A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.
A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.
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基于新的生物素化培养地扩展测定法，用于研究端粒酶抑制剂的人类乳腺癌模型。
DOI：
10.1038/sj.bjc.6690526
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发表时间：
1999-07
影响因子：
8.8
通讯作者：
Von Hoff, DD
中科院分区：
文献类型：
--
作者：
Raymond, E;Sun, D;Izbicka, E;Mangold, G;Silvas, E;Windle, B;Sharma, S;Soda, H;Laurence, R;Davidson, K;Von Hoff, DD
端粒酶是一种依赖于RNA的聚合酶，可合成端粒DNA(TTAGGG)n个重复序列。我们工作的总体目标是建立可用于设计端粒酶抑制剂临床试验的人类癌症模型。这项研究的目的是(1)建立一种人乳腺癌系统，该系统允许使用非扩增的端粒酶试验来评估端粒酶抑制剂在培养细胞中的作用；(2)使用两种影响培养的乳腺癌细胞端粒酶表达的药物(顺铂和TMPyP4)来测试该系统。我们首先使用一种新的生物素化引物延伸试验比较了各种人类乳腺癌细胞株和其他肿瘤类型的端粒酶活性。我们的方法基于非扩增的引物延伸实验，表明端粒酶诱导的~(32)P标记的核苷酸直接掺入到人端粒引物上。32P-dGTP标记的端粒酶延伸5‘-生物素化(TTAGGG)3引物随后可以用链霉亲和素包裹的磁珠分离。与其他非扩增方法相比，我们发现该方法通过减少放射性本底，改善了端粒酶延伸所导致的带型的表征和定量。使用这种方法，我们观察到39个人类癌细胞株的端粒酶活性明显不同。例如，培养的MCF7乳腺癌细胞显示中等端粒酶活性，相当于HeLa细胞(参考细胞系)的33.8±3.4%。同样，不同细胞系的端粒长度也不同，平均为6.24±6.16。没有观察到端粒酶水平与端粒长度之间的相关性，这表明维持端粒不需要高加工能力，在某些细胞系中，另一种端粒延长机制可以维持端粒长度。从这项研究中，我们选择了MCF7和MX1模型，这些模型显示了可重复性的端粒酶活性和相对有限的端粒长度，用于测试潜在的端粒-端粒酶相互作用因子。使用顺铂和一种新的卟啉衍生化合物TMPyP4，我们的模型能够检测到培养中的MCF7细胞和人MX1肿瘤移植瘤中端粒酶活性的下调。基于这些结果，提出了一个评价端粒酶和端粒相互作用因子的乳腺癌模型。©1999癌症研究活动
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. © 1999 Cancer Research Campaign
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影响因子：
11.4
作者：
BRYAN, TM;ENGLEZOU, A;REDDEL, RR
通讯作者：
REDDEL, RR
DOI：
10.1007/bf00132889
发表时间：
1996-01-01
期刊：
Methods in Cell Science
影响因子：
--
作者：
Holt, Shawn E.;Norton, James C.;Shay, Jerry W.
通讯作者：
Shay, Jerry W.
影响因子：
5.6
作者：
LEVY, MZ;ALLSOPP, RC;HARLEY, CB
通讯作者：
HARLEY, CB
DOI：
10.1073/pnas.90.4.1493
发表时间：
1993-02-15
影响因子：
11.1
作者：
PROWSE, KR;AVILION, AA;GREIDER, CW
通讯作者：
GREIDER, CW
影响因子：
4
作者：
HARLEY, CB;VILLEPONTEAU, B
通讯作者：
VILLEPONTEAU, B