Adenoviral Expression of p53 Represses Telomerase Activity through Down-Regulation of Human Telomerase Reverse Transcriptase Transcription

Cell Lines.

SiHa, ME180, TSU-PR1, and DU145 cells were obtained from the American Type Culture Collection (Manassas, VA). Ishikawa cells were kindly provided by Dr. M. Nishida (Department of Obstetrics and Gynecology, Tsukuba University, Tsukuba, Japan). These cells were grown in DMEM with 10% FCS in the presence of 5% CO₂ at 37°C.

Recombinant Adenovirus and Infection.

The recombinant p53 adenoviral vector Ad5CMV-p53 contains the cytomegalovirus promoter, wild-type p53 cDNA, and a SV40 polyadenylation signal in a minigene cassette inserted into the E1-deleted region of modified adenovirus Ad5 (27) . The adenovirus containing the β-gal gene, AdCMV-LacZ, was used to determine transduction efficiencies or was used as a control. Viral stocks were propagated in 293 cells, which were derived from primary embryonal kidney cells transformed by introducing sheared fragments of Ad5 DNA. This cell line contains E1 and is thus highly permissive of the replication of the E1 replication-deficient adenovirus. Virus was purified from this cell to obtain viral stock. Cells were harvested at various time points after infection, pelleted, resuspended in PBS, and lysed by three cycles of freezing and thawing. Cell debris was removed by subjecting the lysed cells to CsCl gradient centrifugation. Concentrated virus was dialyzed and stored at −80°C. Infection was carried out by adding the virus to high-glucose DMEM and to the cell monolayers. The cells were incubated at 37°C for 60 min with constant agitation. Medium was added, and the cells were incubated at 37°C for the desired length of time. The viral titers were determined by plaque assays.

Stretch PCR Assay.

For quantitative analysis of telomerase activity, stretch PCR assays were performed using the Telochaser system according to the manufacturer’s protocol (Toyobo, Tokyo, Japan). The PCR products were electrophoresed on a 7% polyacrylamide gel and visualized with SYBR Green I Nucleic Acid Gel Stain (FMC BioProducts, Rockland, ME). The band intensity of the telomerase ladders was analyzed by NIH Image picture analyzing software.

RNA-PCR Analysis.

Analysis of Bcl-2 mRNA expression was performed by RT-PCR amplification using the primer pair 5′-ACTTGTGGCCCAGATAGGCACCCAG-3′ (forward primer) and 5′-CGACTTCGCCGAGATGTCCAGCCAG-3′ (reverse primer) as described previously (28) . hTERT mRNA was amplified using the primer pair 5′-CGGAAGAGTGTCTGGAGCAA-3′ (LT5) and 5′-GGATGAAGCGGAGTCTGGA-3′(LT6; Ref. 17 ). hTR was amplified using the primer pair 5′-TCTAACCCTAACTGAGAAGGGCGTAG-3′ (F3b) and 5′-GTTTGCTCTAGAATGAACGGTGGAAG-3′ (R3c; Ref. 17 ). Total RNA was isolated from the tissues using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of RNA using the RNA PCR kit version 2 (TaKaRa, Otsu, Japan) with random primers. To amplify the cDNA, 2-μl aliquots of the reverse-transcribed cDNA were subjected to 28 cycles of PCR in 50 μl of 1× buffer [10 mm Tris-HCl (pH 8.3), 2.5 mm MgCl₂, and 50 mm KCl] containing 200 μm each of dATP, dCTP, dGTP, and dTTP; 2.5 units of Taq DNA polymerase (TaKaRa); and 0.2 μm of specific primers. Each cycle consisted of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 90 s. PCR products were electrophoresed in 7% polyacrylamide gel and stained with SYBR green (FMC BioProducts). The efficiency of cDNA synthesis from each sample was estimated by PCR with glyceraldehyde-3-phosphate dehydrogenase-specific primers as described previously (17) .

Plasmid Construction.

The structures of hTERT promoter-LUC plasmids are shown in Fig. 4A.⇓ Various lengths of DNA fragments upstream of the initiating ATG codon of the hTERT gene were PCR-amplified and inserted into LUC reporter vector pGL3-Basic, a promoterless and enhancerless vector (Promega, Madison, WI), in a sense orientation relative to the LUC coding sequence at the Mlu1 and BglII sites. A mutant reporter plasmid (pGL3–181Sp1 MT) containing mutations in five Sp1 binding sites at −109, −88, −56, −36, and −7 was prepared using PCR-based site-specific mutagenesis. As a positive control plasmid, pGL3-Control (Promega) was used, in which the LUC gene is driven by the SV40 LT enhancer/promoter. The names of reporter constructs with serial deletions of the 3.3 kb of hTERT promoter were assigned according to the 5′-end nucleotide numbers of inserted promoter sequences (29) . p53 expression vector (pC53-SN3) was kindly provided by Dr. Bert Vogelstein (30) .

LUC Assay.

Transfection of the plasmids was performed using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) for SiHa cells according to the protocols recommended by the manufacturer or using the calcium phosphate precipitation method for the other cell types (31) . LUC assays were performed using the Dual-Luciferase Reporter Assay System (Promega), in which Renilla LUC plasmids were cotransfected as control plasmids to standardize transcription efficiency. All experiments were performed at least three times for each plasmid, and the mean relative LUC activities were determined.

Introduction and Expression of Exogenous p53 in SiHa Cells.

We introduced the wild-type p53 gene into SiHa cells via the recombinant adenoviral vector Ad5CMV-p53. To determine the adenoviral transduction efficiency, SiHa cells were infected for 2 days with Ad5CMV-LacZ, an adenovirus that expresses the β-gal gene. The transduction efficiency was assessed by scoring 500 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside-positive cells and then determining the percentage of β-gal-positive blue-stained cells. As shown in Fig. 1A,⇓ there was a linear correlation between the number of infected cells and the number of adenovirus particles used for infection. The cells inoculated with a single dose of Ad5CMV-LacZ at a MOI of 12.5 or greater exhibited almost 100% blue-stained cells.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Adenoviral transduction and expression of p53 in SiHa cells. A, transduction efficiency of the p53 gene in SiHa cells. A recombinant β-gal adenovirus, Ad5CMV-LacZ, was used to infect the cells at different MOIs, and the percentages of β-gal-positive cells were determined after scoring 500 cells each on triplicate dishes. Bars, SD. The expression of p53 in SiHa cells infected with Ad5CMV-p53 was examined by Western blot analysis using p53-specific monoclonal antibody 24 h after infection at different MOIs (B) or at various time points after infection at a MOI of 20 (C). Levels of expression of p53 were determined relative to those of actin.

Next, to determine the expression of p53 proteins in the Ad5CMV-p53-infected SiHa cells, Western blot analyses were performed using the mouse antihuman p53 monoclonal antibody DO7. At 24 h after infection, a p53 band recognized by DO7 was observed in cellular extracts isolated from infected SiHa cells (Fig. 1B),⇓ especially at a MOI of 25 or greater. Samples isolated from noninfected SiHa cells exhibited very low levels of p53 protein. The time course of p53 expression in SiHa cells is shown in Fig. 1C.⇓ Cell extracts were prepared at various time points after infection. p53 protein expression peaked 3 days after infection and declined thereafter, but detectable expression was observed even at day 7.

Overexpression of p53 Represses Telomerase Activity with Down-Regulation of hTERT.

To examine the effects of p53 overexpression on telomerase activity, SiHa cells were infected with Ad5CMV-LacZ or Ad5CMV-p53 at various MOIs, and cell extracts were isolated 72 h after infection and examined for telomerase activity by the quantitative stretch PCR assay. As shown in Fig. 2A,⇓ infection by Ad5CMV-p53 decreased telomerase activity at a MOI of 25 and significantly eliminated it at a MOI of 50, whereas infection by Ad5CMV-LacZ had no effect on telomerase activity. Time course experiments were also performed in which repression of telomerase activity became obvious 48 h after infection, and complete loss of telomerase activity was observed 72 h after infection (Fig. 2B).⇓ In contrast, no significant change in telomerase activity was detected in cells infected with Ad5CMV-LacZ as negative controls. We extended analyses on the onset of telomerase repression, monitoring telomerase activity every 6 h, and we found that repression occurred 36 h after the infection (Fig. 2C).⇓ Expression of hTERT and hTR was next examined by RT-PCR analyses. A significant decrease in hTERT mRNA expression was observed 12 h after infection with Ad5CMV-p53, whereas expression of hTR was not altered during the time course of observation (Fig. 2D).⇓ Thus, overexpression of p53 down-regulated hTERT mRNA expression, followed by telomerase repression. A recent study reported that antiapoptotic factor Bcl-2 activates telomerase (32) . Because the expression of Bcl-2 is known to be regulated by p53 in some cell types (33) , the change in Bcl-2 expression after infection by Ad5CMV-p53 was examined by RT-PCR analyses, but no significant change in expression was detected during the time course of observation, suggesting that deregulation of Bcl-2 is not involved in telomerase repression by p53.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Repression of telomerase activity by p53. A, stretch PCR assays to quantify relative telomerase activity in SiHa cells infected with Ad5CMV-p53 or Ad5CMV-LacZ at different MOIs. IC, internal controls to verify the efficacy of PCR amplification. P, positive control samples (C33A cell extracts). N, negative control samples (lysis buffer only). B, time course of telomerase activity in SiHa cells infected with Ad5CMV-p53 or Ad5CMV-LacZ at a MOI of 50. C, time course of telomerase activity between 24 and 42 h after infection. D, expression of hTERT mRNA, hTR, and Bcl-2 mRNA as determined by RT-PCR assays at various time points after infection.

Repression of Telomerase Activity Does Not Accompany Growth Inhibition or Apoptosis of SiHa Cells.

Recent studies have demonstrated a close correlation between telomerase activity and cell proliferation (7, 8, 9, 10, 11, 12) . To examine whether repression of telomerase activity was secondary to growth inhibition induced by p53, cell growth was monitored after infection by Ad5CMV-p53. The growth rate of SiHa cells infected with Ad5CMV-p53 was comparable to that of control cells infected with Ad5CMV-LacZ, and no significant growth inhibition was observed for 42 h after infection (Fig. 3A).⇓

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Repression of telomerase activity does not accompany growth inhibition or apoptosis of SiHa cells. A, growth of SiHa cells after infection with Ad5CMV-p53 or Ad5CMV-LacZ at a MOI of 25. The cells were inoculated at densities of 2.5 × 10⁴ cells/well in 12-well plates 24 h before infection. At each time point, cells were trypsinized and counted. The mean cell counts for triplicate wells were plotted. B, flow cytometric analysis for cell cycle profiles of SiHa cells after infection with Ad5CMV-p53.

p53 is known to induce apoptosis. To examine whether apoptotic changes induced by p53 play a causative role in telomerase repression, the cell cycle profile was examined using flow cytometric analyses. Between 24 and 42 h after infection, no significant change in cell cycle profile was observed, and the sub-G₀-G₁ fraction, a characteristic marker of apoptosis, was not detected, suggesting that apoptosis was not induced during this period of observation (Fig. 3B).⇓ When cells infected with Ad5CMV-p53 were fixed and subjected to a terminal deoxynucleotidyl transferase-mediated nick end labeling assay, no increase in stained cells was detected after infection (data not shown). Taken together, these findings indicate that overexpression of p53 in SiHa cells represses telomerase without altering cellular growth properties or inducing apoptosis.

p53 Represses Transcriptional Activity of hTERT Promoter.

The finding that p53 down-regulates hTERT mRNA suggests that hTERT expression is controlled by p53 at the transcriptional level. In a previous study, we cloned 3.3 kb of the 5′-flanking sequences of the hTERT gene (29) . A computer-asisted homology search revealed two potential p53 binding motifs at −1877 (AGGCTGGTCT) and− 1240 (AGGCCTGTTC) in the 5′-flanking sequences (Fig. 4A).⇓ To examine whether p53 regulates the promoter activity of hTERT, LUC assays were performed using SiHa cells in which p53 expression vectors were cotransfected with 3.3 kb hTERT-promoter LUC reporters (pGL3–3328). As a result, overexpression of p53 significantly repressed the promoter activity of hTERT (Fig. 4B).⇓ Similar inhibition of telomerase activity was observed for other types of cells, such as ME180 and Ishikawa cells (derived from uterine cancers) as well as DU145 and TSU-PR1 cells (derived from prostate cancers).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Repression of the transcriptional activity of hTERT by p53. A, LUC reporter plasmids used in the assays. The 181-bp core promoter region essential for transcriptional activation is shown in the black box in which binding sites for Myc/Max and Sp1 are shown. The transcription start site and the ATG initiation codon are shown as +1 and +78, respectively. Putative p53 consensus binding motifs are shown as blank boxes. Mutations introduced into Sp1 binding sites in pGL3–181Sp1 MT are shown as crossed-out boxes. B, transcriptional activity of the 3.3-kb promoter was examined by LUC assays using a variety of cell lines. p53 expression vectors (pC53-SN3) or blank vectors were cotransfected with pGL3–3328 reporter plasmids into various types of cells, and LUC assays were performed. Relative LUC activities, which were determined by comparison with control activities in samples transfected with blank vectors, are shown. Bars, SD. C, identification of the elements responsible for p53-mediated transcriptional repression. p53 expression vectors or blank vectors were cotransfected with each deletion or substitution mutant reporter plasmid, and LUC assays were performed. Bars, SD.

The Proximal Core Promoter of the hTERT Gene that Binds Cellular Factor Sp1 Is Responsible for p53-mediated Transcriptional Repression.

To identify the elements responsible for the down-regulation of hTERT, serial deletion mutants of reporter plasmids were prepared (Fig. 4A),⇓ and LUC assays were performed. p53 overexpression repressed transcriptional activity not only in wild-type pGL3–3328 but also in its deletion mutants (Fig. 4C).⇓ Interestingly, transcriptional activity in pGL3–1375 and pGL3–1175, which lack one or two potential p53 binding motifs, respectively, was also repressed by p53 overexpression. Repression was further observed in mutants with more extended deletions, and the proximal 181-bp region (pGL3–181) could respond to p53 overexpression. Our previous study demonstrated that this region functions as a core promoter essential for transactivation of hTERT in cancer cells (29) . This core promoter contains five GC-boxes and an E-box that bind transcription factor Sp1 and Myc/Max, respectively (Ref. 29 ; Fig. 4A).⇓ Myc proteins have been found to be critical factors for activation of hTERT transcription (34 , 35) . However, deletion of the E-box (pGL3–150) did not affect the repressive effect of p53, indicating that the remaining 150-bp region containing five GC-boxes was responsible for this repression. Interestingly, even the proximal 32-bp region (pGL3–32) containing one Sp1 site responded to p53 overexpression. In contrast, p53 overexpression failed to repress transcription of mutant p-181 (pGL3–181Sp1 MT), in which all Sp1 sites had been eliminated by substitution mutations.