Carcinogenesis vol.32 no.9 pp.1315–1323, 2011 doi:10.1093/carcin/bgr116 Advance Access publication June 21, 2011
Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells Crystal N.Marconett, Shyam N.Sundar, Min Tseng, Antony S.Tin, Kalvin Q.Tran, Kelly M.Mahuron, Leonard F.Bjeldanes1 and Gary L.Firestone Department of Molecular and Cell Biology and The Cancer Research Laboratory and 1Department of Nutritional Toxicology, 591 LSA, University of California, Berkeley, CA 94720-3200, USA To whom correspondence should be addressed. Tel: þ1 510 642 8319; Fax: þ1 510 643 6791; Email:
[email protected]
Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G1 cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERa) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERa and Sp1 with an estrogen response element–Sp1 composite element within the hTERT promoter. I3C inhibited 17b-estradiol stimulated hTERT expression and stimulated the production of threoninephosphorylated Sp1, which inhibits Sp1–DNA interactions. Exogenous expression of both ERa and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERa- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.
Introduction Telomeres are specialized structures containing (TTAGGG)n repeats that form the protective end-caps of eukaryotic chromosomes (1) and are important for genomic stability by preventing the degradation or fusion of chromosome ends (2). The length of telomere repeats shortens with each cell division because DNA polymerase cannot replicate the ends of double-stranded DNA, and this progressive telomere loss will eventually trigger replicative senescence or aging (3,4). Telomerase, a ribonucleoprotein complex containing reverse transcriptase (RT), counterbalances telomere loss by elongation of the telomeric DNA repeats (5). In normal quiescent adult somatic cells, telomerase is mostly inactive (4,6), and telomerase activity is associated with increased proliferative capacity of certain normal somatic cells and Abbreviations: CDK, cyclin-dependent kinase; DIM, 3,3#-diindolylmethane; DMSO, dimethyl sulfoxide; ERa, estrogen receptor-alpha; ERE, estrogen response element; I3C, indole-3-carbinol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; RT, reverse transcriptase; TBST, Tris-Buffered Saline Tween 20.
has been shown to be sufficient for immortalization of cells (7,8). Elevated levels of telomerase expression and/or activity can be detected in the majority of human cancers (6), which can be highly associated with tumor size, tumor aggressiveness, genomic instability and prognosis of human cancers (9–11). The inhibition of telomerase activity has been shown to enhance responsiveness to the anticancer drugs etoposide and doxorubicin and to radiation therapy in human breast cancer cells (12,13), suggesting that controlling telomerase levels and/or activity may be important for specific classes of anticancer compounds to trigger their antiproliferative responses in human cancer cells. Relatively, little is known about the control of telomerase in human cancer cells by natural plant compounds, which represent a potentially rich source of highly selective anticancer compounds for many cancer types with the potential for favorable therapeutic outcomes with reduced side effects. Indole-3-carbinol (I3C), which is derived by hydrolysis from glycobrassicin produced in cruciferous vegetables of the Brassica genus such as cabbage, broccoli and Brussels sprouts, is a promising anticancer phytochemical (14–18). I3C and its condensation product 3,3#-diindolylmethane (DIM) exhibit potent antitumor effects with negligible levels of toxicity in a wide range of human cancer cells such as lung, liver, colon, melanomas, leukemia, cervical, endometrial, prostate and breast cancers (14–16,19–28). Several of these systems are sensitive to indoles but relatively resistant to conventional therapies (23). Clinical trials have concluded that ingested I3C or DIM have specific anticancer effects in human populations as well as showing beneficial effects on estrogen metabolism and reducing angiogenic estrogen metabolites (29,30). Also, both indoles effect immune cells and/or act as immune modulators that probably contribute to some of their tumor inhibitory effects in vivo (23,31). We and other groups have established that I3C triggers distinct and functionally coordinated sets of transcriptional, cell signaling, enzymatic and metabolic cascades that directly lead to a cell cycle arrest, apoptosis, downregulation of cancer cell migration and metastasis and modulation of hormone receptor signaling (14–16,18–25,27,28,32–45). One conclusion from these studies is that I3C regulates the expression and/ or activity of a variety of transcription factors and cell-signaling components that control the cellular utilization of many critical components of cancer cell proliferative pathways. Conceivably, the I3C cell cycle arrest of indole-sensitive human cancer cells is also associated with the downregulation of cellular telomerase expression and/or activity because high levels of telomerase is considered a growth-promoting factor that directly contributes to the proliferative state of many types of cancer cells (46). Telomerase consists of two critical components, an RNA subunit (hTR) that serves as a template for telomere elongation (47) and a catalytic subunit (hTERT) that provides RT activity (48). hTR is thought to be expressed ubiquitously in all tissues (49), whereas, hTERT messenger RNA expression is highly correlated with telomerase activity and seems to be a limiting determinant of enzyme function (50,51). Regulation of telomerase enzyme expression primarily occurs at the transcriptional level through modulation of hTERT promoter activity. The hTERT gene promoter contains many transcription factor-binding sites including those for estrogen receptor-alpha (ERa), Sp1, Myc, Ets and HIF1 (52,53), which are downstream targets of cellular cascades regulated by growth-promoting factors or antiproliferative compounds. Several of these transcription factors have been shown to be downregulated by I3C in human breast cancer cells, including ERa and the Sp1 transcription factor (34,38). The estrogen response element (ERE) and Sp1-binding sites, in particular the ERE–Sp1 composite element centered around 1105 in the hTERT promoter, are important for basal hTERT expression (54), suggesting that I3C could potentially
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disrupt telomerase activity by downregulating the hTERT promoter activity that is dependent on this composite DNA element. In this study, we demonstrate a role for the I3C downregulation of hTERT expression in the cell cycle arrest of MCF7 estrogen-responsive breast cancer cells, and further demonstrate that this phytochemical mediates this transcriptional effect by disruption of the interactions of both ERa and Sp1 with their composite element within the hTERT promoter. Materials and methods Materials I3C and 17b-estradiol were purchased from Sigma–Aldrich (St Louis, MO). pBABE-hTERT plasmid was a kind gift from Dr Martha Stampfer at Lawrence Berkeley National Laboratory. All media components were purchased from Lonza (Allendale, NJ) and cell culture plates from NUNC-Fischer (Pittsburgh, PA). All other materials were purchased from the specified sources. Cell culture MCF7 human breast cancer cells were obtained from American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco’s Modified Eagle’s Medium from BioWhittaker (Walkersville, MD), supplemented with 10% fetal bovine serum from Mediatech (Manassas, VA), 10 lg/ml insulin, 50 U/ml penicillin, 50 U/ml streptomycin and 2 mM L-glutamine from Sigma (St Louis, MO). Cells were grown to subconfluency in a humidified chamber at 37°C containing 5% CO2. A 200 mM stock stolution of I3C was dissolved in dimethyl sulfoxide (DMSO). I3C was then diluted 1:1000 in media prior to culture plate application. RT–polymerase chain reactions and quantitative polymerase chain reaction Cells were treated with mentioned compounds and harvested in 1 ml of Trireagent (Sigma). RNA was extracted and 1 mg was subjected to RT using random hexamers, followed by polymerase chain reaction (PCR) with the following primers: hTERT forward: 5#-CGGAAGAGTGTCTGGAGCAA-3#, hTERT reverse: 5#-GGATGAAGCGGAGTCTGGA-3#; ERa forward: 5#-AGCACCCAGTGAAGCTACT-3#; hTR forward: 5#-GAAGGGCTGAGGCGC CGTGCTTTTGC-3#, hTR reverse: 5#-GTTTGCTCTAGAATGAACGGTGGAAGG-3#. ERa reverse: 5#-TGAGGCACACAAACTCCT-3#; Sp1 forward: 5#-CACCACAGCTGTCATTTCATCCAT-3#, Sp1 reverse 5#-CCATGGATGA AATGACAGCTGTGGTG-3#; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward 5#-TGAAGGTCGGAGTCAACGGATTTG-3#, GAPDH reverse: 5#-CATGTGGGCCATGAGGTCCACCAC-3#. PCR products were analyzed on 1.2% agarose gel along with 1 kb Plus DNA ladder from Invitrogen (Carlsbad, CA) and the products were visualized with GelRed from Biotium (Hayward, CA). qPCR reactions were subjected to an initial step of 10 min at 95°C to activate the AmpliTaq Gold, followed by 40 cycles consisting of 15 s at 95°C, 45 s at 57°C, 15 s at 70°C. Fluorescence was measured at the end of each elongation step. Data were analyzed using the StepOnePlus RealTime PCR system (Applied Biosystems, Carlsbad, CA), and a threshold cycle value Ct was calculated from the linear phase of each PCR sample. Expression levels of messenger RNAs were calculated and expressed in relative units of SYBR Green fluorescence. Normalization was performed using 2(DDCt) values. Transfections MCF7 cells were transfected with appropriate plasmids (1 mg) using Polyfect transfection reagent (Qiagen, Valencia, CA) as per manufacturer’s instructions in fully serum and antibiotic supplemented media. Twenty-four hours posttransfection cells were exposed to media containing 600 mg/ml G418 sulfate (Gibco, Carlsbad, CA) for selection of stable pools of cells. Empty pBABE-neo plasmid was utilized for the selection controls. Immunoprecipitation After the indicated treatments, immunoprecipitations were performed as described previously (55). Precleared samples were then incubated with 50 lg mouse anti-Sp1 overnight at 4°C. Immunoprecipitated protein was eluted from beads by addition of GLB and heating the sample at 100°C for 5 min. Samples were analyzed by western blot as described previously (38). Western blots After the indicated treatments, western blots were performed as previously indicated (38). Rabbit anti-Sp1 (sc-59), rabbit anti-phospho-threonine (sc-5267) and rabbit anti-hTERT (sc-7212) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and diluted in 1:200 Tris-Buffered Saline Tween 20 (TBST). Hsp90 and actin were used as loading controls. Rabbit anti-actin (#AANO1; Cytoskeleton, Denver, CO) was diluted 1:1000 in TBST and used as a gel-loading control. Hsp90 from BD Transduction laboratories #610419 (Franklin Lakes, NJ)
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was diluted 1:2000 in TBST. Immunoreactive proteins were detected after incubation with horseradish peroxidase-conjugated secondary antibodies diluted 3 104 in 1% nonfat dry milk in TBST. Blots were then treated with enhanced chemiluminescence reagents (Eastman Kodak, Rochester, NY) for visualization on film. Flow cytometry MCF7-neo and MCF7-hTERT cells were seeded on six well plates and treated with 200 lM I3C for 48 h. Cells were then scraped in cold phosphate-buffered saline and spun down. Cells were hypotonically lysed using propidium iodide and subjected to flow cytometry and analyzed as described previously (38). Telomere repeat amplification protocol (TRAP assay) MCF7 cell pellets were subjected to lysis in 300 ll of CHAPS lysis buffer [30 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate] and protein concentration standardization was performed with the BioRad Protein Assay reagent. For each sample, 1.5 lg of cell extract was added to the TRAPeze Telomerase Detection Kit assay (Millipore, Billerica, MA). Telomerase activity was detected by Alexa488 probe binding to specific telomeric sequence, loading was measured by internal sulforophane probe directed to nonspecific DNA sequence, per the TRAPeze protocol. Senescence assay MCF7-neo and MCF7-hTERT cells were grown in six well plates to 50% confluency treated with 200 lM I3C or DMSO vehicle control for 48 h. Cells were then incubated with X-gal-staining solution from the Scenescence Detection Kit (BioVision, Mountain View, CA) overnight. Positive staining cells were visualized using light microscope. Images captured with a 10 megapixel digital camera. Statistical analysis was computed between DMSO and 200 lM I3C treatment with a paired-t-test using Prism v5.0a software (GraphPad Software, La Jolla, CA). Chromatin immunopreciptitation MCF7 cells were grown to 60% subconfluency and treated for 48 h with 200 lM I3C or DMSO vehicle control. Chromatin immunoprecipitations for ERa and Sp1 were performed as described previously (56,57). Primers for ChIP experiments were as follows: p-hTERT forward 5#-GCGTTTGTTAGCATTTCAGTGTTT-3#, p-hTERT reverse 5#-CGGGTTGCTCAAGTTTGGA-3#. These primers frame a 107 bp promoter fragment spanning from 1177 to 1071 upstream of the transcription start site, that includes an ERE–Sp1 composite element, centered at 1105 (reference: University of California, Santa Cruz, genome browser hg18 build). PCR products were visualized on a 1.5% agarose gel buffered with tris base, boric acid, EDTA.
Results I3C downregulation of hTERT transcript expression The potential effects of I3C on the expression of hTERT and hTR transcripts were analyzed in human MCF7 breast cancer cells, which represent an estrogen-responsive early stage cancer type that is highly responsive to the antiproliferative effects of I3C (38,39,58) and express relatively high levels of telomerase (59). Total RNA was isolated from MCF7 cells treated for 48 h over a range of concentrations up to 250 lM I3C, and the levels of hTERT and hTR transcripts assessed by RT–PCR analysis. As shown in Figure 1A, I3C selectively downregulated the level of hTERT transcripts without altering hTR expression. Consistent with our previous studies (34,38,39,58), I3C downregulated the transcript levels of ERa, the ERa target gene progesterone receptor, and the G1 acting cyclin-dependent kinase-6 (CDK6), which occurred at approximately the same dose response as that observed for the effects on hTERT expression (Figure 1A). The levels of CDK4 transcripts and of GAPDH transcripts, which was used as a gel loading control, remained unchanged in I3C-treated cells. The effects of I3C on transcript expression of hTERT, hTR, CDK6, CDK4, ERa and progesterone receptor were quantified by qPCR (Figure 1A, right panel). The results show the strong I3C downregulation of hTERT, CDK6, ERa and progesterone receptor transcipts, whereas, the levels of hTR and CDK4 remained constant. The mechanistic connection between the downregulation of ERa and CDK6 transcripts and of hTERT transcripts is described in later sections. Throughout the same I3C concentration range, the effects of I3C on the number of cells undergoing G1 cell cycle arrest was analyzed by flow cytometery of propidium iodide-stained nuclei. As shown in the
I3C disruption of telomerase promoter activity
Fig. 1. I3C inhibited expression of hTERT correlates with the indole-mediated cell cycle arrest of human breast cancer cells. (A) Hormone-sensitive MCF7 human breast cancer cells were treated with the indicated doses of I3C or the DMSO vehicle control for 48 h. Expression of hTERT, hTR, CDK6, CDK4, ERa and progesterone receptor (PR) transcripts was determined by RT–PCR analysis of total isolated RNA (left panel). The PCR products were visualized on a 1% agarose gel stained with ethidium bromide. GAPDH provided a gel-loading control for the RT–PCR (left panel). Expression of the indicated gene transcripts was also quantified by qPCR (right panel). qPCR data are expressed as fold change after normalization to GAPDH for each treatment and to DMSO for comparative purposes. In a parallel experiment over the same indole concentrations, the I3C-induced G1 cell cycle arrest was analyzed by flow cytometry of propidium stained nuclei. At each I3C concentration, the number of cells with a G1 phase DNA content was plotted in comparison with the densitometric analysis of hTERT and hTR transcript levels. (B) MCF7 cells were treated with DMSO vehicle control, 200 lM I3C, 50 lM of the I3C dimer DIM, 200 lM of the inactive indole tryptophol, 1 lM tamoxifen or 1 nM fulvestrant for 48 h and the levels of hTERT and hTR transcripts were determined by RT–PCR analysis of total isolated RNA. The cell cycle arrest in each treatment condition was monitored by flow cytometry as described above. The G1 arrest of at least 70% of the cells compared with the 55% of cells with a G1 DNA content in control growing cells was considered positive for the cell cycle arrest.
Figure 1A graph, the dose-dependent loss of hTERT transcripts was highly correlated with the dose-dependent increase in G1 phase arrested breast cancer cells. The half maximal response was 125 lM I3C for both the downregulation of hTERT transcripts and the increase in G1 phase arrested cells, and the maximal responses were observed by 200 lM I3C. In order to determine whether the I3C downregulation of hTERT expression is linked to the indole-mediated cell cycle arrest, or is simply an indirect consequence of the loss of cell growth, MCF7 cells were treated with several indole and/or non-indole antiproliferative compounds and levels of hTERT and hTR transcripts analyzed by RT– PCR of total isolated RNA. Tamoxifen is a selective ER modulator that binds to ERa and has estrogen-antagonistic effect in breast tissue (60), whereas, fulvestrant is an ERa antagonist that increases ERa degradation (61). Both compounds induce a G1 cell cycle arrest of MCF7 human breast cancer cells cultured in full medium that contains
estrogen (56,58). Therefore, MCF7 cells were treated with the DMSO vehicle control, 200 lM I3C, 50 lM of the I3C dimer DIM, 200 lM of the inactive indole tryptophol, 1 lM tamoxifen or 1 nM fulvestrant for 48 h. After treatment, one set of cells was subjected to RT–PCR to examine gene expression, and another set of cells was concurrently analyzed by flow cytometery of propidium iodide-stained nuclei for cell cycle analyses. As shown in Figure 1B, I3C and its dimeric condensation product DIM substantially downregulated hTERT transcript levels compared with the DMSO vehicle control, whereas tryptophol, tamoxifen and fulvestrant did not affect hTERT levels. None of the treatment conditions affected hTR transcript expression. Flow cytometry analyses revealed that I3C, DIM, tamoxifen and fulvestrant induced a G1 cell cycle arrest, causing the accumulation of 70% or more cells with a G1 DNA content. Importantly, even though both tamoxifen and fulvestrant induced a G1 cell cycle arrest, neither compound triggered the loss of hTERT transcripts. Therefore, we
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conclude that the I3C downregulation of hTERT expression is not an indirect consequence of the respective G1 cell cycle arrests, but rather a specific downstream target of indole-regulated pathways. Exogenous expression of hTERT prevents the I3C-dependent G1 cell cycle arrest of MCF7 breast cancer cells To functionally test whether the I3C downregulation of hTERT expression directly contributes to the I3C-dependent growth arrest, we examined whether exogenously expressed hTERT driven by a constitutive promoter can override the I3C-induced G1 cell cycle arrest. MCF7 breast cancer cell populations were stably transfected with the pBABE-hTERT expression vector (forming MCF7-hTERT cells) or the pBABE-neo vector control (forming MCF7-neo cells). Western blots demonstrated that significantly higher levels of hTERT protein are expressed in MCF7-hTERT cells compared with the MCF7-neo cells relative to Hsp90 protein levels (Figure 2A). The resultant MCF7-hTERT and MCF7-neo cells were treated with or without 200 lM I3C for 48 h, hypotonically lysed and the cell cycle phase of the propidium iodide stained nuclear DNA was quantified by flow cytometry. Consistent with our previous results using untransfected MCF7 cells (27,28,34,58), in control MCF7-neo cells, I3C induced a G1 cell cycle arrest in that the percentage of cells with a G1 DNA content increased from 51–76% and the number of cells in S phase
Fig. 2. Exogenous expression of hTERT overcomes I3C-dependent G1 cell cycle arrest of MCF7 cells. MCF7 cells were stably transfected with a hTERT expression vector (forming MCF7-hTERT cells or with the neomycin empty expression vector (forming MCF7-neo cells). Western blots of total cell extracts were probed with hTERT antibodies or with hsp90 antibodies as a gel-loading control (top panel). MCF7-neo and MCF7hTERT cells were treated with 200 lM I3C or with the DMSO vehicle control for 48 h. Cells were hypotonically lysed and stained with propidium iodide prior to analysis by coulter cell counter. Cell count versus PI staining is displayed (n 5 10 000) per treatment. Cell cycle phase analyzed using Win-MultiCycle software. All analysis was performed in triplicate.
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decreased from 42 to 14% (Figure 2B, upper panels). In contrast, ectopic expression of hTERT prevented I3C from growth arresting the human breast cancer cells. In the absence of added indole, MCF7hTERT cells displayed an overall shift in the cell cycle distribution of proliferating cells, as evidenced by an increased number of S phase cells and reduced G1 phase cells compared with the MCF7-neo cells (Figure 2B, left panels). Importantly, the number of MCF7-hTERT cells in each phase of cell cycle was unchanged in either the presence or absence of I3C (Figure 2B, lower panels), showing that these cells are resistant to the antiproliferative effects of I3C. Thus, exogenous hTERT expression prevented the I3C-dependent G1 cell cycle arrest of human breast cancer cells, which implicates a direct role for the downregulation of hTERT in the antiproliferative response of this indole. I3C inhibition of telomerase activity is rescued by exogenous expression of hTERT The telomerase ribonucleoprotein complex is ascribed a variety of cellular functions, including blocking cellular senescence as well as extending telomeric repeats to chromosome ends (3–5). These parameters of telomerase activity were analyzed in MCF7-hTERT and MCF7-neo cells to functionally characterize cellular consequences of the I3C downregulation of hTERT expression. Cells were treated with or without I3C for 48 h and the level of telomerase activity accessed using the telomeric repeat amplification protocol (TRAP assay). As shown in Figure 3A, consistent with the downregulation of hTERT expression, I3C decreased telomerase activity in vector transfected control MCF7-neo cells. In contrast, in MCF7-hTERT cells that express exogenous hTERT, the total level of telomerase activity was overall higher and also resistant to the I3C downregulation of telomerase activity. In order to determine if I3C induces cellular senescence through the loss of hTERT expression, MCF7-neo and MCF7-hTERT cells were treated with or without 200 lM I3C for 48 h, followed by incubation with X-gal-staining reagent to measure the amount of b-galactosidase, which is a marker of cellular senescence. As shown in Figure 3B, treatment of MCF7-neo cells with I3C significantly increased b-gal staining (P 5 0.0225). The I3C stimulation of cellular senescence is consistent with a loss of telomerase activity within these cells. In contrast, ectopic expression of hTERT strongly reduced overall b-gal staining and prevented the I3C-dependent increase in b-gal staining and cellular senescence. Taken together, our results demonstrate that I3C downregulation of hTERT expression is the primary mechanism by which I3C inhibits telomerase activity in MCF7 human breast cancer cells. Kinetics of the I3C downregulation of hTERT transcripts in comparison with ERa and CDK6 transcripts To begin to assess the mechanism of I3C downregulation of hTERT transcripts, the kinetics of hTERT transcript regulation was compared with the kinetics of known targets of indole downregulation, specifically ERa and CDK6 transcripts. MCF7 cells were treated with or without 200 lM I3C over a 72 h time course, and hTERT, hTR, ERa and CDK6 transcript levels were analyzed by RT–PCR. As shown in Figure 4A (left panel), I3C rapidly downregulated hTERT transcripts, which was detectable by 12 h of I3C exposure and occurred well before the 48 h of indole treatment needed for a complete G1 cell cycle arrest (28,39). By 24 h, I3C induced a near maximal inhibition of hTERT transcript levels, whereas the levels of hTR messenger RNA was not affected throughout the entire duration of indole treatment. The kinetics of the effects of I3C on expression of hTERT, hTR, CDK6 and ERa transcripts was quantified by qPCR, As shown in Figure 4A (right panel), I3C downregulated ERa and CDK6 transcripts with a more rapid response compared with hTERT expression in that by 12 h of indole exposure, the loss of both transcripts was of a greater magnitude than hTERT. These kinetic results suggest that the I3C control of ERa and CDK6 expression may be mechanistically linked to the disruption of hTERT expression.
I3C disruption of telomerase promoter activity
Fig. 3. I3C-dependent inhibition of telomerase activity is rescued by exogenous expression of hTERT. (A) MCF7-neo and MCF7-hTERT cells were treated with 200 lM I3C or with the DMSO vehicle control for 48 h and then subjected to cellular lysis using CHAPS lysis buffer. Telomeric repeat DNA extention from template DNA was measured using fluorescent probes (telomere extension 5 increased fluorescence) as indicated in Millipore TRAP assay kit. Internal sulforophane probe to nonspecific DNA sequence served as loading control. Analysis was performed in triplicate. (B) MCF7neo and MCF7-hTERT cells were treated with 200 lM I3C or the DMSO vehicle control for 48 h. Cells were incubated with senescence detection reagent (X-gal solution) to measure internal b-galactosidase expression. Analysis was performed in triplicate. Positive staining cells were visualized using light microscope. Images captured with 10 megapixel digital camera. Prism software was used to perform a paired t-test to determine significance of difference between treated and untreated cells (P 5 0.0225).
I3C disrupts binding of endogenous ERa and Sp1 to a composite element within the hTERT promoter and alters estrogen-responsive hTERT expression and Sp1 phosphorylation We previously established in breast cancer cells that I3C inhibits CDK6 expression by disruption of Sp1 interactions with an Sp1– ETS composite element in the CDK6 promoter (34) and that I3C triggers the degradation of the ERa protein, leading to a rapid disruption of a positive transcriptional regulatory loop that causes the downregulation of ERa transcripts (39). The hTERT promoter contains a critical ERa–Sp1 composite element at 1094 to 1117 that controls expression of hTERT transcripts in hormone-sensitive human cancer cells (54), suggesting that the I3C downregulation of hTERT expression in MCF7 cells may be due to the loss of endogenous ERa and Sp1 interactions with this composite element. ChIP analysis was used to directly test this possibility. MCF7 cells were treated with 200 lM I3C for 48 h, and the genomic fragments cross-linked to protein were immunoprecipitated with anti-ERa or anti-Sp1 antibodies or with an IgG control antibody. Primers specific to the hTERT promoter revealed that I3C disrupted endogenous binding of both ERa and Sp1
to the ERE–Sp1 composite element (Figure 4B) as shown by both RT– PCR (upper panel) and qPCR (lower panel). These results implicate the loss of endogenous ERa and Sp1 interactions with the hTERT promoter accounts for the I3C downregulation of hTERT expression. Telomerase levels have been shown to be estrogen responsive in several types of ERa-expressing cancer cells (50,54,62), and the I3C inhibition of ERa binding to the hTERT promoter predicts that this indole should disrupt the estrogen-dependent hTERT expression in MCF7 cells. In addition, I3C downregulation of hTERT protein expression was also observed in different hormone-sensitive human breast cancer cell line, T47D (Figure 4C, upper panel). Taken together, estrogen signaling is implicated in the regulation of hTERT expression in hormone-sensitive breast cancer. To test this possibility, cells were cultured in steroid-deficient medium and treated with or without 10 nM b-estradiol (E2) for 24 h in the presence or absence of 200 lM I3C, and hTERT transcript levels determined by RT–PCR analysis. As shown in Figure 4C, E2 stimulated hTERT transcript levels showing that this gene is steroid inducible in estrogen-responsive MCF7 cells. Treatment with I3C concomitantly with E2 administration abrogated the E2-dependent stimulation of hTERT expression (Figure 4C, lower panel). These results suggest that the I3C-mediated loss of ERa expression accounts for the loss of estrogen-responsive hTERT expression. We previously demonstrated that I3C does not alter Sp1 expression or localization (34); however, the effects of this indole on the Sp1 phosphorylation status have not been established. In order to determine if the phosphorylation state of Sp1 is altered in the presence of I3C, Sp1 protein was immunoprecipitated and subsequently analyzed by western blot for total Sp1 levels as well as threonine-phosphophorylated Sp1 from I3C treated and untreated MCF7 cells. As shown in Figure 4D, I3C treatment caused a significant increase in threoninephosphorylated Sp1 but had no effect on total Sp1 levels. Phosphorylation of Sp1 at Thr-579, which is located within the DNA-binding domain, is known to disrupt the Sp1–DNA interaction (63) and probably accounts for the loss of interactions of the transcription factor with the hTERT promoter. I3C failed to alter phospho-serine or phospho-tyrosine levels of Sp1 (CNM, unpublished data). Exogenous expression of a combination of ERa and Sp1 rescues the I3C downregulation of hTERT expression Combinations of ERa and/or Sp1 were ectopically expressed in MCF7 cells in order to functionally determine whether the I3C downregulation of these transcription factors, critical to the hTERT promoter, was involved in the I3C-dependent ablation of hTERT expression. MCF7 cells were transiently transfected with CMV-driven expression vectors for ERa and/or Sp1, as well as with the CMV-neo transcription control vector, and the level of hTERT transcripts examined in 48 h I3C treated and untreated cells by RT–PCR analyses of total isolated RNA. As shown in Figure 5, MCF7 cells transfected with the CMV-ERa expression vector prevented the I3C downregulation of ERa transcript levels, whereas, cells transfected with the CMV-Sp1 expression vector displayed high levels of Sp1 transcripts in the presence or absence of indole. I3C efficiently downregulated ERa transcript levels in cells expressing exogenous Sp1 showing that exogenous Sp1 per se does not alter the basal or I3C-regulated expression of ERa. Co-expression of both ERa and Sp1 completely rescued the I3C downregulation of hTERT transcripts, whereas, ectopic expression of either ERa or Sp1 alone had no effect on the I3C downregulation of hTERT expression (Figure 5). Therefore, the I3C disrupted interactions of both ERa and Sp1 with the hTERT promoter is required for the indole downregulation of hTERT gene expression. Discussion I3C mediates its anticancer effects by triggering complementary sets of cellular antiproliferative cascades that lead to a cell cycle arrest in indole-sensitive human cancer cells (14–18,42,64). Our results establish that I3C downregulated expression of the hTERT component of the telomerase ribonucleoprotein complex, which significantly
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Fig. 4. I3C inhibited constitutive and estrogen regulated hTERT expression, I3C disruption of endogenous interactions of ERa and Sp1 with the hTERT promoter and indole stimulated Sp1 phosphorylation. (A) MCF7 cells were treated with 200 lM I3C or DMSO vehicle control for a 72 h time course. hTERT, hTR, CDK6 and ERa transcript expression was determined at the indicated time points by RT–PCR analysis of total isolated RNA. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide (left panel). GAPDH provided a gel-loading control for the RT–PCR. Expression of the indicated genes over the 72 h time course was also quantified by qPCR (right panel). qPCR data are expressed as fold change after normalization to GAPDH for each treatment and to T 5 0 h for comparative purposes. (B) ChIP was employed to characterize endogenous ERa and Sp1 interactions with the ERE–Sp1 composite element within the hTERT promoter. Chromatin was isolated from MCF7 cells treated with or without 200 lM I3C for 48 h. ERa or Sp1 was immunoprecipitated from total cell extracts using Sepharose G bound to either anti-ERa antibody or anti-Sp1 antibody and DNA was amplified using the oligonucleotide primers defined in the Materials and Methods (top panel). Input samples represent total genomic DNA from each treatment (loading control). This result was repeated twice. Isolated chromatin from an independent experiment using the same antibodies and conditions was quantified using qPCR at the same loci (bottom panel). qPCR data were normalized to input levels and is expressed as fold change from DMSO. (C) T47D cells were treated with 200 lM I3C or DMSO control for 48 h in full media. Western blots of total cell extracts were probed with hTERT antibody and with ACTIN antibody as a gel-loading control (top panel). MCF7 cells were grown in steroid-deficient media for 24 h and then treated with the indicated combinations of 200 lM I3C and 10 nM b-2-estradiol (E2). hTERT transcript expression was determined by RT– PCR analysis and GAPDH provided a gel loading control for the RT–PCR (lower panel). (D) MCF7 cells were treated with the 200 lM I3C or with the DMSO vehicle control for 48 h. Total cell extracts were immunoprecipitated with mouse anti-Sp1 antibody and electrophoretically fractionated samples blotted with either rabbit anti-Sp1 or rabbit anti-phospho-threonine antibodies (Sp1-THRP).
reduced cellular telomerase activity and is functionally linked to the indole cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. Ecoptic expression of hTERT from a constitutive promoter overrides the I3C cell cycle arrest and downregulation of telomerase activity in MCF7 cells, and constitutive expression of both ERa and
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Sp1 transcriptional regulators rescues the I3C downregulation of hTERT transcript expression. We propose that the combined I3C-mediated inhibition of ERa expression and stimulation of Sp1 threonine phosphorylation, which strongly attenuates Sp1–DNA interaction (63), prevents the interactions of both transcription factors
I3C disruption of telomerase promoter activity
Fig. 5. I3C disrupts endogenous binding of ERa and Sp1 at a critical regulatory site within the hTERT promoter. MCF7 cells were transfected with CMV-ERa, CMV-Sp1, a combination of CMV-ERa and CMV-Sp1, or with the CMV-Neo vector control and treated with or without 200 lM I3C for 48 h. Total RNA was collected and the level of ERa, Sp1 and hTERT transcripts determined by RT–PCR analysis. GAPDH was used as total RNA loading control. PCR products were visualized on a 1% agarose gel stained with ethidium bromide. Result was repeated twice, representative gel shown.
with an ERE–Sp1 composite element in the hTERT promoter. We previously established that I3C strongly inhibited ERa expression by stimulating the ubiquitin-mediated degradation of ERa that leads to the disruption of a positive GATA3-ERa transcriptional cross regulatory loop (39), although no information was previously available on the indole control of Sp1 phosphorylation. ERa and Sp1 form a heterodimer that interacts with the ERE–Sp1 composite elements to regulate target gene promoters (65), potentially including the hTERT promoter in which one such ERE–Sp1 composite element maintains basal transcription of this gene in hormone-sensitive cancer cells (54). Consistent with this mechanism, ectopic expression of either Sp1 or ERa did not alter the I3C downregulation of hTERT expression, suggesting that disrupting the accessibility of both transcription factors is required for the indole downregulation of hTERT expression, however, we cannot preclude the potential involvement of other transcription factors in this processes. We plan to resolve this issue once we are able to functionally assess the effects of I3C on hTERT promoter activity. The I3C regulated transcriptional signaling events that target the hTERT promoter also regulates CDK6 expression and other Sp1 and ERa target genes (34,38,39). Thus, the indole control of hTERT expression could be directly linked to transcriptional regulatory cascades of other critical cellular factors involved in the control of human breast cancer cell proliferation. hTERT expression is regulated by a several transcriptional downstream effectors of proliferative pathways in human cancer cells, and transcription factors that have been shown in different cell systems to interact with the hTERT promoter include ERa, Sp1, myc, Ets and HIF1 (52–54). Estrogens have been shown to increase hTERT expression in other cancer cell systems (50,54,62) and consistent with the interactions of ERa with hTERT promoter, we have observed that hTERT expression is estrogen responsive in MCF7 cells. Our results demonstrated that the combined loss of ERa expression and an altered Sp1 phosphorylation state mediates the I3C downregulation of hTERT expression by disrupting the accessibility and function of two key transcriptional regulators acting on the hTERT gene promoter. In addition to the direct activation of ERa, estrogens can also indirectly regulate hTERT expression by induction of c-Myc expression (54). There are several Sp1 sites in the hTERT promoter (52,53), suggesting that this transcription factor may regulate hTERT expression at sites beyond the ERE–Sp1 composite element. Sp1 phosphorylation at Thr 579 has been previously demonstrated as a critical regulatory mechanism of Sp1 inactivation, as Thr 579 is located within the DNA-binding domain and phosphorylation on this reside is able to disrupt DNA binding (63). Therefore, the I3C stimulated phosphorylation of Sp1 at threonine residues may be responsible for
the disruption of Sp1 binding to the ERE–Sp1 composite element within the ERa promoter as well as to other Sp1 sites in the hTERT promoter. Given the overall transcriptional program regulated by I3C and the importance of downregulating hTERT expression for the cell cycle arrest, it is conceivable that this indole controls hTERT expression at multiple transcriptional sites. Telomerase enzymatic activity is critical to the maintenance of proliferative potential in hormone-sensitive human reproductive cancers. Therapeutic strategies are being developed that disrupt telomerase activity, such as an antisense-based compound that selectively targets and degrades the hTR RNA by formation of double-stranded RNA (66,67). Another avenue being explored is viral introduction of dominant-negative forms of hTERT protein to complex with the available hTR RNA and prevent the ribonucleoprotein complex activity (68). Other strategies include the development of small molecule inhibitors, the most promising of which is 3#-azido-3#deoxythymidine to inhibit RNA–DNA complex formation (69). However, each of these approaches has drawbacks relating to delivery mode, off target effects and nonspecific cytotoxic effects, respectively. The development of chemotherapeutics with reliable activity, target tissue specificity and reduced side effects is critical to the advancement of therapeutic options targeting telomerase components. We propose that the I3C downregulation of endogenous hTERT expression by the targeted disruption of transcription factor interactions with the hTERT promoter represents a new potential therapeutic strategy based on the small molecule inhibition of cellular telomerase activity. In this regard, we have recently observed that 1-benzyl-I3C is the most potent I3C derivative developed to date, which is stable in breast cancer cells treated with this indole carbinol derivative (57), inhibited hTERT expression in MCF7 cells (CNM, unpublished data). Therefore, this I3C derivative is a potential starting point to develop telomerase-targeted therapeutics for indole-sensitive cancer cells. Another intriguing issue will be to examine the effects of combinations of indoles and other therapeutics that target telomerase for their potential effectiveness as therapeutics. We have identified elastase as the only currently documented I3C target protein that can trigger the indole cell cycle arrest of human cancer cells, such as breast cancer cells, that express relatively high levels of this enzyme (42). The I3C inhibition of elastase enzymatic activity leads to the loss of cyclin E protein processing (42) and the inhibition of nuclear factor-kappaB transcriptional activity by the loss of processing of the CD40 member of the tumor necrosis factor receptor gene family (64). We also showed that I3C induces ERa protein degradation in the process that requires the aryl hydrocarbon receptor (39), which could conceivably directly interact with I3C. We have proposed that I3C mediates its pleiotropic anti-proliferative responses through simultaneous actions of multiple target proteins expressed in a given cancer cell type. An important future direction will be to determine which indole target protein(s) triggers the loss of ERa expression and stimulation in Sp1 threonine phosphorylation that directly leads to the downregulation of hTERT gene expression and cellular telomerase activity. Funding NIH Public Service grant CA102360 awarded from the National Cancer Institute. C.N.M. was supported by a dissertation fellowship from the California Breast Cancer Research Program (#13GB-1801). Conflict of Interest Statement: None declared.
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