Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells

Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.     

Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells: screening for effective target sites.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.     

Ribozyme-mediated cleavage of the human survivin mRNA and inhibition of antiapoptotic function of survivin in MCF-7 cells

Survivin is a new member of the inhibitor of apoptosis protein (IAP) family that is implicated in the control of cell proliferation and the regulation of cell life span. This protein is selectively expressed in most human carcinomas but not in normal adult tissues. To down-regulate a human survivin expression as a strategy for cancer gene therapy, we designed two hammerhead ribozymes (RZ-1, RZ-2) targeting human survivin mRNA. RZ-1 and RZ-2 efficiently cleaved the human survivin mRNA at nucleotide positions +279 and +289, which was identified by in vitro cleavage assay using in vitro transcribed ribozymes and truncated survivin mRNA substrate. To investigate the function of the ribozymes in cells, the sequences of the ribozymes were cloned into replication-deficient adenoviral vector and transferred to breast cancer cell, MCF-7. The infection with adenovirus encoding the ribozymes resulted in a significant reduction of survivin mRNA (74% and 73%, respectively) and protein. As revealed by nuclear condensation/ fragmentation and flow cytometry analysis, inhibition of survivin gene by ribozymes increased apoptosis and sensitivity induced by etoposide or serum starvation. Our results suggest that the designed hammerhead ribozymes against survivin mRNA are good candidates for feasible gene therapy in the treatment of cancer.     

Overexpression of multidrug resistance genes MDR1 and cMOAT in human hepatocellular carcinoma and hepatoblastoma cell lines.

Overexpression of either of the multidrug resistance genes MDR1 and MRP is associated with resistance of tumors to multiple chemotherapeutic agents. Overexpression of MDR1 has been reported in some cell lines derived from human hepatocellular carcinomas (HCC) and hepatoblastomas (HB). The human gene for cMOAT (), a homologue of MRP, is thought to mediate hepatobiliary excretion of organic anions and to be associated with cisplatin resistance. In this study, expression levels of MDR1 and cMOAT were examined in 9 human HCC and HB cell lines and 10 other human cancer cell lines. Overexpression of the cMOAT gene was observed in all 9 HCC and HB cell lines and 3 of 10 other cancer cell lines. Co-overexpression of the cMOAT and MDR1 genes was observed in 7 of 9 HCC and HB cell lines, but in none of the 10 other cancer cell lines. Seven of the HCC and HB cell lines that had overexpression of the cMOAT gene were shown to be highly resistant to cisplatin compared to 2 HCC cell lines with low levels of cMOAT expression. These findings suggest that overexpression of cMOAT could contribute to cisplatin resistance in HCC and HB.     

抑制VEGF表达的锤头状核酶系统

目的:建立和评估抑制血管内皮生长因子(VEGF)表达的锤头状核酶(hammerhead ribozyme)技术系统.方法:对VEGF121基因RNA序列进行二级结构分析,选择靶点;设计并构建针对VEGF的分泌肽RNA的可表达锤头状核酶(14)载体系统和VEGF-荧光色素酶融合基因报告质粒;通过试管内切割实验等方法评估核酶对于试管内转录得到的VEGF RNA切割特异性和效率;通过瞬时共转染实验和稳定转染实验评估核酶在细胞内对于VEGF RNA的切割效率.结果:设计和构建了对VEGF RNA二级结构水平上的暴露区( 8, 36和 71位点:核酶1,3和4)和非暴露区( 17位点:核酶2)4个锤头状核酶(14)的两套质粒;试管内切割检测表明,针对 8, 36和 71位点的核酶(1,3和4)可以有效地在试管内对VEGFRNA进行特异性切割,使其水平分别降至对照的61.7%,27.6%和44.8%(荧光色素酶活性)或66.3%,27.0%和30.0%(蛋白质水平);将核酶表达质粒与VEGF-LUC模板质粒瞬时共转染人SMMC-7721肝癌细胞,核酶1,3和4 VEGF-LUC水平分别降低到对照的81.4%,56.6%和69.1%;稳定表达针对VEGF的核酶1,3或4的SMMC-7721细胞株中,内源性VEGF RNA的水平降至对照水平的5%以下.对照未转染的、转染空载体的和转染了核酶2( 17)的SMMC-7721细胞.结论:分别针对VEGF 8, 36和 71位点锤头状核酶可有效地抑制VEGF的RNA水平.     

多单位核酶对慢性粒细胞白血病细胞的增殖抑制及凋亡诱导效应研究

目的：探讨多单位核酶体外净化慢性粒细胞白血病（慢粒）骨髓的可能性,研究多单位核酶的体外切割活性及对慢粒细胞恶性表型的逆转作用。方法：针对慢粒发病中起重要作用的bcrabl融合基因,在融合位点两侧44碱基范围内设计,合成3个核酶,其中2个切割位点位于bcr基因,1个位于abl基因。通过基因重组构建多单位核酶体外转录载体及逆转病毒表达载体,鉴定其体外切割活性。将多单位核酶逆转录病毒表达载体转染K562细胞,应用MTT,3H－TdR掺入,RT－PCR,斑点杂交,流式细胞仪,透射及扫描电镜等方法,检测多单位核酶对慢粒细胞增殖活性及细胞超微结构,细胞周期,凋亡等的影响。结果：多单位核酶的体外切割活性为70．8％,逆转录病毒载体转染慢粒酶转染细胞后能够有效切割细胞RNA,使转染细胞RNA减少1000倍左右,流式细胞仪检测显示多单位核酶转染72h后,18．4％的细胞发生凋亡,大多数细胞被阻滞于G期,分裂期（S期）细胞数减少约41．9％,透射,扫描电镜见转染细胞出现核固缩,凋亡小体等细胞凋亡的特异性表现。结论：多单位核酶不仅具有较高的体外切割活性,而且其逆转录病毒载体能够有效转染慢粒细胞系,在细胞内持续表达,切割细胞RNA,诱导细胞凋亡,抑制细胞增殖,逆转慢粒细胞的恶性表型,在慢粒基因治疗中具有潜在应用的价值。     

Ribozyme对癌基因Ki-ras~(G12V) mRNA的剪切及其特异性

目的 :设计切割ki-rasG12V mRNA的特异性ribozyme(Rz2 17) ,明确其对癌基因ki-rasG12VmRNA的细胞内外切割活性 ,为以ki-rasG12VmRNA为特异性靶分子的基因治疗及癌基因ki-ras的功能研究提拱一种新的途径。方法 :依Symons总结的“锤头结构”原理 ,设计一种能特异性切割ki-rasG12VmRNA的ribozyme ,利用DNA重组技术构建ki-rasG12V外显子 1和ri-bozymeRz2l7的体外转录质粒及ribozymeRz2 17的真核表达质粒 ,体外转录获得ribozymeRz2 17及ki-rasG12V外显子 1mRNA ,在含Mg2 溶液中ribozymeRz2 17对其靶RNA分子进行切割。以RT -PCR对转染ribozymeRz2 17真核表达质粒的细胞ki-rasG12VmRNA进行半定量分析。结果 :ki-rasG12V外显子 1体外转录mRNA分子 ,能被ribozymeRz2 17定点切割而野生型ki-ras外显子 1体外转录mRNA则不被切割 ;转染ribozymeRz2 17的胰癌细胞ki-rasG12VmRNA含量减少 ,而转染ri bozymeRz2 17的肝癌细胞其内源性ki-rasmRNA含量无明显变化。结论 :ribozymeRz2 17无论在细胞内外均能剪切突变型ki-rasmRNA(G12V)而且其切割作用为突变型ki-rasG12VmRNA特异性的。     

Hammerhead ribozyme-mediated inactivation of mutant RET in medullary thyroid carcinoma.

Activating mutations of the RET proto-oncogene cause hereditary medullary thyroid carcinoma. To examine whether selective inactivation of mutant RET could prevent transformation, a hammerhead ribozyme was designed to cleave RET mRNA containing a transforming mutation of codon 634 TGC --> TAC (Cys634Tyr). In vitro RNA cleavage assay demonstrated that the ribozyme selectively cleaved RET RNA with a Cys634Tyr but not Cys634Arg or the normal sequence. Expression of ribozyme in NIH/3T3 cells prevented RET-mediated colony formation in soft agar. This inhibition required catalytically active ribozyme and was specific for the TAC mutation. Therefore, ribozymes designed to selectively target mutant RET RNA may provide an effective therapeutic in the treatment of this syndrome.     

Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified,synthetic ribozyme

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.     

p53 gene mutation in primary human renal cell carcinoma.

We searched for possible mutations in the entire coding region of tumor suppressor gene p53 in primary human renal cell carcinomas using polymerase chain reaction and single-strand conformational polymorphism analysis of RNA. We found p53 mutations in 2 of 21 cases (10%). DNA sequencing of the polymerase chain reaction products verified that the first case included a 17-base deletion at the beginning of exon 6. The second case showed a T to C transition at nucleotide 1328 in exon 7. No clinical or pathological similarity was found in the renal cell carcinomas containing the mutated p53 genes. Present results suggest that p53 mutation is involved at low frequency in human renal cell carcinomas.     

c-erbB-2癌基因特异性核酶的体外切割作用

目的 构建ｃ－ｅｒｂＢ－２特异性核酶基因及其底物基因的体外转录载体并探讨其体外切割作用。方法 在计算机设计的核酶ＲＺＩ基因的３‘端加上新的限制性酶切位点ＥｃｏＲ Ｖ,先合成ＲＺＩ基因并将之与其底物分别克隆入体外转录载体ｐＧＥＭ３Ｚｆ（－）,经用ＥｃｏＲ Ｖ快速初选出含有ＲＥ１基因重组子并测序鉴定。体外转录物用＾３２Ｐ标记。体外切割作用在Ｍｇ＾＋＋的存在下,３７℃反应１小时,进行聚丙烯酰胺凝胶电泳,     

The cyclin H/cdk7/Mat1 kinase activity is regulated by CK2 phosphorylation of cyclin H

Cyclin dependent kinases are regulated by phosphorylation and dephosphorylation of the catalytic cdk subunits, by assembly with specific cyclins and by specific inhibitor molecules. Recently, it turned out that cyclins are also phosphoproteins, which means that they are also potential targets for a regulation by phosphorylation and dephosphorylation. Here, we show that cyclin H was phosphorylated by protein kinase CK2. Like most other CK2 substrates cyclin H was much better phosphorylated by the CK2 holoenzyme than by the alpha-subunit alone. By using point mutants derived from the cyclin H sequence we mapped the CK2 phosphorylation site at threonine 315 at the C-terminal end of cyclin H. Phosphorylation at this position had no influence on the assembly of the cyclin H/cdk7/Mat1 complex. However, phosphorylation at amino acid 315 of cyclin H turned out to be critical for a full cyclin H/cdk7/Mat1 kinase activity when the CTD peptide of RNA polymerase II or cdk2 was used as a substrate.