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Molecular Biology and Genetics |
Bruce Rappaport Faculty of Medicine and Research Institute, Technion Israel Institute of Technology, Haifa 31096, Israel [I. B., C-B., C. S., M. Y-H., K. L. S.]; Laboratory of Molecular Medicine, Department of Nephrology, Rambam Medical Center, Haifa 31096, Israel [Y. R., M. T., K. L. S.]; The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [G. B. M.]; and University of Arizona, Children’s Research Center, Tucson, Arizona 85724 [O. C-B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the binding elements, we identified two E-boxes (CACGTG) as well as a novel element (MT-box), which we identified recently in a number of differentiation systems. Site-directed mutagenesis was used to introduce mutations into this novel transcription factor binding element. These mutations significantly affect the transcriptional activity of hTERT promoter in a cell type-specific manner and suggest that the transcription factors that bind to the E-box and the novel element cooperatively function as major determinants of hTERT expression and telomerase activity in ovarian cancer. Further comparison of promoter activity, telomerase activity, and telomere length among the different ovarian cancer cells indicated that a threshold level of telomerase activity is apparently sufficient to protect telomere integrity and permit the immortal state of the different ovarian cancer cell lines.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the RNA subunit (hTR) of the human telomerase complex is constitutively expressed in both tumor and normal somatic tissues (8 , 9) , expression of the catalytic subunit (hTERT)3 correlates with telomerase activity during cellular differentiation and neoplastic transformation (10 , 11) . A strong correlation is observed between hTERT mRNA expression and telomerase activity in a variety of epithelial cancers including cervical (12) , breast, colon (11 , 13) , ovarian (11 , 14) , and renal (15) carcinomas. Furthermore, ectopic expression of the hTERT gene in normal telomerase-negative human cells is sufficient to reconstitute enzymatic activity (16 , 17) and extend cellular life span (18) . Therefore, expression of hTERT appears to represent a rate-limiting step regulating overall telomerase activity.
The mechanism of regulation of hTERT gene expression is still under intensive investigation. Cloning of the hTERT promoter, enabled investigation of mechanisms controlling the transcriptional activity of hTERT (19, 20, 21, 22, 23, 24) . Transient transfection assays of telomerase positive cells combined with deletion analysis of hTERT promoter-reporter constructs revealed a core region that is sufficient for maximum promoter activity (19, 20, 21 , 23) . This region contains several putative transcription factor binding elements including potential binding sites for Sp1, c-Myc, AP-2, AP-4, and NF-1 (19, 20, 21, 22, 23 , suggesting that hTERT expression may be regulated by different factors in varying cellular contexts. Because the c-Myc proto-oncogene is an ubiquitous transcription factor involved in the control of cellular proliferation and differentiation (25) , this element was considered of particular interest. Indeed, previous studies have shown that overexpression of c-Myc activates the hTERT promoter and increases hTERT mRNA transcription and telomerase activity in normal and tumor cell lines (20 , 22 , 26, 27, 28) . Gel shift analysis also confirmed protein binding to the putative c-Myc binding sites, but corresponding supershifts failed to consistently identify c-Myc as the endogenous E-box binding transcription factor in telomerase-positive nuclear extracts (20 , 22 , 27 , 29) . These and other findings indicate that different factors may be involved in hTERT promoter regulation in different tumor cell lineages.
In ovarian cancer a number of studies demonstrated telomerase activity in the majority of ovarian carcinoma samples but only rarely in benign ovarian tumors or in normal ovary (6 , 30, 31, 32) . However, there is no information to date about hTERT promoter regulation in normal and malignant ovarian cells. Therefore, a detailed comparative characterization of promoter sequences and transcription factors essential for regulating hTERT promoter activation in ovarian cancer cells is still necessary for a more complete understanding of the role and molecular mechanisms of telomerase regulation in ovarian tumorigenesis.
Recently, we have identified a novel transcription factor binding element designated as the MT-box within the hTERT core promoter sequence (24) . This element was shown to undergo a time-dependent modification in transcription factor binding with differentiation of human embryonic stem cells, mouse embryonal carcinoma F9 cells, and HL-60 human promyelocytic leukemia cells (24) . Accordingly, in the current study, we examined the involvement of this novel element and its interaction with the E-box and other transcription factor binding elements of the hTERT promoter in the regulation of the hTERT expression in ovarian carcinoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of Reporter Plasmids.
A BamHI-SacII fragment containing 5790 bp of the 5' flanking sequence of the hTERT gene was subcloned into pBluescript II KS+ vector. Three 5' flanking fragments, 5790-, 3345-, and 283-bp fragments upstream to the TI, were generated using restriction enzymes, filled in, and ligated into the SmaI site of pGL3-basic reporter plasmid (Promega).
Transient Transfection Assays.
Transient transfections were performed in 24-well dishes using FuGENE6 reagent (Boehringer Mannheim), according to the manufacturer’s protocol. The pGL3-basic promoterless vector (Promega) was used as a control. To evaluate transfection efficiency, cells were cotransfected with a reporter vector containing the ß-gal gene driven by the CMV promoter. For each well of a 24-well plate, 6 x 104 cells were transfected with 0.07 µg of promoter-luciferase reporter construct and 0.14 µg of pCMV-ß-galactosidase expression vector. Transfection mixtures were adjusted with pBluescript DNA to a total of 0.3 µg/well. Forty-eight h after transfection, cells were harvested, and cell extracts were prepared. Luciferase activity was measured on a luminometer, and a ß-gal assay was performed according to standard protocols.
RT-PCR Analysis of hTERT Gene.
Tri-reagent (MRC, Cincinnati, OH) was used to extract total RNA from different cell lines. Similar amounts of total RNA were separated on agarose gels and stained by ethidium bromide. The rRNA of all samples was read by a densitometer to calibrate the amount of RNA. Total RNA, oligo (dT)12–18 primer, and reverse transcriptase were used for cDNA production. hTERT cDNA analysis was performed by PCR amplification of a fragment of 377 bp using primers described by Meyerson et al. (11)
. PCR reactions were carried out with increasing quantities of cDNA for increasing number of amplifying cycles to ensure a linear range of amplification. A fragment of the human ß-actin gene was amplified as an internal control. Amplified products were electrophoresed on a 2% agarose gel containing ethidium bromide, visualized under UV light, and analyzed by use of a densitometer.
TRAP Assay.
To determine the enzymatic activity of telomerase in the different cellular extracts, we performed a modified TRAP using the TRAPeze telomerase detection kit (Intergen; Ref. 6
).
Terminal Restriction Fragment Analysis for Telomere Length Measurement.
Genomic DNA was prepared from normal and tumor ovarian-derived cell lines and digested with the restriction enzymes HinfI and RsaI. Ten µg of digested DNA were electrophoresed on a 1% agarose gel, transferred to a nylon membrane, and incubated overnight at 38°C with a 32P-labeled (TTAGGG)3 telomeric probe, washed twice at 45°C with 6x SSC and 0.05% PPi, and subjected to autoradiography at -80°C for 16 h. We estimated the length of TRFs at the peak position of hybridization signal.
Electrophoretic Mobility Shift Assay.
32P-Labeled, double-stranded oligonucleotides containing the consensus SP1/AP2, distal E-box, proximal E-box, and MT-box were assayed for binding to nuclear extracts from the various cell lines, which were prepared as described (35)
. The sequences of the oligonucleotides used as probes were: SP1/AP2, 5'-CCTTTCCGCGGCCCGCCCTCTCCTCGCGG-3' (-20 to +10); distal E-box (oligo-1), 5'-GGACCGCGCTCCCCACGTGGCGGAGGGACTGGG-3' (-177 to -146); proximal E-box (oligo-2), 5'-GTCCTGCTGCGCACGTGGGAAGCCCTGGC-3' (+32 to +58); and MT-box (oligo-2), 5'-GTCCTGCTGCGCACGTGGGAAGCCCTGGC-3' (+32 to +58). Underlined are the sequences within each oligonucleotide corresponding to the respective named binding site.
Binding reactions and native PAGE were carried out as described previously (24) . Briefly, 10 µg of protein were incubated with 300 ng of poly(deoxyinosinic-deoxycytidylic acid) for 10 min at 4°C in a 20-µl reaction volume containing 25 mM HEPES (pH 7.9), 50 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 10% glycerol with or without 200-fold molar excess of unlabeled competitor DNA. After incubation, 1 x 105 cpm of 32P-labeled probe were added, and the reaction was incubated for an additional 20 min at 4°C. The DNA-protein complexes were separated by electrophoresis on a 5.3% polyacrylamide gel and visualized by autoradiography.
Site-directed Mutagenesis.
Site-directed mutagenesis was performed using a promoter-luciferase reporter plasmid and several mutated oligonucleotides, which disrupted binding to the E-box and/or the MT-box in EMSA. We used the Quick-Change Site-Directed Mutagenesis kit (Stratagene) to introduce these mutations in the context of the pGL3-core promoter construct. In brief, we used the supercoiled double-stranded DNA pGL3-core promoter vector and two synthetic oligonucleotide primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, were extended during temperature cycling by using PfuTurbo DNA polymerase (Stratagene). Incorporation of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. The product was treated with DpnI endonuclease, which digests only the parental DNA that is methylated or hemimethylated and then selected for the mutation-containing, synthesized DNA. Sequencing of the constructs confirmed the introduction of the different mutations. Mutations containing pGL3-core promoter constructs were transiently transfected in parallel with the wild-type pGL3-core promoter, together with the pCMV-ß-gal expression vector in the different cells, to monitor the changes in promoter activity attributable to the different mutations.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 1
, transfection of pGL3–3.3 into ovarian cancer cell lines HEY, SKOV-3, OVCAR-3, or into human embryonic kidney cells HEK 293, which were confirmed to be telomerase positive, yielded significant promoter activity compared with transfection with the control pGL3 basic vector. Transfection of the same construct into telomerase-negative cells (NHFs and the nontransformed large T-antigen, semi-immortalized ovarian cell line, IOSE) resulted only in background levels of luciferase activity. Promoter activity was also undetectable in the ovarian carcinoma-derived cell line, OCC-1. Corresponding telomerase activity, as monitored by TRAP assay in these same cell lines, revealed a correlation between hTERT promoter activity and telomerase activity (Fig. 2a)
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. In contrast, the ovarian cancer cell line OCC-1, normal ovarian cells IOSE, and NHFs showed undetectable hTERT mRNA levels. Of note, among the four ovarian cancer cell lines, the lowest levels of hTERT gene expression and promoter activity were observed in the OCC-1 cell line, and the TRAP assay revealed a barely detectable level of telomerase activity. To elucidate whether the OCC-1 cells do not use an alternative mechanism (36)
for maintaining telomere length and whether any relationship exists between telomere lengths and telomerase activity, TRF analysis was performed in the different ovarian derived cells. As shown in Fig. 2c
, TRF size distribution was heterogeneous among the ovarian carcinoma cell lines ranging from 3 to >10 Kbp. The TRF length of the nonmalignant ovarian cell line, IOSE, was estimated as 
5 Kbp. There is little evidence to support an alternative rescue pathway in OCC-1 cells, because TRF analysis revealed very short telomeres (3 Kbp) in this specific cell line. In general, cells using the alternative rescue pathway display very long average telomere length (36)
. Taken together, these findings are consistent with the notion that overall regulation of telomerase activity is determined, at least in part, by regulation of the hTERT promoter, and that the promoter is functional and telomerase is active in the majority of, but not in all, ovarian cancer-derived cell lines. Furthermore, the results in the OCC-1 ovarian cancer cells, with a known aggressive tumorigenic potential,4
indicate that even a very low level of telomerase activity is sufficient to protect telomere integrity and permit the immortalized state of the cells.
Characterization of cis-acting Regulatory Elements in the hTERT Promoter.
To better define the cis-acting sequences responsible for transcriptional activation of hTERT in ovarian cancer cells, a 5'-deletion analysis was performed. Constructs corresponding to promoter fragments of three sizes, 5790, 3345, and 283 bp upstream to the TI site, were fused upstream to the luciferase reporter gene in the pGL3 vector. These constructs also included 78 nucleotides from the hTERT 5'-untranslated region (Fig. 3a)
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yielded high levels of promoter activity in ovarian carcinoma cell lines that express high levels of endogenous telomerase activity but not in OCC-1 or IOSE ovarian epithelial cells that have low or undetectable endogenous telomerase activity (Fig. 3b)
. In HEY, SKOV-3, and OVCAR-3 cells, promoter activity was sufficient to compare the relative activity of the three constructs. This comparison revealed greater promoter activity when the region between -5790 and -3345 was deleted. The pGL3–3.3 and the pGL3–283 (pGL3-core) constructs conferred the highest transcriptional activity. The proximal 283 bp is sufficient for maximal promoter activity in all telomerase-expressing cells and therefore designated as the core promoter. The lowest levels of promoter activity were observed with the largest construct (pGL3–5.7), suggesting the possible presence of inhibitory elements in the region between -3345 and -5790.
To identify the pattern of transcription factor binding elements contributing to hTERT promoter activity, multiple different oligonucleotides corresponding to putative DNA binding elements within the core promoter region were generated for EMSAs, using nuclear extracts from telomerase-positive and telomerase-negative ovarian cell lines. Gel shift analysis using an oligonucleotide corresponding to nucleotide sequences -20 to +10, which includes an SP1/AP2 consensus motif, yielded the electrophoretic mobility shift pattern presented in Fig. 4a
. Competition with excess cold oligonucleotide and supershift with anti-SP1 antibody (Fig. 4)
confirmed specific SP1 binding. No evidence of AP2 binding was observed (Fig. 4b
, Lane 3).
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Further EMSA were carried out using oligonucleotides corresponding to sequences -177 to -146 (oligo-1) and +32 to +58 (oligo-2), each containing an E-box consensus motif (CACGTG). Gel shift analysis using oligo-1, containing the distal E-box, yielded a prominent bandshift using nuclear extracts from the telomerase-positive ovarian cell lines OVCAR-3, HEY, and SKOV-3 (Fig. 5a)
. This binding could be competed by excess of unlabeled oligonucleotide (Fig. 5a)
. Supershift assays using anti c-Myc antibodies failed to demonstrate c-Myc in the complex (data not shown). Nuclear extracts from OCC-1 and IOSE cells yielded bandshifts of much lower intensity, likely indicating lower levels of transcription factor binding. Correspondingly, these cell lines expressed the lowest hTERT mRNA levels, telomerase enzymatic activity, and hTERT promoter activity.
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. However, a clear additional fast-migrating bandshift was also observed, most intensely evident using the HEY cell line nuclear extract. Specificity of binding was confirmed by competition with excess unlabeled oligo-2 (Fig. 5b)
. Of note, in a previous study (24)
, we have observed a similar pattern of transcription factor binding using nuclear extracts derived from the three different predifferentiated experimental model systems: human embryonic stem cells (hES), mouse F9 cells in culture, and human HL-60 promyelocytic cells. We have designated the novel element as the MT-box (CGTGGGAAG; Ref. 24
).
In Fig. 5, a and b
also compare the relative transcription factor binding to the two E-boxes and to the putative novel binding site noted above, among the different ovarian carcinoma-derived cell lines. This comparison reveals differences in protein binding to the putative E-box and MT-box binding motifs, among the various ovarian carcinoma-derived cell lines. Prominent binding was observed to either one of the E-boxes or the MT-box motifs in all of the cell lines, in which the hTERT promoter is active, motivating further functional analysis of these sites using site-directed mutagenesis and transient transfections reporter assays (see below). To determine the specificity and the distinct binding sequences responsible for the fast-migrating band, EMSA analysis was performed using HEY cell nuclear extracts and labeled oligo-2 in which several competitors were included. As shown in Fig. 6
, Lanes 2 and 3, cold excess of oligo-1 (-177) could compete with binding to the E-box but not with binding to the MT-box. In contrast, cold excess of oligo-2 (+32) greatly decreased the binding to both elements. In addition, a series of oligo-2 with systematically mutated nucleotide sequences was generated as described (24)
and used as competitors in EMSA analysis using HEY cells nuclear extracts and labeled wild-type oligo-2. These mutated oligonucleotides, used as competitors, generated the following results: M1, M2, and M3 in which the E-box consensus sequence was disturbed could not compete with the binding to this motif. M1 but not M2 or M3 competed for the binding to the MT-motif, reflected in a marked decrease in the fast-migrating band. M4–M8 oligonucleotides eliminated the binding to the E-box, because the mutations they carry reside downstream to the E-box. M6–M8, but not M4 and M5, competed with binding to the MT-box, suggesting that M4 and M5 are within the MT-box and that M6–M8 do not reside in the MT-box. Thus, M2 and M3 are in the overlapping portion of the E and MT boxes, whereas M4 and M5 are in the MT-box but not in the E-box. The results obtained confirm that the two overlapping transcription factors binding sites, i.e., the E-box (CACGTG) and the MT-box (CGTGGGAAG), also show a pattern of transcription factor binding in which there is distinct binding to each element. An extensive database search revealed no precedent for a similar DNA binding motif other than as was reported by our laboratory in differentiating cell systems (24)
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. Transient transfection assays were performed using wild-type and mutated core promoter constructs in pGL3-basic, as shown in Fig. 7a
, to evaluate the contribution of these sites to hTERT promoter activity. As shown in Fig. 7b
, the M1 mutation that was shown by EMSA to disrupt proximal E-box binding yielded an 100% increase in transcriptional activity compared with the core promoter activity in SKOV-3 cells. A smaller increase (30%) was evident in HEY cells, whereas in OVCAR-3 cells, this mutation had only a marginal effect on transcription, suggesting that the role of this E-box depends on the ovarian cancer cell line assessed. The mutation M3, which disrupts both the binding to the MT-box and the proximal E-box, yielded a 30–75% decrease in promoter activity in HEY and OVCAR-3 cell lines compared with that observed for the core promoter construct. However, no influence was observed in SKOV-3 cells. On the other hand, M4, which disrupts binding to the MT-element but leaves both E-boxes intact, had a variable effect on hTERT promoter activity, which was also cell line specific. Minor changes in luciferase activity were observed in OVCAR-3 and SKOV-3 cells, whereas a 30% reduction (compared with the core promoter activity) was evident in HEY cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the critical differences between normal and tumor cells lies in their state of telomerase activity. Telomerase is activated in the majority of human tumors, making it one of the most prevalent biochemical cancer-specific markers (38 , 39) . Our findings, in agreement with previous studies, revealed telomerase activity in ovarian carcinoma cell lines but not in nontransformed ovarian cells. Thus, discrimination between malignant and nonmalignant ovarian tumors may be assisted by measurement of telomerase activity. However, studies of the association between telomerase activity level in cancers and clinical outcome have yielded conflicting results. Some studies found that high telomerase activity might be closely correlated with a more aggressive tumor phenotype (40 , 41) , whereas others have not found such a correlation (42) . In our study, OCC-1 cells are derived from the most clinically aggressive ovarian cancer and have the greatest tumorigenic potential yet showed a very low level of endogenous telomerase activity by TRAP assay. This finding of aggressive tumorigenic potential despite low levels of telomerase further suggests that a very low level of activity is sufficient to "cap" and protect telomere integrity and permit the immortalized state of the cells. When cancer cells become immortal, telomeres are stabilized at a length that depends on a balance between the "shortening" of telomeric repeats during cell divisions and the "lengthening" effect of telomerase activity. Thus, once telomerase is activated, the telomere length can be stabilized at almost any length, apparently without influencing the viability of the tumor cells (6) .
In agreement with previous reports (6) , no association was observed between terminal restriction fragment length and the level of telomerase activity in the various ovarian cells. This further suggests that the steady state level of TRF length among the different cancer-derived cell lines may be a function of the telomere length at the time of telomerase activation.
Previous studies investigating the pattern of expression of the RNA component (hTR) and catalytic components (hTERT) of human telomerase revealed that telomerase activity is closely correlated with changes in hTERT expression (11 , 43) . In particular, a >80% concordance was found between telomerase activity and hTERT expression in ovarian tumors (30 , 44) . Accordingly, to learn more about the mechanisms regulating hTERT gene expression in ovarian carcinoma, we isolated the hTERT promoter and examined its transcriptional activity in a variety of ovary-derived cell lines. We found that the hTERT promoter is activated in telomerase-expressing ovarian cancer cells and inactive in an ovarian epithelium-derived cell line that does not express telomerase. A striking correlation was evident between hTERT mRNA levels, telomerase activity, and hTERT promoter activity in the various cell lines tested. This suggests that overall regulation of enzymatic activity in ovarian carcinoma is achieved, at least in part, through transcriptional mechanisms acting at the level of the hTERT promoter. Therefore, we further undertook a detailed analysis of the promoter and its regulation in these cell lines. Using deletion analysis in a transient expression system, we observed that the 5' flanking region of the hTERT gene between positions -283 to +78 (ATG) conferred strong transcriptional activity in the three telomerase-positive ovarian carcinoma cells. Although all of these three telomerase-positive ovarian cell lines are derived from epithelial adenocarcinoma, there was significant variability in hTERT promoter activity among the cells.
As with all studies involving established cell lines, it is possible that such variation may reflect modifications resulting from clonal variation in cell culture, other factors related to cell culture conditions, differences in cytogenetic features (33 , 45) , or variable expression patterns of specific transcription factors in the different tumor cell lines. Analysis of transcription factors that bind to the hTERT core promoter region revealed SP1 binding and E-box binding in agreement with previous studies (20 , 22 , 29) . However, in addition, we found binding to an additional DNA transcription factor binding element (MT-box) distinct from the SP-1 and E-box binding regions.
Binding to this novel element, which partly overlaps one of the E-boxes, has also been noted in the undifferentiated state in three different cell differentiation systems (24) . Binding to this element was also present only in the subset of telomerase-positive ovarian cells, suggesting that the transcription factor binding to this element may recapitulate the undifferentiated state and be partly responsible for hTERT gene activation in some forms of ovarian cancer. The present study also demonstrated that the protein(s) that bind to the E-box and to the MT-box regulate hTERT promoter activity in a variable manner among the different cell lines, perhaps reflecting differences in transcription factor abundance and stoichiometry.
Consistent with this view, we indeed found that mutation of the distal E-box both abrogated transcription factor binding and resulted in a significant decrease in transcriptional activity in HEY and OVCAR-3 cells, whereas in SKOV-3 cells, this mutation led to a marked increase in promoter activity. It is thus evident that the effect of this E-box on promoter activity depends on the particular cell line, consistent with previous reports (29) .
Similarly, mutation of the proximal E-box showed cell line-specific effects. Mutations that abolish binding to both the proximal E-box and to the MT-box resulted in a significant decrease in promoter activity in HEY and OVCAR-3 cell lines, but no effect was observed in SKOV-3 cells. Mutation of the MT-box alone decreased the transcriptional activity in HEY cells and had no effect in other cells. This is consistent with HEY lysates showing the highest MT-box binding activity. Of note, the triple mutant, which abolishes transcription factor binding to both E-boxes as well as the MT-box, resulted in a decrease of transcriptional activity in all cell types. This suggests that binding to either the E-box or MT-box per se may suffice to induce telomerase promoter activity. The most plausible explanation is that although the promoter likely defines a consistent pattern for the possibility of transcription factor binding and interaction, variable expression of the transcription factors themselves may account for differences in promoter activity and hence hTERT gene expression and telomerase activity among the different ovarian cancer cell lines.
From the foregoing is evident that this system can be exploited to elucidate the molecular basis for differences in hTERT activity in different cancer cell lines. The cell lines used in this study have been characterized extensively for genetic abnormalities and protein expression (46, 47, 48, 49, 50)
. There was no correlation between Ras, p53, or PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutation, phosphatidylinositol 3-kinase amplification as indicated by 3q26 amplification or p16 or RB protein levels, and binding or activity of the E-box and MT-box. The only obvious correlation was with the Ras mutant in HEY cells and MT-box binding. However, both OVCAR-3 and SKOV-3 cells exhibited high Ras activity in the absence of Ras mutations (see Table 1
).
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We also characterized core promoter elements that appear to govern the variation in telomerase activity. One of these is a novel DNA binding element (MT-box), which binds one or more known or novel transcription factors. Identification of the transcription factors that bind to the E-boxes and to the MT-box will assist in unraveling the transcriptional regulation of the hTERT gene and may be greatly facilitated by exploiting the variation in hTERT regulation in ovarian cancer cell lines and tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Binational Science Foundation and the Israel Cancer Research Foundation. 
2 To whom requests for reprints should be addressed, at Department of Molecular Medicine, B. Rappaport Faculty of Medicine, P. O. Box 9649, Bat Galim, Haifa 31096, Israel. Phone: 972-4-8295277, extension 81; Fax: 972-4-8512380; E-mail: bimaty{at}tx.technion.ac.il
3 The abbreviations used are: hTERT, human telomerase reverse transcriptase; NHF, normal human fibroblast; TI, transcription initiation; CMV, cytomegalovirus; ß-gal, ß-galactosidase; RT-PCR, reverse transcription-PCR; TRAP, telomerase repeat amplification protocol; TRF, terminal restriction fragment; EMSA, electrophoretic mobility shift assay.
Received 1/15/01. Accepted 5/14/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H). The reaction products were separated on a 10% nondenaturating polyacrylamide gel. b, measurement of hTERT mRNA expression by RT-PCR. RT-PCR analysis was performed on total RNA from HEY (Lane 1), SKOV-3 (Lane 2), OVCAR-3 (Lane 3), OCC-1 (Lane 4), IOSE (Lane 5), NHF (Lane 6), and HEK 293 (Lane 7) cell lines, using primers specific for the hTERT and ß-actin genes and the conditions described in "Materials and Methods." c, TRF analysis. Southern blot hybridization was performed using telomere-specific probe (TTAGGG)3 and genomic DNA extracted from normal (IOSE) and cancer (HEY, SKOV-3, OVCAR-3, and OCC-1) ovarian-derived cells for measuring telomere length. Left, positions of molecular size standards in Kbp.
, SP-1 •, and MT-box 
in the core promoter are shown in a. All three constructs were analyzed for their transcriptional activity using transient transfection assays. Constructs were transiently cotransfected with ß-gal expression vector into ovarian cancer-derived cell lines (HEY, SKOV-3, OVCAR-3, and OCC-1) and the normal ovarian-derived cell line (IOSE), as indicated in b. Transfections were performed using FuGENE6 transfection reagent as described in "Materials and Methods." Cells were harvested 48 h after transfection and assayed for luciferase and ß-gal activity. Luciferase activity values were normalized for transfection efficiency by the corresponding ß-gal value. The reported values represent the normalized relative luciferase activity compared with the pGL3-basic plasmid, which was set at a value of unity. Each data point represents the mean of three independent experiments (bars, SE), each performed in quadruplicate.
