人端粒酶逆转录酶(hTERT)特异性siRNA对乳腺癌细胞hTERT水平及细胞生长影响的研究

背景与目的:在肿瘤细胞中端粒酶高水平的表达是肿瘤细胞永生化的重要因素。本研究利用人端粒酶逆转录酶(hTERT)的小干扰RNA(siRNA)特异性地抑制乳腺癌细胞的hTERT,通过研究该效应对细胞生长、凋亡和永生化相关基因的影响,来进一步阐述端粒酶在肿瘤细胞永生化可能的作用。方法:通过实时定量RT-PCR(Real-TimeRT-PCR)测定各乳腺癌细胞系中内源性hTERT的表达水平及测定RNA干扰对与细胞永生化相关基因的调节;通过脂质体转染法将hTERTsiRNA导入MCF7细胞;通过细胞生长曲线和流式细胞仪(FACS)评价该特异性的siRNA所诱导的hTERT水平下调对细胞生长和凋亡的影响。结果:所检测的乳腺癌细胞系中,hTERT均相对高表达。hTERTsiRNA可抑制MCF7中hTERT的水平,并在抑制后的第4天开始表现出显著的细胞生长抑制,并出现凋亡,以及多个细胞永生化相关的基因,如RAC1、PCYT2、FDFT1和ATP5G2,表达水平均下调50%以上。结论:hTERTsiRNA可特异性地抑制hTERTmRNA水平,从而抑制细胞生长,导致细胞凋亡,并下调多个细胞永生化基因。     

人卵巢癌永生化细胞系BUPH:OVSC-2的建立及其生物学特性研究

背景与目的：利用永生化技术建立预期表型的细胞系,已成为建设系的重要手段之一,而人卵巢癌永生化细胞系的建立国内外少有报道。本文介绍人卵巢肉瘤样癌永生化细胞系的建立,以及对其生物学特性的研究。方法：以手术切除的卵巢肉瘤样癌腹壁转移组织为材料,进行体外培养。将永生化基因SV40T抗原基因转染第10代细胞,经筛选、抗性克隆扩大培养,得到永生化细胞系。通过光学显微镜、电子显微镜、生化曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等,研究其生物学特性,并与其来源细胞的生物学特性进行比较。结果：建立一株人卵巢肉瘤样癌永生化细胞系,命名为BUPH：OVSC－2,现已传至90年代。其生物学特性为：形态学观察细胞呈肉瘤样细胞形态,超微结构证实为上皮起源;细胞生长旺盛;具有恶性细胞的特征,恶性度高。通过比较,与其来源细胞的生物学特性无明显差别。结论：BUPH：OVSC－2为一株恶性度高的人卵巢肉瘤样癌永生化细胞系,保留了其来源细胞的生物学特性,可作为卵巢肉瘤样癌研究的实验模型。     

人类输卵管上皮永生化细胞系的建立及鉴定

背景与目的:最近研究表明卵巢高级别浆液性癌可能起源于输卵管上皮。本课题通过基因沉默和端粒酶导入技术将原代输卵管上皮细胞永生化,为以后建立恶性转化细胞系和卵巢癌动物模型奠定基础。方法:分离并培养输卵管上皮细胞,导入p53和pRb shRNA结合hTERTcDNA,建立p53和pRb基因同时沉默并过表达hTERT的永生化细胞系,并对永生化细胞系进行连续传代、沉默基因的蛋白质印迹法(Western blot)测定、SA-β-gal染色和非停泊生长实验及体内致瘤活性鉴定。结果:我们建立了稳定表达p53、pRbshRNA和hTERT的永生化输卵管上皮细胞系:FTE248116/p53i+pRbi+hTERT及FTE312249/p53i+pRbi+hTERT。结论:可以通过导入端粒酶基因及敲除抑癌基因p53和pRb来建立永生化细胞系,并有望于构建恶性转化细胞系和人类高级别浆液性卵巢癌动物模型。     

人端粒酶催化亚单位及其相关蛋白在骨肿瘤中的表达

目的：研究骨肿瘤中端粒酶相关基因中,人端粒酶催化亚单位（human elomerase catalytic subunit,hTERT)和人端粒酶相关蛋白（human telomerase associated protein,TP1)两个重要成分的表达及对评估肿瘤良、恶性的意义。方法：提取50例手术切除骨肿瘤组织、2种人成骨内瘤细胞系Saos－2、OS732和原代培养人成骨细胞的总RNA。用一步RT－PCR方法检测hTERT和TP1mRNA的表达。结果：所有骨肿瘤标本及细胞系均检测到TP1的表达。hTERT在17例良性骨肿瘤、33例骨肉瘤、2种人成骨肉瘤细胞系和人成骨细胞的表达率分别为64．7％、57．6％、0％和0％。良、恶性肌肿瘤间hTERT表达的差异不具有显著性（P＞0．05）,不同分化程度骨肉瘤间hTERT表达的差异也不具有显著性（P＞0．05）。结论：一步RT－PCR是检测端粒酶亚单位的有效方法。hTERT和TP1表达水平的上调在骨肿瘤恶性进展中可能不起主要作用,在骨肿瘤发展和维持中可能有替代性（ALT）机制导致细胞永生化。     

SV40T介导的人胎儿永生化气管成纤维细胞系的建立

建立永生化气管成纤维细胞系为研究细胞恶性转化提供实验模型。用ｐＺＩＰＳＶ４０Ｔ转染原代人胎气管成纤维细胞，Ｇ４１８筛选，挑取阳性克隆传代培养，并进行细胞生物学特性鉴定。结果表明，细胞经ｐＺＩＰＳＶ４０Ｔ转染后，形态较正常细胞略短，排列不规则，当传到５０代时仍生长良好。Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 证实此转化细胞整合了ＳＶ４０Ｔ基因。永生化细胞接种的第４天细胞为指数生长期，染色体核型以非整倍体为主，不     

HPV18 E6 E7基因诱发的人胎儿食管上皮永生化和恶性转化细胞端粒长度和端粒酶活性

目的：探讨端粒长度、端粒酶活性以及端粒酶亚单位组分（hTERT、hTR）的表达与食管上皮细胞永生化和恶性转化之间的关系。方法：对HPV18E6E7基因诱发的人胎儿食管上皮永生化细胞系SHEE和恶性转经细胞系SHEEC,通过Southern blot检测端粒长度（TRF）,TRAP法测定端粒酶活性,RT－PCR研究端粒酶催化亚单位（hTERT)端粒酶RNA成分（hTR）的表达。结果：SHEE细胞和SHEEC细胞的端料平均长度比正常食管上皮细胞的明显缩短,但稳定维持在一定长度范围内。SHEE细胞和SHEEC细胞均具有端料酶活性,并均有hTERT和hTR表达。结论：端粒酶表达活化使端粒维持在一定长度是永生化食管上皮细胞SHEE和恶性转化细胞SHEEC能够稳定分裂增殖的重要因素之一。     

hTERT启动子的克隆及在MCF7乳腺癌细胞中的特异转录活性

背景与目的：利用肿瘤特异性启动子增强肿瘤细胞中治疗基因的转录选择性是达到基因治疗靶向性的有效途径。人端粒酶催化亚基（hTERT）在大部分肿瘤细胞中呈特异性高表达，有可能成为肿瘤特异性启动子。本文扩增了hTERT基因启动子并分别克隆于报告基因重组质粒pEGFP-1和pGL3-Basic，检测并证实hTERT启动子在乳腺癌细胞MCF7中的特异转录活性。方法：采用套式PCR法扩增hTERT启动子，将其分别克隆到含报告基因增强绿色荧光蛋白（EGFP）的重组质粒pEGFP-1和含Luciferase的重组质粒pGL3-Basic，经脂质体分别转染人乳腺正常上皮细胞系HBL100和乳腺癌细胞MCF7，荧光显微镜观察EGFP的表达情况，并测定两种细胞中荧光素酶转录表达差异。结果：与正常细胞HBL100相比，hTERT启动子在MCF7细胞呈现强的绿色荧光表达；荧光素酶活性检测与其一致，hTERT启动子在MCF7乳腺癌细胞的荧光素酶相对活性RLU／U值（33784）约相当于HBL100细胞中（2400）的15倍。结论：hTERT启动子在乳腺癌MCF7细胞中具有很强的特异性，为进一步开发肿瘤的特异性基因治疗奠定实验基础。     

hTERT启动子调控的融合自杀基因CD:UPRT载体的构建及其应用

目的 构建hTERT启动子调控的融合自杀基因CD:UPRT表达载体,研究其对人胃癌细胞SGC7901的体外靶向性杀伤作用。方法 PCR扩增hTERT核心启动子片段,克隆入荧光素酶报告基因质粒pGL3 Basic,检测hTERT启动子在人胃癌细胞SGC7901和人成纤维细胞HLF中的转录活性。构建hTERT启动子调控的CD:UPRT基因表达载体hTERT CD:UPRT,将其和CMV启动子调控的CD:UPRT基因表达载体pcDNA3.1 CD:UPRT用脂质体转染法分别转染入SGC7901和HLF细胞,筛选稳定表达细胞系,用RT PCR和Western blot方法检测CD基因的表达,用MTT法检测5 FC对转染细胞的杀伤作用。结果 成功克隆hTERT核心启动子;荧光素酶活性检测显示,hTERT启动子在SGC7901细胞中的转录活性为阳性对照的(21.50±2.15)%,而在HLF细胞中仅有背景活性。成功构建hTERT启动子调控的CD:UPRT基因表达载体,转染pcDNA3.1 CD:UPRT的SGC7901和HLF细胞以及转染hTERT CD:UPRT的SGC7901细胞在mRNA和蛋白质水平均可检测到CD基因的表达,且对5 FC敏感;而转染hTERT CD:UPRT的HLF细胞未检测到CD基因的表达,对5 FC不敏感。结论 构建的hTERT启动子调控的融合自杀基因系统CD:UPRT/5 FC能在体外靶向性杀伤SGC7901细胞。     

Oxidative stress pathways highlighted in tumor cell immortalization: association with breast cancer outcome

An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.     

Long-term tumor-free survival from treatment with the GFP-TRAIL fusion gene expressed from the hTERT promoter in breast cancer cells

We evaluated anti-tumor activity and toxic effect of an adenoviral vector expressing the GFP/TRAIL fusion gene from the hTERT promoter (designated Ad/gTRAIL) on human breast cancer cell lines and on normal human breast cells. Treatment with Ad/gTRAIL elicited high levels of transgene expression and apoptosis in a variety of breast cancer cell lines. Furthermore, treatment with Ad/gTRAIL was effective in killing breast cancer lines resistant to doxorubicin or soluble TRAIL protein. In contrast, only minimal transgene expression and toxicity was detected in normal human primary mammary epithelial cells after treatment with this vector. An in vivo study further showed that the intralesional administration of Ad/gTRAIL effectively suppressed the growth of human tumor xenografts derived from both doxorubicin-sensitive and doxorubicin-resistant breast cancer lines. Specifically, about 50% of animals bearing doxorubicin-sensitive and doxorubicin-resistant breast cancer xenografts showed complete tumor regression and remained tumor-free for over 5 months. These results suggest that the adenovirus encoding the GFP/TRAIL gene driven by the hTERT promoter has potential application in cancer therapy.     

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background: Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are over-expressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study. Methods: Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method. Results: The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both. Conclusions: The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.     

Gene expression analysis in SV40-immortalized human breast luminal epithelial cells with stem cell characteristics using a cDNA microarray

The epithelial compartment of the human breast comprises two distinct cell types. Type I human breast epithelial cells (HBECs) are expressing luminal epithelial cell markers and stem cell characteristics, whereas Type II HBECs show basal epithelial cell phenotypes. When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell. We had developed simian virus 40-immortalized cell lines from normal HBECs with luminal and stem cell characteristics. To identify molecular changes involved in immortalization, we analyzed the differential gene expression profiles of normal and non-tumorigenic immortalized Type I HBECs using cDNA microarray with 7,448 sequence-verified clones. Out of the 7,448 genes screened, consistent gene expression changes among biological replicates included 67 in Type I HBECs and 86 in Type II HBECs for 4-fold change criteria. Surprisingly, we identified 148 genes (>2.0-fold) as being either up- or down-regulated related to immortalization: 67 genes (MYBL2, UCHL1 et al) were up-regulated, and 81 genes (IGFBP3, CDKN1A et al) were down-regulated significantly. The altered expression levels of the selected genes were subsequently confirmed by semiquantitative RT-PCR. Our studies suggest that the immortalization of Type I HBECs might be an early step in the initiation of a subset of breast cancer. Furthermore, these results will open up an avenue for more detailed understanding of breast stem cell and tumor biology.     

Expression of WNT14 and WNT14B mRNAs in human cancer, up-regulation of WNT14 by IFNgamma and up-regulation of WNT14B by beta-estradiol.

WNT proteins play key roles in carcinogenesis. We have previously cloned and characterized WNT14 and WNT14B/WNT15. WNT14 and WNT3A genes are clustered on human chromosome 1q42, while WNT14B and WNT3 genes are clustered on human chromosome 17q21. Here, we investigated expression of WNT14 and WNT14B mRNAs in human cancer. WNT14 was significantly up-regulated in 1 out of 9 cases of primary breast cancer. WNT14B was not expressed in primary breast, gastric and colorectal cancers. Among 3 human breast cancer cell lines, WNT14 mRNA was expressed in T-47D cells, and weakly expressed in MCF-7 cells. WNT14 mRNA was also detected in 7 out of 7 pancreatic cancer cell lines, 12 out of 12 esophageal cancer cell lines, 4 out of 4 cervical cancer cell lines, and 5 out of 7 brain tumor cell lines by using cDNA-PCR. These results indicate that WNT14 rather than WNT14B is preferentially expressed in various types of human cancer, such as breast cancer, gastric cancer, and pancreatic cancer. WNT14 mRNA was up-regulated by interferon gamma (IFNgamma), but not by tumor necrosis factor alpha (TNFalpha), in MKN45 cells derived from gastric cancer, while expression of WNT14B mRNA was not affected by IFNgamma and TNFalpha in MKN45 cells. Although expression of WNT14 mRNA was not affected by beta-estradiol in MCF-7 cells, WNT14B mRNA was transiently up-regulated by beta-estradiol in MCF-7 cells. These results indicate that WNT14 is a target gene of IFNgamma in MKN45 cells, and that WNT14B is a target gene of estrogen in MCF-7 cells.