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Antisense hTERT Inhibits Thyroid Cancer Cell Growth

Department of Surgery (L.T., M.C.S., C.B.B., T.J.F.), New York Presbyterian Hospital, and Weill Medical College of Cornell University; and Strang Cancer Prevention Center (T.J.F.), New York, New York 10021

Address all correspondence and requests for reprints to: Thomas J. Fahey III, M.D., New York Presbyterian Hospital-Cornell University, Room F-2024, 525 East 68th Street, New York, New York 10021. E-mail: tjfahey{at}mail.med.cornell.edu.

	Abstract

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Activation of telomerase represents an early step in carcinogenesis. Increased telomerase expression in malignant thyroid tumors suggests that inactivation of telomerase may represent a potential chemotherapeutic target. The purpose of this study was to inhibit the protein component of telomerase, hTERT, in a human thyroid cancer cell line in vitro and in vivo using an antisense strategy. A 235-bp fragment of hTERT cDNA was subcloned, and sense and antisense hTERT expression vectors were constructed. These vectors were transfected into a human thyroid carcinoma cell line (FRO). Tumorigenic potential was determined by cellular growth assay, rate of apoptosis, anchorage-independent growth, and tumor growth in a nude mouse model. Significant down-regulation of hTERT expression was seen in the antisense transfected cells, compared with control and those transfected with the sense vector. A decrease in telomerase activity by TRAP assay was observed in the antisense hTERT cells but not in cells transfected with the sense hTERT construct. Inhibition of cell growth was observed after approximately 20 population doublings in the antisense-hTERT clones and was associated with an increase in the rate of apoptosis and a change in cellular morphology. Moreover, anchorage-independent growth was reduced in vitro, and tumor growth rate was diminished in vivo in the antisense hTERT clones. Inhibition of telomerase activity with antisense hTERT in human thyroid cancer cells is achievable and may represent a novel target to inhibit tumor growth.

	Introduction

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TELOMERES ARE THE noncoding termini of eukaryotic chromosomes. The repeated sequence of TTAGGG serves to stabilize the chromosome for replication through cell division. However, at each cell division telomeres are lost, and at a critical point of erosion of the telomeres, cell cycle arrest and cellular senescence occurs (1).

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. The human enzyme is composed of a constitutively expressed RNA subunit (hTR) and a catalytic protein subunit (hTERT). The hTR component contains a region that is complementary to telomeric DNA and serves as template for de novo addition of deoxynucleotides to chromosome termini. The protein subunit hTERT is a reverse transcriptase, and hTERT expression is the rate-limiting component of the telomerase complex and therefore determines telomerase activity (2).

Normal human somatic cells do not have detectable telomerase activity and lack expression of hTERT. Germ line and immortalized and malignant cells frequently have detectable telomerase activity and express hTERT. In fact, introduction of hTERT into normal cells leads to reactivation of telomerase activity and cellular immortality. Expression of hTERT in conjunction with the simian virus 40 large T antigen and oncogenic ras is sufficient to convert normal human cells into transformed, tumorigenic cells (3). These observations indicate that telomerase activity is essential for the continued growth of malignant cells, rather than being a secondary marker of the transformed state.

Telomerase activity has been demonstrated to be up-regulated in malignant thyroid tissue (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Because telomerase is frequently expressed in malignant tissues but uncommonly active in normal or benign tissues, it is attractive as a potential target for anticancer therapy. Inhibition of telomerase theoretically occurs by interfering with either the hTR or hTERT component of the enzymatic complex. Strategies for inhibiting the hTR subunit of telomerase using antisense RNA or peptide nucleic acids have been confirmed as one method for telomerase inhibition in vitro but have not been active in vivo (18, 19, 20). Here we report an antisense strategy, whereby an antisense hTERT vector targeting the protein component of human telomerase was constructed and used to inhibit telomerase activity and thyroid cancer cell growth.

	Materials and Methods

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Materials

RPMI 1640, DMEM, fetal bovine serum (FBS), penicillin, streptomycin, pcDNA3.1(±) mammalian expression vector, and PerFect lipid transfection kit were from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Agarose medium and geneticin (G418) were from Sigma (St. Louis, MO). The GeneAmp RNA PCR kit and random hexamers were from Perkin-Elmer (Norwalk, CT). Restriction enzymes and T7 were from Promega Corp. (Madison, WI). The TRAP-eze telomerase detection kit was from Oncor, Inc. (Gaithersburg, MD). The dimethylthiazoldiphenyltetra-zoliumbromide (MTT) cellular proliferation kit was from Roche Molecular Biochemicals (Indianapolis, IN). RNAzol was from Biotecx Laboratories, Inc. (Houston, TX). The BCA protein assay kit was from Pierce Chemical Co. (Rockford, IL). TaqStart antibody was from CLONTECH Laboratories, Inc. (Palo Alto, CA). The RapidPrep genomic DNA isolation kit was from Pharmacia-Biotech, Inc. (Uppsala, Sweden). Telomere probe, Teloprobe, was obtained from PharMingen (San Diego, CA). The ApopTag kit was from Intergen (Burlington, MA).

Cell culture

The poorly differentiated human thyroid carcinoma cell line FRO (kindly provided by Dr. G. Juillard, UCLA School of Medicine, Los Angeles, CA) was maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml Amphotericin B under an atmosphere of 95% air 5% CO₂ at 37 C. After transfection the cells were cultured in RPMI 1640 containing 10% FBS and 500 µg/ml geneticin.

Construction of hTERT vectors

The human hTERT cDNA was initially isolated from human normal tissue by RT-PCR. A 235-bp fragment of hTERT cDNA covering the sequence of motif 3 of hTERT was amplified using the GeneAmp RNA PCR kit. The primers used for hTERT were: 5'-gccgaattctgccgttgcccaagagg-3' (sense) and 5'-gcgtggatcccaagcagctccaga-3' (antisense). Amplified DNA fragments were cloned into SK(-) vector and sequenced to verify the correct coding sequence. This vector was subjected to restriction digest with BamH I-Hind III. A resulting 265-bp hTERT cDNA fragment was recovered and purified and then subcloned into the multicloning site of pcDNA3.1(±) mammalian expression vector under the control of the cytomegalovirus promoter. The hTERT cDNA fragment was cloned in the 5'-3' (sense) or 3'-5' (antisense) orientation. Confirmation of the correct insert was determined by sequencing using T7 primer.

Transfection

FRO cells were grown to 50–60% confluence. Approximately 5 x 10⁴ cells were transfected in serum-free media for 4 h using the PerFect lipid transfection kit at a ratio of 24 µg lipid solution per 4 µg plasmid DNA. Twenty-four hours after transfection, G418 was added to the medium to a final concentration of 500 µg/ml. G418-resistant colonies were expanded, and the resulting stable clones were screened for the presence of hTERT insert by PCR with T7 and BGH reverse primers.

hTERT expression by RT-PCR

Total RNA was isolated from transfectants and parental control cells using RNAzol. Total RNA (500 ng) from each transfectant was reverse transcribed with random hexamers followed by PCR using two sets of primers, hTERT primers same as mentioned before; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, 5'-TGGTATCGTGGAAGGACTCATGAC-3' (sense) and 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3' (antisense). The PCR cycling conditions were 94 C for 30 sec, 60 C for 30 sec, and 72 C for 1 min for 30 cycles. The amplified products were electrophoresed on a 2% agarose gel and visualized with ethidium bromide.

Telomerase detection assay

The TRAP-eze telomerase detection kit (Oncor, Inc.) was used according to the manufacturer’s protocol with a minor modification. Briefly, cultured cells were harvested and washed with PBS and then homogenized in 200 µl 1x CHAPS lysis buffer on ice for 30 min and centrifuged for 20 min at 12,000 at 4 C. The supernatant liquid was collected and protein concentration was measured by the BCA protein assay. A set of diluted protein aliquots were tested for each sample in the TRAP assay to establish the linearity of the assays as well as assayed after digestion with RNase as a specificity control. To optimize the TRAP assay TaqStart antibody (CLONTECH Laboratories, Inc.) was added, and the telomerase reaction was carried out at 30 C for 30 min, followed by a two-step PCR amplification (94 C, 30 sec and 60 C, 30 sec for 33 cycles). The amplified products were electrophoresed on a 12.5% nondenaturing polyacrylamide gel. The gel was stained with ethidium bromide and analyzed. Enzymatic activity was quantified from all assays within the linear range by normalizing the total amount of reaction products in each lane to the signal obtained from the internal telomerase assay standard present in the same lane, as described in the kit protocol. Levels of telomerase activity were obtained from a minimum of three assays of two independent prepared extracts from each population.

Telomere length estimation

Genomic DNA was isolated from antisense hTERT-transfected cells and controls using the RapidPrep genomic DNA isolation kit (Pharmacia-Biotech, Inc.) and subsequently digested with Ras I and Hinf I. The DNA digests were electrophoresed on a 0.8% agarose gel and transferred onto a nylon membrane by capillary method. The blot was probed with a biotinylated telomere probe and detected by a chemiluminescent reaction. The mean terminal restriction fragment length was calculated by comparison to known standards.

Cell growth property assay

The G418-resistant cells were grown in G418 medium. Seven-day growth curves of G418-resistant cells harboring antisense hTERT were determined by MTT cellular proliferation assay and compared with sense hTERT cells and parental FRO cells. For the soft agar colony formation assay, cells from different clones at passage 30 were grown to subconfluence, detached using trypsin, and counted using a hemacytometer. Approximately 1 x 10³ cells of each clone were seeded into 0.4% agarose medium, supported on a 0.65% agarose medium, and allowed to grow for 3 wk. The colonies were counted by light microscopy.

Apoptosis analysis

To analyze apoptosis, the ApopTag kit (Intergen) was used according to the manufacturer’s protocol. Briefly, hTERT transformed cells and controls were grown in parallel under similar conditions. At subconfluence they were trypsinized and fixed with paraformaldehyde. They were exposed to TdT-Enzyme, digoxigenin, and fluorescein, respectively (provided in the ApopTag kit). The presence of apoptotic cells was quantified by flow cytometry.

Tumorigenicity in nude mice

All animal studies were conducted with approval from the Institutional Animal Care and Use Committee at Weill Medical College of Cornell University and in accordance with accepted standards of human animal care as outlined in the NIH Guide for the care and use of laboratory animals. The hTERT antisense transfectants and controls growing in log phase under identical conditions were trypsinized and harvested. Approximately 10⁶ cells in 0.2 ml PBS were injected sc into each flank of balb/c (/) athymic mice (n = 6). For each mouse, the hTERT antisense transfectants were injected on the left flank and the hTERT sense control or FRO parental cells were injected on the right flank. After 4 wk, these mice were killed and the tumors were harvested and weighed. Tumor tissue was screened for the presence of hTERT insert by PCR, hTERT expression by RT-PCR, and telomerase activity by TRAP assay, as described above.

	Results

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Generation and characterization of modified FRO cell lines. To inhibit endogenous hTERT expression, we constructed a permanent antisense hTERT expression vector to target the highly conserved third reverse transcription motif of hTERT. Plasmids containing sense and antisense hTERT were transfected into FRO cells. No differences in hTERT expression were observed between parental FRO cells and sense hTERT cells by RT-PCR. Three of the 12 clones (hTERT-AS-A, B, and E) had a reduction in the ratio of hTERT expression to GAPDH with respect to parental FRO cells and sense clone (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Figure 1. Expression of hTERT in thyroid cancer cell lines. Representative results of RT-PCR for hTERT expression (top) and GAPDH (bottom) serves as the internal control. Lanes 1–5, hTERT antisense clones; lane 6, hTERT sense clone; lane 7, FRO parental cells.

Telomerase activity was determined in the stable antisense-expressing clones by TRAP assay (Fig. 2). Down-regulation of hTERT in these clones correlated with a decrease telomerase activity by TRAP. Compared with parental FRO cells, telomerase activities in these three transfectants were reduced by approximately 10-fold.

View larger version (68K): [in this window] [in a new window]   Figure 2. The mean terminal restriction fragment by Southern blot analysis for FRO parental cells, hTERT-sense clone, and hTERT-antisense clone.

View larger version (86K): [in this window] [in a new window]   Figure 3. Telomerase activity by TRAP assay for FRO parental cells and hTERT antisense FRO clones. Telomerase activity is determined by the intensity and number of telomeric repeats per reaction. For each cell line, lane 1 is the heat-inactivated negative control, followed by three lanes of serial dilutions of extracts from each cell line. Internal telomerase assay standard is the 36-bp internal control.

Cellular proliferation was determined in the three antisense-expressing clones, the sense clones, and control cell line. A 7-d cell growth curve was performed by MTT assay at passages 5 and 20. At passage 5 there was no difference in growth curves among the three hTERT antisense clones, hTERT sense clone, and control cells (data not shown). After 20 passages, however, the proliferation rates in the hTERT antisense clones were significantly decreased, compared with hTERT sense clones and parental FRO cells (Fig. 4). The morphology of the hTERT antisense transfectant cells was altered after passage 20 (Fig. 5). In addition, the hTERT antisense cells detached more readily upon trypsinization, and the cell borders were more distinct, compared with parental FRO cells. No morphological changes were observed in the hTERT sense clones, compared with parental FRO cells.

View larger version (15K): [in this window] [in a new window]   Figure 4. Seven-day MTT assay of thyroid cancer cell lines; antisense clones (hTERT-AS), sense clone (hTERT-S), and parental cell line (FRO) at passage 20. Statistically significant growth reduction in hTERT-AS, compared with hTERT-S at d 5–7 (P < 0.05).

View larger version (153K): [in this window] [in a new window]   Figure 5. Photomicrographs of FRO cells growing in culture. A, FRO parental cells. Cells appear uniform and cohesive. B, FRO antisense clones (hTERT-AS) at passage 5. Cells are starting to demonstrate some disorganization and evidence of decreased cell viability. C, FRO hTERT-AS at passage 20. Cells are discohesive, and there is evidence for cell death. D, FRO hTERT-AS at passage 30+. Although there is still widespread apoptosis, pockets of cells that appear to have reverted toward the parental FRO phenotype have started to reappear.

To clarify the mechanisms contributing to the decrease in cell growth rate and the altered cellular morphology, we analyzed the apoptotic rate of the hTERT antisense clones, compared with the sense and parental controls. There was a significant increase in the rate of apoptosis in the three hTERT antisense clones after 20–25 passages, compared with both hTERT-sense and FRO control cells (Fig. 6).

View larger version (35K): [in this window] [in a new window]   Figure 6. Percentage apoptosis of FRO parental cells, hTERT-sense clone, and hTERT-antisense clones. At passage 20 there was a significant increase in the percentage of apoptotic cells in the hTERT-antisense clone, compared with hTERT-sense clone (P < 0.05).

To examine the effect of down-regulation of hTERT expression and telomerase activity on the tumorigenicity of FRO cells, soft agar colony formation and tumor growth in nude mice were assessed at passage 30. Both the number of colonies and the size of the colonies were significantly reduced in the antisense hTERT clones, compared with the hTERT sense clones and FRO parental cells (Fig. 7). Moreover, as shown in Fig. 8, the tumor size in balb/c athymic mice in tumor implants from hTERT antisense cells was reduced, compared with tumor implants from hTERT sense cells.

View larger version (18K): [in this window] [in a new window]   Figure 7. Anchorage-independent growth by soft agar colony formation. There was a significant reduction in number of colonies formed by hTERT-antisense clone, compared with hTERT-sense clone (P < 0.05).

View larger version (13K): [in this window] [in a new window]   Figure 8. In vivo tumor implants in a nude mouse model. There was a significant reduction in tumor weight in tumor implants with hTERT-antisense clone, compared with hTERT-sense clone (P < 0.05).

	Discussion

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A number of studies have documented that telomerase inhibition may diminish cancer cell growth in vivo and in vitro. Recently Hahn et al. (21) and Zhang et al. (22) independently reported that inhibition of the catalytic subunit of human telomerase hTERT by a dominant negative construct resulted in complete inhibition of telomerase activity, reduction in telomere length, and death of tumor cells. Moreover, Hahn et al. demonstrated that expression of a mutant telomerase eliminated in vivo tumorigenicity in an immunodeficient murine model.

Reverse transcriptase inhibitors have also been used to inhibit the function of hTERT. The nucleotide analog AZT is preferentially incorporated into DNA and leads to chain termination. Melana et al. (23) demonstrated decreased telomerase activity and decreased telomere length in breast cancer cells after treatment with AZT. A tumor implant model in a murine model also demonstrated decreased tumorigenicity in a cancer cell line treated with AZT (24).

Antisense treatment of hTERT is not without limitations. First, there is a lag phase before demonstration of tumoricidal effects because the biological effect of decreased telomerase activity, progressive erosion of telomeres, requires several cell cycles to become manifest. In our antisense hTERT-transfected cells, it took approximately 20 passages to see differential in vitro growth as compared with parental controls. Resistance may represent a second limitation in antitelomerase therapy. After more than 30 passages, cell lines of hTERT-antisense clones were noted to have isolated subpopulations of cells that regressed to the FRO parental cellular morphology. These cells may represent subpopulations that regain hTERT expression and telomerase activity and may have an alternative mechanism for telomere preservation. Although the majority of antisense hTERT cells remain TRAP negative, analysis of select subpopulations of "escaped cells" revealed TRAP activity similar to parental FRO activity (data not shown). Finally, telomerase is not only expressed in malignant cells but in germ line cells and some types of normal cells such as hematopoietic progenitor cells, intestinal crypt cells, and other cells of highly regenerative capacity. Growth inhibition and potential destruction of these cells is undesirable for obvious reasons.

Several possibilities exist as to why stably transformed FRO antisense hTERT cells resulted in only partial growth inhibition. The antisense technique does not allow for the complete blockade of expression of target genes; previous studies indicate that the range of inhibition is 50% to approximately 99%. Minimal residual telomerase activity, resulting from the incomplete inhibition of hTERT, may be sufficient to maintain telomere length in these tumor cells. Therefore, it is possible that antitelomerase strategies may be best administered in combination with other chemotherapeutic agents. Our observations in vitro demonstrated that some hTERT antisense cells escape from senescence and revert to FRO parental cells on the basis of morphological and biological characteristics (Fig. 4). It is possible that some hTERT antisense cells escape from senescence by loss of the antisense hTERT insert, resulting in recovery of hTERT expression and telomerase activity. Analysis of tumor tissue recovered from tumors in nude mice indicated that the hTERT antisense construct could still be recovered, even though telomerase activity could still be detected in these cells. This suggests that one mechanism of escape from the antisense strategy may be to overwhelm the antisense message with native telomerase mRNA. Additionally, a telomerase-independent mechanism such as recombination or circularization of chromosomes may be partially responsible for maintenance of cell growth.

Activation of telomerase and subsequent telomere stabilization are important and necessary steps in tumorigenesis. Inhibition of hTERT in FRO cells using an antisense strategy resulted in decreased telomerase activity, loss of telomere length, diminished cellular proliferation, and decrease in anchorage-independent growth in vitro. A xenograft murine model of human thyroid carcinoma demonstrated in vivo growth inhibition of hTERT antisense tumors. Taken together, our data indicate that activation of telomerase is important for the maintenance of the malignant growth phenotype of FRO cells and strategies to inhibit the protein component of telomerase may offer a novel mechanism to treat thyroid cancer.

	Footnotes

 
Abbreviations: FBS, Fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, dimethylthiazoldiphenyltetra-zoliumbromide.

Received August 5, 2002.

Accepted December 11, 2002.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Teng, L. Articles by Fahey, T. J. Search for Related Content PubMed PubMed Citation Articles by Teng, L. Articles by Fahey, T. J., III