Affiliations of authors: X. Wu, C. I. Amos, Y. Zhu, H. Zhao, S. Luo, M. R. Spitz (Department of Epidemiology), B. H. Grossman (Department of Urology), W. K. Hong (Department of Thoracic/Head and Neck Medical Oncology), The University of Texas M. D. Anderson Cancer Center, Houston; J. W. Shay, Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, TX.
Correspondence to: Xifeng Wu, MD, PhD, Department of Epidemiology, Box 189, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: xwu{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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Although telomere shortening is inversely associated with age, telomere length has been found to vary considerably in human peripheral blood lymphocytes from individuals of the same age (9). Our hypothesis was that individuals with telomere dysfunction may be at higher risk for developing cancer and more likely to exhibit genetic instability. To test this hypothesis, we investigated whether telomere dysfunction, as assessed by telomere length, was associated with the risk of four smoking-related cancershead and neck, bladder, lung, and renal cell cancerin four ongoing casecontrol studies.
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MATERIALS AND METHODS |
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Four casecontrol studies of head and neck, bladder, lung, and renal cell cancer were included in this study (Table 1). All case patients with bladder, lung, or renal cell carcinoma were recruited from The University of Texas M. D. Anderson Cancer Center through a daily review of computerized appointment schedules. They had been diagnosed within 1 year of recruitment, had histologic confirmation of their cancer, and had had no prior chemotherapy or radiotherapy. Case patients with head and neck cancer were recruited for a chemoprevention program from The University of Texas M. D. Anderson Cancer Centers Community Clinical Oncology Program affiliates and from Radiation Therapy Oncology Group centers throughout the country. There were no age, sex, or stage restrictions. Control subjects with no prior history of cancer were identified from the rosters of Kelsey Seybold, the largest multispecialty physician group in Houston, TX. Control subjects were matched to the case patients by age (±1 year), sex, and ethnicity. Some control subjects for bladder, lung, and renal cell carcinoma were shared across the studies, because they were drawn from the same control subject pool and because the same quantitative fluorescence in situ hybridization laser scanning cytometry (Q-FISHLSC) technique was used to measure telomere length. For instance, 30% of the control subjects were shared between the lung and bladder cancer studies, 11% of the control subjects were shared between the bladder and renal cell carcinoma studies, and 19% of the control subjects were shared between the lung cancer and renal cell carcinoma studies. However, control subjects for the head and neck cancer study could not be shared across studies because a different assay was used to measure telomere length. Signed informed consent was obtained from each individual. All participants were interviewed to collect information regarding demographics, smoking history, alcohol consumption, family cancer history, medical history, and working history (except for the head and neck cancer study). Baseline blood samples were collected after the interview. This research was approved by all relevant review boards and was in accord with an assurance filed with, and approved by, the U.S. Department of Health and Human Services.
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Blood Collection and Lymphocyte Isolation
Forty milliliters of blood was collected in tubes containing sodium heparin. The blood samples were coded before delivery (or mailing for the head and neck cancer study) to the laboratory so that researchers performing the assays were blinded to the status of the samples (i.e., case or control). Lymphocytes were isolated by use of Ficoll-Hypaque centrifugation. An aliquot of 4 x 106 isolated lymphocytes was placed into a vial, frozen in 50% fetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulfoxide (Fisher Scientific, Pittsburgh, PA) and stored in liquid nitrogen.
Telomere Length Measurement by Q-FISHLSC
The frozen lymphocytes were thawed and incubated in RPMI-1640 medium supplemented with 20% fetal bovine serum and phytohemagglutinin (60 µg/mL; Murex Diagnostics, Norcross, GA) at 37 °C for 72 hours. Slides of cell suspensions were prepared and aged for 24 hours. Telomere length was measured by an improved Q-FISHLSC method, which was modified from a Q-FISH flow cytometry method (10) that used a fluorescence-labeled peptide-coupled nucleic acid (PNA) probe (Applied Biosystems, Foster City, CA). Briefly, a mixture of 3 µL of PNA probe and 7 µL of LSI hybridization buffer (Vysis, Downers Grove, IL) was applied to the target area of the slide. Slides were heated on a heating block at 74 ± 1 °C for 4.5 minutes to denature genomic DNA and were placed in a humidified hybridization chamber at room temperature for 2 hours. After incubation, slides were washed in a prewarmed solution of 1x phosphate-buffered saline (PBS; pH 7.4) and 0.1% Tween 20 at 57 ± 1 °C for 30 minutes followed by a 1-minute rinse in a solution of 2x standard saline citrate and 0.1% Tween 20. Propidium iodide was added to the slides for counterstaining the nuclei. The slides were ready for quantification after 15 minutes. The fluorescence signal was measured by laser scanning cytometry (LSC; CompuCyte, Cambridge, MA). At least 2500 cells were measured per sample. The telomere fluorescence signal was defined as the mean fluorescence signal in cells from each sample. The relative telomere length was calculated as the ratio between the telomere signal of each sample and that of the control cell line (LW5770, a lymphoblastoid cell line established in our laboratory).
Telomere Length Measurement by Southern Blot Analysis
One microgram of purified genomic DNA was first digested with HinfI and RsaI, and fragments were separated by agarose gel electrophoresis in 0.8% gels. DNA fragments were then transferred to a nylon membrane and hybridized to a digoxigenin-labeled probe specific to telomeric repeats, followed by incubation with a digoxigenin-specific antibody covalently coupled to alkaline phosphatase. Finally, the immobilized telomere probe was visualized with alkaline phosphatase-metabolizing CDP-Star (Roche Molecular Biochemicals, Mannheim, Germany), a highly sensitive chemiluminescent substrate. The average length of telomere restriction fragments was determined by comparing the signal with a molecular weight standard.
Comet Assay
Blood lymphocyte cultures were established as soon as the blood samples were received. Three separate cultures for each study subject were set up to measure comets from untreated baseline and mutagen-treated cells. After a 72-hour incubation, one culture was processed for the comet assay without any mutagen treatment, and another culture was irradiated at 150 rad with a 137Cs source at room temperature immediately before the comet assay. After a 48-hour incubation, the third culture received 10 µL of 0.4 mM benzo[a]pyrene diol epoxide (BPDE), and the incubation was continued for another 24 hours before the comet assay. Fully frosted slides were precoated on each end of the slide with 50 µL of 1% agarose in PBS and covered with a glass coverslip (22 x 22 mm); 50 µL of blood culture was gently mixed with 150 µL of 0.5% low-melting-point agarose (Invitrogen, Carlsbad, CA) spread onto each end of the precoated slide and then covered with a fresh glass coverslip. A final layer of 0.5% low-melting-point agarose in PBS was applied to the slide and covered with a new glass coverslip. The cells were lysed by submersion in freshly prepared 1x lysis buffer (2.5 M NaCl, 100 mM EDTA, 1% N-lauroylsarcosine sodium salt, and 10 mM Tris, adjusted to pH 10 with NaOH; 10% dimethyl sulfoxide and 1% Triton X-100 were added before use) for 1 hour at 4° C. To allow DNA denaturation, unwinding, and exposure of the alkali-labile sites, the slides were placed in a horizontal electrophoresis box without power that was filled with freshly prepared alkali buffer (300 mM NaOH and 1 mM EDTA at pH>13) for 30 minutes at 4 °C. To separate the damaged DNA from the intact nuclei, a constant electric current of 295300 mA was applied for 23 minutes at 4° C. After electrophoresis, the slides were neutralized in 0.4 M TrisHCl (pH 7.4) for 5 minutes at room temperature and fixed in 100% methanol for 510 minutes. DNA damage in the individual cells was then visualized under a fluorescence microscope and automatically quantified via Komet 4.0.2 (Kinetic Imaging Ltd., Wirral, U.K.) imaging software attached to the microscope. Fifty consecutive cells (25 cells from each end of the slide) were imaged, and comet cells were automatically quantified by using the Olive tail moment parameter [(tail mean head mean) x (% tail DNA/100)], where tail mean is the tail DNA intensity subtracted from background intensity, head mean is the head DNA intensity subtracted from background intensity, and % tail DNA is the fraction of DNA that has migrated from the head. The average Olive tail moments were calculated for each subject.
Statistical Analysis
Telomere length was analyzed as a continuous variable and as a categorical variable. The Wilcoxon rank sum test was used to compare the differences in telomere length between case patients and control subjects as a continuous variable. As a categorical variable, the quartile values of telomere length, according to its distribution in control subjects, were used to compare the differences in telomere length between case patients and control subjects. Additionally, telomere length was dichotomized at the 75% value in control subjects. Unconditional multiple logistic regression analysis was used to control for confounding by sex, age, or smoking status. Spearmans correlation test was used to examine the associations between telomere length and baseline genetic instability, between telomere length and -radiation sensitivity, and between telomere length and BPDE sensitivity, measured by the comet tail moment for case patients and control subjects separately. A never smoker was defined as an individual who had never smoked or had smoked fewer than 100 cigarettes in his or her lifetime. A former smoker was one who had a history of smoking but had stopped at least 1 year before being diagnosed with cancer (or, for control subjects, 1 year before being enrolled in the study). A current smoker was a smoker at the time of enrollment or one who had stopped smoking less than 1 year before being diagnosed with cancer (or, for control subjects, less than 1 year before being enrolled in the study). All statistical tests were two-sided.
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RESULTS |
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DISCUSSION |
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Telomere dysfunction may be one of the molecular causes of genetic instability. Chromosomal rearrangement, generating gains and losses of segments of chromosomes, is an essential step in epithelial carcinogenesis. These extensive and complex rearrangements typically occur at an early stage when telomerase is first activated, suggesting that an early and brief period of telomere dysfunction could contribute to the complex genomic alterations observed in epithelial cancers (11,12). In support of this hypothesis, there is evidence that shortened telomeric DNA results in nonhomologous end joining of telomeric DNA, leading to loss of telomere function and genomic instability (13,14). Moreover, the combination of telomere dysfunction, rather than loss of telomerase itself, and p53 deficiency has been shown to accelerate tumorigenesis in vivo (8,15). Similarly, Chin et al. (16) found that, concurrent with severe telomere shortening and genomic instability, p53 was activated, leading to growth arrest and/or apoptosis. However, deletion of p53 attenuated the adverse cellular and organismal effects of telomere dysfunction during the earliest stages of genetic crisis. Consequently, the loss of telomere function and p53 deficiency appeared to cooperate to initiate the transformation process. Although telomerase is reactivated in most human cancers, telomere shortening and dysfunction might impair chromosomal stability early in carcinogenesis and, consequently, drive the initial carcinogenic process.
In this study, the Southern blot assay was used to measure telomere length in the head and neck cancer casecontrol study, but an improved Q-FISHLSC assay was used in the other three casecontrol studies. Although Southern blot analysis of genomic DNA digested with selected restriction enzymes is the most commonly used approach, contributions of individual sub-telomeric DNA fragments limit the ability of this method to provide accurate telomeric lengths. The Q-FISHLSC approach has several advantages. FISH uses direct labeling of terminal telomeric repeats so that telomere length data can be accurately and quantitatively obtained. This approach correlates well with telomere restriction fragment lengths derived from Southern blot analysis (10). We have demonstrated statistically significant correlations between telomere length measured in the two assays, with a Spearman correlation coefficient of .47 (P = .036; data not shown). An important feature of the Q-FISHLSC protocol is the use of an internal cell line control that provides automatic compensation for potential differences in any steps in the procedure, from fixation to hybridization and DNA staining. The control cell population also serves as an internal telomere length standard for comparing different samples with high precision. This protocol is simple, rapid, and highly reproducible.
Interestingly, we found that short telomeres appeared to play a greater role in patients younger than 55 years or older than 65 years than in patients aged 5565 years, in women than in men, in never or former smokers than in current smokers, and in light smokers than in heavy smokers. These patterns suggest that there are genetically susceptible subgroups and that susceptibility may be less evident in the presence of constant heavy carcinogenic exposures. However, we cannot exclude the possibility that some of these findings in the subsets were caused by random variation. We have provided some preliminary validation of our telomere length assay by correlating the measurements of DNA damage obtained with the comet assay both before and after mutagen exposure. As we had predicted a priori, there was a negative correlation between telomere length and the level of DNA damage at baseline and after exposure to BPDE or radiation.
In support of these findings, Hanson et al. (17) and Kennedy and Hart (18) observed a statistically significant reduction in telomere length in patients with Fanconi anemia, who have increased susceptibility to cancer. Telomere dysfunction is also associated with advanced age when cancer incidence increases exponentially, which could be attributed in part to age-related telomere loss (19,20).
Although the mechanism of how telomere dysfunction accelerates tumor onset is unclear, recent evidence has linked telomeres and DNA damage signaling or repair in cells. The DNA damage response, upon sensing an uncapped telomere or another broken DNA end, recruits repair enzymes to the DNA ends (21,22). Blackburn (21) suggested that telomere function could be pictured as regulating and channeling the active and sensitive surveillance DNA damage response, which can detect a single DNA break in a cell and trigger an appropriate telomere-specific response to maintain telomere integrity. The usual response to the uncapping of a telomere is eliciting telomerase activity (primarily), homologous recombination, or even nonhomologous end joining. If capping fails to occur, the response of a normal cell is to exit the cell cycle or, in certain mammalian cells, to undergo apoptosis. In addition, telomeres are involved in the process of chromosomal repair, as shown by the recruitment or de novo synthesis of telomere repeats at double-stranded breaks and by the ability of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in nonhomologous end joining (2228). An association involving telomere dysfunction, chromosomal instability, impaired DNA repair, and radiosensitivity in a mammalian model system has recently been reported (8,2931). A statistically significantly inverse correlation between telomere length and chromosomal radiosensitivity in lymphocytes from patients with breast cancer and healthy control subjects was also observed (31). Others (32,33) have reported that short telomeres are associated with hypersensitivity to ionizing radiation in mammals. In this study, our results from the phenotypic comet assay showed an inverse association between telomere length and increased levels of radiation-induced DNA damage or chemical-induced DNA damage. These findings indicate that telomere dysfunction could be involved in the development of cancer and also could have an impact on radiotherapeutic strategies for the treatment of cancer. However, in our study, telomere length was measured in a surrogate tissue, peripheral blood lymphocytes, not in cancer cells. Cancer cells have different telomere dynamics, including the reactivation of telomerase that might also modulate genomic instability.
In addition to the association with DNA damage or repair, telomere attrition may also contribute to tumorigenesis by leading to complex cytogenetic abnormalities. One possible consequence is nonhomologous end joining of telomeric DNA that may cause more telomere damage and affect genes located in sub-telomeric regions (15). Artandi et al. (8) demonstrated that telomere attrition promotes the development of epithelial cancers in mice by a process of breakage-fusion-bridge leading to the formation of nonreciprocal translocations, a hallmark of human carcinoma, with oncogenic potential, first by carrying chimeric or deregulated oncogenes at their breakpoints, and second by altering gene dosage. Interestingly, Gonzalez-Suarez et al. (34) found that telomerase-deficient mice with short telomeres were resistant to skin tumorigenesis. This finding suggests that short telomeres are associated with telomerase activity because high telomerase activity results in increased propagation of cells with DNA damage. However, in telomerase-deficient mice with short telomeres, cells may not survive long enough to undergo neoplastic transformation.
In summary, we have shown, to our knowledge for the first time, that telomere length appears to be associated with increased risks for head and neck, bladder, lung, and renal cell carcinomas. Future research should focus on the associations of telomere dynamics, cell cycle checkpoints, apoptosis, the activation of telomerase, and DNA repair capacity, ultimately, with the goal of enhancing our ability to identify high-risk subgroups.
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NOTES |
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Manuscript received December 13, 2002; revised May 30, 2003; accepted June 6, 2003.
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