of colon multistep carcinogenesis, our data indicate that telomeric length and protein expression levels are inversely correlated with the activation of the DDR pathway.
Key words: Carcinogenesis, DNA damage repair, telomeres, telomeric proteins
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Carcinogenesis is a multistep, multifocal process characterized by the accumulation of genetic and molecular abnormalities. These events generally follow exposure to carcinogens and result in the selection of clonal cells with uncontrolled growth [1]. Cancer develops in a stepwise manner, through a series of events, from preinvasive histological changes to invasive disease [2]. The earliest events are mutations, deletions or polysomy at genomic level, but these changes do not always lead to changes in cell morphology or tissue structure [3, 4]. Current knowledge suggests that the progression of cancer from a premalignant to a malignant state is, consistent with a mechanistic model, based on of the principle of natural selection. Tumor cells acquire the hallmarks of cancer during this carcinogenic selection process [5]. Cell immortality is one of the principal features acquired during this process. Immortality involves the stabilization of telomere length, which is achieved by telomerase activation in
80% of human tumors. Interestingly, telomere length abnormality also appears to be one of the earliest and most prevalent genetic alterations in the multistep process of malignant transformation [5–7]. However, it remains unclear whether telomere dysfunction induces chromosomal instability, initiating certain human epithelial malignancies. It has also recently been shown that DNA damage repair is activated early in most human epithelial carcinogenic processes [2, 3]. DNA damage repair mechanisms are thought to optimize cell survival after DNA damage, thereby controlling the proliferation of damaged cells. Two large, highly conserved protein kinases play a central role in the DNA damage response (DDR) mechanism in human cells: ataxia telangiectasia mutated (ATM) and ataxia telangiectasia related (ATR) kinases, which have different, but partly overlapping functions. Recent studies of human solid tumor samples obtained at different stages of carcinogenesis have revealed that activation of the DNA damage machinery is almost universal during the earliest stages of carcinogenesis [2, 3]. Early premalignant lesions (but not normal tissues) commonly express activated DDR markers. These markers include the phosphorylated kinases, ATM and Chk2, and p53. These findings are consistent with activation of an early ATR/ATM-regulated DDR network playing a role in delaying or preventing cancer in human cells. Telomere attrition can result in short telomeres that the cell may detect as DNA damage. It has therefore been suggested that telomere attrition (together with oncogene activation) may lead to DNA damage checkpoint activation [1].
We evaluated telomere length and telomere protein levels and their relationship to DDR pathway activation in the multistep process of colorectal carcinogenesis. We studied colorectal carcinoma because this was the tumor type in which the multistep carcinogenesis model was first established. Tumor progression has been defined in detail in the colon, in which the earliest histological abnormalities are aberrant crypts with a focally atypical epithelium. These aberrant crypts may develop into benign epithelial tumors—low- and high-grade adenomas—which may in turn progress to malignant carcinoma. We evaluated telomere length, telomeric protein levels and DDR marker expression in consecutive tissue sections from matched patients presenting the major histological steps of colorectal carcinogenesis.
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patients and methods |
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patients and tissue samples
Colon tissue samples were obtained from 15 patients, who underwent surgery at Assan Medical Center, Seoul, Republic of Korea. This series was unusual in that we had normal mucosal, low-graph dysplasia (LGD) and high-grade dysplasia (HGD) and invasive carcinoma (IC) samples from each individual patient. None of the patients had received radiotherapy or chemotherapy before surgery. After surgery, each tissue sample was fixed in formal and embedded in paraffin. Consecutive 4-µm thick paraffin tissue sections were mounted on polylysine-coated slides for analysis. Histological diagnoses were established by senior pathologists: SJJ, PN and EB. The Institute for Research in Biomedicine IRB of the Assan Medical Center, Seoul, Republic of Korea, decided that no ethical review of this study was necessary. Normal skin for antibody validation was obtained from a reductive mammoplasty as previously described [8].
antibodies
We used antibodies against Thr 68-phosphorylated Chk2 (#2661 Cell Signaling Technology, Danvers, MA) at a final dilution of 1/100, 
-H2AX (ser139) (#05-636 Upstate Biotechnology, Lake Placid, NY) at a final dilution of 1/200, Ser 1981-phosphorylated ATM (#200-301-500 Rockland Immunochemicals, Gilbertsville, PA) at a final dilution of 1/2500, TRF1 (#ab 10579-50, Abcam, Paris, France) at a final dilution of 1/100 and TRF2 (#05-521, Millipore, Paris, France) at a final dilution of 1/300.
cells and culture
We used HT-29 cells for antibody validation. HT-29 cells (obtained from LGC Promochem, Molsheim, France) were incubated in RPMI medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37°C, under an atmosphere containing 5% carbon dioxide. Cells were grown in 25-cm2 flasks. For irradiation experiments, duplicate flasks of cells at 80% confluence were or were not subjected to irradiation with 3 Gy. The irradiated cells were incubated for 30 min at 37°C under an atmosphere containing 5% carbon dioxide treated with trypsin and harvested by centrifugation (800 g). They were then washed twice in cold phosphate-buffered saline (PBS) (#14190, Invitrogen). Cells were collected by centrifugation. The cell pellet was fixed by incubation in buffered formalin for 48 h and was then embedded in paraffin and cut into 4-µm sections with a microtome. These sections were mounted on polylysine-coated slides for analysis.
immunohistochemistry
Indirect immunoperoxidase staining was carried out on formalin-fixed tissue sections or cultured cells, using the Vectastain Elite kit (#PK-6200, Vector Laboratories, Burlingame, CA) or the Envision plus/horseradish peroxidase detection system (Dako, Carpinteria, CA).
Paraffin was removed from the sections, which were then rehydrated in a series of ethanol solutions with concentrations from 100% to 70%. Antigens were retrieved by heating (95°C) in 0.25 mM EDTA (pH 8) (for H2AX-p, ATM-p, Chk2-p) for 50 min or in Tris buffer (pH 6) (for TRF1, TRF2) for 60 min. Endogenous peroxidase activity was inhibited by immersing the slides in 3% hydrogen peroxide for 5 min. The slides were re-equilibrated with PBS and incubated with 1% normal horse serum or 2% bovine serum albumin, 0.02% normal goat serum and 0.01% Tween 20 for 20 min. Slides were incubated with primary antibody (diluted in 1% horse serum buffer) at room temperature for 2 h or overnight at 4°C. Slides were then incubated with either biotinylated secondary antibody followed by avidin–biotin peroxidase complex (PK-6200, Vector Laboratories) for 30 min each or a horseradish peroxidase-conjugated secondary antibody (K3468, Dako) for 30 min. A color reaction was developed using diaminobenzidine as the substrate. Slides were lightly counterstained with hematoxylin and mounted in permanent mounting medium.
The specificity of each antibody was assessed by western blotting before use to evaluate levels of the corresponding protein in irradiated and nonirradiated cells.
scoring of stained cells
Two investigators, including a senior pathologist, evaluated the immunostaining patterns independently. The percentage of positive tumor nuclei was calculated for each specimen, and a proportion score was assigned (0 if 0%, 0.1 if 1%–9%, 0.5 if 10%–49% and 1.0 if 50% or more). This proportion score was multiplied by the staining intensity of nuclei to obtain a final semiquantitative H score [9].
telomere length assessment by FISH
Telomere FISH was carried out as described by Meeker et al. [10]. Sections were deparaffinized and rehydrated antigens were then retrieved by heating for 15 min at 98°C in Tris buffer (pH 6). The sections were incubated with 0.1% Tween in PBS for 5 min, followed by 10% pepsin in 0.001 M HCl for 20 min at 37°C. Sections were then dehydrated and air dried. We applied 10 µl of a Cy3-labeled-specific peptide nucleic acid-targeting telomeres (Dako) to each sample, which was then covered with a coverslip and denatured by heating for 4 min at 83°C. Slides were placed in the dark for 2 h at 37°C to allow hybridization to occur. They were then washed twice for 15 min with formamide–Tris and three times, for 5 min each, in Tris–NaCl–Tween buffer at room temperature. They were counterstained with Diamidino-4',6-phénylindol-2 dichlorhydrate (1 µg/ml) and mounted in aqueous mounting medium. The intensity of telomere staining, which is linearly related to telomere length, was assessed visually. Qualitative comparisons of staining were made between the epithelial regions of interest and adjacent epithelial cells of normal appearance or, if unavailable, normal adjacent stromal cells, such as lymphocytes, which invariably have robust telomere signals [6, 10, 11]. Nuclear telomeres were scored 0 (undetectable), 1 (intensity of fluorescent signal much less than for lymphocytes), 2 (signal intensity slightly lower than that for lymphocytes) or 3 (signal equivalent to that of normal epithelium or lymphocytes from the stroma).
statistical analysis
Tumor characteristics were compared using Fisher's exact test for categorical variables [the different histopathological stages: normal (N), LGD, HGD and IC] and pairwise Wilcoxon's test for continuous variables [p-ATM (Ser1981), p-Chk2 (Thr 68), -H2AX, TRF1 and TRF2 expressions]. A generalized linear model was used to assess correlations between continuous variables. Differences were considered significant if a two-sided P value <0.05 was obtained. All statistical analyses were carried out with SAS version 9.13 (SAS Institute Inc, Cary, NC).
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antibody validation
Immunostaining was carried out on paraffin-embedded tissues and cultured cells to assess the specificity of staining with the various antibodies used. Normal human skin and human HT-29 colorectal cancer cells were evaluated at baseline and after exposure to radiation. Antibodies against H2AX phosphorylated at S139 (
-H2AX), ATM phosphorylated at S1981 and Chk2 phosphorylated at T68 gave strong staining only with irradiated skin and colorectal cancer cells (Figure 1). -H2AX staining was punctate in irradiated colorectal cancer cells consistent with the development of foci. Staining was almost exclusively nuclear with the exception of weak cytoplasmic staining for ATM pS1981, which was not considered relevant in this study (Figure 1).
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activation of the DDR
Normal colon tissues were only weakly stained for p-ATM, P-Chk2 and
-H2AX. All three markers were significantly more strongly expressed in LGD (N versus LGD, P values: P = 0.001 for p-ATM, P = 0.002 for p-Chk2 and P = 0.02 for -H2AX) (Figure 2) (Table 1). There was also a trend for an increase in staining intensity for these markers between LGD and HGD, but this difference was not statistically significant. By contrast, the level of expression of these markers decreased between the HGD and invasive carcinoma stages (P = 0.03 for p-ATM, P = 0.02 for p-Chk2 and P = 0.37 for -H2AX) (Table 1). p-ATM levels were correlated with p-Chk2 levels (P < 0.0001, at all stages of carcinogenesis) but were only poorly correlated with -H2AX levels (P = 0.065 from normal tissue to HGD and p = 0.067 from HGD to invasive carcinoma) (Tables 2 and 3). -H2AX levels were correlated with p-Chk2 levels (P = 0.0004 from normal tissue to HGD and P = 0.05 from HGD to invasive carcinoma) (Tables 2 and 3). These observations suggest that the DNA damage repair pathway is already activated in LGD, this activation increasing in HGD, before being switched off in invasive tumors.
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TRF1/2 expression
Strong TRF1 and TRF2 staining (level 3) was observed in normal colic tissue sections, particularly in basal epithelial cells. For TRF1 and TRF2, a net gradient of staining was observed in normal cells, from the base of the crypts (strong staining) to the lumen (weaker staining). TRF1 and TRF2 staining was weaker in LGD than in normal tissue (N versus LGD, P values = 0.004 for TRF1 and P < 0.0001 for TRF2) (Figures 3 and 4) (Table 1).
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No significant difference in TRF1 and TRF2 staining was observed between LGD and HGD. However, staining for these two markers was stronger in invasive carcinoma samples than in HGD samples (P = 0.053 and P = 0.046, for TRF1 and TRF2, respectively). We also found a significant correlation between TRF1 and TRF2 levels (P < 0.0001 from normal tissue to HGD and P = 0.0014 from HGD to invasive carcinoma) (Tables 2 and 3).
measurement of telomere length by telomere FISH
Histological analysis of telomere signals showed that telomere length depended on stage. The nuclei of normal basal epithelial cells and stromal lymphocytes contained a large number of fluorescent telomeric spots. These cells were assigned a score of 3. By contrast, LGD and, to an even greater extent, HGD cells were more weakly stained than internal controls. The decrease in staining with the transition from normal tissue to LGD and with that from LGD to HGD was statistically significant (P < 0.0001 and P = 0.012, respectively) (Figures 4 and 5; Table 1). During later stages of the carcinogenic process, staining increased with invasiveness, reaching the levels observed in normal cells in invasive carcinoma. This increase in staining associated with the transition from HGD to IC was also statistically significant (P = 0.006). The weakest telomeric signals were those obtained for HGD lesions. Moreover, as shown above, TRF1 and TRF2 levels were significantly correlated both with each other and with telomere lengthening during carcinogenesis (P < 0.0001 and P < 0.0001 for TRF1 and TRF2, respectively, for the transition from N to HGD and P = 0.0104 and P = 0.0004 for the transition from HGD to IC; Tables 2 and 3).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Consistent with published data [6, 8, 12, 13], our results show that telomere attrition occurs early in carcinogenic progression in the colon model, with telomere shortening observed in LGD. This attrition peaks in HGD, and it is only when the full invasive potential of the tumor has been reached that telomere length returns to levels close to those observed in normal tissue. We hypothesize that this telomere attrition may have a protective effect against cancer because critically short telomeres are known to induce a cell cycle arrest commonly known as replicative senescence.
We also demonstrate for the first time that levels of the two major telomere-associated proteins—TRF1 and TRF2—are correlated, both with each other and with telomere length during colon carcinogenesis. This finding is consistent with the partially overlapping roles of TRF1/2. The loss of TRF1 and TRF2 accompanied by telomere shortening in LGD and HGD provides support for the notion that telomere attrition occurs in vivo and may disturb telomeric homeostasis.
We also observed signs of DDR activation, including the phosphorylation of H2AX, Chk2 and ATM, during colorectal carcinogenesis. This DDR activation, which is barely detectable in normal crypts, is induced at some point during the transition from normal tissue to LGD. The DDR was maximal in HGD. By contrast, only low levels of DDR activation were observed in fully invasive cells, suggesting that neoplastic cells may have avoided apoptosis by impairing DDR.
Both telomere length and TRF1/2 levels were inversely correlated with DDR activation. There are three possible explanations for this inverse correlation: (i) DDR may be oncogene induced and related to DNA replication stress [2]. In this case, telomere attrition would be the consequence of unregulated cell proliferation. (ii) Telomeres may play an indirect role in DDR activation. Indeed, once they reach a critical size, telomeres lose their normal three-dimensional (3-D) structure. This may result in telomeric fusions and subsequent break fusion bridge cycles. These events may lead to general genomic instability, activating the DDR. (iii) The DDR may be directly activated by telomeres that are too short. Indeed, it has been shown that very short telomeres are associated with telomeric epigenetic modifications and H2AX phosphorylation at telomeres leading to DDR activation [1, 14]. This hypothesis is consistent with the decrease in TRF1/2 levels accompanying telomere attrition. Indeed, previous studies have demonstrated that TRF2 has ATM dephosphorylation activity [15–17]. Thus, following the 3-D stabilization of telomeres, which is thought to protect the ends of the chromosomes from DNA damage sensors, TRF2 may be directly involved in ensuring that telomeres are not recognized as double strand breaks (DSBs). In this case, once cells go beyond replicative senescence, their telomeres would be too short to be protected and would therefore be recognized as DSBs. However, demonstrations of the colocalization of short telomeres and -H2AX on the same tissue sample are required to confirm this hypothesis. Such a demonstration would be very difficult technically. Telomere visualization on paraffin tissue sections generally requires a protease treatment of slides, leading to the partial or complete destruction of -H2AX foci and problems identifying the remaining observable foci as such. Moreover, under this hypothesis, only the shortest telomeres would be recognized as DSBs and carry H2AX foci, and isolated short telomeres are extremely difficult to visualize in human paraffin tissue sections. It might be simpler to work on fresh tissues or frozen material, but this would make it difficult to obtain a cohort of this type, with samples from LGD to invasive carcinoma for the same patients.
A recent study [8] has shown that DDR activation may be tissue specific. By contrast to our observations and those of other groups [18–20], TRF1 and TRF2 have been shown to be overproduced in neoplastic lung lesions [21]. It would therefore be of great interest to determine whether the telomere/DDR activation correlation observed here depends on histological type. Analyses of lung, prostate and breast tissues (together with colon, corresponding to the four most common cancers worldwide), in particular, would be of great value.
Several drugs that target the telomeres and their related proteins are tested in preclinical models and in early clinical trials these days, and one of the suggested mechanisms of action is the induction of progressive telomere shortening and a direct damage of the structure of individual telomeres [22, 23]. While their clinical impact has not been yet demonstrated in patients with an invasive disease, some in vivo models have shown that a pretreatment of tumor cells with such inhibitors led to a marked reduction in their tumorigenic potential [22]. Our demonstration that telomere shortening occurs in LGD and HGD raises the hypothesis that telomere interacting agents could have a role not only at the time of invasive disease but also earlier in the carcinogenic progression of colon cancer, favoring the telomere attrition and the activation of apoptosis in the dysplastic cell. In fact, this approach of reversion of premalignant lesions has already been tested with other targeted molecules in in vivo lung cancer multistep models [24, 25].
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TELINCA and RISC-RAD (FI6R-CT2003-508842). Christophe Raynaud is a doctoral fellow funded by a CEA-Lilly fellowship.
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The work carried out in the L.S. laboratory. We would like to thank Dr Fabrizio D'adda di Fagagna for helpful discussions and exchanges.
Received for publication February 26, 2008. Revision received May 26, 2008. Accepted for publication May 28, 2008.
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