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ENTPD5 Expression Correlates with AKT Activation in Human Cancer Cell Lines and Primary Tumor Samples PTEN mutation and AKT activation are common features for human cancer. To check whether what was observed in PTEN null MEFs is also true for human cancer cells, we screened a panel of human cancer cell lines for the expression of PTEN, activated AKT, and ENTPD5. As shown in Figure 7A, AKT activation was seen in human prostate cancer lines C42 and LNCaP cells, and in these two cell lines, elevated ENTPD5 expression was also observed. We also examined ENTPD5 expression and AKT activation in primary human tumor samples by staining two adjacent sections from a formalin-fixed, paraffin-embedded human primary prostate cancer sample with rabbit monoclonal antibodies against human ENTPD5 and phosphoAKT, respectively. The specificity of this anti-ENTPD5 antibody was verified by western blotting analysis using LNCaP cell lines with or without their ENTPD5 knocked down (Figure S6A). The staining intensity for ENTPD5 in tumor was significantly greater compared with adjacent normal tissue and was correlated with pAKT staining (Figure 7B). Out of 10 samples from patients between age 57 and 76, only one tumor sample from a 57-year-old patient and another sample collected from a patient who had just gone through therapy did not show strongENTPD5staining, and the same tumors were also negative for pAKT (Figure S6B2 and Figure S6B10). The remaining eight samples all showed greater tumor staining of pAKT and ENTPD5 (Figure 7B and Figures S6B3–S6B9). Because microarray data of many tumors are publicly available, we also analyzed a group of recently publicized microarray data from human prostate cancer samples (Bermudo et al., 2008). We found that ENTPD5 is highly expressed in all 20 tumor samples compared to normal prostate epithelium cells (Figure S7). In addition, after clustering all gene expression profiles from prostate tumor microarray data using SOM (self-organization method), we identified dozens of genes associated with AKT activation, including Her-3, PI3KCB, Ras, S6 kinase, CD36, IL8, EGF, osteropontin, and FoxO1, which are significantly coregulated with ENTPD5 (Figure S7). ENTPD5 Is Important for Cancer Cell Growth To verify the functional significance of ENTPD5 expression in human cancer cells, we generated a cell line from the original LNCaP cells in which an shRNA against human ENTPD5 could be induced by Dox. In these cells, knockdown of ENTPD5 by adding Dox to the culture media also lowered N-glycosylation (Figure 7C, comparing lanes 7 and 8, 9 and 10, and 11 and 12) and induced BiP expression (Figure 7C, lanes 8, 10, and 12). These phenotypes were rescued by the expression of a wildtype shRNA-resistant ENTPD5 transgene, but not by the active site mutant ENTPD5 (Figure 7C, lanes 13–24). Several growth factor receptors were also checked in these cell lines after Dox treatment. As shown in Figure 7D, EGFR and Her2/ErbB-2 were significantly down, and IGFRb was slightly down (Figure 7D, lanes 1–4). They were restored to the normal level by the expression of shRNA-resistant ENTPD5 transgene (Figure 7D, lanes 5 and 6), but not the active site mutant (Figure 7D, lanes 7 and 8). Consistently, when the cell number was measured after 4 day knockdown of ENTPD5, only about half of LNCaP cells were there, compared to a control knockdown cell line, and the defect was rescued by expression of wild-type ENTPD5 transgene, but not active site mutant (Figure 7E). To test whether knocking down ENTPD5 in LNCaP cells also has an effect on their growth in vivo, we implanted the LNCaP cells bearing a Dox-inducible shRNA targeting ENTPD5 in matrigel in nude mice. As a control, LNCaP cells with a Dox-inducible shRNA targeting GFP were also implanted. After the xenograft tumors reached the size of 500 mm3, a cohort of seven mice were fed with Dox-containing water. The level of ENTPD5 in these tumors was measured after 6 weeks. Compared with mice fed with normal water, the ENTPD5 levels in ENTPD5-targeting shRNA containing tumors from mice fed with Dox-containing water were significantly lower except in one mouse (Figure 7F). Whereas ENTPD5-targeting shRNA containing tumors in mice fed with normal water continued to grow, the tumors in mice fed with Dox-containing water shrank (Figure 7G). Amazingly, when these tumor samples were analyzed under a microscope after fixing and staining with hematoxylin and eosin, there were very few tumor cells left in the matrigel in tumors grown in Dox-fed mice, whereas in mice fed with normal water, the matrigel was filled with tumor cells (Figure 7H). The GFP shRNA-containing tumors did not respond to Dox treatment and continued to grow during the period of experiment. |
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4楼2010-12-03 08:57:59
2楼2010-11-25 15:43:40
前世今生(金币+5, 翻译EPI+1):因为别人盗用了我的号，所以不是本人发帖，痛斥这种行为，不能按照发帖来发放，但是鉴于bibiaomian 的辛苦翻译还是~~~~ 2010-12-03 09:04:26
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ENTPD5表达与AKT的活化 人肿瘤细胞株和原肿瘤样本 PTEN基因突变和Akt的活化是共同的特征 人类癌症。要检查是否观察中PTEN基因是什么 空MEFs细胞也是真正的人类癌症细胞，我们筛选 人类癌症中PTEN表达的细胞系， 激活AKT和ENTPD5。如图7A条，Akt的活化 被认为在人类前列腺癌细胞系C42单元和LNCaP 细胞，并在这两个细胞系，表达升高ENTPD5 也有人。 我们还研究ENTPD5在表达和Akt的活化 主要由两个相邻的部分染色人类肿瘤样本 从福尔马林固定，石蜡包埋人前列腺癌 癌症样品与兔单克隆抗体 人类ENTPD5和phosphoAKT分别。特异性 这种反ENTPD5抗体免疫印迹验证 分析或不使用前列腺癌细胞LNCaP细胞株的ENTPD5 撞倒（图物S6A）。染色强度为ENTPD5 在肿瘤较显着提高与邻近正常 相关组织，并与染色pAKT（图7b）。出于 10 57和76岁之间，只有一个肿瘤患者的样本 样本来自一名57岁的病人，另一个样品采集 从谁刚刚通过治疗病人并没有显示了 strongENTPD5staining，同样的肿瘤也不利 为pAKT（图S6B2和图S6B10）。其余八 所有样品都表现出更大的肿瘤pAKT和ENTPD5染色 （图7B和图S6B3 - S6B9）。 由于多种肿瘤基因芯片数据的公布， 我们还分析了最近公布芯片组 从人类前列腺癌样本数据（Bermudo等。 2008年）。我们发现，ENTPD5高度表达在所有20个肿瘤 样品相比，正常前列腺上皮细胞（图 七）。此外，在所有基因的表达谱聚类 前列腺肿瘤基因芯片数据使用索姆（自组织 法），我们确定了几十个相关的基因 AKT的活化，包括她的- 3，PI3KCB，拉斯，S6激酶， CD36的，IL8，表皮生长因子，osteropontin和FoxO1基因，这是显着 协同调节与ENTPD5（图七）。 ENTPD5是癌症细胞生长的重要 为了验证ENTPD5表达的功能意义 人类癌细胞中，我们产生了从原始细胞系 LNCaP细胞中，是对人类ENTPD5 shRNA能 阿霉素可诱导。在这些细胞中，以击倒ENTPD5 增加阿霉素的培养基也降低的N -糖基化 （图7c，比较泳道7和8，9，10，11和12） 和诱导BiP的表达（图7c，泳道8，10和12）。 这些表型救出了一个野生表达 shRNA的耐ENTPD5转基因，但不是由活跃 网站（图7c，泳道13-24）突变ENTPD5。多种生长 因子受体还检查了在这些细胞株后，阿霉素 治疗。如图7D的，EGFR和Her2/ErbB-2 均显着下降，IGFRb是小幅下降（图7D的， 泳道1-4）。他们恢复到正常水平的表达 的shRNA耐ENTPD5转基因（图7D的，带5 6），而不是突变的活性部位（图7D的，泳道7 8）。一致，当后测定细胞数 4 ENTPD5天击倒，只有大约一半的LNCaP细胞 在那里，而控制击倒细胞系， 缺陷被救出的野生型ENTPD5表达 转基因，但不活跃的网站突变（图7E）。 为了测试是否撞倒在LNCaP细胞ENTPD5也 有它们在体内生长的影响，我们植入前列腺癌细胞LNCaP 细胞基底膜一轴承霉素诱导的shRNA靶向ENTPD5 在裸鼠体内。作为对照，LNCaP细胞与阿霉素诱导 GFP的shRNA的目标也植入。移植后 肿瘤达到五○○立方毫米的大小，七小鼠队列 阿霉素饲喂含有水。水平的ENTPD5 这些肿瘤的6周后测定。相比 与正常水喂养老鼠，在ENTPD5的ENTPD5水平定位 shRNA的含肿瘤与阿霉素喂食含 水除在一显着降低小鼠 （图7楼）。鉴于ENTPD5定位shRNA的含 水与正常喂养小鼠肿瘤继续增长， 在小鼠体内肿瘤霉素饲喂含水分下跌（图7代）。 令人惊讶的是，当这些肿瘤样本进行了分析下 固定后，与苏木染色和显微镜 曙红，有极少数肿瘤细胞的基底膜留在 生长在阿霉素喂养的小鼠，在小鼠与正常喂养，而肿瘤 水，基底膜弥漫着肿瘤细胞（图7小时）。该 GFP的shRNA的含肿瘤没有回应霉素治疗 并继续增长，在实验期。 |
3楼2010-12-03 08:50:53