端粒和端粒酶与肿瘤干细胞的研究现状

目的:总结国内外对端粒、端粒酶与肿瘤起源、肿瘤干细胞的病理研究现状。方法:应用检索MED-LINE及CHKD期刊全文数据库检索系统,以"端粒、端粒酶和肿瘤干细胞"为关键词,检索1997-2008年有关文献。纳入标准:端粒、端粒酶与肿瘤干细胞的论著性文章。根据标准,纳入分析24篇参考文献。结果:端粒酶激活和端粒稳定对肿瘤干细胞演进是必需的,端粒酶激活是肿瘤干细胞自我更新和不定向分化的必要条件,端粒的动力学代表肿瘤干细胞恶性来源和有丝分裂历史,分析肿瘤干细胞端粒长度,端粒酶活性和细胞遗传学特性有助于揭示肿瘤干细胞起源和肿瘤形成历史,从而深化对肿瘤病理的认识,为恶性肿瘤治疗提供依据。结论:端粒和端粒酶在肿瘤干细胞中表达,是消灭肿瘤理想的靶标,有待进一步研究总结。     

端粒酶活性在乳腺癌中的表达及临床意义

目的　研究乳腺癌中端粒酶活性的表达 ,探讨端粒酶活化与乳腺癌发生发展的关系及其作为乳腺癌肿瘤标志物的可能性。方法　应用TRAP 银染法及TRAP ELISA方法对 2 7例乳腺癌组织及 18例相应癌旁组织中的端粒酶活性进行了定性及定量测定。结果　在 2 7例乳腺癌组织中 ,发现有 2 3例端粒酶表达阳性 ,阳性率为 85 .2 % ;而 18例相应癌旁组织中只有 1例表达阳性 ;乳腺癌组织中的端粒酶活性 (A值 )为 0 .5 85± 0 .2 86;而相应癌旁组织则为 0 .0 95±0 .0 62 ,两者比较有显著性差异 (P <0 .0 1)。乳腺癌肿块直径≥ 2cm及淋巴结转移阳性者端粒酶活性水平显著高于 <2cm及淋巴结转移阴性者 (P <0 .0 5 ) ;而端粒酶活性与肿瘤的病理类型无明显相关性 (P >0 .0 5 )。结论　端粒酶活化在乳腺癌的发生发展中起重要作用 ,有可能成为乳腺癌早期诊断的理想肿瘤标志物。     

Telomeres and telomerase in Candida albicans

Telomeres are the nucleoprotein structures at the ends of linear chromosomes and maintain the genomic integrity through multiple cell divisions. Telomeres protect the chromosome ends from degradation, end-to-end fusion and abnormal recombination and they also promote the end replication. The budding yeast Saccharomyces cerevisiae is the most well-studied model system with regard to telomere and telomerase regulation. Recently, the opportunistic fungal pathogen Candida albicans has emerged as an attractive model system for investigating telomere biology. Candida underwent rapid evolutionary divergence with respect to telomere sequences. Concomitant with the evolutionary divergence of telomere sequences, telomere repeat binding factors and telomerase components have also evolved, leading to differences in their functions and domain structures. Thus, the comparative analysis of the telomeres and telomerase-related factors in the budding yeast has provided a better understanding on both conserved and variable aspects of telomere regulation. In this review, I will discuss telomeres and telomerase-related factors and their functions in telomere and telomerase regulation in C. albicans.     

A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.

Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)n repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that affect the telomerase expression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other tumour types using a new biotinylated-primer extension assay. Our method, based on a non-amplified primer extension assay shows the direct incorporation of 32P-labelled nucleotides induced by telomerase on human telomeric primers. The 32P-dGTP labelled telomerase-extended 5'-biotinylated (TTAGGG)3 primer can subsequently be separated using streptavidin-coated magnetic beads. As compared to other non-amplified method, we showed that this procedure improved the characterization and the quantification of the banding pattern resulting from telomerase extension by reducing the radioactive background. Using this method, we observed that telomerase activity varies markedly in a panel of 39 human cancer cell lines. For example, MCF7 breast cancer cells in culture showed intermediate telomerase activity corresponding to 33.8+/-3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24+/-6.16). No correlation between the level of telomerase and telomere length was observed, suggesting that a high processivity is not required to maintain telomeres and that, in some cell lines, another mechanism of telomere elongation can maintain telomere length. From this study, we selected MCF7 and MX1 models that showed reproducible telomerase activity and a relatively limited telomere length for the testing of potential telomere-telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we showed that our model was able to detect a down-regulation of the telomerase activity in MCF7 cells in culture and in a human MX1 tumour xenografts. Based on these results, a breast cancer model for evaluating telomerase and telomere interactive agents is proposed.     

端粒、端粒酶及端粒酶催化亚基在大肠癌中的作用

目的:探讨端粒、端粒酶活性及端粒酶催化亚基蛋白(hTERT)在大肠癌发生发展、侵袭转移中的作用。方法:应用Southern blot、端粒重复序列扩增(TRAP)和免疫组织化学方法检测端粒长度(terminal restrictionfragments,TRFs)、端粒酶活性及hTERT表达水平。结果:大肠癌TRFs明显缩短,且随大肠癌Dukes分期的进展进一步缩短;端粒酶活性及hTERT在大肠癌组织中的阳性表达率分别为83.33%及76.67%,显著高于其他组织(P<0.05);大肠癌组织端粒酶与淋巴结转移关系密切,伴淋巴结转移大肠癌组织中的端粒酶活性及hTERT阳性表达率为80%和70%,显著高于无淋巴结转移者的0%和5%(P<0.05);相关分析显示端粒酶活性与hTERT蛋白表达存在显著相关性(P<0.05)。结论:端粒的短缩及端粒酶的活化与大肠癌的发生发展密切相关,hTERT的表达对端粒酶的激活可能起着重要作用。     

Gastrokine 1 induces senescence and apoptosis through regulating telomere length in gastric cancer

The present study aims to investigate whether gastrokine 1 (GKN1) induces senescence and apoptosis in gastric cancer cells by regulating telomere length and telomerase activity. Telomere length, telomerase activity, and hTERT expression decreased significantly in AGS^GKN1 and MKN1^GKN1 cells. Both stable cell lines showed increased expression of TRF1 and reduced expression of the hTERT and c-myc proteins. In addition, TRF1 induced a considerable decrease in cell growth, telomerase activity, and expression of hTERT mRNA and protein. GKN1 completely counteracted the effects of c-myc on cell growth, telomere length, and telomerase activity. Interestingly, GKN1 directly bound to c-myc and down-regulated its expression as well as inhibited its binding to the TRF1 protein and a hTERT promoter. Furthermore, GKN1 triggered senescence, followed by apoptosis via up-regulating the p53, p21, p27, and p16 proteins and down-regulating Skp2. Telomere length in 35 gastric cancers was shortened significantly compared with the corresponding gastric mucosae, whereas GKN1 expression was inversely correlated with telomere length and c-myc and hTERT mRNA expression. Taken together, these results suggest that GKN1 may shorten telomeres by acting as a potential c-myc inhibitor that eventually leads to senescence and apoptosis in gastric cancer cells.     

Tumour-cell apoptosis after cisplatin treatment is not telomere dependent

Cisplatin is a major chemotherapeutic agent, especially for the treatment of neuroblastoma. Telomeres with their sequence (TTAGGG)n are probable targets for cisplatin intrastrand cross-linking, but the role of telomeres in mediating cisplatin cytotoxicity is not clear. After exposure to cisplatin as single dose or continuous treatment, we found no loss of telomeres in either SHSY5Y neuroblastoma cells (telomere length, approximately 4 kbp), HeLa 229 cells (telomere length, 20 kbp) or in the acute lymphoblastic T cell line 1301 (telomere length, approximately 80 kbp). There was no induction of telomeric single strand breaks, telomeric overhangs were not degraded and telomerase activity was down-regulated only after massive onset of apoptosis. In contrast, cisplatin induced a delayed formation of DNA strand breaks and induced DNA damage foci containing gamma-H2A.X at nontelomeric sites. Interstitial DNA damage appears to be more important than telomere loss or telomeric damage as inducer of the signal pathway towards apoptosis and/or growth arrest in cisplatin-treated tumour cells.     

靶向端粒端粒酶的抗肿瘤治疗研究进展*

端粒是人类染色体末端由重复核酸序列组成的保护性结构,会随着细胞成功的分裂进行性的缩短。超过85% 的肿瘤细胞通过激活在大多数正常体细胞中被抑制的端粒酶来阻止端粒的无限缩短,维持细胞的永生化。肿瘤细胞跟正常细胞相比,有着更短的端粒和被重新激活的端粒酶,这些简单却又特殊的生物学差异促进了靶向端粒/ 端粒酶抗肿瘤治疗的发展。近年来许多成功的治疗药物经过临床前的筛选,在多种肿瘤中取得Ⅰ/ Ⅱ期临床试验的成功,GRN 163L 和GV1001等药物已进入Ⅲ期临床试验。联合传统药物治疗是目前的发展方向,未来靶向端粒/ 端粒酶治疗联合放射治疗可能在取得抗肿瘤疗效叠加的同时,提高治疗的安全性。     

Shortened telomere length and increased telomerase activity in hamster pancreatic duct adenocarcinomas and cell lines

Recently, shortened telomere length and increased telomerase activity have been demonstrated in various human cancers. In the study reported here, we ascertained whether gene changes are characteristic of pancreatic cancers. Hamster duct carcinomas and cell lines were investigated by Southern blot analysis for telomere restriction fragment (TRF) length and by the telomeric repeat amplification protocol (TRAP) assay for telomerase activity. Comparison with normal pancreas and spleen revealed shortened TRF length and markedly increased telomerase activity in primary pancreatic duct carcinomas induced by the rapid-production model as well as in a transplantable carcinoma and the cell lines. The enzyme level was 86.0–215.7 times the low levels found in control pancreas and spleen tissues. Late-passage Syrian hamster embryo cells, known to be immortalized and tumorigenic, had shorter TRFs than the original cells in primary culture did. These results indicate that hamster pancreatic duct carcinoma cells are immortalized, with the potential for proliferation ad infinitum, and provide a model for basic therapeutic research into the substances targeting telomerase. Mol. Carcinog. 18:153–159, 1997. © 1997 Wiley-Liss, Inc.     

Risk of multiple myeloma is associated with polymorphisms within telomerase genes and telomere length

Compelling biological and epidemiological evidences point to a key role of genetic variants of the TERT and TERC genes in cancer development. We analyzed the genetic variability of these two gene regions using samples of 2,267 multiple myeloma (MM) cases and 2,796 healthy controls. We found that a TERT variant, rs2242652, is associated with reduced MM susceptibility (OR = 0.81; 95% CI: 0.72–0.92; p = 0.001). In addition we measured the leukocyte telomere length (LTL) in a subgroup of 140 cases who were chemotherapy‐free at the time of blood donation and 468 controls, and found that MM patients had longer telomeres compared to controls (OR = 1.19; 95% CI: 0.63–2.24; p_trend = 0.01 comparing the quartile with the longest LTL versus the shortest LTL). Our data suggest the hypothesis of decreased disease risk by genetic variants that reduce the efficiency of the telomerase complex. This reduced efficiency leads to shorter telomere ends, which in turn may also be a marker of decreased MM risk.     

急性白血病端粒酶检测及临床相关分析

目的　检测急性白血病细胞端粒酶活性与临床相关分析。方法　端粒重复扩增法 (TRAP)结合光密度定量分析端粒酶活性和临床病例资料分析。结果　在急性白血病初诊期或复发期均表现很高的端粒酶活性 ,缓解期病例活性降低 ,骨髓增生异常综合征转变为急性白血病者及慢性粒细胞白血病急性变后端粒酶活性明显增高。结论　急性白血病较高的端粒酶活性与临床病情演变相关 ,分析白血病细胞端粒酶活性 ,对白血病临床诊断和治疗具有一定意义。     

Targeting telomerase for cancer therapeutics

One of the hallmarks of advanced malignancies is continuous cell growth and this almost universally correlates with the reactivation of telomerase. Although there is still much we do not understand about the regulation of telomerase, it remains a very attractive and novel target for cancer therapeutics. Several clinical trials have been initiated, and in this review we highlight some of the most promising approaches and conclude by speculating on the role of telomerase in cancer stem cells.     

胃癌细胞中端粒酶活性的表达研究

Objective To study the relationship between telomerase activity and biological behavior in human gastric cancer cells and appraise the clinical significance of detecting telomerase activity. Methods The telomerase activity in 47 gastric cancer tissue samples, their matched normal tissues, 7 gastric ulcer and 2 gastric cancer cell lines was detected using a PCR-based non-radioisotopic telomeric repeat amplification protocol (TRAP) assay. Results None of the 47 samples from normal gastric tissues expressed telomerase activity. The 41 of 47 cases of gastric cancer presented telomerase activity with an 87.2% positive rate (P<0.001). 2/2 gastric cancer cell lines and 0/7 gastric ulcer line were also positive for telomerase activity. The activity of telomerase was associated with the pathological differentiation of gastric cancer. Conclusion Telomerase activity may be related to the biological behavior of gastric cancer and can help in assessing the malignant potential of gastric cancer. Telomeras activity will be a good diagnostic marker for the detection of gastric cancer.     

Preclinical and clinical evaluation of farnesyltransferase inhibitors

Farnesylation of Ras, a protooncogene that is frequently mutated in a number of malignancies, is critical for its biologic function. This observation has led to the development of several agents that inhibit farnesyltransferase, known as farnesyltransferase inhibitors (FTIs). The antiproliferative and antitumor effects of these agents have been demonstrated in preclinical and clinical studies. Interestingly, FTI activity does not necessarily rely on ras mutational status, indicating that Ras is not the only FTI target. Clinical data suggest that FTIs, alone and in combination with other agents, have antitumor activity. Further study is needed to determine the precise mechanism of FTI antitumor activity as well as how and where FTIs will be best used clinically.     

Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length

Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression.