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Telomerase reverse transcriptase regulates the expression of a key cell cycle regulator, cyclin D1
BBRC Biochemical and Biophysical Research Communications 347 (2006) 774–780 www.elsevier.com/locate/ybbrc
Telomerase reverse transcriptase regulates the expression of a key cell cycle regulator, cyclin D1 Shankar Jagadeesh, Partha P. Banerjee
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Department of Biochemistry, Molecular and Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20057, USA Received 26 June 2006 Available online 10 July 2006
Abstract Cells require a mechanism to maintain telomere stability in order to overcome replicative senescence and telomerase catalyzes the synthesis and extension of telomeric DNA, therefore, be a rate-limiting step in cellular immortality and oncogenesis. However, some studies have raised questions about whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis. To elucidate non-canonical functions of human telomerase reverse transcriptase (hTERT), we used loss-of-function and gain-of-function studies. We demonstrated that hTERT shRNA down-regulated and hTERT overexpression up-regulated the expression and transcriptional activity of a key cell cycle regulator, cyclin D1, in human prostate epithelial cell lines, DU-145 and BPH-1. Based on these observations, we propose that in addition to its well-defined function in telomere length regulation, hTERT has a novel role in the modulation of cyclin D1 expression. 2006 Elsevier Inc. All rights reserved. Keywords: Telomerase; hTERT; Cyclin D1; Cell cycle; Prostate cancer; DU-145; BPH-1
Telomerase, a ribonucleoprotein enzyme with specialized reverse transcriptase activity, catalyzes the synthesis and extension of telomeric DNA [1]. Telomerase is present in germline cells, cancer-derived cell lines, and spontaneously immortalized cells in culture. It is activated in 85–90% of malignant tumors but usually absent in normal somatic cells which results in the progressive loss of telomeres with each cell division [2]. Activation of telomerase allows cells to overcome replicative senescence, therefore, be a rate-limiting step in cellular immortality and oncogenesis [3]. The telomerase complex is composed of telomerase reverse transcriptase (TERT) [4], telomerase RNA component (TERC) [5], telomerase associated proteins (TEP1) [6], and chaperone proteins (p23, Hsp90) [7]. TERC, TEP1, p23, and Hsp90 are expressed in a wide variety of cells, irrespective of the
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Corresponding author. E-mail address: [email protected] (P.P. Banerjee).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.172
presence or absence of telomerase activity. On the other hand, a strong correlation is observed between hTERT mRNA expression and telomerase activity in a variety of epithelial cancers, including cervical [8], breast, colon [9], ovarian [10], and renal [11] carcinomas, indicating that the expression levels of hTERT could be important during tumorigenesis. However, some studies have raised questions about whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis [12–15]. Recent evidence shows that telomerase might have extratelomeric functions in cancer and in various post-mitotic cells [16,17]. To investigate a possible extraneous role for hTERT in prostate epithelial cells, we used loss-of-function and gain-of-function studies. In this study, we demonstrate for the first time that hTERT regulates transcriptional activity of a key cell cycle regulator, cyclin D1. The biological significance of this study will be enormous because of the cell cycle regulatory role of hTERT particularly in cancer.
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Materials and methods Cell culture. Human prostate (DU-145) cancer cell line was purchased from the American Type Cell Culture Collection (Manassas, VA). BPH-1 cells were obtained from Dr. Simon Hayward, Vanderbilt University Medical Center, Nashville, TN. Cells were grown in complete growth medium (IMEM without phenol red; Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Quality Biologicals, Gaithersburg, MD), 2 mM glutamine, 100 U/ml penicillin G sodium, and 100 lg/ml streptomycin sulfate (Sigma Chemicals, St. Louis, MO) in the presence of 5% CO2 at 37 C. Western blot analysis. Protein lysates were prepared from DU-145 and BPH-1 cells that were transfected with various plasmids. The lysates were resolved on 12% SDS–PAGE and transferred to nitrocellulose membranes. The membranes were probed with 1:1000 dilutions of hTERT (Novocastra Laboratories, Newcastle upon Tyne, UK), 1:1000 dilution of cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C. Each blot was re-probed with 1:10,000 dilution of b-actin (Sigma Chemicals, St. Louis, MO). Images of the membranes were captured using a Fuji LAS-1000 Imager (Tokyo, Japan) and imported into Adobe Photoshop. Band intensities were quantified by utilizing ImageJ software (NIH, Bethesda, MD). Immunofluorescence staining. DU-145 and BPH-1 cells were plated onto chamber slides and transfected with hTERT shRNA or hTERT expression vectors for 3 days. Appropriate negative control plasmids were transfected in the control cells. The cells were fixed in methanol, air-dried, and re-hydrated with PBS. 0.2% BSA was used for blocking and then the cells were incubated with the primary antibody (hTERT and cyclin D1; 1:100 and 1:200 dilution, respectively) overnight at 4 C. The cells were washed three times with PBS and incubated with 4 lg/ml Alexa Fluor 488 labeled donkey anti-mouse IgG and Alexa Fluor 594 donkey anti-rabbit IgG (Molecular probes, Invitrogen, Carlsbad, CA), for 1 h. The cells were washed again three times with PBS and subsequently incubated with 2 mg/ ml DAPI (Sigma) for 5 min. The cells were washed once more with PBS, mounted with 50% glycerol, and viewed under a fluorescent microscope (ZEISS AxioPlan2 Imaging System, Jena, Germany). Images were imported into Adobe Photoshop. Reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was extracted from DU-145 cells with TRIzol solution as suggested by the manufacturer (Invitrogen, Carlsbad, CA). Genes of interest were amplified using 500 ng of total RNA reverse transcribed to cDNA using a Superscript II kit (Invitrogen) with random hexamers. Human specific primers were designed using the Primer Quest program and purchased from Integrated DNA Technologies, Inc (Coralville, IA). Their sequences and product band sizes are: hTERT forward primer 5 0 -AGA ACG TTC CGC AGA GAA AA-3 0 , hTERT reverse primer 5 0 -ATG TAC GGC TGG AGG TCT GT-3 0 (392 bp), cyclin D1 forward primer 5 0 -CAC ACG GAC TAC AGG GGA GT-3 0 , cyclin D1 reverse primer 5 0 -AGG AAG CGG TCC AGG TAG TT-3 0 (475 bp), cyclin A1 forward primer 5 0 -AAGGAGTGTGCGTCAGGACT-3 0 , cyclin A1 reverse primer 5 0 -CAA CGT GCA GAA GCC TAT GA-3 0 (413 bp), cyclin B1 forward primer 5 0 -CGG GAA GTC ACT GGA AAC AT-3 0 cyclin B1 reverse primer 5 0 -CCG ACC CAG ACC AAA GTT TA-3 0 (315 bp) and cyclin E1 forward primer 5 0 -AAG TGG ATG GTT CCA TTT GC-3 0 cyclin E1 reverse primer 5 0 -TTT GAT GCC ATC CAC AGA AA-3 0 (399 bp) and GAPDH forward primer: 5 0 -CCA CCC ATG GCA AAT TCC ATG GCA-3 0 , GAPDH reverse primer: 5 0 -TCT AGA CGG CAG GTC AGG TCC ACC-3 0 (598 bp). PCRs were initiated at 94 C for 2 min, followed by 28 cycles of 94 C for 1 min, 1 min annealing temperature, 72 C for 1 min, and final extension at 72 C for 5 min. The annealing temperature for cyclin B1 and cyclin E1 was 55 C, for hTERT was 58 and 60 C for cyclin D1, cyclin A1, and GAPDH. After amplification, PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide fluorescence using the Fuji LAS-1000 Imager. Images were captured and imported to Adobe Photoshop. Band intensities were quantified by using ImageJ software (NIH, Bethesda, MD). Cyclin D1 promoter activity assay. DU-145 and BPH-1 cells were transfected with a cyclin D1 promoter luciferase (1745-cyclin D1-Luc)
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construct using GeneJammer transfection reagent (Stratagene, La Jolla, CA). DU-145 cells were also co-transfected with 100 ng hTERT shRNA plasmids or negative control shRNA plasmids (Origene, Rockville, MD). BPH-1 cells were also co-transfected with 100 ng hTERT expression vector plasmids or empty vector plasmids. After 48 h, luciferase activity was measured in cell lysates by a microplate luminometer using the Dual Luciferase Assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. Luciferase activity was normalized to Renilla luciferase activity by co-transfection of pRL-TK plasmid (20 ng). Telomeric repeat amplification protocol (TRAP) assays. Detection of telomerase activity from cell extracts was performed in a two-step process: (1) telomerase-mediated extension of an oligonucleotide (TS: 5 0 -AAT CCG TCG AGC AGA GTT-3 0 ) and (2) PCR amplification of the resultant product with forward (TS) and reverse (ACX: 5 0 -GCG CGG CTT ACC CTT ACC CTT ACC CTA ACC-3 0 ) primers. In addition, as internal controls, NT: 5 0 -ATC GCT TCT CGG CCT TTT-3 0 and TSNT: 5 0 -AAT CCG TCG AGC AGA GTT AAA AGG CCG AGA AGC GAT-30 primers were used. Telomeric DNA products were separated by a 10% nondenaturing PAGE in 0.5· Tris–borate–EDTA buffer, pH 8.3, at 300 V for 2.5 h. Gels were then stained with SYBR Green I and images were captured with a Fuji LAS-1000 Imager. TRAP products were quantified by using ImageJ software (NIH, Bethesda, MD). Statistical analyses. All data were derived from at least three independent experiments and statistical analyses were conducted using the Prism 3 GraphPad software. Values were presented as means ± SEM. Significance level was calculated using the t test, with an assigned confidence interval of 95%. A p-value <0.05 was considered significant.
Results and discussion hTERT regulates telomerase activity in human prostate epithelial cells To investigate a possible extraneous role for hTERT in prostate epithelial cells, we used loss-of-function and gainof-function studies. First, to confirm that telomerase activity is tightly regulated by the expression of hTERT, endogenous hTERT was repressed by the ectopic expression of hTERT shRNA (short hairpin RNA) containing plasmids in a human prostate cancer cell line, DU-145, that expresses very high levels of telomerase activity. Down-regulation of hTERT by the transfection of hTERT shRNA plasmids decreased telomerase activity more than 3-fold (Fig. 1A). On the other hand, ectopic expression of hTERT in BPH-1 cells that have very low levels of TERT increased telomerase activity 3-fold (Fig. 1B). Using reverse transcriptase-PCR (RT-PCR) we evaluated the levels of hTERT message in both DU-145 cells and BPH-1 cells transfected with hTERT shRNA and hTERT expression plasmids, respectively. As expected, we observed that ectopic expression of hTERT shRNA reduced the levels of hTERT message more than 5-fold (Fig. 2A), whereas ectopic expression of hTERT increased the levels of hTERT message in BPH-1 cells over 3-fold (Fig. 2B). hTERT regulates cyclin D1 expression in prostate epithelial cells but not other cyclins Most interestingly, we observed that decreased hTERT expression in DU-145 cells was associated with a significant (p < 0.0002) decrease in cyclin D1 message (Fig. 2A),
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whereas increased hTERT expression in BPH-1 cells upregulated the expression of cyclin D1 significantly (p < 0.006). This modulation of cyclin D1 expression by hTERT is not an isolated event, as we also observed a similar effect on variety of cancer cells such as skin (A431), pancreas (AsPC-1), mammary (MCF7), colon (HT-29), and ovarian (OVCAR-3) cancer cells (data not shown), which are highly telomerase positive (data not shown). Although we observed modulation of cyclin D1 expression in prostate epithelial cells by the hTERT, we did not find any change in the expression levels of other cyclins such as cyclin A1, B1, and E1 (Fig. 2C). These results suggest that the regulation of cyclin D1 by hTERT is specific. hTERT regulates cyclin D1 transcriptional activity in prostate epithelial cells
Fig. 1. hTERT regulates telomerase activity in human prostate epithelial cells. (A) Assessment of telomerase activity in DU-145 cells after transient expression of hTERT shRNA (+) or negative control shRNA () vector for 3 days. (B) Assessment of telomerase activity in BPH-1 cells after transient expression of empty vector () or hTERT expression vector (+) for 3 days. Cell pellets were collected from each experiment and subjected to TRAP assay. NC: negative control using lysis buffer only. Quantitative estimations of telomerase activity in DU-145 and BPH-1 cells were determined by densitometric measurement of TRAP products from three independent experiments. Columns, mean; bars, SE. *p < 0.001 for DU-145 and p < 0.004 for BPH-1, significantly different from control.
Since we observed that hTERT regulates cyclin D1 expression, it was important to know whether the alteration of cyclin D1 expression is transcriptionally regulated. To evaluate this, a full-length cyclin D1 promoter luciferase construct was transfected in DU-145 and BPH-1 cells along with either hTERT-shRNA or hTERT expression plasmids. We observed that transfection of cyclin D1-promoter luciferase plasmid alone increased the cyclin D1 transcriptional activity in DU-145 cells approximately 100-fold compared to the control plasmids (basic-luciferase plasmid). Co-transfection of the negative control (negative control shRNA plasmid) plasmid with the cyclin D1promoter luciferase plasmid did not alter the transcriptional activity of cyclin D1, whereas co-transfection of hTERT shRNA plasmid decreased cyclin D1 transcriptional
Fig. 2. hTERT regulates the expression of cyclin D1. (A,C) DU-145 cells were transiently transfected with hTERT shRNA (+) or negative control shRNA () vector for 3 days. (B) BPH-1 cells were transiently transfected with empty vector () or hTERT expression vector (+) for 3 days. RNA was extracted, and RT-PCR assays were performed to detect hTERT, cyclin D1, and GAPDH mRNAs. Representative photograph from an experiment that was repeated thrice. Quantitative estimations of relative levels of hTERT and cyclin D1 mRNAs (right panels) were determined by densitometric measurements of RT-PCR gels from three independent experiments after normalization with GAPDH. (C) RT-PCR analyses of cyclin A1, cyclin B1, cyclin E1, and GAPDH in DU-145 cells. Columns, mean; bars, SE. *p < 0.001, significantly different from control.
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activity approximately 5-fold (Fig. 3A). Similarly, the transfection of the cyclin D1-promoter luciferase plasmid alone in BPH-1 cells increased the cyclin D1 transcriptional activity approximately 6-fold over the control (basic-luciferase plasmid). Co-transfection of the empty vector control with the cyclin D1-promoter luciferase construct did not change the transcriptional activity of cyclin D1, but the co-transfection of hTERT plasmid increased cyclin D1 transcriptional activity approximately 4-fold over the level with cyclin D1-promoter luciferase alone (Fig. 3B). These results are consistent with the down-regulation and up-regulation of the levels of cyclin D1 mRNAs observed in RTPCR studies with the ectopic expression of hTERTshRNA and hTERT expression vectors in DU-145 and BPH-1 cells (Fig. 2), and re-confirm that hTERT is capable of regulating the transcription of cyclin D1 in these cells. We do not know whether the transcriptional regulation of cyclin D1 by hTERT is a direct or indirect effect and future investigations will be necessary to evaluate this issue. The transcription of the cyclin D1 gene is induced through distinct DNA sequences in the promoter by diverse mitogenic and oncogenic signaling pathways of Ras, Src, Stat3, Stat5, and Erbb2 [18]. Several transcription factors such as CREB, AP-1, and b-catenin/Tcf-1 have been identified to interact with the cyclin D1 promoter [19,20]. It is possible that one or more of these signaling pathways and transcription factors could be involved in the transcriptional regulation of cyclin D1 by hTERT. In this study, we observed that ectopic expression of hTERT shRNA in DU-145 cells down-regulates cyclin D1, but not cyclin A1, B1, and E1. It is interesting to note that some earlier study has shown that IKKa selectively and directly induces cyclin D1 but not cyclin E or cyclin A [21]. This study also demonstrated that the induction of cyclin D1 expression and promoter activity requires b-catenin/Tcf activity. Although we do not know whether Akt and/or IKKa are regulated by hTERT, it is possible
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that these signaling pathways could induce the transcriptional activity of cyclin D1. Detailed future experiments would be necessary to determine the involvement of the Akt, NFjB, and b-catenin signaling pathways in hTERT induced cyclin D1 expression. hTERT regulates cyclin D1 protein in prostate epithelial cells To this point, we have examined the message levels and/ or the transcriptional activity of cyclin D1; however, we do not know whether cyclin D1 protein levels were altered. Therefore, we examined the levels of cyclin D1 protein after down-regulation or up-regulation of hTERT message in prostate epithelial cells. We observed that the transfection of hTERT shRNA in DU-145 cells decreased the protein expression of hTERT as well as cyclin D1 significantly (p < 0.001) (Fig. 4A). On the other hand, a gain-of-function by the transfection of hTERT expression vector increased both hTERT and cyclin D1 proteins significantly (p < 0.01 and 0.005 for hTERT and cyclin D1, respectively) (Fig. 4B). These results suggest that cyclin D1 protein levels are dependent on the transcriptional regulation by hTERT. Since hTERT regulates the levels of cyclin D1 protein, it was important to know where cyclin D1 proteins are localized within the cell because cyclin D1 has to be in the nucleus to regulate cell cycle and/or proliferation. Using double immunolabeling with hTERT and cyclin D1 antibodies, we observed that in control DU-145 cells both telomerase (hTERT) and cyclin D1 are predominantly localized in the nucleus. Whereas, the transfection of hTERT shRNA down-regulated hTERT protein and its nuclear localization substantially. As a result, the levels of cyclin D1 also decreased dramatically and the nuclear localization of cyclin D1 was completely abolished (Fig. 5A). However, minimal staining of cyclin D1 was observed in the cytoplasm of hTERT shRNA transfected DU-145 cells. On
Fig. 3. hTERT regulates transcriptional activity of cyclin D1. (A) DU-145 cells were transfected with 200 ng of full-length cyclin D1 promoter luciferase plasmids (1745-cyclin D1-Luc) or basic luciferase (PA3-Luc) plasmids and 10 ng of Renilla luciferase (pRL-TK-Luc) plasmids with 200 ng of hTERT shRNA or negative control shRNA for 48 h in normal growth media. (B) BPH-1 cells were transfected with 200 ng of full-length cyclin D1 promoter luciferase plasmids or basic luciferase plasmids and 10 ng of Renilla luciferase (pRL-TK-Luc) plasmids with 200 ng of hTERT or empty vector for 48 h in normal growth media. After treatment, cells were harvested, and luciferase assays were performed. Relative cyclin D1 promoter activity was determined after normalization with Renilla luciferase activity. Luciferase activities in basic vector transfected cells were considered as 1.0. Columns, mean of three independent experiments with quadruplicate samples; bars, SE. *p < 0.0005, significantly different from control.
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Fig. 4. hTERT regulates the expression of cyclin D1 protein. (A) DU-145 cells were transiently transfected with hTERT shRNA (+) or negative control shRNA () vector for 3 days. (B) BPH-1 cells were transiently transfected with empty vector () or hTERT expression vector (+) for 3 days. Protein lysates (50 lg) from DU-145 and BPH-1 cells treated with hTERT shRNA or hTERT for 3 days were resolved on 12% SDS–PAGE, and immunoblots were probed with antibodies to hTERT and cyclin D1. All immunoblots were re-probed with b-actin antibodies to ensure equal loading. Representative photographs from an experiment that was repeated thrice. Quantitative analyses of relative levels of hTERT and cyclin D1 proteins are shown on the right panels. Columns, mean of three independent experiments; bars, SE. *p < 0.005, significantly different from control.
the other hand, control BPH cells expressed very little hTERT protein in the nucleus and as a result of the over-expression of hTERT, both hTERT and cyclin D1 staining intensity increased in the nucleus (Fig. 5B). The question might arise whether the constant presence of cyclin D1 would impair cell cycle progression. Interestingly, a published report suggests that in the presence of growth factors cyclin D1 levels remain relatively high and constant through the cell cycle, although a new burst of cyclin D1 synthesis occurs every time cell enters G1 after mitosis [22]. These results suggest that telomerase (hTERT) not only regulates the expression of cyclin D1, but also regulates the levels and localization of cyclin D1. Since the formation of the active complex of cyclin D1/cdk4/6 is important for the nuclear localization of cyclin D1, it is possible that hTERT could have effects on cdk4/6 and that would work along with the transcriptional regulation of cyclin D1. It is too speculative at this point and future experiments would be necessary to determine if hTERT also modulates the expression of cdks and/or its complex formation with cyclin D1. The activation of cyclin D1 gene transcription is dependent on the activation of Ras, Raf, mitogen-activated protein kinase-kinases (MEK1 and MEK2), Akt and the sustained activation of extracellular signal regulated protein kinases (ERKs) [18]. On the other hand, cyclin D1 degradation is mediated by phosphorylation-triggered, ubiquitin-dependent proteolysis [23]. Glycogen synthase kinase 3b (GSK-3b) catalyzes the phosphorylation of cyclin D1 on Thr286 and redirects the protein from the nucleus to the cytoplasm [23]. It would be interesting to investigate whether hTERT regulates the activation of
GSK3b which determines the nuclear localization of cyclin D1. Overexpression of cyclin D1 is a common event in various forms of cancer including prostate cancer [24–26]. The overexpression of cyclin D1 leads to enhanced organ growth in mice [27]. Transient transfection of hepatocytes with cyclin D1 leads to vigorous proliferation and a more than 50% increase in liver mass within 6 days [28]. Conversely, cyclin D1 knockout mice are smaller than wildtype mice, and mice with the homozygous deletion of the p27 gene (which inhibits cyclin D1/Cdk4/6 complexes) show gigantism and enhanced organ size [29]. Moreover, the expression of cyclin D1 modulates invasive ability by increasing matrix metalloproteinase (MMP-2 and MMP-9) activity and motility in glioma cells [30]. Furthermore, some studies show that over-expression of cyclin D1 is associated with metastatic prostate cancer to bone [31]. These findings suggest that in addition to its well-defined role in cell cycle progression, cyclin D1 may also play a role in the regulation of cell growth and metastasis. Therefore, the up-regulation of cyclin D1 by hTERT would allow telomerase to modulate telomere length as well as cell proliferation and/or its invasive potential. In summary, using both loss-of-function and gain-offunction studies, we were able to define a novel function of hTERT that is unrelated to the telomere length regulation and provide additional evidence that telomerase has more than one function in epithelial cells. Therefore, the expression of hTERT could have potentially important biological consequences, particularly in cancer cells, where it can control the stabilization of chromosome ends as well as cell proliferation, survival, and metastasis. On the other
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Fig. 5. hTERT decreases the nuclear localization of cyclin D1. (A) DU-145 cells were plated on chamber slides and transfected with 200 ng of shRNA negative control or hTERT shRNA for 3 days. (B) BPH-1 cells were plated on chamber slides and transfected with 200 ng of empty vector or hTERT expression vector for 3 days. Cells were then fixed in methanol, incubated with hTERT and cyclin D1 antibodies overnight, Alexa Fluor-conjugated secondary antibodies for 1 h, and counter stained with DAPI. Slides were then mounted and examined using a fluorescence microscope. Photographs were taken at the same magnification (20·) and then transported to Photoshop. Representative photographs from an experiment that was repeated twice.
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Telomerase reverse transcriptase regulates the expression of a key cell cycle regulator, cyclin D1 Shankar Jagadeesh, Partha P. Banerjee
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Department of Biochemistry, Molecular and Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20057, USA Received 26 June 2006 Available online 10 July 2006
Abstract Cells require a mechanism to maintain telomere stability in order to overcome replicative senescence and telomerase catalyzes the synthesis and extension of telomeric DNA, therefore, be a rate-limiting step in cellular immortality and oncogenesis. However, some studies have raised questions about whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis. To elucidate non-canonical functions of human telomerase reverse transcriptase (hTERT), we used loss-of-function and gain-of-function studies. We demonstrated that hTERT shRNA down-regulated and hTERT overexpression up-regulated the expression and transcriptional activity of a key cell cycle regulator, cyclin D1, in human prostate epithelial cell lines, DU-145 and BPH-1. Based on these observations, we propose that in addition to its well-defined function in telomere length regulation, hTERT has a novel role in the modulation of cyclin D1 expression. 2006 Elsevier Inc. All rights reserved. Keywords: Telomerase; hTERT; Cyclin D1; Cell cycle; Prostate cancer; DU-145; BPH-1
Telomerase, a ribonucleoprotein enzyme with specialized reverse transcriptase activity, catalyzes the synthesis and extension of telomeric DNA [1]. Telomerase is present in germline cells, cancer-derived cell lines, and spontaneously immortalized cells in culture. It is activated in 85–90% of malignant tumors but usually absent in normal somatic cells which results in the progressive loss of telomeres with each cell division [2]. Activation of telomerase allows cells to overcome replicative senescence, therefore, be a rate-limiting step in cellular immortality and oncogenesis [3]. The telomerase complex is composed of telomerase reverse transcriptase (TERT) [4], telomerase RNA component (TERC) [5], telomerase associated proteins (TEP1) [6], and chaperone proteins (p23, Hsp90) [7]. TERC, TEP1, p23, and Hsp90 are expressed in a wide variety of cells, irrespective of the
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Corresponding author. E-mail address: [email protected] (P.P. Banerjee).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.172
presence or absence of telomerase activity. On the other hand, a strong correlation is observed between hTERT mRNA expression and telomerase activity in a variety of epithelial cancers, including cervical [8], breast, colon [9], ovarian [10], and renal [11] carcinomas, indicating that the expression levels of hTERT could be important during tumorigenesis. However, some studies have raised questions about whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis [12–15]. Recent evidence shows that telomerase might have extratelomeric functions in cancer and in various post-mitotic cells [16,17]. To investigate a possible extraneous role for hTERT in prostate epithelial cells, we used loss-of-function and gain-of-function studies. In this study, we demonstrate for the first time that hTERT regulates transcriptional activity of a key cell cycle regulator, cyclin D1. The biological significance of this study will be enormous because of the cell cycle regulatory role of hTERT particularly in cancer.
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Materials and methods Cell culture. Human prostate (DU-145) cancer cell line was purchased from the American Type Cell Culture Collection (Manassas, VA). BPH-1 cells were obtained from Dr. Simon Hayward, Vanderbilt University Medical Center, Nashville, TN. Cells were grown in complete growth medium (IMEM without phenol red; Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Quality Biologicals, Gaithersburg, MD), 2 mM glutamine, 100 U/ml penicillin G sodium, and 100 lg/ml streptomycin sulfate (Sigma Chemicals, St. Louis, MO) in the presence of 5% CO2 at 37 C. Western blot analysis. Protein lysates were prepared from DU-145 and BPH-1 cells that were transfected with various plasmids. The lysates were resolved on 12% SDS–PAGE and transferred to nitrocellulose membranes. The membranes were probed with 1:1000 dilutions of hTERT (Novocastra Laboratories, Newcastle upon Tyne, UK), 1:1000 dilution of cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C. Each blot was re-probed with 1:10,000 dilution of b-actin (Sigma Chemicals, St. Louis, MO). Images of the membranes were captured using a Fuji LAS-1000 Imager (Tokyo, Japan) and imported into Adobe Photoshop. Band intensities were quantified by utilizing ImageJ software (NIH, Bethesda, MD). Immunofluorescence staining. DU-145 and BPH-1 cells were plated onto chamber slides and transfected with hTERT shRNA or hTERT expression vectors for 3 days. Appropriate negative control plasmids were transfected in the control cells. The cells were fixed in methanol, air-dried, and re-hydrated with PBS. 0.2% BSA was used for blocking and then the cells were incubated with the primary antibody (hTERT and cyclin D1; 1:100 and 1:200 dilution, respectively) overnight at 4 C. The cells were washed three times with PBS and incubated with 4 lg/ml Alexa Fluor 488 labeled donkey anti-mouse IgG and Alexa Fluor 594 donkey anti-rabbit IgG (Molecular probes, Invitrogen, Carlsbad, CA), for 1 h. The cells were washed again three times with PBS and subsequently incubated with 2 mg/ ml DAPI (Sigma) for 5 min. The cells were washed once more with PBS, mounted with 50% glycerol, and viewed under a fluorescent microscope (ZEISS AxioPlan2 Imaging System, Jena, Germany). Images were imported into Adobe Photoshop. Reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was extracted from DU-145 cells with TRIzol solution as suggested by the manufacturer (Invitrogen, Carlsbad, CA). Genes of interest were amplified using 500 ng of total RNA reverse transcribed to cDNA using a Superscript II kit (Invitrogen) with random hexamers. Human specific primers were designed using the Primer Quest program and purchased from Integrated DNA Technologies, Inc (Coralville, IA). Their sequences and product band sizes are: hTERT forward primer 5 0 -AGA ACG TTC CGC AGA GAA AA-3 0 , hTERT reverse primer 5 0 -ATG TAC GGC TGG AGG TCT GT-3 0 (392 bp), cyclin D1 forward primer 5 0 -CAC ACG GAC TAC AGG GGA GT-3 0 , cyclin D1 reverse primer 5 0 -AGG AAG CGG TCC AGG TAG TT-3 0 (475 bp), cyclin A1 forward primer 5 0 -AAGGAGTGTGCGTCAGGACT-3 0 , cyclin A1 reverse primer 5 0 -CAA CGT GCA GAA GCC TAT GA-3 0 (413 bp), cyclin B1 forward primer 5 0 -CGG GAA GTC ACT GGA AAC AT-3 0 cyclin B1 reverse primer 5 0 -CCG ACC CAG ACC AAA GTT TA-3 0 (315 bp) and cyclin E1 forward primer 5 0 -AAG TGG ATG GTT CCA TTT GC-3 0 cyclin E1 reverse primer 5 0 -TTT GAT GCC ATC CAC AGA AA-3 0 (399 bp) and GAPDH forward primer: 5 0 -CCA CCC ATG GCA AAT TCC ATG GCA-3 0 , GAPDH reverse primer: 5 0 -TCT AGA CGG CAG GTC AGG TCC ACC-3 0 (598 bp). PCRs were initiated at 94 C for 2 min, followed by 28 cycles of 94 C for 1 min, 1 min annealing temperature, 72 C for 1 min, and final extension at 72 C for 5 min. The annealing temperature for cyclin B1 and cyclin E1 was 55 C, for hTERT was 58 and 60 C for cyclin D1, cyclin A1, and GAPDH. After amplification, PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide fluorescence using the Fuji LAS-1000 Imager. Images were captured and imported to Adobe Photoshop. Band intensities were quantified by using ImageJ software (NIH, Bethesda, MD). Cyclin D1 promoter activity assay. DU-145 and BPH-1 cells were transfected with a cyclin D1 promoter luciferase (1745-cyclin D1-Luc)
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construct using GeneJammer transfection reagent (Stratagene, La Jolla, CA). DU-145 cells were also co-transfected with 100 ng hTERT shRNA plasmids or negative control shRNA plasmids (Origene, Rockville, MD). BPH-1 cells were also co-transfected with 100 ng hTERT expression vector plasmids or empty vector plasmids. After 48 h, luciferase activity was measured in cell lysates by a microplate luminometer using the Dual Luciferase Assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. Luciferase activity was normalized to Renilla luciferase activity by co-transfection of pRL-TK plasmid (20 ng). Telomeric repeat amplification protocol (TRAP) assays. Detection of telomerase activity from cell extracts was performed in a two-step process: (1) telomerase-mediated extension of an oligonucleotide (TS: 5 0 -AAT CCG TCG AGC AGA GTT-3 0 ) and (2) PCR amplification of the resultant product with forward (TS) and reverse (ACX: 5 0 -GCG CGG CTT ACC CTT ACC CTT ACC CTA ACC-3 0 ) primers. In addition, as internal controls, NT: 5 0 -ATC GCT TCT CGG CCT TTT-3 0 and TSNT: 5 0 -AAT CCG TCG AGC AGA GTT AAA AGG CCG AGA AGC GAT-30 primers were used. Telomeric DNA products were separated by a 10% nondenaturing PAGE in 0.5· Tris–borate–EDTA buffer, pH 8.3, at 300 V for 2.5 h. Gels were then stained with SYBR Green I and images were captured with a Fuji LAS-1000 Imager. TRAP products were quantified by using ImageJ software (NIH, Bethesda, MD). Statistical analyses. All data were derived from at least three independent experiments and statistical analyses were conducted using the Prism 3 GraphPad software. Values were presented as means ± SEM. Significance level was calculated using the t test, with an assigned confidence interval of 95%. A p-value <0.05 was considered significant.
Results and discussion hTERT regulates telomerase activity in human prostate epithelial cells To investigate a possible extraneous role for hTERT in prostate epithelial cells, we used loss-of-function and gainof-function studies. First, to confirm that telomerase activity is tightly regulated by the expression of hTERT, endogenous hTERT was repressed by the ectopic expression of hTERT shRNA (short hairpin RNA) containing plasmids in a human prostate cancer cell line, DU-145, that expresses very high levels of telomerase activity. Down-regulation of hTERT by the transfection of hTERT shRNA plasmids decreased telomerase activity more than 3-fold (Fig. 1A). On the other hand, ectopic expression of hTERT in BPH-1 cells that have very low levels of TERT increased telomerase activity 3-fold (Fig. 1B). Using reverse transcriptase-PCR (RT-PCR) we evaluated the levels of hTERT message in both DU-145 cells and BPH-1 cells transfected with hTERT shRNA and hTERT expression plasmids, respectively. As expected, we observed that ectopic expression of hTERT shRNA reduced the levels of hTERT message more than 5-fold (Fig. 2A), whereas ectopic expression of hTERT increased the levels of hTERT message in BPH-1 cells over 3-fold (Fig. 2B). hTERT regulates cyclin D1 expression in prostate epithelial cells but not other cyclins Most interestingly, we observed that decreased hTERT expression in DU-145 cells was associated with a significant (p < 0.0002) decrease in cyclin D1 message (Fig. 2A),
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whereas increased hTERT expression in BPH-1 cells upregulated the expression of cyclin D1 significantly (p < 0.006). This modulation of cyclin D1 expression by hTERT is not an isolated event, as we also observed a similar effect on variety of cancer cells such as skin (A431), pancreas (AsPC-1), mammary (MCF7), colon (HT-29), and ovarian (OVCAR-3) cancer cells (data not shown), which are highly telomerase positive (data not shown). Although we observed modulation of cyclin D1 expression in prostate epithelial cells by the hTERT, we did not find any change in the expression levels of other cyclins such as cyclin A1, B1, and E1 (Fig. 2C). These results suggest that the regulation of cyclin D1 by hTERT is specific. hTERT regulates cyclin D1 transcriptional activity in prostate epithelial cells
Fig. 1. hTERT regulates telomerase activity in human prostate epithelial cells. (A) Assessment of telomerase activity in DU-145 cells after transient expression of hTERT shRNA (+) or negative control shRNA () vector for 3 days. (B) Assessment of telomerase activity in BPH-1 cells after transient expression of empty vector () or hTERT expression vector (+) for 3 days. Cell pellets were collected from each experiment and subjected to TRAP assay. NC: negative control using lysis buffer only. Quantitative estimations of telomerase activity in DU-145 and BPH-1 cells were determined by densitometric measurement of TRAP products from three independent experiments. Columns, mean; bars, SE. *p < 0.001 for DU-145 and p < 0.004 for BPH-1, significantly different from control.
Since we observed that hTERT regulates cyclin D1 expression, it was important to know whether the alteration of cyclin D1 expression is transcriptionally regulated. To evaluate this, a full-length cyclin D1 promoter luciferase construct was transfected in DU-145 and BPH-1 cells along with either hTERT-shRNA or hTERT expression plasmids. We observed that transfection of cyclin D1-promoter luciferase plasmid alone increased the cyclin D1 transcriptional activity in DU-145 cells approximately 100-fold compared to the control plasmids (basic-luciferase plasmid). Co-transfection of the negative control (negative control shRNA plasmid) plasmid with the cyclin D1promoter luciferase plasmid did not alter the transcriptional activity of cyclin D1, whereas co-transfection of hTERT shRNA plasmid decreased cyclin D1 transcriptional
Fig. 2. hTERT regulates the expression of cyclin D1. (A,C) DU-145 cells were transiently transfected with hTERT shRNA (+) or negative control shRNA () vector for 3 days. (B) BPH-1 cells were transiently transfected with empty vector () or hTERT expression vector (+) for 3 days. RNA was extracted, and RT-PCR assays were performed to detect hTERT, cyclin D1, and GAPDH mRNAs. Representative photograph from an experiment that was repeated thrice. Quantitative estimations of relative levels of hTERT and cyclin D1 mRNAs (right panels) were determined by densitometric measurements of RT-PCR gels from three independent experiments after normalization with GAPDH. (C) RT-PCR analyses of cyclin A1, cyclin B1, cyclin E1, and GAPDH in DU-145 cells. Columns, mean; bars, SE. *p < 0.001, significantly different from control.
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activity approximately 5-fold (Fig. 3A). Similarly, the transfection of the cyclin D1-promoter luciferase plasmid alone in BPH-1 cells increased the cyclin D1 transcriptional activity approximately 6-fold over the control (basic-luciferase plasmid). Co-transfection of the empty vector control with the cyclin D1-promoter luciferase construct did not change the transcriptional activity of cyclin D1, but the co-transfection of hTERT plasmid increased cyclin D1 transcriptional activity approximately 4-fold over the level with cyclin D1-promoter luciferase alone (Fig. 3B). These results are consistent with the down-regulation and up-regulation of the levels of cyclin D1 mRNAs observed in RTPCR studies with the ectopic expression of hTERTshRNA and hTERT expression vectors in DU-145 and BPH-1 cells (Fig. 2), and re-confirm that hTERT is capable of regulating the transcription of cyclin D1 in these cells. We do not know whether the transcriptional regulation of cyclin D1 by hTERT is a direct or indirect effect and future investigations will be necessary to evaluate this issue. The transcription of the cyclin D1 gene is induced through distinct DNA sequences in the promoter by diverse mitogenic and oncogenic signaling pathways of Ras, Src, Stat3, Stat5, and Erbb2 [18]. Several transcription factors such as CREB, AP-1, and b-catenin/Tcf-1 have been identified to interact with the cyclin D1 promoter [19,20]. It is possible that one or more of these signaling pathways and transcription factors could be involved in the transcriptional regulation of cyclin D1 by hTERT. In this study, we observed that ectopic expression of hTERT shRNA in DU-145 cells down-regulates cyclin D1, but not cyclin A1, B1, and E1. It is interesting to note that some earlier study has shown that IKKa selectively and directly induces cyclin D1 but not cyclin E or cyclin A [21]. This study also demonstrated that the induction of cyclin D1 expression and promoter activity requires b-catenin/Tcf activity. Although we do not know whether Akt and/or IKKa are regulated by hTERT, it is possible
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that these signaling pathways could induce the transcriptional activity of cyclin D1. Detailed future experiments would be necessary to determine the involvement of the Akt, NFjB, and b-catenin signaling pathways in hTERT induced cyclin D1 expression. hTERT regulates cyclin D1 protein in prostate epithelial cells To this point, we have examined the message levels and/ or the transcriptional activity of cyclin D1; however, we do not know whether cyclin D1 protein levels were altered. Therefore, we examined the levels of cyclin D1 protein after down-regulation or up-regulation of hTERT message in prostate epithelial cells. We observed that the transfection of hTERT shRNA in DU-145 cells decreased the protein expression of hTERT as well as cyclin D1 significantly (p < 0.001) (Fig. 4A). On the other hand, a gain-of-function by the transfection of hTERT expression vector increased both hTERT and cyclin D1 proteins significantly (p < 0.01 and 0.005 for hTERT and cyclin D1, respectively) (Fig. 4B). These results suggest that cyclin D1 protein levels are dependent on the transcriptional regulation by hTERT. Since hTERT regulates the levels of cyclin D1 protein, it was important to know where cyclin D1 proteins are localized within the cell because cyclin D1 has to be in the nucleus to regulate cell cycle and/or proliferation. Using double immunolabeling with hTERT and cyclin D1 antibodies, we observed that in control DU-145 cells both telomerase (hTERT) and cyclin D1 are predominantly localized in the nucleus. Whereas, the transfection of hTERT shRNA down-regulated hTERT protein and its nuclear localization substantially. As a result, the levels of cyclin D1 also decreased dramatically and the nuclear localization of cyclin D1 was completely abolished (Fig. 5A). However, minimal staining of cyclin D1 was observed in the cytoplasm of hTERT shRNA transfected DU-145 cells. On
Fig. 3. hTERT regulates transcriptional activity of cyclin D1. (A) DU-145 cells were transfected with 200 ng of full-length cyclin D1 promoter luciferase plasmids (1745-cyclin D1-Luc) or basic luciferase (PA3-Luc) plasmids and 10 ng of Renilla luciferase (pRL-TK-Luc) plasmids with 200 ng of hTERT shRNA or negative control shRNA for 48 h in normal growth media. (B) BPH-1 cells were transfected with 200 ng of full-length cyclin D1 promoter luciferase plasmids or basic luciferase plasmids and 10 ng of Renilla luciferase (pRL-TK-Luc) plasmids with 200 ng of hTERT or empty vector for 48 h in normal growth media. After treatment, cells were harvested, and luciferase assays were performed. Relative cyclin D1 promoter activity was determined after normalization with Renilla luciferase activity. Luciferase activities in basic vector transfected cells were considered as 1.0. Columns, mean of three independent experiments with quadruplicate samples; bars, SE. *p < 0.0005, significantly different from control.
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Fig. 4. hTERT regulates the expression of cyclin D1 protein. (A) DU-145 cells were transiently transfected with hTERT shRNA (+) or negative control shRNA () vector for 3 days. (B) BPH-1 cells were transiently transfected with empty vector () or hTERT expression vector (+) for 3 days. Protein lysates (50 lg) from DU-145 and BPH-1 cells treated with hTERT shRNA or hTERT for 3 days were resolved on 12% SDS–PAGE, and immunoblots were probed with antibodies to hTERT and cyclin D1. All immunoblots were re-probed with b-actin antibodies to ensure equal loading. Representative photographs from an experiment that was repeated thrice. Quantitative analyses of relative levels of hTERT and cyclin D1 proteins are shown on the right panels. Columns, mean of three independent experiments; bars, SE. *p < 0.005, significantly different from control.
the other hand, control BPH cells expressed very little hTERT protein in the nucleus and as a result of the over-expression of hTERT, both hTERT and cyclin D1 staining intensity increased in the nucleus (Fig. 5B). The question might arise whether the constant presence of cyclin D1 would impair cell cycle progression. Interestingly, a published report suggests that in the presence of growth factors cyclin D1 levels remain relatively high and constant through the cell cycle, although a new burst of cyclin D1 synthesis occurs every time cell enters G1 after mitosis [22]. These results suggest that telomerase (hTERT) not only regulates the expression of cyclin D1, but also regulates the levels and localization of cyclin D1. Since the formation of the active complex of cyclin D1/cdk4/6 is important for the nuclear localization of cyclin D1, it is possible that hTERT could have effects on cdk4/6 and that would work along with the transcriptional regulation of cyclin D1. It is too speculative at this point and future experiments would be necessary to determine if hTERT also modulates the expression of cdks and/or its complex formation with cyclin D1. The activation of cyclin D1 gene transcription is dependent on the activation of Ras, Raf, mitogen-activated protein kinase-kinases (MEK1 and MEK2), Akt and the sustained activation of extracellular signal regulated protein kinases (ERKs) [18]. On the other hand, cyclin D1 degradation is mediated by phosphorylation-triggered, ubiquitin-dependent proteolysis [23]. Glycogen synthase kinase 3b (GSK-3b) catalyzes the phosphorylation of cyclin D1 on Thr286 and redirects the protein from the nucleus to the cytoplasm [23]. It would be interesting to investigate whether hTERT regulates the activation of
GSK3b which determines the nuclear localization of cyclin D1. Overexpression of cyclin D1 is a common event in various forms of cancer including prostate cancer [24–26]. The overexpression of cyclin D1 leads to enhanced organ growth in mice [27]. Transient transfection of hepatocytes with cyclin D1 leads to vigorous proliferation and a more than 50% increase in liver mass within 6 days [28]. Conversely, cyclin D1 knockout mice are smaller than wildtype mice, and mice with the homozygous deletion of the p27 gene (which inhibits cyclin D1/Cdk4/6 complexes) show gigantism and enhanced organ size [29]. Moreover, the expression of cyclin D1 modulates invasive ability by increasing matrix metalloproteinase (MMP-2 and MMP-9) activity and motility in glioma cells [30]. Furthermore, some studies show that over-expression of cyclin D1 is associated with metastatic prostate cancer to bone [31]. These findings suggest that in addition to its well-defined role in cell cycle progression, cyclin D1 may also play a role in the regulation of cell growth and metastasis. Therefore, the up-regulation of cyclin D1 by hTERT would allow telomerase to modulate telomere length as well as cell proliferation and/or its invasive potential. In summary, using both loss-of-function and gain-offunction studies, we were able to define a novel function of hTERT that is unrelated to the telomere length regulation and provide additional evidence that telomerase has more than one function in epithelial cells. Therefore, the expression of hTERT could have potentially important biological consequences, particularly in cancer cells, where it can control the stabilization of chromosome ends as well as cell proliferation, survival, and metastasis. On the other
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Fig. 5. hTERT decreases the nuclear localization of cyclin D1. (A) DU-145 cells were plated on chamber slides and transfected with 200 ng of shRNA negative control or hTERT shRNA for 3 days. (B) BPH-1 cells were plated on chamber slides and transfected with 200 ng of empty vector or hTERT expression vector for 3 days. Cells were then fixed in methanol, incubated with hTERT and cyclin D1 antibodies overnight, Alexa Fluor-conjugated secondary antibodies for 1 h, and counter stained with DAPI. Slides were then mounted and examined using a fluorescence microscope. Photographs were taken at the same magnification (20·) and then transported to Photoshop. Representative photographs from an experiment that was repeated twice.
hand, hTERT could be a potential target for chemotherapeutic intervention of a variety of cancers including prostate cancer. Acknowledgments We thank Dr. Richard Pestell (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA) and Dr. Robert Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA) for their generous gifts of the fulllength cyclin D1 promoter luciferase construct and hTERT expression vector, respectively. This work was supported by NIH Grant R01 DK060875 (P.P.B.). References [1] C.W. Greider, E.H. Blackburn, A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis, Nature 337 (1989) 331–337. [2] N.W. Kim, M.A. Piatyszek, K.R. Prowse, et al., Specific association of human telomerase activity with immortal cells and cancer, Science 266 (1994) 2011–2015. [3] C.B. Harley, B. Villeponteau, Telomeres and telomerase in aging and cancer, Curr. Opin. Genet. Dev. 5 (1995) 249–255.
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