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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 500-506 |
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doi:10.1111/j.1745-7270.2006.00182.x |
Inhibition of Proliferation and
Induction of Apoptosis in Human Renal Carcinoma Cells by Anti-telomerase Small
Interfering RNAs
Jun-Nian ZHENG1,3#*,
Ya-Feng SUN2#,
Dong-Sheng PEI2,
Jun-Jie LIU1,
Jia-Cun CHEN1,
Wang LI1,
Xiao-Qing SUN1,
Qi-Duo SHI3,
Rui-Fa HAN3,
and Teng-Xiang MA3
1 Laboratory
of Urology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002,
China;
2 Research
Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou
221002, China;
3 Institute
of Urology, Tianjin Medical University, Tianjin 300070, China
Received: January
9, 2006
Accepted: April 20,
2006
This work was
supported by the grants from the National Natural Science Foundation of China (No.
30570385), the Jiangsu Science and Technology Department (No. BK2005429) and
the Health Department of China (No. 2005-2-026)
# These authors
contributed equally to this work
*Corresponding
author: Tel, 86-516-5802027; E-mail, [email protected]
Abstract Telomerase is an attractive molecular target for cancer
therapy because it is present in most malignant cells but is undetectable in
most normal somatic cells. Human telomerase consists of two subunits, an RNA
component (hTR) and a human telomerase reverse transcriptase component (hTERT).
Small interfering RNA (siRNA), one kind of RNA interferences, has been
demonstrated to be an effective method to inhibit target gene expression in
human cells. We investigated the effects of siRNA targeting at both hTR and
hTERT mRNA on the inhibition of telomerase activity in human renal carcinoma
cells (HRCCs). The proliferation and apoptosis of HRCCs were examined. The
treatment of HRCCs using hTR and hTERT siRNAs resulted in significant decrease
of hTR mRNA, hTERT mRNA and hTERT protein. The siRNA can also inhibit the
telomerase activity and the proliferation of HRCCs. Moreover, they can induce
apoptotic cell death in a dose-dependent manner. From these findings, we propose
that the inhibition of telomerase activity using siRNA targeting hTR and hTERT
might be a rational approach in renal cancer therapy.
Key words hTERT; hTR; telomerase; siRNA; renal cell carcinoma;
proliferation; apoptosis
The replication of linear chromosomes using DNA polymerases fails to completely duplicate the telomeres, the ends of the chromosomes. To complete the replication of telomeres, cells have evolved a specialized reverse transcriptase, telomerase, which can add 5'-TTAGGG-3' repeats into the telomeres [1]. Human telomerase consists of two subunits, an RNA component (hTR) containing the template for the telomere sequence, and a telomerase reverse transcriptase component (hTERT), which is a protein component and catalyzes telomeric repeat addition at the ends of chromosomes [2]. The essential role of telomerase is to ensure chromosome integrity. The ability of a cell to replicate indefinitely has been linked to telomerase expression.
Many kinds of tumor cells that have immortality characteristic show telomerase activity. The ubiquitous expression of telomerase in various human tumor cells has supported the hypothesis that this enzyme is involved in cellular immortality and carcinogenesis. The notion that telomerase is essential for the generation of human tumor cells has been supported by the following findings: mutation of the hTR leads to the decrease of cell proliferation [3]; and expression of antisense RNA complementary to the hTR component causes the decrease of proliferation of HeLa cells after 23 to 26 doublings [4]. Conversely, transfection of cells with the gene encoding hTERT and subsequent expression of active telomerase have been shown to extend the lifespan of human fibroblastoma epithelial cells [5-7]. Thus, the inhibition of telomerase gene expression seems to limit the growth of human cancer cells [8].
The telomerase is considered as a potential target for cancer therapy with few side effects. Inhibition of telomerase activity using conventional phosphorothioate-modified oligonucleotides (ODNs) has been reported previously [9]. However, the poor sequence selectivity of ODNs has led to the application of the second generational oligonucleotides, peptide nucleic acids (PNAs). The PNAs targeted at human telomerase could inhibit the activity of the telomerase with 10-50-fold more efficiency than that of analogous ODNs [10]. Although solid evidence of antisense effects of PNAs has been demonstrated, further investigation of PNAs as gene therapy has been hampered by the poor intrinsic absorbability of PNA in living cells [11]. Recently, the demonstration that RNA interference (RNAi) can be used to inhibit gene expression in mammal cells opens new avenues for gene-targeted therapies [12].
RNAi is a sequence-specific, post-transcriptional gene silencing mechanism that uses the introduction of small interfering RNA (siRNA), a hybrid consisting of a sense and antisense strand homologous in sequence to the silenced gene [13]. siRNA, 21-nt RNA with 2-nt 3' overhang, can mediate strong and specific suppression of gene expression [14]. Many examples have demonstrated that siRNA is an effective method to inhibit oncogene expression [15,16]. RNAi technology, especially using chemically synthesized siRNA, is currently evaluated as a potentially useful method to develop highly specific gene-silencing therapies. Because renal cancers are highly refractory to conventional anticancer treatments and telomerase reactivation has been detected in a high percentage of renal cancers, the possibility to inhibit renal carcinoma cell growth using RNAi against telomerase appears reasonable.
Here, our data shows that RNAi could be used to down-regulate telomerase components, resulting in the decrease of telomerase activity in human renal carcinoma cells.
Materials and Methods
siRNA preparation
The siRNA duplex sequences were synthesized, purified and annealed by Ambion (Austin, USA). The siRNA for hTR targeted at the region containing the telomere repeat template sequence (bolded): hTR siRNA sense sequence 5'-UUGUCUAACCCUAACUGAGTT-3', and antisense sequence 3'-TTAACAGAUUGGGAUUGACUC-5' . For hTERT, the siRNA targeted at the region containing nt 2127-2145 of the complementary hTERT DNA (GenBank accession No. AB085628) [17]: hTERT siRNA sense sequence 5'-CAAGGUGGAUGUGACGGGCTT-3', and antisense sequence 3'-TTGUUCCACCUACACUGCCCG-5'. The selected sequences were submitted to BLAST (http://www.ncbi.nlm.nih.gov/blast/) to ensure that the selected genes were targeted at specially. A scrambled siRNA was purchased from Ambion (Silencer negative control siRNA #3) and used as the control.
Cell culture and transfection
The human renal carcinoma cell line 786-0 was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 mM penicillin and 100 mM streptomycin. Cells were regularly passaged to maintain exponential growth. The day before transfection, cells were trypsinized, diluted with fresh medium and transferred to 24-well plates. Transfection of siRNA was carried out using siPORT lipids (Ambion). siPORT lipids and siRNA were diluted into Opti-MEM I (Invitrogen, Carlsbad, USA). Diluted siPORT lipids were mixed with diluted siRNA and the mixture was incubated for 20 min at room temperature for the complex formation. After adding Opti-MEM I into each cell well to a total volume of 200 ml, the entire mixture was added to the cells in one well resulting in a final concentration of 10, 50 or 100 nM of the siRNA. Cells were harvested and assayed 24, 48 or 72 h after transfection. All experiments were repeated at least six times.
Reverse
transcription-polymerase chain reaction (RT-PCR)
Analysis of RNA levels of hTR and hTERT was carried out using RT-PCR. Total RNA was extracted using the Total RNA isolation system (Promega, Madison, USA) and RT-PCR was carried out using the Access RT-PCR system (Promega, Madison, USA). The primers for hTR were 5'-CTGGGAGGGGTGGTGGCCATTT-3' and 5'-CGAACGGGCCAGCAGCTGACAT-3'. Reaction parameters were 94 șC for 20 s, 50 șC for 20 s, 72 șC for 30 s, 25 cycles. The primers for hTERT were 5'-GCCAGAACGTTCCGCAGAGAAAA-3' and 5'-AATCATCCACCAAACGCAGGAGC-3'. Reaction parameters were 94 șC for 20 s, 48 șC for 30 s, 72 șC for 30 s, 30 cycles. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control to ensure accuracy. Quantitation was carried out with an image analyzer (LabWorks Software, version 3.0; UVP, Upland, USA).
Western blot analysis
For determination of hTERT protein levels by Western blot, cellular extracts were prepared as described previously [18]. hTERT protein was separated using 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the b-actin control was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electrotransferred onto a nitrocellulose membrane, after blocking for 3 h in 3% bovine serum albumin. Membranes were incubated overnight at 4 șC with hTERT primary antibody (1:250; Roche, Basel, Switzerland), then washed and incubated with alkaline phosphatase conjugated secondary antibody (1:1000; DAKO, Glostrup, Denmark) in TBST for 2 h and developed using NBT/BCIP color substrate (Promega). The bands on the membrane were scanned for the density and analyzed with the image analyzer (LabWorks Software).
Telomerase activity
Telomerase activity assay was carried out according to a PCR-based telomeric repeat amplification protocol as described by Bhaduri [19] using the Telomerase PCR ELISA kit (Roche). The Telomerase PCR ELISA combines the highly specific amplification of telomerase DNA with the detection of those PCR products by a photometric enzyme immunoassay. Briefly, the PCR amplification product is immobilized on the well of a streptavidin-coated microplate and hybridized to a digoxigenin (DIG)-labeled probe that is specific for the telomeric repeat sequence. Finally, the DIG-labeled hybrids are visualized with a peroxidase-conjugated anti-DIG antibody and a colorimetric peroxidase substrate. The relative telomerase activity can be calculated according to the relative absorbance at 450 nm.
Terminal deoxynucleotidyl
transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay
To detect and quantitate apoptotic cell death, TUNEL assay was carried out using an In situ cell death detection kit (Roche Diagnostics, Indianapolis, USA) according to the provider's instructions. Briefly, chamber slides were fixed with 4% paraformaldehyde for 30 min and permeabilized in 0.1% Triton-100 and 0.1% sodium citrate at 4 șC for 2 min. The slides were incubated with TUNEL reaction mixture for 1 h at 37 șC. After washing with phosphate-buffered saline, the slides were incubated with peroxidase-conjugated streptavidin for 30 min at 37 șC and were developed with a 3,3'-diaminobenzidine-tetrachloride system. Under a microscopy, six fields were randomly selected for each sample and 100 cells were randomly selected in each field, and the rate was calculated as following:
Cell proliferation assay
Cellular proliferation was assayed using 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay. The MTT assay is a colorimetric assay system that measures the reduction of a tetrazolium component into an insoluble formazan product by the mitochondria of viable cells. In brief, 786-0 cells (2104 cells/well) were incubated in a 96-well plate, in the absence or presence of siRNA, at 37 șC in a humidified atmosphere containing 5% CO2. At the end of the experiment, 20 ml of 5 mg/ml MTT (Sigma, St. Louis, USA) was added to each well. Four hours later, 100 ml of DMSO was added to each well and the absorption at 570 nm (UA) were determined on an ELX-800 spectrometer reader (Bio-Tek Instruments, Winooski, USA). The proliferation inhibition rate is calculated as following:
where UAE is the average UA value of the experimental group, and UAC is the average UA value of the control group.
Cellular growth curve
To evaluate cell numbers, cells were trypsinized with appropriate times, stained with trypan blue and counted using a hemocytometer. Each experimental condition was carried out six times, and the average value for each group was determined to compose the growth curve.
Statistical analysis
Values were expressed as mean±standard deviation and obtained from at least six independent groups (nł6). Statistical analysis of the results was carried out by one-way anova followed by Duncan's new multiple range method or the Newman-Keuls test. P<0.05 was considered significant.
Results
Effects of siRNA treatment on
hTR and hTERT mRNA expression
hTR and hTERT mRNA expression were examined using RT-PCR. As shown in Fig. 1, cells treated with hTR siRNA and hTERT siRNA (50 or 100 nM) had a significant decrease of hTR and hTERT mRNA expression compared with 786-0 cells treated with control siRNA. An approximately 60% reduction of both hTR and hTERT mRNA was observed at the concentration of 100 nM.
Effects of hTERT siRNA
treatment on hTERT protein expression
The effects of the hTERT siRNA on hTERT protein expression were evaluated by Western blot 24 h after treatment. An approximately 50% reduction in immunodetectable hTERT protein was observed in lysates of the cells transfected with hTERT siRNA at a concentration of 100 nM (Fig. 2). These results indicate significant knockdown of hTERT protein expression by its siRNA.
Effects of siRNA treatment on
telomerase activity
The effects of the hTR and hTERT siRNA on telomerase activity were evaluated by telomeric repeat amplification protocol assay. Results throughout are reported as a percentage of telomerase activity of untreated cells (control). As shown in Fig. 3, hTR and hTERT siRNA depressed the telomerase activity of 786-0 cells to the same degree. Cells treated for 48 h with hTR or hTERT siRNA (50 or 100 nM) showed a significant decrease in telomerase activity compared with 786-0 cells treated with control siRNA. The maximum effect was observed in 786-0 cells was that the telomerase activity decreased to 33% of untreated cell activity for 100 nM hTR siRNA and to 35% of untreated cell activity for 100 nM hTERT siRNA.
Apoptotic cell death
The results of 786-0 cell apoptosis evaluation by TUNEL showed approximately 10% of cells cultured with negative control siRNA manifested evident apoptotic change after 72 h treatment. In contrast, a significantly great proportion (approximately 39%) of 786-0 cells treated with 100 nM hTR siRNA, and approximately 46% of 786-0 cells treated with 100 nM hTERT siRNA, were TUNEL positive (Fig. 4).
Antiproliferative effects of
siRNA treatment
The results of MTT assay of 786-0 cell proliferation showed that the proliferation activities of 786-0 cells decreased by 64% when treated with hTERT siRNA (100 nM), and by 63% with hTR siRNA (100 nM), compared with the control group, respectively [Fig. 5(A)]. The cell numbers were determined on day 1-3 after siRNA treatment. Both hTERT siRNA (100 nM) and hTR siRNA (100 nM) treatment resulted in a marked inhibition of cell proliferation during day 1-3. Cell growth was not influenced significantly by treatment with control siRNA and transfection reagent control [Fig. 5(B)].
Discussion
Telomerase is expressed in cancer cells but not most normal cells, leading to the hypothesis that telomerase inhibitors might be a powerful approach to cancer therapy. Telomerase activity was reported to be inhibited using conventional antisense oligonucleotide targeting hTR [9]. Kraemer et al. also reported that inhibition of telomerase activity by antisense oligonucleotide targeting hTERT caused significant inhibition of proliferation and induction of apoptosis in bladder cancer cells [20]. Recently, many reports have demonstrated that specific hTERT siRNA could successfully inhibit telomerase activity in several cancer cell lines. Nakamura et al. reported that cells lacking hTERT showed a significantly increased sensitivity, compared with control cells, to ionizing radiation or chemotherapeutics [21]. RNAi could inhibit hTERT gene expression and proliferation of hepatocalullar carcinoma SMMC-7721 cells with specificity [22]. Furthermore, the application of hTERT RNAi can effectively inhibit the growth of laryngeal squamous carcinoma [23] and prove feasible as a treatment for gastric cancer [24]. Thus, application of RNAi on the human telomerase reverse transcriptase provides a possible new approach for neoplasm gene therapy.
In the present study, we evaluated the ability of both hTR and hTERT siRNA to reduce gene expression in 786-0 cells. Our results showed that the mRNA level of hTR and the mRNA and protein levels of hTERT were decreased by siRNA treatment in a dose-dependent fashion. Moreover, the effects of hTR and hTERT siRNA in inhibiting gene expression were sequence-specific. hTR siRNA did not inhibit hTERT expression and hTERT siRNA did not inhibit hTR expression. Our results also showed that telomerase activity in human cancer cells could be inhibited by siRNA targeted at telomerase components and the inhibition was also dose-dependent. The maximum effect observed in 786-0 cells was the telomerase activity decreased to 33% of untreated cell activity for hTR siRNA and to 35% of untreated cell activity for hTERT siRNA. When the cells were treated with hTR and hTERT siRNA simultaneously, the effects of depressing telomerase activity did not exceed that observed separately in each (data not shown). Similar results in HCT-15 human colon carcinoma cells and HeLa human cervical carcinoma cells using siRNA targeting telomerase components were observed by Kosciolek et al. [25]. The study also showed that the down-regulation of telomerase activity and apoptosis were well correlated. Cells transfected with high concentration siRNA for hTR or hTERT showed low telomerase activity and high degree of apoptosis. Low concentration siRNA had minor effects on telomerase activity and apoptosis. Moreover, we found that application of siRNA for hTR and hTERT could depress the proliferation of 786-0 cells.
The catalytic activity of telomerase is detected in most cancerous tissues and highly regenerative organs, suggesting the significance of telomere maintenance in highly proliferating cells. In this regard, the inhibition of telomerase activity inevitably induces telomere shortening over a long period, and results in apoptosis in tumor cell lines or proliferating cell lines. In the present study, we observed the immediate inhibitory effects of siRNA on cell growth, which might be based on a different mechanism, beyond its roles associated with telomeres. Even though definitive experiments are lacking, the evidence emerging regarding the non-telomeric role of telomerase in various cell types is intriguing. More direct clues show that telomerase might participate in anti-apoptotic roles in targeting both mitochondrial dysfunction and caspase activation [26,27]. It is obvious that the precise mechanisms underlying the apoptosis of 786-0 cells in this study remain to be further elucidated.
Overall, our results suggest that telomerase plays an essential role in cell proliferation and viability control of human renal carcinoma cells. RNAi represents a new and powerful gene silencing approach that is currently believed to be more efficacious, selective and specific than antisense technology. Moreover, the novel role of telomerase will facilitate the development of drugs for prevention or therapy of various cancers and aging-related diseases.
Acknowledgements
We thank G. Y. ZHENG for very useful comments and Y. Y. ZONG for technical assistance.
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