端粒酶基因多态与儿童急性淋巴细胞白血病易感性的分子流行病学研究
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摘要
急性淋巴细胞白血病（acute lymphoblastic leukemia, ALL）是儿童最常见的肿瘤，约占儿童肿瘤的1/3。美国“监测、流行病学和最终结果”（surveillence,epidemiology, and end results, SEER）显示每年大约有3,250儿童诊断为白血病，中国每年新增ALL儿童12,000人，ALL成为威胁全球儿童健康的重要疾病之一。如何识别ALL的高危人群，降低儿童ALL的发病率，是摆在我们面前的重要问题。儿童ALL的具体病因目前尚不完全清楚，发病机制也十分复杂。流行病学、分子生物学和遗传学的研究证实，儿童ALL是环境因素和遗传因素共同作用的疾病。宏观流行病学研究表明，个体因素（如免疫系统缺陷性疾病）和环境暴露因素（如家庭装修、母亲孕期感染、长期接触放射环境）是ALL发病的危险因素。但不同遗传背景的个体在相同的环境危险因素暴露下发生ALL的危险性不同，说明遗传背景的差异影响了个体癌症易感性的高低。因此，识别ALL的易感基因是鉴别ALL高危人群的关键。
目前已知，染色体和基因重排是白血病的重要遗传学标志。染色体的末端异常是导致基因重排的重要原因并参与了白血病的发病。端粒位于染色体的末端，由DNA重复序列和蛋白组成，产生一个称为端粒体的特殊结构来保护染色体末端防止融合和降解。端粒包括端粒酶和端粒相关蛋白（shelterin）。端粒酶以自身RNA（TERC）为模板，在端粒酶反转录酶（TERT）的作用下，在染色体末端合成端粒TTAGGG重复序列，维持端粒长度的稳定。端粒酶活性在大多数体细胞中是缺失的，但在90%人类肿瘤中可以检测到端粒酶过表达。端粒酶的异常表达导致细胞生命的延长和永生，从而导致癌变，并且在肿瘤形成过程中，端粒酶还参与DNA损伤修复、染色质结构调节和基因表达调控。目前研究发现，对于大约10%~15%不表达TERT或端粒酶的细胞，选择性地延长端粒（alternative lengthening of telomeres, ALT）通路是维持端粒长度的机制，且为正常细胞死亡逃逸和恶性转化的机制。最新的meta分析发现，缩短的端粒与多种肿瘤的发病风险有关，并且成人ALL患者的端粒长度比正常骨髓细胞短，伴随着端粒酶活性的增高。因此，端粒酶的遗传变异与儿童ALL的发生发展可能存在很大的关联，但是目前尚未见相关研究报道。
人类基因组计划（Human Genome Project, HGP）研究表明，基因组中的遗传变异会影响基因的结构和功能，而单核苷酸多态性（single nucleotidepolymorphism, SNP）是遗传变异中最常见的一种类型。目前有多个全基因组关联研究（gemome-wide association study, GWAS）结果发现TERT基因内部多个SNPs与多种肿瘤易感性有关、TERC基因附近SNPs与端粒长度有关。
近年来儿童ALL遗传药理学的研究成为热点，而单核苷酸多态性是遗传药理学所研究的重要的遗传变异。大剂量甲氨喋呤（high dose methotrexate,HDMTX）联合亚叶酸钙（calcium folinate, CF）是儿童ALL常见的治疗方法，成为儿童ALL遗传药理学研究的经典模型。端粒酶已被证实为影响药物代谢的候选基因。
本研究旨在通过宏观流行病学和分子流行病学相结合的手段，采用病例-对照的研究方法，评价端粒酶主要组成基因（TERT和TERC基因）多态变异及端粒长度与儿童ALL发生风险间的关系，并在此基础上分析两基因多态变异与端粒长度及大剂量甲氨喋呤代谢敏感性之间的关系，该研究结果为进一步揭示儿童ALL发病机制提供重要的理论依据，并为筛选与中国人群ALL发生风险相关的易感性生物标志物以用于今后筛查高危人群或易感个体，实施目标明确的个体化预防、干预和临床治疗等方面提供新的策略和思路。
第一部分
TERT基因多态与儿童急性淋巴细胞白血病易感性的分子流行病学研究
TERT在维持端粒长度、染色体稳定和细胞凋亡/死亡等方面起到重要作用。有研究表明白血病患者端粒长度缩短，端粒酶活性增高，因此TERT基因遗传变异可能与儿童ALL的发生发展有关。
本部分研究对象包括570例经骨髓涂片、免疫分型确诊的儿童ALL病例和673例在年龄和性别上与病例匹配的对照。采用标签SNPs的选点策略，筛选出11个标签SNPs以覆盖整个TERT基因（包括上下游各2,000bp），探讨TERT遗传变异与儿童ALL发病风险的关系。
单个SNP位点Logistic回归结果显示，TERT启动子区rs2736109和rs2735940，内含子区（rs2736100、rs2853676、rs10069690和rs4246742）与儿童ALL的发病风险有关。在联合分析中，我们发现携带6～11个危险等位基因的个体比携带0～5个危险等位基因的个体更易罹患ALL（调整OR=1.44，95%CI=1.13-1.84），并且这种风险在B-ALL中更明显。值得注意的是，TERT第2内含子的两个SNPs与端粒长度有关，即rs2736100CC基因型比野生基因型增加61%的机会获得长端粒，rs2853676AG/AA基因型比野生基因型增加27%的机会获得长端粒。但未发现这些标签SNPs与TERT mRNA表达水平和端粒酶活性的关系。
上述结果显示，TERT遗传变异与儿童ALL的易感性有关，可能影响了端粒长度的自身平衡，从而参与了ALL的发病。
第二部分
TERC基因多态与儿童急性淋巴细胞白血病易感性的分子流行病学研究
端粒在维持染色体稳定、细胞凋亡/死亡和肿瘤发生中起到重要作用。端粒长度缩短和/或端粒功能障碍是肿瘤形成的重要原因。端粒的延长依赖于TERC编码的自身RNA模板，在TERT编码的逆转录酶的作用下进行。最近的GWAS发现包含TERC的3q26的两个SNPs位点（rs12696304和rs16847897）与端粒长度有关。因此，TERC的遗传变异可能与儿童ALL的发生发展有关。本部分研究对象包括570例儿童ALL病例和673例在年龄和性别上与病例匹配的对照。对这两个点采用TaqMan基因分型法，探讨TERC遗传变异与儿童ALL发病风险的关系。结果发现与野生CC基因型相比，携带rs16847897CG基因型的儿童罹患ALL的风险明显增加（调整OR=1.29,95%CI=1.00-1.67），而rs12696304多态变异与儿童ALL的发病风险无关。结果还发现，这两个SNPs位点与端粒长度无关。上述结果表明，TERC基因与中国汉族儿童ALL发病风险升高有关，其中rs16847897CG杂合子可能为危险基因型。
第三部分端粒长度与儿童急性淋巴细胞白血病易感性的关联研究
端粒长度具有遗传特性，不同的个体、不同的细胞、不同的组织间均存在差异。端粒长度的缩短在肿瘤的发生和发展中起到重要作用。多项流行病学研究发现，缩短的端粒长度与多种肿瘤的发生风险有关，但目前未见有关中国汉族儿童ALL与端粒长度的研究报道。本研究针对570例儿童ALL病例和673例健康对照，采用T/S相对定量PCR法进行相对端粒长度的检测，并进行了相关危险因素的流行病学研究。结果发现，与最短的端粒长度组（1分组）相比，适中的端粒长度（2分组和3分组）与ALL发病风险降低有关，表现为保护作用，调整的OR值和95%可信区间分别为0.65（0.47-0.91）和0.56（0.40-0.79）。该研究结果提示，极端的端粒长度可能是影响儿童ALL发病风险的重要生物标志物。
第四部分
端粒酶基因多态与大剂量甲氨蝶呤排泄延迟的关联研究
大剂量甲氨蝶呤联合亚叶酸钙解救是目前预防儿童ALL髓外白血病和全身巩固治疗的有效措施，但治疗时若发生排泄延迟，常发生严重的毒副作用（如口腔黏膜、肝肾功能损害等），且个体间存在较大差异。端粒酶基因多态可能通过影响MTX药物凋亡通路而与MTX代谢敏感性存在关联。
本部分研究对象为123名接受HDMTX治疗的ALL患儿，进行HDMTX治疗开始后44h和68h时间点的MTX代谢及肝脏毒性与端粒酶主要基因（TERT和TERC）多态变异的研究。
研究发现，123例接受大剂量MTX治疗的患儿，44h监测点MTX血清平均浓度为0.73±0.84μmol/L，68h为0.21±0.27μmol/L，一共有69例（56.1%）发生了MTX排泄延迟，41例（33.3%）发生肝功能损伤，其中有18例（14.6%）MTX排泄延迟的患儿发生了肝功能损伤。位于TERT启动子区的rs2735940CT/TT基因型个体发生HDMTX44h排泄延迟的机率比携带CC基因型个体增加1.89倍（OR=2.89，95%CI=1.08-7.77），而位于TERT第2内含子的rs2736100AC/CC基因型个体发生HDMTX44h排泄延迟的机率比携带AA基因型个体增加1.64倍（OR=2.64，95%CI=1.01-6.87），但是这两个SNPs与68h MTX排泄延迟没有关联，亦与肝功能损伤无关联。
上述结果提示TERT基因多态与HDMTX治疗后44h血药浓度及排泄延迟之间存在关联，这为MTX的遗传药理学研究提供参考，为儿童ALL的临床个体化治疗提供新的理论依据。
Acute lymphoblastic leukemia (ALL) is the most common malignant disorder inchildhood accounting for approximately three-quarters of all childhood leukemiadiagnoses and the incidence is increasing worldwide. According to the report ofsurveillence, epidemiology, and end results (SEER) in USA, each year, around3,250children are diagnosed with leukemia, of which about2,400are ALL. Approximately12,000new ALL cases were diagnosed each year in China. Despite extensive studies,little is known about the pathogenesis of ALL, although both inherited susceptibilityand specific environmental exposure (such as house painting, infection duringpregnancy, irradiation) are supposed to play a role in this process.
Chromosome and gene rearrangements are the hallmark of leukemia. Abnormalchromosome extremities favor such rearrangements and are thereby involved inleukemogenesis. Telomere-driven genome instability is a widespread cause ofgenome instabiliy. Telomere is a complex DNA-protein structure located at the end ofall linear eukaryotic chromosomes. Telomerase consists of a catalytic protein subunit,human telomerase reverse transcriptase (TERT), the RNA component of thetelomerase (TERC) and the shelterin complex. The major role of human telomerase isto catalyze the addition of telomeric repeat sequences TTAGGG onto chromosomeends for stabilization of telomere length in attaining cellular immortality and maytherefore be a critical step in carcinogenesis. Moreover, telomerase is involved inDNA damage repair, chromatin regulation and genes regulation. It is known thattelomerase is active in90%of tumors, while in those cells which do not expressTERT or telomerase at all, the alternative lengthening of telomeres (ALT) pathway is assumed to preserve the length of telomeres, which are obligate for senescencebypass and for neoplastic transformation of normal cells. Recent meta-analyses havereported an association between short telomeres and increased risk of several types ofhuman cancers. Moreover, telomere length was shorter in ALL subjects than innormal bone marrow mononuclear cells. And compared to healthy individuals,telomerase activity was higher in acute leukemia children. Therefore, genetic variantsof telomerase may play important roles in the development of childhood ALL,however, there were not any studies about it.
With the completion of Human Genome Projict (HGP), it is known that geneticvariantions play an important role in gene structure and function, and singlenucleotide polymorphism (SNP) is the most common type of genetic variants. In theprevious years, genome-wide association studies (GWAS) have successfullydiscovered multiple SNPs at TERT associated with risk for multiple cancers andSNPs near TERC are associated with telomere length.
Moreover, in recent years, pharmacogenetics in childhood ALL has become amajor field of research and SNPs is of particular importance for drugs with a verynarrow therapeutic index, such as Methotrexate (MTX). The pharmacologic effects ofMTX are well characterized, and telomerase has emerged as one drug resistancecandidate and may be involved in drug metabolism.
In the present study, we conducted a hospital-based case-control study toinvestigate the associations between genetic variants of telomerase and childhoodALL susceptibility, telomere length and the childhood ALL susceptibility and thepharmacogenetics of MTX. Our study searched for potential biomarkers forchildhood ALL susceptibility and provides clues of MTX for the childhood ALLpharmacogenetics study, which may provide a “proof-of-principle” approach forindividualized prevention, clinical decision and individualized treatment.
Part1
A Molecular Epidemiology Study on the Associationsbetween the TERT Polymorphisms and GeneticSusceptibility of Childhood Acute Lymphoblastic Leukemia
Human TERT is essential for the maintenance of telomere DNA length,chromosomal stability, and cellular immortality. We hypothesized that TERTpolymorphisms are associated with risk of childhood acute lymphoblastic leukemia(ALL). In a case-control study of570cases and673cancer-free controls of Chinese,we genotyped TERT tagging single-nucleotide polymorphisms (tSNPs) to evaluatethe association and further examined the association among these polymorphisms andtelomerase activity, telomere length. We found that two TERT promoter region tSNPs(rs2735940and rs2736109) and four intron region tSNPs (rs2736100, rs2853676,rs10069690and rs4246742) were associated with risk of childhood ALL. When thesetSNPs were evaluated together by the number of putative risk alleles, a statisticallyincreased risk of ALL was associated with6-11risk alleles, compared with those with0-5risk alleles [adjusted odds ratio (OR)=1.44,95%confidence interval (95%CI)=1.13-1.84] and this association was more pronounced in B-ALL. Notably, TERTintron2polymorphisms (rs2736100and rs2853676) are associated with longertelomeres. Compared with the wild genotype, CC genotype of rs2736100increased61%and AG/AA genotype of rs2853676increased27%susceptibility of longertelomere length. However, no effect modifications were found for variant alleles andtelomerase activity. Our findings suggested that TERT genetic variants are associatedwith the susceptibility of childhood ALL. They may influence the telomere lengthhomeostasis and contributed to the development of childhood ALL.
Part2
A Molecular Epidemiology Study on the Associationsbetween the TERC Polymorphisms and GeneticSusceptibility of Childhood Acute Lymphoblastic Leukemia
Telomeres are involved in maintaining chromosomal stability, cellularimmortality and tumorigenesis. A recent GWAS has identified an associationbetween telomere length and two common variants (rs12696304and rs16847897) at3q26that includes TERC. We hypothesized that the two variants and relativetelomere length (RTL) would be a predictor of the risk of childhood acutelymphoblastic leukemia (ALL). A case-control study of570cases and673cancer-free controls of Chinese children was performed. We found that rs16847897CG genotype increased risk of childhood ALL compared with the CC genotype by29%. However, there was no association between this polymorphism and telomerelength. Our findings indicated that TERC variants are associated with elevated risk ofALL in Chinese children and rs16847897heterozygote may be the risk genotype ofchildhood ALL.
Part3
Telomere Length Contribute to Childhood AcuteLymphoblastic Leukemia Risk
Telomere length is a heritable trait, and is variable among different types of cells,tissues and individuals. Telomere shortening is involved in initiation and progressionof malignancies. A series of epidemiological studies have examined the associationbetween shortened telomeres and risk of cancers, but there was no study aboutchildhood ALL in Chinese Han population. In a case-control study of570cases and673cancer-free controls of Chinese children, we examined relative telomere length. We found that there was a protective relationship between second and third quartilesof RTL and risk of ALL [adjusted OR with95%CI by quartile:0.65(0.47-0.91) and0.56(0.40-0.79)], compared with the first quartile (shortest) RTL. Our findingsindicated that extreme telomere length may be a potential predictor for future risk ofALL.
Part4
Telomerase Polymorphisms Are Associated withMethotrexate Elimination after High-Dose Infusion
High-dose MTX (HDMTX) with calcium folinate (CF) rescue has been widelyused in the treatment of ALL. The high doses and therefore of a high systemicexposure to MTX was shown to significantly improve the outcome in children withALL. Nevertheless, HDMTX administration is responsible for toxic effects includingdermatitis and mucositis, which are related to serum concentrations. Inter-individualvariability in MTX disposition is well known. Genetic variants of telomerase may beinvolved in drug metabolism and are associated with the MTX metabolism.123casesof childhood ALL undergoing HDMTX chemotherapy with CF rescue were includedin the study and their genomic DNA was genotyped to identify polymorphisms attelomerase. We obtained the values of their plasma MTX concentration and liverenzymes at the same time. As a result, we found the serum MTX level was0.73±0.84μmol/L at44h after HDMTX treatment,0.21±0.27μmol/L at68h. The overallnumber with delayed MTX elimination was69(56.1%) and MTX hepatotoxicity was41(33.3%). Those cases both with delayed MTX elimination and hepatotoxicity were18(14.6%). Furthermore, TERT promoter SNP rs2735940CT/TT genotype increasedthe risk of delayed MTX elimination at44h by1.89fold compared with CC genotypeand TERT intron2SNP rs2736100AC/CC genotype increased the risk of delayedMTX elimination at44h by1.64fold compared with AA genotype. Our resultsindicated that the TERT polymorphisms may be involved in the clearance of serum MTX and act as a biological marker for the MTX treatment.
目前已知，染色体和基因重排是白血病的重要遗传学标志。染色体的末端异常是导致基因重排的重要原因并参与了白血病的发病。端粒位于染色体的末端，由DNA重复序列和蛋白组成，产生一个称为端粒体的特殊结构来保护染色体末端防止融合和降解。端粒包括端粒酶和端粒相关蛋白（shelterin）。端粒酶以自身RNA（TERC）为模板，在端粒酶反转录酶（TERT）的作用下，在染色体末端合成端粒TTAGGG重复序列，维持端粒长度的稳定。端粒酶活性在大多数体细胞中是缺失的，但在90%人类肿瘤中可以检测到端粒酶过表达。端粒酶的异常表达导致细胞生命的延长和永生，从而导致癌变，并且在肿瘤形成过程中，端粒酶还参与DNA损伤修复、染色质结构调节和基因表达调控。目前研究发现，对于大约10%~15%不表达TERT或端粒酶的细胞，选择性地延长端粒（alternative lengthening of telomeres, ALT）通路是维持端粒长度的机制，且为正常细胞死亡逃逸和恶性转化的机制。最新的meta分析发现，缩短的端粒与多种肿瘤的发病风险有关，并且成人ALL患者的端粒长度比正常骨髓细胞短，伴随着端粒酶活性的增高。因此，端粒酶的遗传变异与儿童ALL的发生发展可能存在很大的关联，但是目前尚未见相关研究报道。
人类基因组计划（Human Genome Project, HGP）研究表明，基因组中的遗传变异会影响基因的结构和功能，而单核苷酸多态性（single nucleotidepolymorphism, SNP）是遗传变异中最常见的一种类型。目前有多个全基因组关联研究（gemome-wide association study, GWAS）结果发现TERT基因内部多个SNPs与多种肿瘤易感性有关、TERC基因附近SNPs与端粒长度有关。
近年来儿童ALL遗传药理学的研究成为热点，而单核苷酸多态性是遗传药理学所研究的重要的遗传变异。大剂量甲氨喋呤（high dose methotrexate,HDMTX）联合亚叶酸钙（calcium folinate, CF）是儿童ALL常见的治疗方法，成为儿童ALL遗传药理学研究的经典模型。端粒酶已被证实为影响药物代谢的候选基因。
本研究旨在通过宏观流行病学和分子流行病学相结合的手段，采用病例-对照的研究方法，评价端粒酶主要组成基因（TERT和TERC基因）多态变异及端粒长度与儿童ALL发生风险间的关系，并在此基础上分析两基因多态变异与端粒长度及大剂量甲氨喋呤代谢敏感性之间的关系，该研究结果为进一步揭示儿童ALL发病机制提供重要的理论依据，并为筛选与中国人群ALL发生风险相关的易感性生物标志物以用于今后筛查高危人群或易感个体，实施目标明确的个体化预防、干预和临床治疗等方面提供新的策略和思路。
第一部分
TERT基因多态与儿童急性淋巴细胞白血病易感性的分子流行病学研究
TERT在维持端粒长度、染色体稳定和细胞凋亡/死亡等方面起到重要作用。有研究表明白血病患者端粒长度缩短，端粒酶活性增高，因此TERT基因遗传变异可能与儿童ALL的发生发展有关。
本部分研究对象包括570例经骨髓涂片、免疫分型确诊的儿童ALL病例和673例在年龄和性别上与病例匹配的对照。采用标签SNPs的选点策略，筛选出11个标签SNPs以覆盖整个TERT基因（包括上下游各2,000bp），探讨TERT遗传变异与儿童ALL发病风险的关系。
单个SNP位点Logistic回归结果显示，TERT启动子区rs2736109和rs2735940，内含子区（rs2736100、rs2853676、rs10069690和rs4246742）与儿童ALL的发病风险有关。在联合分析中，我们发现携带6～11个危险等位基因的个体比携带0～5个危险等位基因的个体更易罹患ALL（调整OR=1.44，95%CI=1.13-1.84），并且这种风险在B-ALL中更明显。值得注意的是，TERT第2内含子的两个SNPs与端粒长度有关，即rs2736100CC基因型比野生基因型增加61%的机会获得长端粒，rs2853676AG/AA基因型比野生基因型增加27%的机会获得长端粒。但未发现这些标签SNPs与TERT mRNA表达水平和端粒酶活性的关系。
上述结果显示，TERT遗传变异与儿童ALL的易感性有关，可能影响了端粒长度的自身平衡，从而参与了ALL的发病。
第二部分
TERC基因多态与儿童急性淋巴细胞白血病易感性的分子流行病学研究
端粒在维持染色体稳定、细胞凋亡/死亡和肿瘤发生中起到重要作用。端粒长度缩短和/或端粒功能障碍是肿瘤形成的重要原因。端粒的延长依赖于TERC编码的自身RNA模板，在TERT编码的逆转录酶的作用下进行。最近的GWAS发现包含TERC的3q26的两个SNPs位点（rs12696304和rs16847897）与端粒长度有关。因此，TERC的遗传变异可能与儿童ALL的发生发展有关。本部分研究对象包括570例儿童ALL病例和673例在年龄和性别上与病例匹配的对照。对这两个点采用TaqMan基因分型法，探讨TERC遗传变异与儿童ALL发病风险的关系。结果发现与野生CC基因型相比，携带rs16847897CG基因型的儿童罹患ALL的风险明显增加（调整OR=1.29,95%CI=1.00-1.67），而rs12696304多态变异与儿童ALL的发病风险无关。结果还发现，这两个SNPs位点与端粒长度无关。上述结果表明，TERC基因与中国汉族儿童ALL发病风险升高有关，其中rs16847897CG杂合子可能为危险基因型。
第三部分端粒长度与儿童急性淋巴细胞白血病易感性的关联研究
端粒长度具有遗传特性，不同的个体、不同的细胞、不同的组织间均存在差异。端粒长度的缩短在肿瘤的发生和发展中起到重要作用。多项流行病学研究发现，缩短的端粒长度与多种肿瘤的发生风险有关，但目前未见有关中国汉族儿童ALL与端粒长度的研究报道。本研究针对570例儿童ALL病例和673例健康对照，采用T/S相对定量PCR法进行相对端粒长度的检测，并进行了相关危险因素的流行病学研究。结果发现，与最短的端粒长度组（1分组）相比，适中的端粒长度（2分组和3分组）与ALL发病风险降低有关，表现为保护作用，调整的OR值和95%可信区间分别为0.65（0.47-0.91）和0.56（0.40-0.79）。该研究结果提示，极端的端粒长度可能是影响儿童ALL发病风险的重要生物标志物。
第四部分
端粒酶基因多态与大剂量甲氨蝶呤排泄延迟的关联研究
大剂量甲氨蝶呤联合亚叶酸钙解救是目前预防儿童ALL髓外白血病和全身巩固治疗的有效措施，但治疗时若发生排泄延迟，常发生严重的毒副作用（如口腔黏膜、肝肾功能损害等），且个体间存在较大差异。端粒酶基因多态可能通过影响MTX药物凋亡通路而与MTX代谢敏感性存在关联。
本部分研究对象为123名接受HDMTX治疗的ALL患儿，进行HDMTX治疗开始后44h和68h时间点的MTX代谢及肝脏毒性与端粒酶主要基因（TERT和TERC）多态变异的研究。
研究发现，123例接受大剂量MTX治疗的患儿，44h监测点MTX血清平均浓度为0.73±0.84μmol/L，68h为0.21±0.27μmol/L，一共有69例（56.1%）发生了MTX排泄延迟，41例（33.3%）发生肝功能损伤，其中有18例（14.6%）MTX排泄延迟的患儿发生了肝功能损伤。位于TERT启动子区的rs2735940CT/TT基因型个体发生HDMTX44h排泄延迟的机率比携带CC基因型个体增加1.89倍（OR=2.89，95%CI=1.08-7.77），而位于TERT第2内含子的rs2736100AC/CC基因型个体发生HDMTX44h排泄延迟的机率比携带AA基因型个体增加1.64倍（OR=2.64，95%CI=1.01-6.87），但是这两个SNPs与68h MTX排泄延迟没有关联，亦与肝功能损伤无关联。
上述结果提示TERT基因多态与HDMTX治疗后44h血药浓度及排泄延迟之间存在关联，这为MTX的遗传药理学研究提供参考，为儿童ALL的临床个体化治疗提供新的理论依据。
Acute lymphoblastic leukemia (ALL) is the most common malignant disorder inchildhood accounting for approximately three-quarters of all childhood leukemiadiagnoses and the incidence is increasing worldwide. According to the report ofsurveillence, epidemiology, and end results (SEER) in USA, each year, around3,250children are diagnosed with leukemia, of which about2,400are ALL. Approximately12,000new ALL cases were diagnosed each year in China. Despite extensive studies,little is known about the pathogenesis of ALL, although both inherited susceptibilityand specific environmental exposure (such as house painting, infection duringpregnancy, irradiation) are supposed to play a role in this process.
Chromosome and gene rearrangements are the hallmark of leukemia. Abnormalchromosome extremities favor such rearrangements and are thereby involved inleukemogenesis. Telomere-driven genome instability is a widespread cause ofgenome instabiliy. Telomere is a complex DNA-protein structure located at the end ofall linear eukaryotic chromosomes. Telomerase consists of a catalytic protein subunit,human telomerase reverse transcriptase (TERT), the RNA component of thetelomerase (TERC) and the shelterin complex. The major role of human telomerase isto catalyze the addition of telomeric repeat sequences TTAGGG onto chromosomeends for stabilization of telomere length in attaining cellular immortality and maytherefore be a critical step in carcinogenesis. Moreover, telomerase is involved inDNA damage repair, chromatin regulation and genes regulation. It is known thattelomerase is active in90%of tumors, while in those cells which do not expressTERT or telomerase at all, the alternative lengthening of telomeres (ALT) pathway is assumed to preserve the length of telomeres, which are obligate for senescencebypass and for neoplastic transformation of normal cells. Recent meta-analyses havereported an association between short telomeres and increased risk of several types ofhuman cancers. Moreover, telomere length was shorter in ALL subjects than innormal bone marrow mononuclear cells. And compared to healthy individuals,telomerase activity was higher in acute leukemia children. Therefore, genetic variantsof telomerase may play important roles in the development of childhood ALL,however, there were not any studies about it.
With the completion of Human Genome Projict (HGP), it is known that geneticvariantions play an important role in gene structure and function, and singlenucleotide polymorphism (SNP) is the most common type of genetic variants. In theprevious years, genome-wide association studies (GWAS) have successfullydiscovered multiple SNPs at TERT associated with risk for multiple cancers andSNPs near TERC are associated with telomere length.
Moreover, in recent years, pharmacogenetics in childhood ALL has become amajor field of research and SNPs is of particular importance for drugs with a verynarrow therapeutic index, such as Methotrexate (MTX). The pharmacologic effects ofMTX are well characterized, and telomerase has emerged as one drug resistancecandidate and may be involved in drug metabolism.
In the present study, we conducted a hospital-based case-control study toinvestigate the associations between genetic variants of telomerase and childhoodALL susceptibility, telomere length and the childhood ALL susceptibility and thepharmacogenetics of MTX. Our study searched for potential biomarkers forchildhood ALL susceptibility and provides clues of MTX for the childhood ALLpharmacogenetics study, which may provide a “proof-of-principle” approach forindividualized prevention, clinical decision and individualized treatment.
Part1
A Molecular Epidemiology Study on the Associationsbetween the TERT Polymorphisms and GeneticSusceptibility of Childhood Acute Lymphoblastic Leukemia
Human TERT is essential for the maintenance of telomere DNA length,chromosomal stability, and cellular immortality. We hypothesized that TERTpolymorphisms are associated with risk of childhood acute lymphoblastic leukemia(ALL). In a case-control study of570cases and673cancer-free controls of Chinese,we genotyped TERT tagging single-nucleotide polymorphisms (tSNPs) to evaluatethe association and further examined the association among these polymorphisms andtelomerase activity, telomere length. We found that two TERT promoter region tSNPs(rs2735940and rs2736109) and four intron region tSNPs (rs2736100, rs2853676,rs10069690and rs4246742) were associated with risk of childhood ALL. When thesetSNPs were evaluated together by the number of putative risk alleles, a statisticallyincreased risk of ALL was associated with6-11risk alleles, compared with those with0-5risk alleles [adjusted odds ratio (OR)=1.44,95%confidence interval (95%CI)=1.13-1.84] and this association was more pronounced in B-ALL. Notably, TERTintron2polymorphisms (rs2736100and rs2853676) are associated with longertelomeres. Compared with the wild genotype, CC genotype of rs2736100increased61%and AG/AA genotype of rs2853676increased27%susceptibility of longertelomere length. However, no effect modifications were found for variant alleles andtelomerase activity. Our findings suggested that TERT genetic variants are associatedwith the susceptibility of childhood ALL. They may influence the telomere lengthhomeostasis and contributed to the development of childhood ALL.
Part2
A Molecular Epidemiology Study on the Associationsbetween the TERC Polymorphisms and GeneticSusceptibility of Childhood Acute Lymphoblastic Leukemia
Telomeres are involved in maintaining chromosomal stability, cellularimmortality and tumorigenesis. A recent GWAS has identified an associationbetween telomere length and two common variants (rs12696304and rs16847897) at3q26that includes TERC. We hypothesized that the two variants and relativetelomere length (RTL) would be a predictor of the risk of childhood acutelymphoblastic leukemia (ALL). A case-control study of570cases and673cancer-free controls of Chinese children was performed. We found that rs16847897CG genotype increased risk of childhood ALL compared with the CC genotype by29%. However, there was no association between this polymorphism and telomerelength. Our findings indicated that TERC variants are associated with elevated risk ofALL in Chinese children and rs16847897heterozygote may be the risk genotype ofchildhood ALL.
Part3
Telomere Length Contribute to Childhood AcuteLymphoblastic Leukemia Risk
Telomere length is a heritable trait, and is variable among different types of cells,tissues and individuals. Telomere shortening is involved in initiation and progressionof malignancies. A series of epidemiological studies have examined the associationbetween shortened telomeres and risk of cancers, but there was no study aboutchildhood ALL in Chinese Han population. In a case-control study of570cases and673cancer-free controls of Chinese children, we examined relative telomere length. We found that there was a protective relationship between second and third quartilesof RTL and risk of ALL [adjusted OR with95%CI by quartile:0.65(0.47-0.91) and0.56(0.40-0.79)], compared with the first quartile (shortest) RTL. Our findingsindicated that extreme telomere length may be a potential predictor for future risk ofALL.
Part4
Telomerase Polymorphisms Are Associated withMethotrexate Elimination after High-Dose Infusion
High-dose MTX (HDMTX) with calcium folinate (CF) rescue has been widelyused in the treatment of ALL. The high doses and therefore of a high systemicexposure to MTX was shown to significantly improve the outcome in children withALL. Nevertheless, HDMTX administration is responsible for toxic effects includingdermatitis and mucositis, which are related to serum concentrations. Inter-individualvariability in MTX disposition is well known. Genetic variants of telomerase may beinvolved in drug metabolism and are associated with the MTX metabolism.123casesof childhood ALL undergoing HDMTX chemotherapy with CF rescue were includedin the study and their genomic DNA was genotyped to identify polymorphisms attelomerase. We obtained the values of their plasma MTX concentration and liverenzymes at the same time. As a result, we found the serum MTX level was0.73±0.84μmol/L at44h after HDMTX treatment,0.21±0.27μmol/L at68h. The overallnumber with delayed MTX elimination was69(56.1%) and MTX hepatotoxicity was41(33.3%). Those cases both with delayed MTX elimination and hepatotoxicity were18(14.6%). Furthermore, TERT promoter SNP rs2735940CT/TT genotype increasedthe risk of delayed MTX elimination at44h by1.89fold compared with CC genotypeand TERT intron2SNP rs2736100AC/CC genotype increased the risk of delayedMTX elimination at44h by1.64fold compared with AA genotype. Our resultsindicated that the TERT polymorphisms may be involved in the clearance of serum MTX and act as a biological marker for the MTX treatment.
引文
[1] Kaatsch, P. Epidemiology of childhood cancer. Cancer Treat Rev.2010;36:277-85.
[2] Onciu, M. Acute lymphoblastic leukemia. Hematol Oncol Clin North Am.2009;23:655-74.
[3] Pui, CH, WL Carroll, S Meshinchi and RJ Arceci. Biology, risk stratification,and therapy of pediatric acute leukemias: an update. J Clin Oncol.2011;29:551-65.
[4] Liu, R, L Zhang, CM McHale and SK Hammond. Paternal smoking and riskof childhood acute lymphoblastic leukemia: systematic review andmeta-analysis. J Oncol.2011;2011:854584.
[5] Eden, T. Aetiology of childhood leukaemia. Cancer Treat Rev.2010;36:286-97.
[6] Greaves. Science, medicine, and the future: childhood leukaemia. BritishMedical Journal.2002;324:283-287.
[7] Belson, M, B Kingsley and A Holmes. Risk factors for acute leukemia inchildren: a review. Environ Health Perspect.2007;115:138-45.
[8] Van Maele-Fabry, G, AC Lantin, P Hoet and D Lison. Childhood leukaemiaand parental occupational exposure to pesticides: a systematic review andmeta-analysis. Cancer Causes Control.2010;21:787-809.
[9] Shi, JY, ZH Ren, B Jiao, et al. Genetic variations of DNA repair genes andtheir prognostic significance in patients with acute myeloid leukemia. Int JCancer.2011;128:233-8.
[10] Salisbury, BA, M Pungliya, JY Choi, et al. SNP and haplotype variation in thehuman genome. Mutat Res.2003;526:53-61.
[11] Sachidanandam, R, D Weissman, SC Schmidt, et al. A map of human genomesequence variation containing1.42million single nucleotide polymorphisms.Nature.2001;409:928-33.
[12] Davidsen, ML, K Dalhoff and K Schmiegelow. Pharmacogenetics influencetreatment efficacy in childhood acute lymphoblastic leukemia. J PediatrHematol Oncol.2008;30:831-49.
[13] Palm, W and T de Lange. How shelterin protects mammalian telomeres. AnnuRev Genet.2008;42:301-34.
[14] Olovnikov, AM. A theory of marginotomy. The incomplete copying oftemplate margin in enzymic synthesis of polynucleotides and biologicalsignificance of the phenomenon. J Theor Biol.1973;41:181-90.
[15] Allsopp, RC, H Vaziri, C Patterson, et al. Telomere length predicts replicativecapacity of human fibroblasts. Proc Natl Acad Sci U S A.1992;89:10114-8.
[16] Szostak, JW and EH Blackburn. Cloning yeast telomeres on linear plasmidvectors. Cell.1982;29:245-55.
[17] Greider, CW and EH Blackburn. Identification of a specific telomere terminaltransferase activity in Tetrahymena extracts. Cell.1985;43:405-13.
[18] De Semir, D, M Nosrati, S Li and M Kashani-Sabet. Telomerase: goingbeyond the ends. Cell Cycle.2007;6:546-9.
[19] Cong, Y and JW Shay. Actions of human telomerase beyond telomeres. CellRes.2008;18:725-32.
[20] Gigek, CO, MF Leal, PN Silva, et al. hTERT methylation and expression ingastric cancer. Biomarkers.2009;14:630-6.
[21] Divella, R, S Tommasi, R Lacalamita, et al. Circulating hTERT DNA in earlybreast cancer. Anticancer Res.2009;29:2845-9.
[22] Xian, L, H Zhou, X Zhang, et al.[The Expression of hTERT mRNA and p16Protein in Non-small Cell Lung Cancer.]. Zhongguo Fei Ai Za Zhi.2009;12:1005-8.
[23] Capraro, V, L Zane, D Poncet, et al. Telomere deregulations possesscytogenetic, phenotype, and prognostic specificities in acute leukemias. ExpHematol.2011;39:195-202e2.
[24] Turnbull, C, EA Rapley, S Seal, et al. Variants near DMRT1, TERT andATF7IP are associated with testicular germ cell cancer. Nat Genet.2010;42:604-7.
[25] Landi, MT, N Chatterjee, K Yu, et al. A genome-wide association study oflung cancer identifies a region of chromosome5p15associated with risk foradenocarcinoma. Am J Hum Genet.2009;85:679-91.
[26] Rafnar, T, P Sulem, SN Stacey, et al. Sequence variants at theTERT-CLPTM1L locus associate with many cancer types. Nat Genet.2009;41:221-7.
[27] Shete, S, FJ Hosking, LB Robertson, et al. Genome-wide association studyidentifies five susceptibility loci for glioma. Nat Genet.2009;41:899-904.
[28] Haiman, CA, GK Chen, CM Vachon, et al. A common variant at theTERT-CLPTM1L locus is associated with estrogen receptor-negative breastcancer. Nat Genet.2011;43:1210-4.
[29] Kratz, CP, SS Han, PS Rosenberg, et al. Variants in or near KITLG, BAK1,DMRT1, and TERT-CLPTM1L predispose to familial testicular germ celltumour. J Med Genet.2011;48:473-6.
[30] Zhang, Q, NK Kim and J Feigon. Architecture of human telomerase RNA.Proc Natl Acad Sci U S A.2011;108:20325-32.
[31] Zvereva, MI, DM Shcherbakova and OA Dontsova. Telomerase: structure,functions, and activity regulation. Biochemistry (Mosc).2010;75:1563-83.
[32] Soder, AI, SF Hoare, S Muir, et al. Amplification, increased dosage and insitu expression of the telomerase RNA gene in human cancer. Oncogene.1997;14:1013-21.
[33] Vulliamy, T, A Marrone, R Szydlo, et al. Disease anticipation is associatedwith progressive telomere shortening in families with dyskeratosis congenitadue to mutations in TERC. Nat Genet.2004;36:447-9.
[34] Yamaguchi, H, GM Baerlocher, PM Lansdorp, et al. Mutations of the humantelomerase RNA gene (TERC) in aplastic anemia and myelodysplasticsyndrome. Blood.2003;102:916-8.
[35] Armanios, MY, JJ Chen, JD Cogan, et al. Telomerase mutations in familieswith idiopathic pulmonary fibrosis. N Engl J Med.2007;356:1317-26.
[36] Codd, V, M Mangino, P van der Harst, et al. Common variants near TERC areassociated with mean telomere length. Nat Genet.2010;42:197-9.
[37] Levy, D, SL Neuhausen, SC Hunt, et al. Genome-wide association identifiesOBFC1as a locus involved in human leukocyte telomere biology. Proc NatlAcad Sci U S A.2010;107:9293-8.
[38] Wentzensen, IM, L Mirabello, RM Pfeiffer and SA Savage. The association oftelomere length and cancer: a meta-analysis. Cancer Epidemiol BiomarkersPrev.2011;20:1238-50.
[39] Ma, H, Z Zhou, S Wei, et al. Shortened telomere length is associated withincreased risk of cancer: a meta-analysis. PLoS One.2011;6:e20466.
[40] Gancarcikova, M, Z Zemanova, J Brezinova, et al. The role of telomeres andtelomerase complex in haematological neoplasia: the length of telomeres as amarker of carcinogenesis and prognosis of disease. Prague Med Rep.2010;111:91-105.
[41] Park, JI, AS Venteicher, JY Hong, et al. Telomerase modulates Wnt signallingby association with target gene chromatin. Nature.2009;460:66-72.
[42] Indran, IR, MP Hande and S Pervaiz. hTERT overexpression alleviatesintracellular ROS production, improves mitochondrial function, and inhibitsROS-mediated apoptosis in cancer cells. Cancer Res.2011;71:266-76.
[43] Yamada, O, K Kawauchi, M Akiyama, et al. Leukemic cells with increasedtelomerase activity exhibit resistance to imatinib. Leuk Lymphoma.2008;49:1168-77.
[44] Yamada, O, K Ozaki, T Furukawa, et al. Activation of STAT5confersimatinib resistance on leukemic cells through the transcription of TERT andMDR1. Cell Signal.2011;23:1119-27.
[45] Nakayama, J, H Tahara, E Tahara, et al. Telomerase activation by hTRT inhuman normal fibroblasts and hepatocellular carcinomas. Nat Genet.1998;18:65-8.
[46] Morales, CP, SE Holt, M Ouellette, et al. Absence of cancer-associatedchanges in human fibroblasts immortalized with telomerase. Nat Genet.1999;21:115-8.
[47] Artandi, SE, S Alson, MK Tietze, et al. Constitutive telomerase expressionpromotes mammary carcinomas in aging mice. Proc Natl Acad Sci U S A.2002;99:8191-6.
[48] Hahn, WC, SA Stewart, MW Brooks, et al. Inhibition of telomerase limits thegrowth of human cancer cells. Nat Med.1999;5:1164-70.
[49] Kim, S, H Youn, MG Song, et al. Complementary treatment of siTERT forimproving the antitumor effect of TERT-specific I-131therapy. Cancer GeneTher.2012;
[50] Xu, D, A Gruber, C Peterson and P Pisa. Telomerase activity and theexpression of telomerase components in acute myelogenous leukaemia. Br JHaematol.1998;102:1367-75.
[51] Hartmann, U, TH Brummendorf, S Balabanov, et al. Telomere length andhTERT expression in patients with acute myeloid leukemia correlates withchromosomal abnormalities. Haematologica.2005;90:307-16.
[52] Swiggers, SJ, MA Kuijpers, MJ de Cort, HB Beverloo and JM Zijlmans.Critically short telomeres in acute myeloid leukemia with loss or gain of partsof chromosomes. Genes Chromosomes Cancer.2006;45:247-56.
[53] Cogulu, O, B Kosova, E Karaca, et al. Evaluation of telomerase mRNA(hTERT) in childhood acute leukemia. Leuk Lymphoma.2004;45:2477-80.
[54] Campbell, LJ, C Fidler, H Eagleton, et al. hTERT, the catalytic component oftelomerase, is downregulated in the haematopoietic stem cells of patients withchronic myeloid leukaemia. Leukemia.2006;20:671-9.
[55] Drummond, MW, SF Hoare, A Monaghan, et al. Dysregulated expression ofthe major telomerase components in leukaemic stem cells. Leukemia.2005;19:381-9.
[56] Gabet, AS, F Mortreux, P Charneau, et al. Inactivation of hTERTtranscription by Tax. Oncogene.2003;22:3734-41.
[57] Hara, T, Y Matsumura-Arioka, K Ohtani and M Nakamura. Role of humanT-cell leukemia virus type I Tax in expression of the human telomerasereverse transcriptase (hTERT) gene in human T-cells. Cancer Sci.2008;99:1155-63.
[58] Poncet, D, A Belleville, C t'kint de Roodenbeke, et al. Changes in theexpression of telomere maintenance genes suggest global telomeredysfunction in B-chronic lymphocytic leukemia. Blood.2008;111:2388-91.
[59]中华医学会儿科学分会血液学组，中华儿科杂志编辑委员会.儿童急性淋巴细胞白血病诊疗建议(第三次修订草案).中华儿科杂志.2006;44:392-395.
[60] Pieters, R and WL Carroll. Biology and treatment of acute lymphoblasticleukemia. Hematol Oncol Clin North Am.2010;24:1-18.
[61] Matsubara, Y, M Murata, T Yoshida, et al. Telomere length of normalleukocytes is affected by a functional polymorphism of hTERT. BiochemBiophys Res Commun.2006;341:128-31.
[62] Matsubara, Y, M Murata, K Watanabe, et al. Coronary artery disease and afunctional polymorphism of hTERT. Biochem Biophys Res Commun.2006;348:669-72.
[63] Nordfjall, K, P Osterman, O Melander, P Nilsson and G Roos. hTERT(-1327)T/C polymorphism is not associated with age-related telomere attritionin peripheral blood. Biochem Biophys Res Commun.2007;358:215-8.
[64] Beesley, J, HA Pickett, SE Johnatty, et al. Functional polymorphisms in theTERT promoter are associated with risk of serous epithelial ovarian and breastcancers. PLoS One.2011;6:e24987.
[65] Shen, J, MD Gammon, HC Wu, et al. Multiple genetic variants in telomerepathway genes and breast cancer risk. Cancer Epidemiol Biomarkers Prev.2010;19:219-28.
[66] Hsu, CP, NY Hsu, LW Lee and JL Ko. Ets2binding site single nucleotidepolymorphism at the hTERT gene promoter--effect on telomerase expressionand telomere length maintenance in non-small cell lung cancer. Eur J Cancer.2006;42:1466-74.
[67] Raynaud, CM, L Sabatier, O Philipot, KA Olaussen and JC Soria. Telomerelength, telomeric proteins and genomic instability during the multistepcarcinogenic process. Crit Rev Oncol Hematol.2008;66:99-117.
[68] Mooi, WJ and DS Peeper. Oncogene-induced cell senescence--halting on theroad to cancer. N Engl J Med.2006;355:1037-46.
[69] Kim, AS, DA Eastmond and RJ Preston. Childhood acute lymphocyticleukemia and perspectives on risk assessment of early-life stage exposures.Mutat Res.2006;613:138-60.
[70] Yang, JJ, C Cheng, W Yang, et al. Genome-wide interrogation of germlinegenetic variation associated with treatment response in childhood acutelymphoblastic leukemia. JAMA.2009;301:393-403.
[71] Papaemmanuil, E, FJ Hosking, J Vijayakrishnan, et al. Loci on7p12.2,10q21.2and14q11.2are associated with risk of childhood acutelymphoblastic leukemia. Nat Genet.2009;41:1006-10.
[72] Trevino, LR, W Yang, D French, et al. Germline genomic variants associatedwith childhood acute lymphoblastic leukemia. Nat Genet.2009;41:1001-5.
[73] Sherborne, AL, FJ Hosking, RB Prasad, et al. Variation in CDKN2A at9p21.3influences childhood acute lymphoblastic leukemia risk. Nat Genet.2010;42:492-4.
[74] Ellinghaus, E, M Stanulla, G Richter, et al. Identification of germlinesusceptibility loci in ETV6-RUNX1-rearranged childhood acutelymphoblastic leukemia. Leukemia.2011;
[75] Han, S, KM Lee, SK Park, et al. Genome-wide association study of childhoodacute lymphoblastic leukemia in Korea. Leuk Res.2010;34:1271-4.
[76] Chen, JL, MA Blasco and CW Greider. Secondary structure of vertebratetelomerase RNA. Cell.2000;100:503-14.
[77] Greider, CW and EH Blackburn. A telomeric sequence in the RNA ofTetrahymena telomerase required for telomere repeat synthesis. Nature.1989;337:331-7.
[78] Chu, C, K Qu, FL Zhong, SE Artandi and HY Chang. Genomic maps of longnoncoding RNA occupancy reveal principles of RNA-chromatin interactions.Mol Cell.2011;44:667-78.
[79] Shen, Q, Z Zhang, L Yu, et al. Common variants near TERC are associatedwith leukocyte telomere length in the Chinese Han population. Eur J HumGenet.2011;19:721-3.
[80] Kobe, B and AV Kajava. The leucine-rich repeat as a protein recognitionmotif. Curr Opin Struct Biol.2001;11:725-32.
[81] Blasco, MA. The epigenetic regulation of mammalian telomeres. Nat RevGenet.2007;8:299-309.
[82] Autexier, C and NF Lue. The structure and function of telomerase reversetranscriptase. Annu Rev Biochem.2006;75:493-517.
[83] Donate, LE and MA Blasco. Telomeres in cancer and ageing. Philos Trans RSoc Lond B Biol Sci.2011;366:76-84.
[84] Andrew, T, A Aviv, M Falchi, et al. Mapping genetic loci that determineleukocyte telomere length in a large sample of unselected female sibling pairs.Am J Hum Genet.2006;78:480-6.
[85] Vasa-Nicotera, M, S Brouilette, M Mangino, et al. Mapping of a major locusthat determines telomere length in humans. Am J Hum Genet.2005;76:147-51.
[86] Harley, CB. Human ageing and telomeres. Ciba Found Symp.1997;211:129-39; discussion139-44.
[87] Blackburn, EH. Switching and signaling at the telomere. Cell.2001;106:661-73.
[88] von Zglinicki, T, V Serra, M Lorenz, et al. Short telomeres in patients withvascular dementia: an indicator of low antioxidative capacity and a possiblerisk factor? Lab Invest.2000;80:1739-47.
[89] Panossian, LA, VR Porter, HF Valenzuela, et al. Telomere shortening in Tcells correlates with Alzheimer's disease status. Neurobiol Aging.2003;24:77-84.
[90] Wiemann, SU, A Satyanarayana, M Tsahuridu, et al. Hepatocyte telomereshortening and senescence are general markers of human liver cirrhosis.FASEB J.2002;16:935-42.
[91] Benetos, A, JP Gardner, M Zureik, et al. Short telomeres are associated withincreased carotid atherosclerosis in hypertensive subjects. Hypertension.2004;43:182-5.
[92] Kinouchi, Y, N Hiwatashi, M Chida, et al. Telomere shortening in the colonicmucosa of patients with ulcerative colitis. J Gastroenterol.1998;33:343-8.
[93] Brouilette, S, RK Singh, JR Thompson, AH Goodall and NJ Samani. Whitecell telomere length and risk of premature myocardial infarction. ArteriosclerThromb Vasc Biol.2003;23:842-6.
[94] Blasco, MA. Telomeres and human disease: ageing, cancer and beyond. NatRev Genet.2005;6:611-22.
[95] Smogorzewska, A, B van Steensel, A Bianchi, et al. Control of humantelomere length by TRF1and TRF2. Mol Cell Biol.2000;20:1659-68.
[96] von Zglinicki, T, R Pilger and N Sitte. Accumulation of single-strand breaksis the major cause of telomere shortening in human fibroblasts. Free RadicBiol Med.2000;28:64-74.
[97] Blasco, MA, HW Lee, MP Hande, et al. Telomere shortening and tumorformation by mouse cells lacking telomerase RNA. Cell.1997;91:25-34.
[98] Artandi, SE, S Chang, SL Lee, et al. Telomere dysfunction promotesnon-reciprocal translocations and epithelial cancers in mice. Nature.2000;406:641-5.
[99] Joshua, AM, B Vukovic, I Braude, et al. Telomere attrition in isolatedhigh-grade prostatic intraepithelial neoplasia and surrounding stroma ispredictive of prostate cancer. Neoplasia.2007;9:81-9.
[100] Kammori, M, K Takubo, K Nakamura, et al. Telomerase activity and telomerelength in benign and malignant human thyroid tissues. Cancer Lett.2000;159:175-81.
[101] Wong, KK, RS Maser, RM Bachoo, et al. Telomere dysfunction and Atmdeficiency compromises organ homeostasis and accelerates ageing. Nature.2003;421:643-8.
[102] Kim, HR, YJ Kim, HJ Kim, SK Kim and JH Lee. Telomere length changes incolorectal cancers and polyps. J Korean Med Sci.2002;17:360-5.
[103] Sommerfeld, HJ, AK Meeker, MA Piatyszek, et al. Telomerase activity: aprevalent marker of malignant human prostate tissue. Cancer Res.1996;56:218-22.
[104] Stewart, SA and RA Weinberg. Telomeres: cancer to human aging. Annu RevCell Dev Biol.2006;22:531-57.
[105] Capper, R, B Britt-Compton, M Tankimanova, et al. The nature of telomerefusion and a definition of the critical telomere length in human cells. GenesDev.2007;21:2495-508.
[106] Svenson, U, K Nordfjall, B Stegmayr, et al. Breast cancer survival isassociated with telomere length in peripheral blood cells. Cancer Res.2008;68:3618-23.
[107] Borssen, M, I Cullman, U Noren-Nystrom, et al. hTERT promotermethylation and telomere length in childhood acute lymphoblastic leukemia:associations with immunophenotype and cytogenetic subgroup. Exp Hematol.2011;39:1144-51.
[108] Ghaffari, SH, N Shayan-Asl, AH Jamialahmadi, K Alimoghaddam and AGhavamzadeh. Telomerase activity and telomere length in patients with acutepromyelocytic leukemia: indicative of proliferative activity, diseaseprogression, and overall survival. Ann Oncol.2008;19:1927-34.
[109] Kubuki, Y, M Suzuki, H Sasaki, et al. Telomerase activity and telomerelength as prognostic factors of adult T-cell leukemia. Leuk Lymphoma.2005;46:393-9.
[110] Wang, Y, M Fang, X Sun and J Sun. Telomerase activity and telomere lengthin acute leukemia: correlations with disease progression, subtypes and overallsurvival. Int J Lab Hematol.2010;32:230-8.
[111] Epel, ES, EH Blackburn, J Lin, et al. Accelerated telomere shortening inresponse to life stress. Proc Natl Acad Sci U S A.2004;101:17312-5.
[112] Mirabello, L, K Yu, P Kraft, et al. The association of telomere length andgenetic variation in telomere biology genes. Hum Mutat.2010;31:1050-8.
[113] Hoffmann, J, Y Erben, AM Zeiher, S Dimmeler and I Spyridopoulos.Telomere length-heterogeneity among myeloid cells is a predictor forchronological ageing. Exp Gerontol.2009;44:363-6.
[114] Jones, AM, AD Beggs, L Carvajal-Carmona, et al. TERC polymorphisms areassociated both with susceptibility to colorectal cancer and with longertelomeres. Gut.2012;61:248-54.
[115] Treon, SP and BA Chabner. Concepts in use of high-dose methotrexatetherapy. Clin Chem.1996;42:1322-9.
[116] Pitman, SW and E Frei,3rd. Weekly methotrexate-calcium leucovorin rescue:effect of alkalinization on nephrotoxicity; pharmacokinetics in the CNS; anduse in CNS non-Hodgkin's lymphoma. Cancer Treat Rep.1977;61:695-701.
[117] Krajinovic, M and A Moghrabi. Pharmacogenetics of methotrexate.Pharmacogenomics.2004;5:819-34.
[118]张春燕，顾健，李玉珍，卢炜.大剂量甲氨喋呤治疗329例儿童急性淋巴细胞白血病的群体药物动力学研究.中国实验血液学杂志.2008;16:106-110.
[119]汪洋，张华年，宋新文，梅艳.急性淋巴细胞白血病患儿HD-MTX-CF化疗方案中血药浓度的监测.华西药学杂志.2010;25:219-221.
[120]周际昌,实用肿瘤内科学.人民卫生出版社:北京.2003;100-101.
[121]周莉，陆勤，扬引，方拥军.大剂量甲氨喋呤治疗儿童急性淋巴细胞白血病.江苏医药.2007;33:1275.
[122]许静，李庆平，王斌，陆勤，方拥军，黄婕，徐康康，罗琳. GSTT1、GSTM1基因多态性与大剂量甲氨喋呤血药浓度及消化道黏膜损伤的关系研究.安徽医药.2010;14:1336-1338.
[123] Neuman, MG, RG Cameron, JA Haber, et al. Inducers of cytochrome P4502E1enhance methotrexate-induced hepatocytoxicity. Clin Biochem.1999;32:519-36.
[124] Chladek, J, J Martinkova and L Sispera. An in vitro study on methotrexatehydroxylation in rat and human liver. Physiol Res.1997;46:371-9.
[125] Galivan, J and M Balinska. Studies of formation and efflux of methotrexatepolyglutamates with cultured hepatic cells. Adv Exp Med Biol.1983;163:235-46.
[126] Kamen, BA, PA Nylen, BM Camitta and JR Bertino. Methotrexateaccumulation and folate depletion in cells as a possible mechanism of chronictoxicity to the drug. Br J Haematol.1981;49:355-60.
[127] Yamamoto, N, LC Lopes, AP Campello and ML Kluppel. Methotrexate:studies on cellular metabolism. II--Effects on mitochondrial oxidativemetabolism and ion transport. Cell Biochem Funct.1989;7:129-34.
[128] Meeker, ND, JJ Yang and JD Schiffman. Pharmacogenomics of pediatricacute lymphoblastic leukemia. Expert Opin Pharmacother.2010;11:1621-32.
[129] Trevino, LR, N Shimasaki, W Yang, et al. Germline genetic variation in anorganic anion transporter polypeptide associated with methotrexatepharmacokinetics and clinical effects. J Clin Oncol.2009;27:5972-8.
[130]王来成，薛天阳，吕冬梅，韩强.小儿急性淋巴细胞白血病HD-MTX-CF治疗方案的血药浓度监测分析.儿科药学杂志.2008;14:5-7.
[131] Odoul, F, C Le Guellec, JP Lamagnere, et al. Prediction of methotrexateelimination after high dose infusion in children with acute lymphoblasticleukaemia using a population pharmacokinetic approach. Fundam ClinPharmacol.1999;13:595-604.
[132] Borsi, JD and PJ Moe. Systemic clearance of methotrexate in the prognosis ofacute lymphoblastic leukemia in children. Cancer.1987;60:3020-4.
[1] Palm, W and T de Lange. How shelterin protects mammalian telomeres. AnnuRev Genet.2008;42:301-34.
[2] Blackburn, EH. Switching and signaling at the telomere. Cell.2001;106:661-73.
[3] Blasco, MA, HW Lee, MP Hande, et al. Telomere shortening and tumorformation by mouse cells lacking telomerase RNA. Cell.1997;91:25-34.
[4] Cosme-Blanco, W and S Chang. Dual roles of telomere dysfunction ininitiation and suppression of tumorigenesis. Exp Cell Res.2008;314:1973-9.
[5] Cayuela, ML, JM Flores and MA Blasco. The telomerase RNA componentTerc is required for the tumour-promoting effects of Tert overexpression.EMBO Rep.2005;6:268-74.
[6] Flores, I, ML Cayuela and MA Blasco. Effects of telomerase and telomerelength on epidermal stem cell behavior. Science.2005;309:1253-6.
[7] Tomas-Loba, A, I Flores, PJ Fernandez-Marcos, et al. Telomerase reversetranscriptase delays aging in cancer-resistant mice. Cell.2008;135:609-22.
[8] Stewart, SA and AA Bertuch. The role of telomeres and telomerase in cancerresearch. Cancer Res.2010;70:7365-71.
[9] Celli, GB and T de Lange. DNA processing is not required for ATM-mediatedtelomere damage response after TRF2deletion. Nat Cell Biol.2005;7:712-8.
[10] Hockemeyer, D, JP Daniels, H Takai and T de Lange. Recent expansion of thetelomeric complex in rodents: Two distinct POT1proteins protect mousetelomeres. Cell.2006;126:63-77.
[11] Martinez, P, M Thanasoula, P Munoz, et al. Increased telomere fragility andfusions resulting from TRF1deficiency lead to degenerative pathologies andincreased cancer in mice. Genes Dev.2009;23:2060-75.
[12] Else, T, A Trovato, AC Kim, et al. Genetic p53deficiency partially rescuesthe adrenocortical dysplasia phenotype at the expense of increasedtumorigenesis. Cancer Cell.2009;15:465-76.
[13] Tejera, AM, M Stagno d'Alcontres, M Thanasoula, et al. TPP1is required forTERT recruitment, telomere elongation during nuclear reprogramming, andnormal skin development in mice. Dev Cell.2010;18:775-89.
[14] Munoz, P, R Blanco, JM Flores and MA Blasco. XPF nuclease-dependenttelomere loss and increased DNA damage in mice overexpressing TRF2resultin premature aging and cancer. Nat Genet.2005;37:1063-71.
[15] Cesare, AJ and RR Reddel. Alternative lengthening of telomeres: models,mechanisms and implications. Nat Rev Genet.2010;11:319-30.
[16] Olovnikov, AM. A theory of marginotomy. The incomplete copying oftemplate margin in enzymic synthesis of polynucleotides and biologicalsignificance of the phenomenon. J Theor Biol.1973;41:181-90.
[17] Greider, CW and EH Blackburn. Identification of a specific telomere terminaltransferase activity in Tetrahymena extracts. Cell.1985;43:405-13.
[18] Flores, I, A Canela, E Vera, et al. The longest telomeres: a general signatureof adult stem cell compartments. Genes Dev.2008;22:654-67.
[19] Marion, RM, K Strati, H Li, et al. Telomeres acquire embryonic stem cellcharacteristics in induced pluripotent stem cells. Cell Stem Cell.2009;4:141-54.
[20] Collado, M, MA Blasco and M Serrano. Cellular senescence in cancer andaging. Cell.2007;130:223-33.
[21] Armanios, MY, JJ Chen, JD Cogan, et al. Telomerase mutations in familieswith idiopathic pulmonary fibrosis. N Engl J Med.2007;356:1317-26.
[22] Mitchell, JR, E Wood and K Collins. A telomerase component is defective inthe human disease dyskeratosis congenita. Nature.1999;402:551-5.
[23] Yamaguchi, H, RT Calado, H Ly, et al. Mutations in TERT, the gene fortelomerase reverse transcriptase, in aplastic anemia. N Engl J Med.2005;352:1413-24.
[24] Gonzalo, S, I Jaco, MF Fraga, et al. DNA methyltransferases control telomerelength and telomere recombination in mammalian cells. Nat Cell Biol.2006;8:416-24.
[25] Garcia-Cao, M, R O'Sullivan, AH Peters, T Jenuwein and MA Blasco.Epigenetic regulation of telomere length in mammalian cells by the Suv39h1and Suv39h2histone methyltransferases. Nat Genet.2004;36:94-9.
[26] Benetti, R, S Gonzalo, I Jaco, et al. Suv4-20h deficiency results in telomereelongation and derepression of telomere recombination. J Cell Biol.2007;178:925-36.
[27] Benetti, R, S Gonzalo, I Jaco, et al. A mammalian microRNA cluster controlsDNA methylation and telomere recombination via Rbl2-dependent regulationof DNA methyltransferases. Nat Struct Mol Biol.2008;15:998.
[28] Azzalin, CM, P Reichenbach, L Khoriauli, E Giulotto and J Lingner.Telomeric repeat containing RNA and RNA surveillance factors atmammalian chromosome ends. Science.2007;318:798-801.
[29] Schoeftner, S and MA Blasco. Developmentally regulated transcription ofmammalian telomeres by DNA-dependent RNA polymerase II. Nat Cell Biol.2008;10:228-36.
[30] Takahashi, K and S Yamanaka. Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors. Cell.2006;126:663-76.
[31] de Lange, T. Shelterin: the protein complex that shapes and safeguards humantelomeres. Genes Dev.2005;19:2100-10.
[32] Court, R, L Chapman, L Fairall and D Rhodes. How the human telomericproteins TRF1and TRF2recognize telomeric DNA: a view fromhigh-resolution crystal structures. EMBO Rep.2005;6:39-45.
[33] Loayza, D and T De Lange. POT1as a terminal transducer of TRF1telomerelength control. Nature.2003;423:1013-8.
[34] Kim, SH, C Beausejour, AR Davalos, et al. TIN2mediates functions of TRF2at human telomeres. J Biol Chem.2004;279:43799-804.
[35] Ye, JZ, D Hockemeyer, AN Krutchinsky, et al. POT1-interacting protein PIP1:a telomere length regulator that recruits POT1to the TIN2/TRF1complex.Genes Dev.2004;18:1649-54.
[36] Liu, D, A Safari, MS O'Connor, et al. PTOP interacts with POT1andregulates its localization to telomeres. Nat Cell Biol.2004;6:673-80.
[37] Chen, LY, D Liu and Z Songyang. Telomere maintenance through spatialcontrol of telomeric proteins. Mol Cell Biol.2007;27:5898-909.
[38] Xin, H, D Liu, M Wan, et al. TPP1is a homologue of ciliate TEBP-beta andinteracts with POT1to recruit telomerase. Nature.2007;445:559-62.
[39] Li, B and T de Lange. Rap1affects the length and heterogeneity of humantelomeres. Mol Biol Cell.2003;14:5060-8.
[40] Sfeir, A, S Kabir, M van Overbeek, GB Celli and T de Lange. Loss of Rap1induces telomere recombination in the absence of NHEJ or a DNA damagesignal. Science.2010;327:1657-61.
[41] Martinez, P, M Thanasoula, AR Carlos, et al. Mammalian Rap1controlstelomere function and gene expression through binding to telomeric andextratelomeric sites. Nat Cell Biol.2010;12:768-80.
[42] Hanahan, D and RA Weinberg. The hallmarks of cancer. Cell.2000;100:57-70.
[43] Lengauer, C, KW Kinzler and B Vogelstein. Genetic instabilities in humancancers. Nature.1998;396:643-9.
[44] Artandi, SE and RA DePinho. Telomeres and telomerase in cancer.Carcinogenesis.2010;31:9-18.
[45] Meeker, AK, JL Hicks, CA Iacobuzio-Donahue, et al. Telomere lengthabnormalities occur early in the initiation of epithelial carcinogenesis. ClinCancer Res.2004;10:3317-26.
[46] Chin, L, SE Artandi, Q Shen, et al. p53deficiency rescues the adverse effectsof telomere loss and cooperates with telomere dysfunction to acceleratecarcinogenesis. Cell.1999;97:527-38.
[47] de Lange, T. How telomeres solve the end-protection problem. Science.2009;326:948-52.
[48] Rai, R, H Zheng, H He, et al. The function of classical and alternativenon-homologous end-joining pathways in the fusion of dysfunctionaltelomeres. EMBO J.2010;29:2598-610.
[49] Espejel, S, S Franco, S Rodriguez-Perales, et al. Mammalian Ku86mediateschromosomal fusions and apoptosis caused by critically short telomeres.EMBO J.2002;21:2207-19.
[50] Murnane, JP. Telomere loss as a mechanism for chromosome instability inhuman cancer. Cancer Res.2010;70:4255-9.
[51] Gilson, E and V Geli. How telomeres are replicated. Nat Rev Mol Cell Biol.2007;8:825-38.
[52] Cacchione, S, MA Cerone and M Savino. In vitro low propensity to formnucleosomes of four telomeric sequences. FEBS Lett.1997;400:37-41.
[53] Verdun, RE and J Karlseder. The DNA damage machinery and homologousrecombination pathway act consecutively to protect human telomeres. Cell.2006;127:709-20.
[54] Rochette, PJ and DE Brash. Human telomeres are hypersensitive toUV-induced DNA Damage and refractory to repair. PLoS Genet.2010;6:e1000926.
[55] Zhu, XD, L Niedernhofer, B Kuster, et al. ERCC1/XPF removes the3'overhang from uncapped telomeres and represses formation of telomericDNA-containing double minute chromosomes. Mol Cell.2003;12:1489-98.
[56] Rajagopalan, H and C Lengauer. Aneuploidy and cancer. Nature.2004;432:338-41.
[57] Sotillo, R, E Hernando, E Diaz-Rodriguez, et al. Mad2overexpressionpromotes aneuploidy and tumorigenesis in mice. Cancer Cell.2007;11:9-23.
[58] Davoli, T, EL Denchi and T de Lange. Persistent telomere damage inducesbypass of mitosis and tetraploidy. Cell.2010;141:81-93.
[59] Denchi, EL and T de Lange. Protection of telomeres through independentcontrol of ATM and ATR by TRF2and POT1. Nature.2007;448:1068-71.
[60] Li, B, S Oestreich and T de Lange. Identification of human Rap1: implicationsfor telomere evolution. Cell.2000;101:471-83.
[61] Shore, D and K Nasmyth. Purification and cloning of a DNA binding proteinfrom yeast that binds to both silencer and activator elements. Cell.1987;51:721-32.
[62] Imai, S, CM Armstrong, M Kaeberlein and L Guarente. Transcriptionalsilencing and longevity protein Sir2is an NAD-dependent histone deacetylase.Nature.2000;403:795-800.
[63] Buchman, AR, WJ Kimmerly, J Rine and RD Kornberg. Two DNA-bindingfactors recognize specific sequences at silencers, upstream activatingsequences, autonomously replicating sequences, and telomeres inSaccharomyces cerevisiae. Mol Cell Biol.1988;8:210-25.
[64] Teo, H, S Ghosh, H Luesch, et al. Telomere-independent Rap1is an IKKadaptor and regulates NF-kappaB-dependent gene expression. Nat Cell Biol.2010;12:758-67.
[65] Martinez, P and MA Blasco. Role of shelterin in cancer and aging. Aging Cell.2010;9:653-66.
[66] Bodnar, AG, M Ouellette, M Frolkis, et al. Extension of life-span byintroduction of telomerase into normal human cells. Science.1998;279:349-52.
[67] Shay, JW and WE Wright. Telomerase therapeutics for cancer: challenges andnew directions. Nat Rev Drug Discov.2006;5:577-84.
[68] Shete, S, FJ Hosking, LB Robertson, et al. Genome-wide association studyidentifies five susceptibility loci for glioma. Nat Genet.2009;41:899-904.
[69] Petersen, GM, L Amundadottir, CS Fuchs, et al. A genome-wide associationstudy identifies pancreatic cancer susceptibility loci on chromosomes13q22.1,1q32.1and5p15.33. Nat Genet.2010;42:224-8.
[70] McKay, JD, RJ Hung, V Gaborieau, et al. Lung cancer susceptibility locus at5p15.33. Nat Genet.2008;40:1404-6.
[71] Rafnar, T, P Sulem, SN Stacey, et al. Sequence variants at theTERT-CLPTM1L locus associate with many cancer types. Nat Genet.2009;41:221-7.
[72] Wang, Y, P Broderick, E Webb, et al. Common5p15.33and6p21.33variantsinfluence lung cancer risk. Nat Genet.2008;40:1407-9.
[73] Zhang, A, S Maner, R Betz, et al. Genetic alterations in cervical carcinomas:frequent low-level amplifications of oncogenes are associated with humanpapillomavirus infection. Int J Cancer.2002;101:427-33.
[74] Kang, JU, SH Koo, KC Kwon, JW Park and JM Kim. Gain at chromosomalregion5p15.33, containing TERT, is the most frequent genetic event in earlystages of non-small cell lung cancer. Cancer Genet Cytogenet.2008;182:1-11.
[75] Sarin, KY, P Cheung, D Gilison, et al. Conditional telomerase inductioncauses proliferation of hair follicle stem cells. Nature.2005;436:1048-52.
[76] Choi, J, LK Southworth, KY Sarin, et al. TERT promotes epithelialproliferation through transcriptional control of a Myc-and Wnt-relateddevelopmental program. PLoS Genet.2008;4:e10.
[77] Cong, Y and JW Shay. Actions of human telomerase beyond telomeres. CellRes.2008;18:725-32.
[78] Santos, JH, JN Meyer, M Skorvaga, LA Annab and B Van Houten.Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage.Aging Cell.2004;3:399-411.
[79] Santos, JH, JN Meyer and B Van Houten. Mitochondrial localization oftelomerase as a determinant for hydrogen peroxide-induced mitochondrialDNA damage and apoptosis. Hum Mol Genet.2006;15:1757-68.
[80] Indran, IR, MP Hande and S Pervaiz. hTERT overexpression alleviatesintracellular ROS production, improves mitochondrial function, and inhibitsROS-mediated apoptosis in cancer cells. Cancer Res.2011;71:266-76.
[81] Vidal-Cardenas, SL and CW Greider. Comparing effects of mTR and mTERTdeletion on gene expression and DNA damage response: a criticalexamination of telomere length maintenance-independent roles of telomerase.Nucleic Acids Res.2010;38:60-71.
[82] Barolo, S. Transgenic Wnt/TCF pathway reporters: all you need is Lef?Oncogene.2006;25:7505-11.
[83] Nabetani, A and F Ishikawa. Alternative lengthening of telomeres pathway:recombination-mediated telomere maintenance mechanism in human cells. JBiochem.2011;149:5-14.
[84] Harley, CB. Telomerase and cancer therapeutics. Nat Rev Cancer.2008;8:167-79.
[85] Kim, S, H Youn, MG Song, et al. Complementary treatment of siTERT forimproving the antitumor effect of TERT-specific I-131therapy. Cancer GeneTher.2012;19:263-70.
[86] Folini, M, L Venturini, G Cimino-Reale and N Zaffaroni. Telomeres as targetsfor anticancer therapies. Expert Opin Ther Targets.2011;15:579-93.
[2] Onciu, M. Acute lymphoblastic leukemia. Hematol Oncol Clin North Am.2009;23:655-74.
[3] Pui, CH, WL Carroll, S Meshinchi and RJ Arceci. Biology, risk stratification,and therapy of pediatric acute leukemias: an update. J Clin Oncol.2011;29:551-65.
[4] Liu, R, L Zhang, CM McHale and SK Hammond. Paternal smoking and riskof childhood acute lymphoblastic leukemia: systematic review andmeta-analysis. J Oncol.2011;2011:854584.
[5] Eden, T. Aetiology of childhood leukaemia. Cancer Treat Rev.2010;36:286-97.
[6] Greaves. Science, medicine, and the future: childhood leukaemia. BritishMedical Journal.2002;324:283-287.
[7] Belson, M, B Kingsley and A Holmes. Risk factors for acute leukemia inchildren: a review. Environ Health Perspect.2007;115:138-45.
[8] Van Maele-Fabry, G, AC Lantin, P Hoet and D Lison. Childhood leukaemiaand parental occupational exposure to pesticides: a systematic review andmeta-analysis. Cancer Causes Control.2010;21:787-809.
[9] Shi, JY, ZH Ren, B Jiao, et al. Genetic variations of DNA repair genes andtheir prognostic significance in patients with acute myeloid leukemia. Int JCancer.2011;128:233-8.
[10] Salisbury, BA, M Pungliya, JY Choi, et al. SNP and haplotype variation in thehuman genome. Mutat Res.2003;526:53-61.
[11] Sachidanandam, R, D Weissman, SC Schmidt, et al. A map of human genomesequence variation containing1.42million single nucleotide polymorphisms.Nature.2001;409:928-33.
[12] Davidsen, ML, K Dalhoff and K Schmiegelow. Pharmacogenetics influencetreatment efficacy in childhood acute lymphoblastic leukemia. J PediatrHematol Oncol.2008;30:831-49.
[13] Palm, W and T de Lange. How shelterin protects mammalian telomeres. AnnuRev Genet.2008;42:301-34.
[14] Olovnikov, AM. A theory of marginotomy. The incomplete copying oftemplate margin in enzymic synthesis of polynucleotides and biologicalsignificance of the phenomenon. J Theor Biol.1973;41:181-90.
[15] Allsopp, RC, H Vaziri, C Patterson, et al. Telomere length predicts replicativecapacity of human fibroblasts. Proc Natl Acad Sci U S A.1992;89:10114-8.
[16] Szostak, JW and EH Blackburn. Cloning yeast telomeres on linear plasmidvectors. Cell.1982;29:245-55.
[17] Greider, CW and EH Blackburn. Identification of a specific telomere terminaltransferase activity in Tetrahymena extracts. Cell.1985;43:405-13.
[18] De Semir, D, M Nosrati, S Li and M Kashani-Sabet. Telomerase: goingbeyond the ends. Cell Cycle.2007;6:546-9.
[19] Cong, Y and JW Shay. Actions of human telomerase beyond telomeres. CellRes.2008;18:725-32.
[20] Gigek, CO, MF Leal, PN Silva, et al. hTERT methylation and expression ingastric cancer. Biomarkers.2009;14:630-6.
[21] Divella, R, S Tommasi, R Lacalamita, et al. Circulating hTERT DNA in earlybreast cancer. Anticancer Res.2009;29:2845-9.
[22] Xian, L, H Zhou, X Zhang, et al.[The Expression of hTERT mRNA and p16Protein in Non-small Cell Lung Cancer.]. Zhongguo Fei Ai Za Zhi.2009;12:1005-8.
[23] Capraro, V, L Zane, D Poncet, et al. Telomere deregulations possesscytogenetic, phenotype, and prognostic specificities in acute leukemias. ExpHematol.2011;39:195-202e2.
[24] Turnbull, C, EA Rapley, S Seal, et al. Variants near DMRT1, TERT andATF7IP are associated with testicular germ cell cancer. Nat Genet.2010;42:604-7.
[25] Landi, MT, N Chatterjee, K Yu, et al. A genome-wide association study oflung cancer identifies a region of chromosome5p15associated with risk foradenocarcinoma. Am J Hum Genet.2009;85:679-91.
[26] Rafnar, T, P Sulem, SN Stacey, et al. Sequence variants at theTERT-CLPTM1L locus associate with many cancer types. Nat Genet.2009;41:221-7.
[27] Shete, S, FJ Hosking, LB Robertson, et al. Genome-wide association studyidentifies five susceptibility loci for glioma. Nat Genet.2009;41:899-904.
[28] Haiman, CA, GK Chen, CM Vachon, et al. A common variant at theTERT-CLPTM1L locus is associated with estrogen receptor-negative breastcancer. Nat Genet.2011;43:1210-4.
[29] Kratz, CP, SS Han, PS Rosenberg, et al. Variants in or near KITLG, BAK1,DMRT1, and TERT-CLPTM1L predispose to familial testicular germ celltumour. J Med Genet.2011;48:473-6.
[30] Zhang, Q, NK Kim and J Feigon. Architecture of human telomerase RNA.Proc Natl Acad Sci U S A.2011;108:20325-32.
[31] Zvereva, MI, DM Shcherbakova and OA Dontsova. Telomerase: structure,functions, and activity regulation. Biochemistry (Mosc).2010;75:1563-83.
[32] Soder, AI, SF Hoare, S Muir, et al. Amplification, increased dosage and insitu expression of the telomerase RNA gene in human cancer. Oncogene.1997;14:1013-21.
[33] Vulliamy, T, A Marrone, R Szydlo, et al. Disease anticipation is associatedwith progressive telomere shortening in families with dyskeratosis congenitadue to mutations in TERC. Nat Genet.2004;36:447-9.
[34] Yamaguchi, H, GM Baerlocher, PM Lansdorp, et al. Mutations of the humantelomerase RNA gene (TERC) in aplastic anemia and myelodysplasticsyndrome. Blood.2003;102:916-8.
[35] Armanios, MY, JJ Chen, JD Cogan, et al. Telomerase mutations in familieswith idiopathic pulmonary fibrosis. N Engl J Med.2007;356:1317-26.
[36] Codd, V, M Mangino, P van der Harst, et al. Common variants near TERC areassociated with mean telomere length. Nat Genet.2010;42:197-9.
[37] Levy, D, SL Neuhausen, SC Hunt, et al. Genome-wide association identifiesOBFC1as a locus involved in human leukocyte telomere biology. Proc NatlAcad Sci U S A.2010;107:9293-8.
[38] Wentzensen, IM, L Mirabello, RM Pfeiffer and SA Savage. The association oftelomere length and cancer: a meta-analysis. Cancer Epidemiol BiomarkersPrev.2011;20:1238-50.
[39] Ma, H, Z Zhou, S Wei, et al. Shortened telomere length is associated withincreased risk of cancer: a meta-analysis. PLoS One.2011;6:e20466.
[40] Gancarcikova, M, Z Zemanova, J Brezinova, et al. The role of telomeres andtelomerase complex in haematological neoplasia: the length of telomeres as amarker of carcinogenesis and prognosis of disease. Prague Med Rep.2010;111:91-105.
[41] Park, JI, AS Venteicher, JY Hong, et al. Telomerase modulates Wnt signallingby association with target gene chromatin. Nature.2009;460:66-72.
[42] Indran, IR, MP Hande and S Pervaiz. hTERT overexpression alleviatesintracellular ROS production, improves mitochondrial function, and inhibitsROS-mediated apoptosis in cancer cells. Cancer Res.2011;71:266-76.
[43] Yamada, O, K Kawauchi, M Akiyama, et al. Leukemic cells with increasedtelomerase activity exhibit resistance to imatinib. Leuk Lymphoma.2008;49:1168-77.
[44] Yamada, O, K Ozaki, T Furukawa, et al. Activation of STAT5confersimatinib resistance on leukemic cells through the transcription of TERT andMDR1. Cell Signal.2011;23:1119-27.
[45] Nakayama, J, H Tahara, E Tahara, et al. Telomerase activation by hTRT inhuman normal fibroblasts and hepatocellular carcinomas. Nat Genet.1998;18:65-8.
[46] Morales, CP, SE Holt, M Ouellette, et al. Absence of cancer-associatedchanges in human fibroblasts immortalized with telomerase. Nat Genet.1999;21:115-8.
[47] Artandi, SE, S Alson, MK Tietze, et al. Constitutive telomerase expressionpromotes mammary carcinomas in aging mice. Proc Natl Acad Sci U S A.2002;99:8191-6.
[48] Hahn, WC, SA Stewart, MW Brooks, et al. Inhibition of telomerase limits thegrowth of human cancer cells. Nat Med.1999;5:1164-70.
[49] Kim, S, H Youn, MG Song, et al. Complementary treatment of siTERT forimproving the antitumor effect of TERT-specific I-131therapy. Cancer GeneTher.2012;
[50] Xu, D, A Gruber, C Peterson and P Pisa. Telomerase activity and theexpression of telomerase components in acute myelogenous leukaemia. Br JHaematol.1998;102:1367-75.
[51] Hartmann, U, TH Brummendorf, S Balabanov, et al. Telomere length andhTERT expression in patients with acute myeloid leukemia correlates withchromosomal abnormalities. Haematologica.2005;90:307-16.
[52] Swiggers, SJ, MA Kuijpers, MJ de Cort, HB Beverloo and JM Zijlmans.Critically short telomeres in acute myeloid leukemia with loss or gain of partsof chromosomes. Genes Chromosomes Cancer.2006;45:247-56.
[53] Cogulu, O, B Kosova, E Karaca, et al. Evaluation of telomerase mRNA(hTERT) in childhood acute leukemia. Leuk Lymphoma.2004;45:2477-80.
[54] Campbell, LJ, C Fidler, H Eagleton, et al. hTERT, the catalytic component oftelomerase, is downregulated in the haematopoietic stem cells of patients withchronic myeloid leukaemia. Leukemia.2006;20:671-9.
[55] Drummond, MW, SF Hoare, A Monaghan, et al. Dysregulated expression ofthe major telomerase components in leukaemic stem cells. Leukemia.2005;19:381-9.
[56] Gabet, AS, F Mortreux, P Charneau, et al. Inactivation of hTERTtranscription by Tax. Oncogene.2003;22:3734-41.
[57] Hara, T, Y Matsumura-Arioka, K Ohtani and M Nakamura. Role of humanT-cell leukemia virus type I Tax in expression of the human telomerasereverse transcriptase (hTERT) gene in human T-cells. Cancer Sci.2008;99:1155-63.
[58] Poncet, D, A Belleville, C t'kint de Roodenbeke, et al. Changes in theexpression of telomere maintenance genes suggest global telomeredysfunction in B-chronic lymphocytic leukemia. Blood.2008;111:2388-91.
[59]中华医学会儿科学分会血液学组，中华儿科杂志编辑委员会.儿童急性淋巴细胞白血病诊疗建议(第三次修订草案).中华儿科杂志.2006;44:392-395.
[60] Pieters, R and WL Carroll. Biology and treatment of acute lymphoblasticleukemia. Hematol Oncol Clin North Am.2010;24:1-18.
[61] Matsubara, Y, M Murata, T Yoshida, et al. Telomere length of normalleukocytes is affected by a functional polymorphism of hTERT. BiochemBiophys Res Commun.2006;341:128-31.
[62] Matsubara, Y, M Murata, K Watanabe, et al. Coronary artery disease and afunctional polymorphism of hTERT. Biochem Biophys Res Commun.2006;348:669-72.
[63] Nordfjall, K, P Osterman, O Melander, P Nilsson and G Roos. hTERT(-1327)T/C polymorphism is not associated with age-related telomere attritionin peripheral blood. Biochem Biophys Res Commun.2007;358:215-8.
[64] Beesley, J, HA Pickett, SE Johnatty, et al. Functional polymorphisms in theTERT promoter are associated with risk of serous epithelial ovarian and breastcancers. PLoS One.2011;6:e24987.
[65] Shen, J, MD Gammon, HC Wu, et al. Multiple genetic variants in telomerepathway genes and breast cancer risk. Cancer Epidemiol Biomarkers Prev.2010;19:219-28.
[66] Hsu, CP, NY Hsu, LW Lee and JL Ko. Ets2binding site single nucleotidepolymorphism at the hTERT gene promoter--effect on telomerase expressionand telomere length maintenance in non-small cell lung cancer. Eur J Cancer.2006;42:1466-74.
[67] Raynaud, CM, L Sabatier, O Philipot, KA Olaussen and JC Soria. Telomerelength, telomeric proteins and genomic instability during the multistepcarcinogenic process. Crit Rev Oncol Hematol.2008;66:99-117.
[68] Mooi, WJ and DS Peeper. Oncogene-induced cell senescence--halting on theroad to cancer. N Engl J Med.2006;355:1037-46.
[69] Kim, AS, DA Eastmond and RJ Preston. Childhood acute lymphocyticleukemia and perspectives on risk assessment of early-life stage exposures.Mutat Res.2006;613:138-60.
[70] Yang, JJ, C Cheng, W Yang, et al. Genome-wide interrogation of germlinegenetic variation associated with treatment response in childhood acutelymphoblastic leukemia. JAMA.2009;301:393-403.
[71] Papaemmanuil, E, FJ Hosking, J Vijayakrishnan, et al. Loci on7p12.2,10q21.2and14q11.2are associated with risk of childhood acutelymphoblastic leukemia. Nat Genet.2009;41:1006-10.
[72] Trevino, LR, W Yang, D French, et al. Germline genomic variants associatedwith childhood acute lymphoblastic leukemia. Nat Genet.2009;41:1001-5.
[73] Sherborne, AL, FJ Hosking, RB Prasad, et al. Variation in CDKN2A at9p21.3influences childhood acute lymphoblastic leukemia risk. Nat Genet.2010;42:492-4.
[74] Ellinghaus, E, M Stanulla, G Richter, et al. Identification of germlinesusceptibility loci in ETV6-RUNX1-rearranged childhood acutelymphoblastic leukemia. Leukemia.2011;
[75] Han, S, KM Lee, SK Park, et al. Genome-wide association study of childhoodacute lymphoblastic leukemia in Korea. Leuk Res.2010;34:1271-4.
[76] Chen, JL, MA Blasco and CW Greider. Secondary structure of vertebratetelomerase RNA. Cell.2000;100:503-14.
[77] Greider, CW and EH Blackburn. A telomeric sequence in the RNA ofTetrahymena telomerase required for telomere repeat synthesis. Nature.1989;337:331-7.
[78] Chu, C, K Qu, FL Zhong, SE Artandi and HY Chang. Genomic maps of longnoncoding RNA occupancy reveal principles of RNA-chromatin interactions.Mol Cell.2011;44:667-78.
[79] Shen, Q, Z Zhang, L Yu, et al. Common variants near TERC are associatedwith leukocyte telomere length in the Chinese Han population. Eur J HumGenet.2011;19:721-3.
[80] Kobe, B and AV Kajava. The leucine-rich repeat as a protein recognitionmotif. Curr Opin Struct Biol.2001;11:725-32.
[81] Blasco, MA. The epigenetic regulation of mammalian telomeres. Nat RevGenet.2007;8:299-309.
[82] Autexier, C and NF Lue. The structure and function of telomerase reversetranscriptase. Annu Rev Biochem.2006;75:493-517.
[83] Donate, LE and MA Blasco. Telomeres in cancer and ageing. Philos Trans RSoc Lond B Biol Sci.2011;366:76-84.
[84] Andrew, T, A Aviv, M Falchi, et al. Mapping genetic loci that determineleukocyte telomere length in a large sample of unselected female sibling pairs.Am J Hum Genet.2006;78:480-6.
[85] Vasa-Nicotera, M, S Brouilette, M Mangino, et al. Mapping of a major locusthat determines telomere length in humans. Am J Hum Genet.2005;76:147-51.
[86] Harley, CB. Human ageing and telomeres. Ciba Found Symp.1997;211:129-39; discussion139-44.
[87] Blackburn, EH. Switching and signaling at the telomere. Cell.2001;106:661-73.
[88] von Zglinicki, T, V Serra, M Lorenz, et al. Short telomeres in patients withvascular dementia: an indicator of low antioxidative capacity and a possiblerisk factor? Lab Invest.2000;80:1739-47.
[89] Panossian, LA, VR Porter, HF Valenzuela, et al. Telomere shortening in Tcells correlates with Alzheimer's disease status. Neurobiol Aging.2003;24:77-84.
[90] Wiemann, SU, A Satyanarayana, M Tsahuridu, et al. Hepatocyte telomereshortening and senescence are general markers of human liver cirrhosis.FASEB J.2002;16:935-42.
[91] Benetos, A, JP Gardner, M Zureik, et al. Short telomeres are associated withincreased carotid atherosclerosis in hypertensive subjects. Hypertension.2004;43:182-5.
[92] Kinouchi, Y, N Hiwatashi, M Chida, et al. Telomere shortening in the colonicmucosa of patients with ulcerative colitis. J Gastroenterol.1998;33:343-8.
[93] Brouilette, S, RK Singh, JR Thompson, AH Goodall and NJ Samani. Whitecell telomere length and risk of premature myocardial infarction. ArteriosclerThromb Vasc Biol.2003;23:842-6.
[94] Blasco, MA. Telomeres and human disease: ageing, cancer and beyond. NatRev Genet.2005;6:611-22.
[95] Smogorzewska, A, B van Steensel, A Bianchi, et al. Control of humantelomere length by TRF1and TRF2. Mol Cell Biol.2000;20:1659-68.
[96] von Zglinicki, T, R Pilger and N Sitte. Accumulation of single-strand breaksis the major cause of telomere shortening in human fibroblasts. Free RadicBiol Med.2000;28:64-74.
[97] Blasco, MA, HW Lee, MP Hande, et al. Telomere shortening and tumorformation by mouse cells lacking telomerase RNA. Cell.1997;91:25-34.
[98] Artandi, SE, S Chang, SL Lee, et al. Telomere dysfunction promotesnon-reciprocal translocations and epithelial cancers in mice. Nature.2000;406:641-5.
[99] Joshua, AM, B Vukovic, I Braude, et al. Telomere attrition in isolatedhigh-grade prostatic intraepithelial neoplasia and surrounding stroma ispredictive of prostate cancer. Neoplasia.2007;9:81-9.
[100] Kammori, M, K Takubo, K Nakamura, et al. Telomerase activity and telomerelength in benign and malignant human thyroid tissues. Cancer Lett.2000;159:175-81.
[101] Wong, KK, RS Maser, RM Bachoo, et al. Telomere dysfunction and Atmdeficiency compromises organ homeostasis and accelerates ageing. Nature.2003;421:643-8.
[102] Kim, HR, YJ Kim, HJ Kim, SK Kim and JH Lee. Telomere length changes incolorectal cancers and polyps. J Korean Med Sci.2002;17:360-5.
[103] Sommerfeld, HJ, AK Meeker, MA Piatyszek, et al. Telomerase activity: aprevalent marker of malignant human prostate tissue. Cancer Res.1996;56:218-22.
[104] Stewart, SA and RA Weinberg. Telomeres: cancer to human aging. Annu RevCell Dev Biol.2006;22:531-57.
[105] Capper, R, B Britt-Compton, M Tankimanova, et al. The nature of telomerefusion and a definition of the critical telomere length in human cells. GenesDev.2007;21:2495-508.
[106] Svenson, U, K Nordfjall, B Stegmayr, et al. Breast cancer survival isassociated with telomere length in peripheral blood cells. Cancer Res.2008;68:3618-23.
[107] Borssen, M, I Cullman, U Noren-Nystrom, et al. hTERT promotermethylation and telomere length in childhood acute lymphoblastic leukemia:associations with immunophenotype and cytogenetic subgroup. Exp Hematol.2011;39:1144-51.
[108] Ghaffari, SH, N Shayan-Asl, AH Jamialahmadi, K Alimoghaddam and AGhavamzadeh. Telomerase activity and telomere length in patients with acutepromyelocytic leukemia: indicative of proliferative activity, diseaseprogression, and overall survival. Ann Oncol.2008;19:1927-34.
[109] Kubuki, Y, M Suzuki, H Sasaki, et al. Telomerase activity and telomerelength as prognostic factors of adult T-cell leukemia. Leuk Lymphoma.2005;46:393-9.
[110] Wang, Y, M Fang, X Sun and J Sun. Telomerase activity and telomere lengthin acute leukemia: correlations with disease progression, subtypes and overallsurvival. Int J Lab Hematol.2010;32:230-8.
[111] Epel, ES, EH Blackburn, J Lin, et al. Accelerated telomere shortening inresponse to life stress. Proc Natl Acad Sci U S A.2004;101:17312-5.
[112] Mirabello, L, K Yu, P Kraft, et al. The association of telomere length andgenetic variation in telomere biology genes. Hum Mutat.2010;31:1050-8.
[113] Hoffmann, J, Y Erben, AM Zeiher, S Dimmeler and I Spyridopoulos.Telomere length-heterogeneity among myeloid cells is a predictor forchronological ageing. Exp Gerontol.2009;44:363-6.
[114] Jones, AM, AD Beggs, L Carvajal-Carmona, et al. TERC polymorphisms areassociated both with susceptibility to colorectal cancer and with longertelomeres. Gut.2012;61:248-54.
[115] Treon, SP and BA Chabner. Concepts in use of high-dose methotrexatetherapy. Clin Chem.1996;42:1322-9.
[116] Pitman, SW and E Frei,3rd. Weekly methotrexate-calcium leucovorin rescue:effect of alkalinization on nephrotoxicity; pharmacokinetics in the CNS; anduse in CNS non-Hodgkin's lymphoma. Cancer Treat Rep.1977;61:695-701.
[117] Krajinovic, M and A Moghrabi. Pharmacogenetics of methotrexate.Pharmacogenomics.2004;5:819-34.
[118]张春燕，顾健，李玉珍，卢炜.大剂量甲氨喋呤治疗329例儿童急性淋巴细胞白血病的群体药物动力学研究.中国实验血液学杂志.2008;16:106-110.
[119]汪洋，张华年，宋新文，梅艳.急性淋巴细胞白血病患儿HD-MTX-CF化疗方案中血药浓度的监测.华西药学杂志.2010;25:219-221.
[120]周际昌,实用肿瘤内科学.人民卫生出版社:北京.2003;100-101.
[121]周莉，陆勤，扬引，方拥军.大剂量甲氨喋呤治疗儿童急性淋巴细胞白血病.江苏医药.2007;33:1275.
[122]许静，李庆平，王斌，陆勤，方拥军，黄婕，徐康康，罗琳. GSTT1、GSTM1基因多态性与大剂量甲氨喋呤血药浓度及消化道黏膜损伤的关系研究.安徽医药.2010;14:1336-1338.
[123] Neuman, MG, RG Cameron, JA Haber, et al. Inducers of cytochrome P4502E1enhance methotrexate-induced hepatocytoxicity. Clin Biochem.1999;32:519-36.
[124] Chladek, J, J Martinkova and L Sispera. An in vitro study on methotrexatehydroxylation in rat and human liver. Physiol Res.1997;46:371-9.
[125] Galivan, J and M Balinska. Studies of formation and efflux of methotrexatepolyglutamates with cultured hepatic cells. Adv Exp Med Biol.1983;163:235-46.
[126] Kamen, BA, PA Nylen, BM Camitta and JR Bertino. Methotrexateaccumulation and folate depletion in cells as a possible mechanism of chronictoxicity to the drug. Br J Haematol.1981;49:355-60.
[127] Yamamoto, N, LC Lopes, AP Campello and ML Kluppel. Methotrexate:studies on cellular metabolism. II--Effects on mitochondrial oxidativemetabolism and ion transport. Cell Biochem Funct.1989;7:129-34.
[128] Meeker, ND, JJ Yang and JD Schiffman. Pharmacogenomics of pediatricacute lymphoblastic leukemia. Expert Opin Pharmacother.2010;11:1621-32.
[129] Trevino, LR, N Shimasaki, W Yang, et al. Germline genetic variation in anorganic anion transporter polypeptide associated with methotrexatepharmacokinetics and clinical effects. J Clin Oncol.2009;27:5972-8.
[130]王来成，薛天阳，吕冬梅，韩强.小儿急性淋巴细胞白血病HD-MTX-CF治疗方案的血药浓度监测分析.儿科药学杂志.2008;14:5-7.
[131] Odoul, F, C Le Guellec, JP Lamagnere, et al. Prediction of methotrexateelimination after high dose infusion in children with acute lymphoblasticleukaemia using a population pharmacokinetic approach. Fundam ClinPharmacol.1999;13:595-604.
[132] Borsi, JD and PJ Moe. Systemic clearance of methotrexate in the prognosis ofacute lymphoblastic leukemia in children. Cancer.1987;60:3020-4.
[1] Palm, W and T de Lange. How shelterin protects mammalian telomeres. AnnuRev Genet.2008;42:301-34.
[2] Blackburn, EH. Switching and signaling at the telomere. Cell.2001;106:661-73.
[3] Blasco, MA, HW Lee, MP Hande, et al. Telomere shortening and tumorformation by mouse cells lacking telomerase RNA. Cell.1997;91:25-34.
[4] Cosme-Blanco, W and S Chang. Dual roles of telomere dysfunction ininitiation and suppression of tumorigenesis. Exp Cell Res.2008;314:1973-9.
[5] Cayuela, ML, JM Flores and MA Blasco. The telomerase RNA componentTerc is required for the tumour-promoting effects of Tert overexpression.EMBO Rep.2005;6:268-74.
[6] Flores, I, ML Cayuela and MA Blasco. Effects of telomerase and telomerelength on epidermal stem cell behavior. Science.2005;309:1253-6.
[7] Tomas-Loba, A, I Flores, PJ Fernandez-Marcos, et al. Telomerase reversetranscriptase delays aging in cancer-resistant mice. Cell.2008;135:609-22.
[8] Stewart, SA and AA Bertuch. The role of telomeres and telomerase in cancerresearch. Cancer Res.2010;70:7365-71.
[9] Celli, GB and T de Lange. DNA processing is not required for ATM-mediatedtelomere damage response after TRF2deletion. Nat Cell Biol.2005;7:712-8.
[10] Hockemeyer, D, JP Daniels, H Takai and T de Lange. Recent expansion of thetelomeric complex in rodents: Two distinct POT1proteins protect mousetelomeres. Cell.2006;126:63-77.
[11] Martinez, P, M Thanasoula, P Munoz, et al. Increased telomere fragility andfusions resulting from TRF1deficiency lead to degenerative pathologies andincreased cancer in mice. Genes Dev.2009;23:2060-75.
[12] Else, T, A Trovato, AC Kim, et al. Genetic p53deficiency partially rescuesthe adrenocortical dysplasia phenotype at the expense of increasedtumorigenesis. Cancer Cell.2009;15:465-76.
[13] Tejera, AM, M Stagno d'Alcontres, M Thanasoula, et al. TPP1is required forTERT recruitment, telomere elongation during nuclear reprogramming, andnormal skin development in mice. Dev Cell.2010;18:775-89.
[14] Munoz, P, R Blanco, JM Flores and MA Blasco. XPF nuclease-dependenttelomere loss and increased DNA damage in mice overexpressing TRF2resultin premature aging and cancer. Nat Genet.2005;37:1063-71.
[15] Cesare, AJ and RR Reddel. Alternative lengthening of telomeres: models,mechanisms and implications. Nat Rev Genet.2010;11:319-30.
[16] Olovnikov, AM. A theory of marginotomy. The incomplete copying oftemplate margin in enzymic synthesis of polynucleotides and biologicalsignificance of the phenomenon. J Theor Biol.1973;41:181-90.
[17] Greider, CW and EH Blackburn. Identification of a specific telomere terminaltransferase activity in Tetrahymena extracts. Cell.1985;43:405-13.
[18] Flores, I, A Canela, E Vera, et al. The longest telomeres: a general signatureof adult stem cell compartments. Genes Dev.2008;22:654-67.
[19] Marion, RM, K Strati, H Li, et al. Telomeres acquire embryonic stem cellcharacteristics in induced pluripotent stem cells. Cell Stem Cell.2009;4:141-54.
[20] Collado, M, MA Blasco and M Serrano. Cellular senescence in cancer andaging. Cell.2007;130:223-33.
[21] Armanios, MY, JJ Chen, JD Cogan, et al. Telomerase mutations in familieswith idiopathic pulmonary fibrosis. N Engl J Med.2007;356:1317-26.
[22] Mitchell, JR, E Wood and K Collins. A telomerase component is defective inthe human disease dyskeratosis congenita. Nature.1999;402:551-5.
[23] Yamaguchi, H, RT Calado, H Ly, et al. Mutations in TERT, the gene fortelomerase reverse transcriptase, in aplastic anemia. N Engl J Med.2005;352:1413-24.
[24] Gonzalo, S, I Jaco, MF Fraga, et al. DNA methyltransferases control telomerelength and telomere recombination in mammalian cells. Nat Cell Biol.2006;8:416-24.
[25] Garcia-Cao, M, R O'Sullivan, AH Peters, T Jenuwein and MA Blasco.Epigenetic regulation of telomere length in mammalian cells by the Suv39h1and Suv39h2histone methyltransferases. Nat Genet.2004;36:94-9.
[26] Benetti, R, S Gonzalo, I Jaco, et al. Suv4-20h deficiency results in telomereelongation and derepression of telomere recombination. J Cell Biol.2007;178:925-36.
[27] Benetti, R, S Gonzalo, I Jaco, et al. A mammalian microRNA cluster controlsDNA methylation and telomere recombination via Rbl2-dependent regulationof DNA methyltransferases. Nat Struct Mol Biol.2008;15:998.
[28] Azzalin, CM, P Reichenbach, L Khoriauli, E Giulotto and J Lingner.Telomeric repeat containing RNA and RNA surveillance factors atmammalian chromosome ends. Science.2007;318:798-801.
[29] Schoeftner, S and MA Blasco. Developmentally regulated transcription ofmammalian telomeres by DNA-dependent RNA polymerase II. Nat Cell Biol.2008;10:228-36.
[30] Takahashi, K and S Yamanaka. Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors. Cell.2006;126:663-76.
[31] de Lange, T. Shelterin: the protein complex that shapes and safeguards humantelomeres. Genes Dev.2005;19:2100-10.
[32] Court, R, L Chapman, L Fairall and D Rhodes. How the human telomericproteins TRF1and TRF2recognize telomeric DNA: a view fromhigh-resolution crystal structures. EMBO Rep.2005;6:39-45.
[33] Loayza, D and T De Lange. POT1as a terminal transducer of TRF1telomerelength control. Nature.2003;423:1013-8.
[34] Kim, SH, C Beausejour, AR Davalos, et al. TIN2mediates functions of TRF2at human telomeres. J Biol Chem.2004;279:43799-804.
[35] Ye, JZ, D Hockemeyer, AN Krutchinsky, et al. POT1-interacting protein PIP1:a telomere length regulator that recruits POT1to the TIN2/TRF1complex.Genes Dev.2004;18:1649-54.
[36] Liu, D, A Safari, MS O'Connor, et al. PTOP interacts with POT1andregulates its localization to telomeres. Nat Cell Biol.2004;6:673-80.
[37] Chen, LY, D Liu and Z Songyang. Telomere maintenance through spatialcontrol of telomeric proteins. Mol Cell Biol.2007;27:5898-909.
[38] Xin, H, D Liu, M Wan, et al. TPP1is a homologue of ciliate TEBP-beta andinteracts with POT1to recruit telomerase. Nature.2007;445:559-62.
[39] Li, B and T de Lange. Rap1affects the length and heterogeneity of humantelomeres. Mol Biol Cell.2003;14:5060-8.
[40] Sfeir, A, S Kabir, M van Overbeek, GB Celli and T de Lange. Loss of Rap1induces telomere recombination in the absence of NHEJ or a DNA damagesignal. Science.2010;327:1657-61.
[41] Martinez, P, M Thanasoula, AR Carlos, et al. Mammalian Rap1controlstelomere function and gene expression through binding to telomeric andextratelomeric sites. Nat Cell Biol.2010;12:768-80.
[42] Hanahan, D and RA Weinberg. The hallmarks of cancer. Cell.2000;100:57-70.
[43] Lengauer, C, KW Kinzler and B Vogelstein. Genetic instabilities in humancancers. Nature.1998;396:643-9.
[44] Artandi, SE and RA DePinho. Telomeres and telomerase in cancer.Carcinogenesis.2010;31:9-18.
[45] Meeker, AK, JL Hicks, CA Iacobuzio-Donahue, et al. Telomere lengthabnormalities occur early in the initiation of epithelial carcinogenesis. ClinCancer Res.2004;10:3317-26.
[46] Chin, L, SE Artandi, Q Shen, et al. p53deficiency rescues the adverse effectsof telomere loss and cooperates with telomere dysfunction to acceleratecarcinogenesis. Cell.1999;97:527-38.
[47] de Lange, T. How telomeres solve the end-protection problem. Science.2009;326:948-52.
[48] Rai, R, H Zheng, H He, et al. The function of classical and alternativenon-homologous end-joining pathways in the fusion of dysfunctionaltelomeres. EMBO J.2010;29:2598-610.
[49] Espejel, S, S Franco, S Rodriguez-Perales, et al. Mammalian Ku86mediateschromosomal fusions and apoptosis caused by critically short telomeres.EMBO J.2002;21:2207-19.
[50] Murnane, JP. Telomere loss as a mechanism for chromosome instability inhuman cancer. Cancer Res.2010;70:4255-9.
[51] Gilson, E and V Geli. How telomeres are replicated. Nat Rev Mol Cell Biol.2007;8:825-38.
[52] Cacchione, S, MA Cerone and M Savino. In vitro low propensity to formnucleosomes of four telomeric sequences. FEBS Lett.1997;400:37-41.
[53] Verdun, RE and J Karlseder. The DNA damage machinery and homologousrecombination pathway act consecutively to protect human telomeres. Cell.2006;127:709-20.
[54] Rochette, PJ and DE Brash. Human telomeres are hypersensitive toUV-induced DNA Damage and refractory to repair. PLoS Genet.2010;6:e1000926.
[55] Zhu, XD, L Niedernhofer, B Kuster, et al. ERCC1/XPF removes the3'overhang from uncapped telomeres and represses formation of telomericDNA-containing double minute chromosomes. Mol Cell.2003;12:1489-98.
[56] Rajagopalan, H and C Lengauer. Aneuploidy and cancer. Nature.2004;432:338-41.
[57] Sotillo, R, E Hernando, E Diaz-Rodriguez, et al. Mad2overexpressionpromotes aneuploidy and tumorigenesis in mice. Cancer Cell.2007;11:9-23.
[58] Davoli, T, EL Denchi and T de Lange. Persistent telomere damage inducesbypass of mitosis and tetraploidy. Cell.2010;141:81-93.
[59] Denchi, EL and T de Lange. Protection of telomeres through independentcontrol of ATM and ATR by TRF2and POT1. Nature.2007;448:1068-71.
[60] Li, B, S Oestreich and T de Lange. Identification of human Rap1: implicationsfor telomere evolution. Cell.2000;101:471-83.
[61] Shore, D and K Nasmyth. Purification and cloning of a DNA binding proteinfrom yeast that binds to both silencer and activator elements. Cell.1987;51:721-32.
[62] Imai, S, CM Armstrong, M Kaeberlein and L Guarente. Transcriptionalsilencing and longevity protein Sir2is an NAD-dependent histone deacetylase.Nature.2000;403:795-800.
[63] Buchman, AR, WJ Kimmerly, J Rine and RD Kornberg. Two DNA-bindingfactors recognize specific sequences at silencers, upstream activatingsequences, autonomously replicating sequences, and telomeres inSaccharomyces cerevisiae. Mol Cell Biol.1988;8:210-25.
[64] Teo, H, S Ghosh, H Luesch, et al. Telomere-independent Rap1is an IKKadaptor and regulates NF-kappaB-dependent gene expression. Nat Cell Biol.2010;12:758-67.
[65] Martinez, P and MA Blasco. Role of shelterin in cancer and aging. Aging Cell.2010;9:653-66.
[66] Bodnar, AG, M Ouellette, M Frolkis, et al. Extension of life-span byintroduction of telomerase into normal human cells. Science.1998;279:349-52.
[67] Shay, JW and WE Wright. Telomerase therapeutics for cancer: challenges andnew directions. Nat Rev Drug Discov.2006;5:577-84.
[68] Shete, S, FJ Hosking, LB Robertson, et al. Genome-wide association studyidentifies five susceptibility loci for glioma. Nat Genet.2009;41:899-904.
[69] Petersen, GM, L Amundadottir, CS Fuchs, et al. A genome-wide associationstudy identifies pancreatic cancer susceptibility loci on chromosomes13q22.1,1q32.1and5p15.33. Nat Genet.2010;42:224-8.
[70] McKay, JD, RJ Hung, V Gaborieau, et al. Lung cancer susceptibility locus at5p15.33. Nat Genet.2008;40:1404-6.
[71] Rafnar, T, P Sulem, SN Stacey, et al. Sequence variants at theTERT-CLPTM1L locus associate with many cancer types. Nat Genet.2009;41:221-7.
[72] Wang, Y, P Broderick, E Webb, et al. Common5p15.33and6p21.33variantsinfluence lung cancer risk. Nat Genet.2008;40:1407-9.
[73] Zhang, A, S Maner, R Betz, et al. Genetic alterations in cervical carcinomas:frequent low-level amplifications of oncogenes are associated with humanpapillomavirus infection. Int J Cancer.2002;101:427-33.
[74] Kang, JU, SH Koo, KC Kwon, JW Park and JM Kim. Gain at chromosomalregion5p15.33, containing TERT, is the most frequent genetic event in earlystages of non-small cell lung cancer. Cancer Genet Cytogenet.2008;182:1-11.
[75] Sarin, KY, P Cheung, D Gilison, et al. Conditional telomerase inductioncauses proliferation of hair follicle stem cells. Nature.2005;436:1048-52.
[76] Choi, J, LK Southworth, KY Sarin, et al. TERT promotes epithelialproliferation through transcriptional control of a Myc-and Wnt-relateddevelopmental program. PLoS Genet.2008;4:e10.
[77] Cong, Y and JW Shay. Actions of human telomerase beyond telomeres. CellRes.2008;18:725-32.
[78] Santos, JH, JN Meyer, M Skorvaga, LA Annab and B Van Houten.Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage.Aging Cell.2004;3:399-411.
[79] Santos, JH, JN Meyer and B Van Houten. Mitochondrial localization oftelomerase as a determinant for hydrogen peroxide-induced mitochondrialDNA damage and apoptosis. Hum Mol Genet.2006;15:1757-68.
[80] Indran, IR, MP Hande and S Pervaiz. hTERT overexpression alleviatesintracellular ROS production, improves mitochondrial function, and inhibitsROS-mediated apoptosis in cancer cells. Cancer Res.2011;71:266-76.
[81] Vidal-Cardenas, SL and CW Greider. Comparing effects of mTR and mTERTdeletion on gene expression and DNA damage response: a criticalexamination of telomere length maintenance-independent roles of telomerase.Nucleic Acids Res.2010;38:60-71.
[82] Barolo, S. Transgenic Wnt/TCF pathway reporters: all you need is Lef?Oncogene.2006;25:7505-11.
[83] Nabetani, A and F Ishikawa. Alternative lengthening of telomeres pathway:recombination-mediated telomere maintenance mechanism in human cells. JBiochem.2011;149:5-14.
[84] Harley, CB. Telomerase and cancer therapeutics. Nat Rev Cancer.2008;8:167-79.
[85] Kim, S, H Youn, MG Song, et al. Complementary treatment of siTERT forimproving the antitumor effect of TERT-specific I-131therapy. Cancer GeneTher.2012;19:263-70.
[86] Folini, M, L Venturini, G Cimino-Reale and N Zaffaroni. Telomeres as targetsfor anticancer therapies. Expert Opin Ther Targets.2011;15:579-93.