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Cell Growth & Differentiation Vol. 12, 119-127, February 2001
© 2001 American Association for Cancer Research

Autocrine Transforming Growth Factor ß Suppresses Telomerase Activity and Transcription of Human Telomerase Reverse Transcriptase in Human Cancer Cells1

Hua Yang, Satoru Kyo, Masahiro Takatura and LuZhe Sun2

Department of Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 [H. Y., L. S.], and Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Ishikawa 920-0934, Japan [S. K., M. T.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Because autocrine transforming growth factor ß (TGF-ß) can suppress carcinogenesis, which is often associated with telomerase activation, we studied whether autocrine TGF-ß inhibits telomerase activity. Restoration of autocrine TGF-ß activity in human colon carcinoma HCT116 cells after reexpression of its type II receptor (RII) led to a significant reduction of telomerase activity and the mRNA level of telomerase reverse transcriptase (hTERT), whereas suppression of the autocrine TGF-ß activity with a dominant negative RII without the cytoplasmic domain ({Delta}RII) in human breast cancer MCF-7 cells led to a significant increase of telomerase activity and hTERT mRNA level. This appears to be due to repression of hTERT mRNA transcription because exogenous TGF-ß treatment of MCF-7 cells transiently transfected with a hTERT promoter-reporter construct significantly repressed the hTERT promoter activity in a dose-dependent manner. Furthermore, the hTERT promoter activity was significantly decreased in HCT116 RII cells and increased in MCF-7 {Delta}RII cells when compared with their respective controls. Therefore, autocrine TGF-ß appears to target hTERT promoter to inhibit telomerase activity.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Telomerase, a ribonucleoprotein complex, uses its own RNA as a template to add hexanucleotide repeats to the ends of replicating chromosomes, compensating for the loss of telomeric DNA that occurs with DNA replication (1) . Telomerase activity is inactivated or repressed in the majority of human somatic tissue, and telomeres shorten with successive cell division. In contrast, telomerase activity is present in germ line and the majority of immortal and cancer cells (2 , 3) . Because continual loss of telomeric DNA leads to the eventual halt of cell proliferation, activation of telomerase in cancer cells may represent an important step in acquisition of the cell immortalization that occurs during tumorigenesis (4 , 5) . The human active telomerase requires at least two components for its activity: (a) hTR3 template, which is the template for reverse transcription; and (b) hTERT, which is the enzyme’s catalytic subunit (6 , 7) . Studies on the hTR and a telomerase-associated protein, TP1, showed that they are expressed in both normal cells and tumor cells and that they are usually not limiting factors for telomerase activity (8 , 9) . hTERT is a human homologue of the yeast Saccharomyces cerevisiae gene EST2 that codes the catalytic subunit of the yeast telomerase (7) . Several reports have shown that induction of hTERT expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression (10 , 11) . The transfection of hTERT gene into telomerase-negative, normal human fibroblasts resulted in the expression of hTERT, reconstitution of telomerase activity, and maintenance of telomeres (12) . In addition, the hTERT gene was shown to be down-regulated in differentiated cells in vitro and up-regulated after immortalization (10) . These results suggest hTERT is the rate-limiting component for regulation of telomerase activity in human cells. However, the molecular mechanisms by which hTERT expression is regulated in human cells remain largely elusive.

Several recent reports indicate that telomerase activity is growth regulated in normal human tissues in vivo. Primitive hematopoietic cells have recently been shown to express low levels of telomerase activity (13) . When activated to divide, lymphocytes exhibit a significant increase in telomerase activity (14 , 15) . Telomerase activity in endometrial tissue is tightly correlated with proliferation during the menstrual cycle, and the proliferative zone of intestinal crypts also shows telomerase activity (16 , 17) . Thus, telomerase activity appears to correlate with cell proliferative potential, as suggested by some investigators (18 , 19) . This scenario implies that proliferation-inhibitory agents such as TGF-ß would suppress telomerase activity.

TGF-ß is a multifunctional cytokine that plays an important role in the regulation of growth of various cell types (20) . It exerts its function by binding to two different cell surface serine/threonine kinase receptors called RI and RII. The formation of the ligand-receptor complex starts with the binding of TGF-ß to RII, which in turn recruits and transphosphorylates RI. The activated RI then phosphorylates intracellular Smad proteins to propagate the signal (21 , 22) . TGF-ß induces a growth arrest at late G1 phase in epithelial cells as well as other types of cells by regulating the expression and activity of various proteins that control cell cycle progression from G1 to S phase (23) . Whereas TGF-ß isoforms are secreted in a latent form, a small percentage of them become activated in the extracellular domain of almost all types of cells. The mature, active TGF-ß has been shown in a number of studies to act as an autocrine negative growth factor in epithelial cells (24, 25, 26) . Inactivation or attenuation of the autocrine TGF-ß growth-inhibitory loop has been shown to promote tumorigenesis and progression in a number of systems (27, 28, 29, 30) . On the other hand, restoration or enhancement of the autocrine TGF-ß activity can suppress the malignancy of various types of cancer cells (24 , 31, 32, 33) . These studies demonstrate that autocrine TGF-ß plays a vital role in preventing neoplastic progression.

Because both activation of telomerase activity and inactivation/attenuation of autocrine TGF-ß activity are associated with tumorigenesis and progression, we examined whether telomerase activity and hTERT expression are regulated by autocrine TGF-ß. Here we report that restoration of autocrine TGF-ß activity by reconstitution of RII in human colon cancer HCT116 cells reduced telomerase activity and expression of hTERT mRNA. On the other hand, introduction of a kinase-defective dominant negative RII into human breast cancer MCF-7 cells resulted in increased expression of hTERT mRNA and telomerase activity. In addition, we found that down-regulation of hTERT expression and telomerase activity by autocrine TGF-ß is apparently due to repression of hTERT transcription, suggesting that autocrine TGF-ß activity contributes to the regulation of telomerase activity in human cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Activity of a TGF-ß-responsive Promoter in MCF-7 Cell Line Transfected with a Dominant Negative RII.
Previous studies have shown that expression of wild-type RII can restore autocrine TGF-ß activity in HCT116 cells (24) . On the other hand, expression of a dominant-negative RII ({Delta}RII) without the cytoplasmic kinase domain can render MCF-7 cells insensitive to exogenous TGF-ß, whereas the control MCF-7 cells are growth inhibited by exogenous TGF-ß (34) . To confirm that the expression of {Delta}RII in MCF-7 cells also attenuated autocrine TGF-ß activity, we compared TGF-ß-responsive promoter activity in MCF-7 control vector-transfected (Neo) and {Delta}RII expression vector-transfected cells using the p3TP-Lux construct, whose luciferase expression was shown to be inducible by TGF-ß signaling due to the presence of three consecutive TPA response elements and a portion of the plasminogen activator inhibitor-1 promoter region (35) . Transient transfection of this construct into MCF-7 Neo and {Delta}RII cells revealed that the luciferase activity was 5-fold lower in the {Delta}RII cells than in the Neo cells, suggesting that autocrine TGF-ß loop in MCF-7 cells is operational and that {Delta}RII expression significantly attenuated its activity (Fig. 1)Citation . The {Delta}RII cells were also found to have a higher proliferation rate than the control cells (data not shown). These observations are consistent with the previous observation in this model system that the expression of a TGF-ß-responsive gene, fibronectin, decreased after {Delta}RII expression (34) .



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Fig. 1. Suppression of TGF-ß-responsive promoter activity in MCF-7 cells transfected with the kinase-defective dominant negative RII ({Delta}RII). MCF-7 Neo and {Delta}RII cells were transiently cotransfected with p3TP-Lux and a ß-galactosidase expression plasmid using Tfx-20 reagent as described in "Materials and Methods." Luciferase activity is reported in arbitrary units normalized by ß-galactosidase activity. Values are presented as the mean ± SE from three transfections. The luciferase activity is significantly lower (P < 0.01) in {Delta}RII cells than in Neo cells, as indicated by *.

 
Effect of Autocrine TGF-ß on Telomerase Activity.
To examine whether telomerase activity is regulated by autocrine TGF-ß, we used the two pairs of cell lines with altered autocrine TGF-ß activity as mentioned above. The human colon carcinoma HCT116 cell line does not have autocrine TGF-ß activity due to the mutation of its RII. Reexpression of RII by transfection restored autocrine TGF-ß activity as evidenced by increased expression of fibronectin and restored sensitivity to a TGF-ß1-neutralizing antibody treatment (24) . The restoration of the autocrine TGF-ß activity also led to reduced telomerase activity measured with the TRAPeze Telomerase Detection Kit from Intergen Company, which is a PCR-based TRAP (Fig. 2A)Citation . The characteristic bands of a telomeric ladder are visible in the autoradiograph when 10 and 100 ng of cell extract protein from the control cells (Neo) were used in the assay. In contrast, the telomeric ladder is not visible when 10 ng of protein from the RII cells were used. Incubation of the cell extract (1000 ng of protein) at 90°C for 15 min (lanes labeled Ht) before the TRAP assay eliminated these bands, suggesting that telomerase was inactivated by the heat. To quantitate the telomerase activity, we obtained the ratio of background-corrected cumulative radioactivity of all telomeric bands in each lane to that of its internal control (IC) with a PhosphorImager. According to the manufacturer’s instruction, relative telomerase activity shown in Fig. 2, B and DCitation , is the percentage of the sample ratio over the positive control (+C) ratio. As shown in Fig. 2BCitation , the telomerase activity in HCT116 RII is only 55% of that of HCT116 control (Neo) cells at both input protein levels. On the other hand, blockade of autocrine TGF-ß signaling with the {Delta}RII increased telomerase activity by 44% (Fig. 2, C and D)Citation .



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Fig. 2. Regulation of the telomerase activity in HCT116 and MCF-7 cell lines with altered autocrine TGF-ß activity. Logarithmically growing cells were lysed using the lysis buffer in the TRAPeze Telomerase Detection Kit. Cell extracts containing 10 or 100 ng of protein were used in the assays. Cell extracts containing 1 µg of protein were heated at 90°C for 15 min and used as negative control (Ht). The telomerase quantitation control (+C) and the negative control (-C) from the detection kit were also included in the assays for the calculation of relative telomerase activity. The PCR band from internal control for PCR efficiency is indicated as IC. The radioactive PCR products were electrophoresed on a 12% polyacrylamide gel and visualized after autoradiography as shown in A for HCT116 cells and in C for MCF-7 cells. The total radioactivity of all PCR-amplified telomeric bands and the IC band in each lane was quantified separately with a PhosphorImager, and the relative telomerase activity was calculated according to the manufacturer’s instructions and plotted in B for HCT116 cells and in D for MCF-7 cells.

 
To evaluate whether the altered telomerase activity by autocrine TGF-ß was statistically significant, we also measured telomerase activity with a TRAP ELISA kit from Roche Molecular Biochemicals, which allowed multiple quantitative determinations of TRAP products in a 96-well plate reader. The results obtained with this method confirmed those presented in Fig. 2Citation in that the telomerase activity was significantly (P < 0.01) lower in HCT116 RII cells than in HCT116 Neo cells (Fig. 3A)Citation . On the other hand, suppression of autocrine TGF-ß activity by {Delta}RII expression significantly (P < 0.01) increased telomerase activity in MCF-7 {Delta}RII cells when compared with MCF-7 Neo cells (Fig. 3B)Citation . We have consistently observed the same phenomenon in human colon carcinoma CBS and FET cell lines as well as in breast carcinoma MDA-MB-231 cell line with altered autocrine TGF-ß activity.4 Additional repression of the telomerase activity was also obtained when we treated HCT116 RII and MCF-7 Neo, the cell lines that have operational autocrine TGF-ß activity, with exogenous TGF-ß1 (Fig. 3C)Citation . This is significant because the growth of HCT116 RII cells was not inhibited by exogenous TGF-ß according to previous studies (24) . Thus, TGF-ß can apparently suppress telomerase activity independent of its growth-inhibitory pathway.



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Fig. 3. Effect of altered autocrine TGF-ß activity or exogenous TGF-ß1 on telomerase activity of HCT116 and MCF-7 cells. Cell extracts containing 10 ng of protein were analyzed by the TRAP-ELISA assays for HCT116 Neo and RII cells (A) and MCF-7 Neo and {Delta}RII cells (B). The levels of telomerase activity were expressed as absorbance. For C, HCT116 RII or MCF-7 Neo cells were treated with or without 5 ng/ml TGF-ß1 for 48 h before their extracts were used in TRAP-ELISA assays. The telomerase activity for C was expressed as relative activity, in that the telomerase activities of the two cell lines not treated with TGF-ß were presented as 100. The values are presented as the means ± SE from four separate determinations for the data in A and B and from three separate determinations for the data in C. The bars bearing * indicate a significant difference (P < 0.01) from their respective controls.

 
Effect of Autocrine TGF-ß on hTERT mRNA Expression.
Because telomerase activity is related more to the expression of hTERT than to the expression of other components of telomerase complex in many cases (11 , 36 , 37) , we investigated whether the changes of telomerase activity in the cells with altered autocrine TGF-ß activity were due to changes in hTERT expression. To compare the hTERT mRNA levels between the control and experimental cell lines, we initially used a RNase protection assay to measured hTERT mRNA levels in the paired cell lines. Fig. 4Citation shows that HCT116 Neo cells expressed a low level of hTERT mRNA and that restoration of the autocrine TGF-ß activity suppressed hTERT mRNA expression to an undetectable level in a 4-µg poly(A)+ RNA sample. Because the hTERT mRNA expression was too low to be detected with our RNase protection assay in the 5-µg poly(A)+ RNA sample from the MCF-7 Neo cells, the enhancement of hTERT mRNA expression by {Delta}RII expression in the MCF-7 {Delta}RII cells, although visible on the autoradiograph, could not be photographically reproduced for publication. For this reason, we developed a competitive RT-PCR assay according to the method described by Siebert and Larrick (38) . A known amount of total cellular RNA (1 µg) was reverse-transcribed to generate cDNA. The cDNA was first used with a pair of ß-actin primers in a PCR to verify that the input total RNA and the efficiency of RT were comparable for the paired samples as shown in Fig. 5BCitation . The mean intensity of the triplicate bands of the ß-actin PCR products from paired cDNA samples was statistically the same as that quantitated with the PhosphorImager after SYBR Green I staining (data not shown). We then mixed a constant amount of cDNA with decreasing amounts of a competitor DNA. A series of PCRs were performed to simultaneously amplify the hTERT cDNA (target) and the competitor with the same pair of primers. The coamplified PCR products were resolved with agarose gel electrophoresis (Fig. 5A)Citation and quantitated with a PhosphorImager after SYBR Green I staining. The ratio of the target:competitor as a function of the amount of competitor was plotted in a double logarithmic scale (Fig. 5C)Citation . The ratio of MCF-7 Neo was consistently lower than that of MCF-7 {Delta}RII at each competitor concentration, suggesting that the MCF-7 {Delta}RII cells have a higher hTERT mRNA level. Statistical analyses of the paired regression lines in Fig. 5CCitation revealed that whereas the slopes were statistically the same, the elevations were significantly (P < 0.05) different between the paired MCF-7 cell lines. Consistent with the RNase protection result in Fig. 4Citation , we also detected a significantly lower hTERT mRNA level in HCT116 RII cells than in HCT116 Neo cells using the competitive RT-PCR method (data not shown). Taken together, these results suggest that the repression of telomerase activity by the autocrine TGF-ß appears to be due to the down-regulation of hTERT mRNA expression.



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Fig. 4. Repression of hTERT mRNA expression by autocrine TGF-ß in HCT116 cells. hTERT and actin mRNA were detected in 4 µg of poly(A)+ RNA extracted from HCT116 Neo and RII cells with a RNase protection assay. Human actin mRNA was used for verification of equal sample loading. Yeast tRNA was included in the assay as a negative control.

 


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Fig. 5. Stimulation of hTERT mRNA expression in MCF-7 cells after expression of {Delta}RII. Total RNA (1 µg) extracted from MCF-7 Neo and {Delta}RII cell lines was used as a template for cDNA synthesis. The comparable input of total RNA and the efficiency of cDNA synthesis for the paired cell lines were confirmed by PCR in triplicates with ß-actin-specific primers (B). Serial dilutions (left to right in A) of the competitor DNA were mixed with a constant amount of cDNA from the paired cell lines and coamplified by PCR with hTERT-specific primers. After amplification, the PCR products were analyzed by 2% agarose gel electrophoresis (A). The relative intensity of the target and competitor bands in each lane was determined by Phosphor-Imager scanning. The ratio of target to competitor as a function of the amount of competitor was plotted in a double logarithmic scale for each cell line (C). The best-fitted lines for each data set were drawn after linear regression analyses with a {gamma}2 value of 0.98. The assay for paired cell lines was repeated two times with similar results.

 
Effect of TGF-ß on Transcriptional Activity of hTERT Promoter.
Although many studies have shown that hTERT mRNA expression is a rate-limiting step for the enzymatic activity of telomerase, little is known concerning how the expression of hTERT mRNA is regulated. We investigated whether the changes in hTERT mRNA expression in the cells with altered autocrine TGF-ß activity were due to the regulation of hTERT transcription. To observe a concentration-dependent and a time-dependent effect of TGF-ß on hTERT promoter activity, we initially studied the effect of exogenous TGF-ß treatment on hTERT promoter activity in MCF-7 cells. MCF-7 parental cells were transiently cotransfected with pGL3-1375, which contained a 1375-bp promoter fragment of hTERT fused with the luciferase reporter gene in pGL3-Basic vector from Promega, and a pCMV-ß-galactosidase plasmid. In the first experiment, the transfected cells were treated with human recombinant TGF-ß1 at concentrations of 0, 0.5, 1.0, and 5.0 ng/ml for 44 h. The normalized luciferase activity in cell lysates indicated that the transcriptional activity of hTERT promoter was repressed in a dose-dependent manner by exogenous TGF-ß1 in MCF-7 cells (Fig. 6A)Citation . In the second experiment, the transiently transfected cells were treated with or without 5 ng/ml TGF-ß1 for 6, 12, 24, and 48 h. The normalized luciferase activity was inhibited by 26% after 12 h and by 35% after 24 and 48 h of TGF-ß1 treatment (Fig. 6B)Citation .



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Fig. 6. Suppression of the transcriptional activity of the hTERT promoter by exogenous TGF-ß1 in MCF-7 cells. The MCF-7 parental cell line was transiently cotransfected with a 1375-bp promoter fragment of hTERT fused to luciferase reporter gene plasmid and pCMV-ß-galactosidase plasmid. The transfected cells were treated with different concentrations of recombinant TGF-ß1 for 44 h as shown in A. In a separate experiment, the transfected cells were treated with 5 ng/ml TGF-ß1 for different periods of time as shown in B. Luciferase activity is presented in arbitrary units normalized to ß-galactosidase activity. The values are presented as the mean ± SE from four transfections.

 
Next we transiently cotransfected pGL3-1375 and pCMV-ß-galactosidase plasmids into the paired HCT116 and MCF-7 cells to examine whether hTERT promoter activity is coupled to the autocrine TGF-ß activity. As shown in Fig. 7ACitation , the transcriptional activity of the hTERT promoter in HCT116 RII cells was significantly (P < 0.05) lower than that in HCT116 Neo cells, whereas it was significantly (P < 0.05) higher in MCF-7 {Delta}RII cells than in MCF-7 Neo cells (Fig. 7B)Citation . Thus, these results are all consistent with our observations of changes in telomerase activity and hTERT expression in the cells with altered autocrine TGF-ß activity and suggest that the hTERT promoter is a transcriptional target of TGF-ß and that the down-regulation of the telomerase activity and hTERT expression by autocrine TGF-ß is due at least in part to the transcriptional repression of the hTERT gene.



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Fig. 7. Effect of altered autocrine TGF-ß activity on the transcriptional activity of hTERT promoter in HCT116 and MCF-7 cell lines. The hTERT promoter-luciferase reporter construct and ß-galactosidase expression plasmid were transiently cotransfected into HCT116 Neo and RII cell lines (A) and MCF-7 Neo and {Delta}RII cell lines (B) as described in "Materials and Methods." Luciferase activity is presented in arbitrary units normalized by ß-galactosidase activity. The values are presented as the mean ± SE from three transfections. The bars bearing * indicate a significant difference (P < 0.05) from their respective controls.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGF-ß is a multifunctional polypeptide growth factor. It can regulate many biological processes such as cell proliferation, differentiation, extracellular matrix formation, angiogenesis, and so forth (39) . Most notably, it is a potent natural inhibitor of epithelial cell proliferation in vitro and in vivo. Certain human carcinoma cells as well as cell lines are unresponsive to the inhibitory effect of TGF-ß due to mutations of its receptors or the intracellular signaling proteins (21 , 40) . Thus, it is believed that inactivation of autocrine TGF-ß signaling could cause neoplastic transformation. Because unlimited proliferation of transformed cells requires maintenance of telomeric length by telomerase or other alternative mechanisms, and telomerase activity is increased in many types of immortalized and transformed cells, we tested our hypothesis that telomerase activity may be regulated by autocrine TGF-ß. By taking advantage of previously established systems, we found that restoration of autocrine TGF-ß activity in human colon carcinoma HCT116 cells significantly suppressed telomerase activity (Figs. 2, A and BCitation , and 3A)Citation , whereas disruption of autocrine TGF-ß activity with a dominant negative RII in the human breast cancer MCF-7 cells significantly increased telomerase activity (Figs. 2, C and DCitation , and 3B)Citation . The same observation was also made in human colon carcinoma CBS and FET cells and in breast carcinoma MDA-MB-231 cells with altered autocrine TGF-ß activity.4 Thus, the tumor-suppressive activity of autocrine TGF-ß loop in normal epithelial cells may reside not only in its growth-inhibitory function but also in its suppression of telomerase activity. Disruption of the autocrine TGF-ß loop permits uncontrolled cell proliferation and telomerase activation that can in turn maintain telomeric length, a prerequisite for unlimited proliferation.

Multiple levels of antagonization of TGF-ß signal transduction have been reported due to the complexity of its signaling pathway (21) . As such, loss of the growth-inhibitory activity of TGF-ß can be a gradual process taking place at multiple levels along the TGF-ß signaling cascade and is correlated with cell proliferation rate and tumor progression (21 , 40) . Interestingly, the expression of hTERT appears to increase gradually during the progression of human colon and breast cancers, both in the amount of hTERT mRNA present in individual cells and in the number of positive cells within a neoplastic lesion (11) . A more recent study also showed that hTERT promoter activity increased gradually as normal human fibroblasts became experimentally immortalized, transformed in vitro, and eventually tumorigenic in vivo (41) . As such, the TGF-ß signaling pathway may couple telomerase activity to cell proliferation rate so that telomeres can be maintained as the cell proliferation rate increases during tumor progression. If this is true, the 30–50% up- or down-regulation of telomerase activity we observed in the human colon and breast cancer cells with abrogated or enhanced autocrine TGF-ß activity may be sufficient for the cells to maintain telomere homeostasis as their proliferation rate is altered by autocrine TGF-ß signaling. We will test this hypothesis in our future studies.

Human telomerase activity can be reconstituted in vitro in the presence of hTR and hTERT (42) . Recently, a number of studies have shown that telomerase activity is associated with hTERT expression in normal and transformed human cells, suggesting that hTERT is probably the limiting component for the expression of telomerase activity in most cases (11 , 36 , 37) . Therefore, to investigate how autocrine TGF-ß suppresses telomerase activity, we also focused our attention on the effect of autocrine TGF-ß on hTERT expression. RNase protection assays and competitive RT-PCR assays showed that hTERT mRNA levels were inversely related to autocrine TGF-ß activity (Figs. 4Citation and 5)Citation , suggesting that the suppression of telomerase activity by autocrine TGF-ß appeared to be due to the down-regulation of hTERT mRNA expression.

The antimitotic activity of TGF-ß is primarily due to its ability to block the G1 to S-phase transition by up-regulating cell cycle inhibitors such as p15 and p21 and by down-regulating cell cycle stimulators such as cyclin-dependent kinase 4, cyclin-dependent kinase 6, and c-myc (23) . Because telomerase activation is generally associated with cell proliferative activity (19) , negative growth regulators would be a priori inhibitors of telomerase. Indeed, in addition to TGF-ß, retinoic acid, another growth inhibitor, was also shown to suppress telomerase activity in HL60 leukemia cells (43 , 44) . However, one of the issues to be resolved about telomerase regulation by the growth inhibitors is whether the inhibition of telomerase activity and hTERT expression is secondary to the growth inhibition or whether the inhibitors can initiate the telomerase-inhibitory pathway independent of the growth-inhibitory pathway. An earlier study reported that telomerase activity was highest in S-phase cells and lowest in G2-M-phase cells (45) . Consequently, the authors attributed the inhibition of telomerase activity in human breast cancer MDA-MB-435 cells by exogenous TGF-ß to its activity in blocking G1-S-phase transition and increasing the G1-phase cell population. However, a later study reported that telomerase activity has a half-life of >24 h and is related to population doubling time but not to any specific cell cycle stage in a number of immortalized or transformed human cells (46) .

In the current study, we have deliberately used logarithmically growing cells to examine the effect of autocrine TGF-ß on telomerase activity. Autocrine TGF-ß is known to predominantly increase population doubling time and lengthen the lag phase of newly plated cultures. As a culture enters logarithmic growth, the effect of autocrine TGF-ß on doubling time is diminished as observed in the human colon carcinoma HCT116 and FET cells, presumably due to the increasing levels of autocrine-stimulatory growth factors secreted by the culture (24 , 47) . Indeed, alteration of autocrine TGF-ß activity in both HCT116 and MCF-7 cell lines had no effect on cell cycle distribution when we performed cell cycle analysis of the logarithmically growing cells using flow cytometry (data not shown). This suggests that the autocrine TGF-ß-induced suppression of telomerase activity and hTERT expression in the HCT116 and MCF-7 cells was not due to altered cell cycle distribution. Another important observation we made was the inhibition of telomerase activity in HCT116 RII cells by exogenous TGF-ß treatment (Fig. 3C)Citation . Previous studies showed that reexpression of RII in HCT116 restored autocrine TGF-ß growth-inhibitory activity, but its growth was not inhibited by exogenous TGF-ß treatment. In contrast, the expression of a TGF-ß-regulated gene, fibronectin, was enhanced after reexpression of RII and further stimulated by exogenous TGF-ß (24) . Similarly, we also observed a significant additional inhibition of telomerase activity by exogenous TGF-ß treatment in HCT116 RII cells whose telomerase activity was already inhibited by autocrine TGF-ß activity (Fig. 3, A and C)Citation . These results suggest that the growth-inhibitory pathway and the telomerase-inhibitory pathway diverge along TGF-ß signal transduction pathway. Whereas the former was apparently saturated by the endogenous TGF-ß secreted by the HCT116 RII cells, the latter was not saturated and was still responsive to exogenous TGF-ß treatment.

Recent studies have begun to elucidate the mechanisms of telomerase activation at the molecular level. For example, c-myc expression was shown to activate telomerase in normal human mammary epithelial cells and fibroblasts by inducing hTERT mRNA expression (48) . On the other hand, treatment with antisense c-myc oligonucleotides suppressed telomerase activity in human leukemia cell lines (49) . These studies are consistent with our data in that TGF-ß is known to be a suppressor of c-myc expression (50 , 51) . The cloning of the hTERT gene promoter revealed that the transcription of hTERT mRNA may be regulated by a number of cis regulatory elements located in the promoter region including the SP1 sites and E box, a c-myc binding site (41 , 52 , 53) . Our present study shows that hTERT promoter activity is inversely affected by autocrine TGF-ß activity (Fig. 7)Citation . Thus, the inhibition of hTERT mRNA expression by autocrine TGF-ß in the HCT116 and MCF-7 cells may be mediated in part by its suppression of c-myc expression, resulting in down-regulation of the hTERT promoter activity, although other cis and trans elements may also be involved. Additional studies are needed to elucidate the pathways by which autocrine TGF-ß regulates hTERT transcription.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
The human colon carcinoma HCT116 Neo cell line and TGF-ß RII-transfected cell line (cl.17) have been described previously (24) . The human breast cancer MCF-7 Neo cell line and dominant-negative RII ({Delta}RII)-transfected cell line (ME24t6) were kindly provided by Dr. Michael G. Brattain (University of Texas Health Science Center, San Antonia, TX; Ref. 34 ). HCT116 Neo and RII cells were maintained in McCoy’s 5A serum-free medium supplemented with pyruvate, vitamins, amino acids, antibiotics, 10 ng/ml epidermal growth factor, 20 ng/ml insulin, and 4 µg/ml transferrin. MCF-7 Neo and {Delta}RII cells were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum. All cell lines were cultured in a 5% CO2 humidified atmosphere at 37°C.

Telomerase Assay.
Cell extracts were obtained from logarithmically growing cells treated or not treated with exogenous TGF-ß1 according to the manufacturer’s (Intergen Company, Purchase, NY) instructions regarding the TRAPeze Telomerase Detection Kit. The extracts were assayed for total protein content using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Telomerase activity in the cell lines was first measured using the kit from Intergen Company, which uses PCR-based TRAP (4) . Certain amounts of cell extract protein were incubated with telomerase substrates to generate telomerase products, which were amplified by PCR. The 32P-labeled PCR products were electrophoresed in a 12% polyacrylamide gel, visualized by autoradiography, and quantitated using a PhosphorImager scanner (Molecular Dynamics). For each sample including the positive (quantitation) control and heat-inactivated negative control, the radioactivity in all PCR-amplified telomeric bands, and the internal control band was separately quantitated and used for the calculation of relative telomerase activity according to the manufacturer’s instruction.

To statistically assess the role of autocrine TGF-ß activity in controlling telomerase activity, we used a TRAP ELISA kit (Roche Molecular Biochemicals) to measure telomerase activity according to the manufacturer’s instructions. A constant amount of total cell extract protein was used from each cell line to compare the telomerase activities. In our preliminary experiments, we found that for HCT116 and MCF-7 cells, the amount of PCR-amplified telomerase product detected with the TRAP ELISA kit increased linearly as a function of logarithmic input of total cell extract protein from 1–100 ng, suggesting that the assay is sensitive to detection of changes of telomerase activity. To detect both increases and decreases of telomerase activity as a result of altered autocrine TGF-ß activity, we chose a middle point of 10 ng of cell extract protein for our subsequent experiments using the TRAP ELISA assay.

Construction of Plasmids Containing hTERT cDNA.
A 235-bp hTERT gene cDNA fragment was generated by RT-PCR using sense primer 5'-TCACCTCGAGGGTGAAGGCACTGTT-3' and antisense primer 5'-ATGCTGGCGATGACCTCCGTGA-3'. The fragment corresponds to the hTERT motif A region as described by Nakamura et al. (7) . The PCR was performed with a starting temperature at 94°C for 2 min, followed by 35 cycles at 94°C for 45 s, 54°C for 30 s, and 72°C for 1 min. The PCR fragment was cloned into the pBSK(-) vector at its SmaI site and sequenced using T7 primer. The recombinant vector was then linearized as DNA template to synthesize a radioactive antisense hTERT probe for the RNase protection assay. We also deleted a 60-bp fragment within the inserted 235-bp hTERT cDNA fragment using PCR. The resulting 175-bp fragment was used as a competitive template in competitive PCR assays.

RNase Protection Assay.
Total RNA from cell lines was isolated by guanidine thiocyanate homogenization and acidic phenol extraction (54) . Purification of poly(A)+ RNA from total RNA was performed with Oligotex mRNA Spin-Column (Qiagen). To measure the hTERT mRNA levels in paired cell lines, we first used a RNase protection assay as described previously (31) . Briefly, 32P-labeled antisense hTERT and human actin riboprobes were hybridized with 4 µg of poly(A)+ RNA extracted from the cell lines at room temperature overnight. The samples were digested with RNase A and T1, followed by proteinase K. The protected fragment of the probe was analyzed by urea-PAGE and visualized by autoradiography.

Competitive PCR Analysis of hTERT mRNA.
Because hTERT mRNA was almost undetectable in the MCF-7 cells with the RNase protection assay, we also used a competitive PCR method described by Siebert and Larrick (38 , 55) to measure hTERT mRNA levels. The cDNA for quantification by competitive PCR was synthesized in a RT reaction mixture containing 1 µg of DNase-treated total RNA, 600 ng of random hexamers, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The input of total RNA and the efficiency of RT in paired samples were confirmed to be comparable by PCR with ß-actin-specific primers and quantitation of the ß-actin PCR product with SYBR Green I (Molecular Probes) staining and the PhosphorImager scanning. A constant amount of cDNA was then coamplified with known concentrations of the competitor by PCR using 200 µM of the above-mentioned hTERT gene-specific primers. The PCR products were resolved by 2% agarose gel electrophoresis, visualized by ethidium bromide staining, and photographed. For quantitation of hTERT mRNA, a replicate gel was stained with SYBR Green I and quantitated using the PhosphorImager scanner. The logarithmic ratios of the densities of the corresponding PCR product pair (235-bp hTERT PCR product to 175-bp competitor PCR product) were plotted against the logarithmic concentration of the competitor DNA. A best fitted line was then generated by a linear regression analysis and used to make comparisons of hTERT mRNA levels in paired cell lines.

Transient Transfection and Luciferase Assay.
A TGF-ß-responsive promoter-luciferase construct called p3TP-Lux (35) and a hTERT promoter-luciferase construct called pGL3-1375 (41) were used in our study to measure autocrine TGF-ß activity and hTERT transcriptional activity, respectively, in the paired cell lines. p3TP-Lux is a luciferase reporter construct whose luciferase expression was shown to be inducible by TGF-ß signaling due to the presence of three consecutive TPA response elements and a portion of the plasminogen activator inhibitor-1 promoter region (35) . pGL3-1375 contained a 1375-bp promoter fragment of hTERT fused with the luciferase reporter gene in pGL3-Basic vector from Promega. Transient transfection of these luciferase reporter plasmids was performed using Tfx-20 reagent (Promega) according to the manufacturer’s protocol, with some modifications. Briefly, a Tfx-20 and DNA mixture containing 3 µg of one of the luciferase reporter constructs and 1 µg of pCMV-ß-galactosidase reporter construct was incubated for 15 min at room temperature. Cells (7 x 105) plated in a 60-mm tissue culture dish 1 day before the transfection were exposed to the Tfx-20 and DNA mixture for 4 h at 37°C. The transfection mixture was then aspirated and replaced with the culture medium for an additional 44-h incubation. For the experiments with exogenous TGF-ß treatment, the transfection mixture was replaced with culture medium containing various concentrations of recombinant human TGF-ß1 (R&D Systems), and the cultures were incubated for an additional 44 h for a dose-dependent study. For a time-dependent study, the transfected cells were treated with 5 ng/ml TGF-ß1 and incubated for various periods of time. Cells were harvested in 500 µl of luciferase lysate buffer [100 mM K2HPO4 (pH 7.8) and 1 mM DTT], and cell extracts were obtained after three cycles of freezing and thawing. The activities of luciferase and ß-galactosidase in the cell extracts were assayed using procedures described previously (56 , 57) . Luciferase activity was normalized to ß-galactosidase activity and expressed as relative luciferase activity.

Statistical Analysis.
Two-tailed Student’s t tests were used to examine significant differences of telomerase activity and hTERT promoter activity between paired cell lines. Evaluation of statistical differences between two linear regression lines was performed according to the method described by Zar (58) .


    Acknowledgments
 
We thank Dr. J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY) for the p3TP-Lux plasmid and Dr. Michael G. Brattain for the MCF-7 cell lines. We also thank Liwei Bao for excellent technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA63480 and CA75253. Back

2 To whom requests for reprints should be addressed, at Department of Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Mail Code7743, San Antonio, TX 78229-3900. Phone: (210) 567-5746; Fax: (210) 567-4664; E-mail: SUNL{at}UTHSCSA.EDU Back

3 The abbreviations used are: hTR, human telomerase RNA; hTERT, human telomerase reverse transcriptase; TGF-ß, transforming growth factor ß; TRAP, telomeric repeat amplification protocol; RII, TGF-ß type II receptor; RI, TGF-ß type I receptor; RT-PCR, reverse transcription-PCR; poly(A)+ RNA, polyadenylated RNA; CMV, cytomegalovirus. Back

4 Unpublished data. Back

Received for publication 7/ 7/00. Revision received 12/ 5/00. Accepted for publication 12/18/00.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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