GROWTH INHIBITION OF BEL-7404 HUMAN HEPATOMA CELLS BY EXPRESSION OF MUTANT TELOMERASE REVERSE TRANSCRIPTASE

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1 Int. J. Cancer: 97, (2002) 2002 Wiley-Liss, Inc. Publication of the International Union Against Cancer GROWTH INHIBITION OF BEL-7404 HUMAN HEPATOMA CELLS BY EXPRESSION OF MUTANT TELOMERASE REVERSE TRANSCRIPTASE Rugang ZHANG, Xingwang WANG, Lixia GUO and Hong XIE* Department of Biotherapy, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People s Republic of China Human hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia and Africa. Human telomerasereversetranscriptase(htert)isexpressedinhcc but absent in normal human liver cells, which is consistent with the expression pattern of telomerase. In the present study, expression of a dominant-negative form of htert (DN-hTERT) resulted in inhibition of telomerase activity and decreased mean telomeric length of BEL-7404 human hepatoma cells, whereas expression of wild-type htert (WThTERT) and control vector had no such effects. Cell growth was inhibited by this mutant (DN-hTERT), which was consistent with the changes in telomerase level. Flattened large cells were found in late generations with the DN-hTERT treatment. When mean telomeric length of DN-hTERT transfected cells reached a critical length (about 1.7 kb), apoptosis was induced. Tumorigenicity of DN-hTERT expressing cells was eliminated in vivo. These data indicated that htert was essential for the growth of hepatoma cells. htert can also be used as an important target for anti-hcc drug screening Wiley-Liss, Inc. Key words: hepatocellular carcinoma; telomerase; DN-hTERT; cell growth; tumorigenicity The telomere is aspecialized DNA protein complex located at the eukaryotic chromosome terminus, which consists of highly conserved, repeated hexanucleotide (TTAGGG) n. 1,2 The telomere is proposed to be responsible for chromosomal stabilization by protecting the chromosome from enzymatic end degradation and preventing its fusion with other chromosomes. In every celldivision cycle, telomeres continually shorten by about nucleotides, and when they reach acritical length, the cell stops dividing. This means that maintaining the telomere at aconstant length is necessary for cell immortalization and tumor cell division. 3 Telomerase is aspecialized reverse transcriptase that adds telomeric repeats to telomeres, counteracting the progressive loss of DNA that occurs during replication and maintaining karyotypic stability. Because telomerase is present in 85 90% of cancer specimens but absent in most normal tissues, it is thought to be a good candidate for targeted therapy of cancer. 4,5 Human telomerase is aribonucleoprotein complex composed of an RNA component (htr) that contains the RNA template, acatalytic reversetranscriptase subunit (htert) and an associated protein subunit (TEP1). 6 8 HCC is one of the most frequent fatal malignancies in Asia and Africa. 9 InChina, it is the second major cause of cancer death in males and the third in females. 10,11 Unfortunately, no great progressinitspreventionandtreatmenthasbeenmade.ithasbeen reported that HCC exhibits ahigh incidence of telomerase activity andthattelomeraseactivityincreasesinaccordancewithdegreeof histologic undifferentiation of HCC but is absent in normal liver tissue. 12,13 Other reports have revealed that the htert expression level is the rate-limiting determinant of HCC telomerase activity Asaresult, htert is an excellent target for HCC gene therapy. In the present study, we expressed DN-hTERT; mutant htert, which was created by substituting the aspartic acid and valine residues at positions 710 and 711 in the third RT motif of htert with alanine and isoleucine, 17,18 respectively; and WT-hTERT in BEL-7404 human hepatoma cells, to assess the effects of telomerase modulation on malignant behavior. MATERIAL AND METHODS Cell and culture condition TheBEL-7404humanhepatomacellline,fromtheCellBankof the Chinese Academy of Sciences, 19 was cultured in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated newborn calf serum, at 37 C in ahumidified CO 2 incubator containing 5% CO 2 and 95% air. Assessment of cell proliferation Cells were seeded at /well in a96-well plate. The cell numberwasassessedevery12hrovera96hrperiodbythetrypan blue exclusion method using ahemocytometer. 20 Toavoid bias, counting was done blindly for each sample by 2 individuals. Results are means SE from triplicate wells. Stable transfection DN-hTERT and WT-hTERT genes were obtained from Dr. R.A. Weinberg (Massachusetts Institute of Technology, Cambridge, MA) and subcloned into the EcoRI/SalI site of the eukaryotic stable expression pcmv-script Vector (Stratagene, La Jolla, CA). Final constructs were stably transfected into BEL-7404 human hepatoma cells using lipofectamine reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer s instructions. Stably transfected cells were selected after 2 weeks with 400 g/ml G-418 (Life Technologies). RT-PCR Total cellular RNA was extracted from cells using Trizol (Life Technologies)accordingtotheinstructionsofthemanufacturer.In each reaction, 1 g of total RNA was reverse-transcribed into cdna using M-Mlv reverse transcriptase (Promega, Madison, WI). Vector-encoded htert and -actin were amplified for 30 and 24 cycles, respectively (94 C, 45 sec; 60 C, 45 sec; 72 C, 90 sec).primersequencesforvector-encodedhtert,givingrisetoa 264 bp fragment, were 5 -GACTCGACACCGTGTCACCTAC-3 and 5 -GTAATACGACTCACTCACTATAGGGC-3 ; 21 primer Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1- propane sulfonic acid; CMV, cytomegalovirus; DIG, digoxigenin; HCC, hepatocellularcarcinoma;htert,humantelomerasereversetranscriptase; DN-hTERT, dominant negative form of htert; PD, population doubling; PI, propidium iodide; TRAP, telomeric repeat amplification protocol; TUNEL, TdT-mediated dutp nick end labeling; WT-hTERT, wild-type htert. *Correspondence to: Department of Biotherapy, Institute of Biochemistry and Cell Biology (Cell Building), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai , PRC. Fax: xiehong@sunm.shcnc.ac.cn Received 16 February 2001; Revised 6June, 27 July 2001; Accepted 6 August 2001

2 174 ZHANG ET AL. FIGURE 1 Expression of transfected WT-hTERT and DN-hTERT (Ectopic htert). (a) Constructs used to express WT-hTERT and DN-hTERT. Arrows, primers used for RT-PCR. One primer was designed on the ectopic htert gene behind the mutation sites; the other primer was designed on the vector ahead of the polyadenylation signal site. Motifs labeled as described; motif 3 has also been called motif A. T, region conserved among the TERTs but not among other reverse transcriptases. 8 (b) Expression pattern of ectopic htert. V, vector control; WT2 and WT8, 2 clones expressing WT-hTERT; DN1, DN3, DN4 and DN8, 4 clones expressing DN-hTERT. -Actin was used as standard. FIGURE 3 Effects of DN-hTERT transfection on telomeric length of BEL-7404 human hepatoma cells. Mr, marker; P, parental cells; V, vector control (PD 10). (a) Telomeric length of WT3 (WT-hTERT expression clone 3, PD 10); DN1 and DN3, DN-hTERT expression clone 1 (PD 10) and clone 3 (PD 7). (b) Telomeric length of WThTERT expression clone 2 and DN-hTERT expression clone 4 at different PDs. FIGURE 2 Effects of WT-hTERT and DN-hTERT on telomerase activity of BEL-7404 human hepatoma cells. C, lysis buffer control; P, parental cells; V, vector control; WT2, WT3 and WT8, 3 clones expressing WT-hTERT; DN1, DN3, DN4 and DN8, 4 clones expressing DN-hTERT. sequences for -actin (539 bp) were 5 -GTGGGGCGCCCCAG- GCACCA-3 and 5 -GTCCTTAATGTCACGCACGATTTC-3. Telomerase assay Telomerase activity was assayed using a PCR-based TRAP assay, as previously described. 22,23 Briefly, cells were washed with PBS, lysed in 1 CHAPS buffer, incubated on ice for 30 min and centrifuged at 12,000g for 30 min. Protein concentration was determined by the Coomassie protein assay. Each TRAP reaction contained 1 g of total protein. The reaction mixture was incubated at 30 C for 30 min, heated at 94 C for 5 min and subjected to 28 PCR cycles: 94 C for 30 sec and 60 C for 30 sec. PCR products were separated by electrophoresis on 12% nondenaturing polyacrylamide gels and stained with SYBR green I (FMC Bioproducts, Rockland, ME) for 15 min and visualized by the UVP system. In every experiment, a negative control (1 l CHAPS lysis buffer) was included. All experiments were repeated at least twice. FIGURE 4 Cell growth curves of DN-hTERT expression clones, WT-hTERT expression clones and vector control clone. Values are means SE for triplicate wells, as described in Material and Methods. All clones used for tests were at PD 6, except DN-hTERT expression clone 3, used at PD 5. Telomeric length assay Telomeric length was detected by the TeloTAGGG telomeric length assay (Roche, Nutley, NJ) according to the manufacturer s protocol. Briefly, genomic DNA samples were prepared as described. 24 For each sample, 1 g of genomic DNA was digested with RsaI/HinfI, separated on a 0.8% agarose gel, transferred to a nylon membrane (Hybond-N ; Amersham, Arlington Heights, IL) and hybridized with a telomere-specific, DIG-labeled probe, which was incubated with anti-dig-alkaline phosphatase and detected by chemiluminescence. Apoptosis analysis by flow cytometry Cells were harvested and resuspended in solution containing 40 mm sodium citrate, 250 mm sucrose and 5% DMSO. The suspension was stored at 20 C for 20 min, then thawed rapidly at room temperature and centrifuged to collect the cells. Cells were resuspended in a solution containing RNase A ( units/g, 50

3 HEPATOMA GROWTH INHIBITION BY DN-HTERT 175 FIGURE 5 Morphologic changes induced by DN-hTERT expression. Compared with (a) cells expressing control vector, (b) large flat cells in crisis constituted the later generation of DN-hTERT expression clones, (c) whereas WT-hTERT expression clones had normal cell morphology. mg/l) and 20 mg/l PI. The percentage of apoptotic cells was determined by the fluorescence of individual cells, measured by flow cytometry. 25 FIGURE 6 Apoptotic nuclear morphologic changes induced by DNhTERT expression. (a) Ultrastructures of DN-hTERT expressing BEL-7404 human hepatoma cells showed apoptotic nuclear characteristics, (b) whereas the nuclei of vector control and (c) WT-hTERT expressing cells were normal in shape. Scale bar 1 m.

4 176 ZHANG ET AL. FIGURE 7 Flow-cytometric analysis of apoptotic (sub-g 1 ) cells in later generation of DN-hTERT expression clone 3 (PD 7), WT-hTERT expression clone 3 (PD 10) and control vector clone (PD 10). TUNEL assay TUNEL assay was done using an in situ cell death detection kit (Roche). Briefly, cells were grown on coverslips and fixed for 1 hr at room temperature in 4% paraformaldehyde, incubated with blocking solution (3% H 2 O 2 in methanol) for 10 min at room temperature and permeabilized for 2 min with 0.1% Triton X-100 in 0.1% sodium citrate on ice. Then, cells were added to 50 l TUNEL reaction mixture, incubated in a humidified chamber for 1 hr at 37 C and analyzed under a fluorescence microscope. Negative control was established using 50 l of label solution (without terminal transferase) instead of the TUNEL reaction mixture, and positive control was established by treating fixed and permeabilized cells with 5 g/ml DNase I for 10 min at room temperature to induce DNA strand breaks. Microscopic observation Cells were fixed with 25 g/l glutaraldehyde and postfixed with 20 g/l osmium tetroxide. After dehydration, samples were embedded in Epon 812 epoxy resin and then sectioned. Sections were routinely stained. The apoptotic morphology of cells was examined under electron microscopes. 26 Tumorigenicity assays Immunodeficient Balb/c nude mice were maintained in pathogen-free conditions. Cells ( ) were injected s.c. into mice and observed for tumor formation. 21 Tumorigenicity was evaluated using 5 animals in each group. Five weeks after cell inoculation, animals were killed using isofluorane-containing chambers. All procedures involving animals and their care were in accordance with institutional ethical guidelines and national and international laws and policies. RESULTS Effect of DN-hTERT on telomerase activity We introduced DN-hTERT, WT-hTERT or a control vector with a drug-resistance marker into BEL-7404 human hepatoma cells. After transfection, cells were selected in G-418, grown to confluence and cloned by limiting dilution and ring cloning. PD 0 was defined after this point. The process of obtaining clonal isolates required approximately 25 PDs before the designation of PD 0. After detection of telomerase activity of transfected cells every PD until PD 5, 3 WT-hTERT expression clones (2, 3 and 8), 4 DN-hTERT expression clones (1, 3, 4 and 8) and 1 control vector expression clone were selected for the following experiments. All selected clones were stable during the selection process. Expression of transfected DN- and WT-hTERT was confirmed by RT- PCR with primers specific for the transfected genes (Fig. 1). Expression of DN-hTERT inhibited telomerase activity of BEL human hepatoma cells to different levels, consistent with the transfected DN-hTERT expression levels, while expression of WT-hTERT slightly increased its activity (Fig. 2). Effect of DN-hTERT on telomeric length The mean telomeric length of cells transfected with WT-hTERT or vector was stable during PD compared with parental cells. In contrast, cells expressing DN-hTERT showed decreased telomeres (Fig. 3). The speed of telomeric loss was consistent with the levels of telomerase inhibition. The rate of telomeric loss of telomerasenegative DN-hTERT expression clone 3 was about 70 to 80 bp/pd. Effect of DN-hTERT on cell proliferation The growth properties of cells transfected with DN-hTERT, WT-hTERT and control vector were characterized at PD 6, except for DN-hTERT expression clone 3, which was characterized at PD 5. DN-hTERT expression clones showed slow cell growth, consistent with the inhibitory levels of telomerase activity. When the parental BEL-7404 human hepatoma cells received sufficient DNhTERT to inhibit telomerase activity completely, cells grew very slowly and eventually stopped proliferating. This indicated that cell proliferation correlated with telomerase activity directly. In contrast, the growth kinetics of cells transfected with WT-hTERT did not differ markedly from cells transfected with control vector only (Fig. 4). Effect of DN-hTERT on cell apoptosis When the mean telomeric length of cells transfected with DNhTERT reached a critical point (about 1.7 kb) in their late passages, they stopped proliferating and showed the morphologic characteristics associated with crisis. Flattened large cells predominated at first in culture, then widespread cell death occurred (Fig. 5). To investigate whether these cells had the hallmarks of apoptosis, electron microscopic nuclear phenotype, TUNEL assay of DNA fragmentation and flow-cytometric apoptosis analyses were done. These cells exhibited cytoplasmic blebbing, chromatin condensation and fragmented nucleus, features of apoptosis, while WT-hTERT and control vector-transfected cells had normal nuclear morphology (Fig. 6). Flow-cytometric analysis showed that apoptosis of these cells reached 42.18% compared to about 6% of cells expressing WT-hTERT or control vector (Fig. 7). Results of the TUNEL assay of DNA fragmentation confirmed the above observation (Fig. 8). Our results revealed that inhibition of telomerase leads to telomeric shortening and eventually to induction of apoptosis. Effect of DN-hTERT on tumorigenicity DN-hTERT expression clone 3 at PD 7 and clone 1 at PD 10, WT-hTERT expression clones 3 and 8 at PD 10, vector control

5 HEPATOMA GROWTH INHIBITION BY DN-HTERT 177 FIGURE 8 BEL-7404 human hepatoma cells expressing (a) DN-hTERT, (b) WT-hTERT or (c) control vector, analyzed by the TUNEL assay for apoptosis. (d) Negative and (e) positive controls. expression clone at PD 10 and parental BEL-7404 human hepatoma cells were injected s.c. into immunodeficient nude mice. Parental cells and cells expressing control vector or WT-hTERT formed tumors rapidly. In contrast, 2 DN-hTERT expressing clones did not form tumors (Table I). DISCUSSION Telomerase activity has been shown to be inhibited by treatment with antisense oligonucleotides, hammerhead ribozyme or peptide nucleic acids against htr. 23,27 29 However, htr expression was found in both HCCs and noncancerous tissues, and there was no close correlation between htr expression and telomerase activity, 15 though htr contained the essential template region specifying the addition of telomerase. In contrast, expression of htert correlated well with telomerase activity and degree of undifferentiation of HCC Our results showed that expression of DNhTERT could inhibit telomerase activity of hepatoma cells completely, confirming that htert is essential for telomerase activity of HCC cells. Inhibition of telomerase is a promising therapy for cancer because telomerase is detected in the vast majority of tumor cells but not in most normal cells. 4,5 Introduction of single amino acid substitutions within the reverse-transcriptase motifs of Est2 protein led to telomerase inhibition, telomeric shortening and senescence in yeast. 30,31 Suppression of telomerase activity by hammerhead ribozyme or expression of mutant htert was found to lead to apoptosis. 32,33 Small-molecule reverse transcriptase inhibitors (e.g., azidothymidine) were shown to be effective in telomerase activity and growth inhibition in several kinds of tumor. 34 Our observation of growth inhibition and apoptosis of BEL-7404 hu-

6 178 ZHANG ET AL. TABLE I EFFECT OF DN-HTERT ON TUMORIGENICITY IN BEL-7404 HUMAN HEPATOMA CELLS PD Number of tumors/ number of injections Mean telomeric length (kb) Parental cells 5/5 4.1 Vector control 10 5/5 4.1 WT-hTERT, clone /5 4.1 WT-hTERT, clone /5 ND DN-hTERT, clone /5 3.2 DN-hTERT, clone 3 7 0/5 1.7 ND, not determined. PD and mean telomeric length of cells determined at the time of injection. man hepatoma cells by expressing mutant htert is in agreement with those reports, indicating that htert could be an ideal target in HCC gene therapy and anti-hcc drug screening. Related investigations have shown that apoptosis induced by telomerase inhibition was telomeric length-dependent. 32,33,35 The presence of chromosome end fusion was observed in the latter experiments, indicating that sensors of DNA damage might be involved in the process. The signal to activate apoptosis is not known, but this system is p53-independent. 18,33 Understanding the exact mechanisms of apoptosis induction by telomerase inhibition may enhance the therapeutic effect of telomerase targeting, and further investigations are necessary. Previous reports have shown that alteration of the carboxyl terminus of htert did not affect telomerase enzymatic activity but impeded the ability of this enzyme to maintain telomeres 17 and that telomerase RNA component knockout telomerase-negative mouse cells could generate tumors during early generations but exhibited defective spermatogenesis, with increased apoptosis and decreased proliferation in the tests, during the late generation coinciding with substantial erosion of telomeres. 36,37 From all of these investigations, we could deduce that the telomere might be a more direct and effective target for cancer therapy using telomerase inhibition. The present study confirmed this deduction. Although DN-hTERT expressing clone 1 still had telomerase activity, it could not form tumors in nude mice. The shortened telomere might account for this result. It might be because the telomeres of the cells became critically short and telomere length-dependent apoptosis occurred after several divisions of the cells injected into nude mice. The increased apoptosis during PD confirmed this deduction (data not shown). Our results indicated that the telomere could be a potential target of anticancer agents, especially for telomerase-negative tumors. Telomeric DNA contains a singlestranded, G-rich DNA overhang, which may adopt a G-quadruplex structure. Several G-quadruplex interactive small molecules have been proven effective at telomerase inhibition and antiproliferation. 38 In conclusion, DN-hTERT transfection of BEL-7404 human hepatoma cells inhibited telomerase activity, which resulted in shortening of telomeres and inhibition of growth. 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