Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 500-506
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.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 ?C
for 20 s, 50 ?C for 20 s, 72 ?C for 30 s,
25 cycles. The primers for hTERT were 5‘-GCCAGAACGTTCCGCAGAGAAAA-3‘
and 5‘-AATCATCCACCAAACGCAGGAGC-3‘. Reaction parameters were 94
?C for 20 s, 48 ?C for 30 s, 72 ?C 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 ?C 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 ?C for 2 min. The slides were
incubated with TUNEL reaction mixture for 1 h at 37 ?C. After washing with
phosphate-buffered saline, the slides were incubated with
peroxidase-conjugated streptavidin for 30 min at 37 ?C 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 ?C 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 (n?6). 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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