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Molecular Biology and Genetics |
Departments of Pathology [A. A., L. W. E. H. L. F., B. A. A., E. D. M., J. L. W., S. E. H.], and Human Genetics [J. L. W., S. E. H.], Massey Cancer Center, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298, and Department of Human Biology/Program in Genomics, The Fred Hutchison Cancer Center, Seattle Washington 98109 [P. S. N.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal somatic cells do not express telomerase activity and, therefore, shorten their telomeres with each cell division. The catalytic subunit of telomerase, hTERT,3 has been cloned (6 , 7) and has been shown to be capable of restoring telomerase activity by expression in normal cells (8 , 9) . Stable expression of telomerase in a variety of normal cells devoid of activity results in maintenance of telomere lengths and extension of cellular life span (10 , 11) , implicating telomere attrition as a major cause of cellular senescence.
Telomerase is a ribonucleoprotein that is minimally comprised of an integral RNA template (hTR) and a reverse transcriptase protein component (hTERT; Refs. 6, 7, 8 , 12 ). The molecular size of the telomerase complex has been determined previously with telomerase from lower eukaryotes (13) and humans (14) . In Euplotes crassus, the size of telomerase complexes varies greatly, depending on levels of activity. In addition, Euplotes data indicate that higher-order complexes are more active than the smallest active complex at about Mr 550,000 (13) , similar to the size suggested for purification of active telomerase from human cells (14) . These data suggest that additional proteins or complexes associated with the telomerase holoenzyme are able to modulate its activity. Although there has been identification of a telomerase-associated protein (TEP-1; Refs. 15 , 16 ), it does not appear required for catalysis nor is its exact telomerase-related function known. However, we have recently identified the hsp90 chaperone complex as regulatory elements necessary for telomerase assembly and function both in vitro and in vivo (17) . The hsp90 chaperone complex has been shown to facilitate the folding of glucocorticoid receptors as well as other reverse transcriptases from viral origins (18, 19, 20, 21) . The hsp90 foldosome is minimally composed of hsp90, p23, hsp70, HOP, and hsp40/ydj (18 , 19) .
Importantly for our study, only about 10% of BPH displays detectable telomerase activity, whereas advanced prostate carcinoma expresses high levels of telomerase (
90%; Ref. 5
). Moreover, those rare BPH specimens with telomerase have significantly lower levels of activity than advanced prostate cancers. Using an experimental system for cancer development consisting of human nontumorigenic, tumorigenic, and metastatic prostate epithelial cells derived from the same lineage (22, 23, 24)
, we have observed a significant increase in telomerase activity levels as immortalized prostate epithelial cells progress from the nontumorigenic to tumorigenic phenotype without any observable change in the expression of the telomerase template RNA (hTR) or the catalytic subunit (hTERT). Because transcriptional regulation remains unchanged for the critical telomerase core components, we investigated the role of the hsp90 chaperone complex, the only known set of proteins to associate functionally with telomerase (17)
. We found that the hsp90-associated chaperones (hsp90, p23, hsp70, and hsp40/ydj) are dramatically increased in parallel with tumorigenic progression, which suggests an enhanced assembly of telomerase as the mechanism for the activity increase. To our knowledge, this is the first report that links the importance of a chaperone-mediated telomerase increase to tumorigenic conversion.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cells were cultured in defined media (RPMI 1640) supplemented as previously described (22, 23, 24)
. DU145 cells were cultured in DMEM with 10% cosmic calf serum (Hyclone, Logan, UT) and 25 µg/ml gentamicin (Life Technologies, Inc., Gaithersburg, MD).
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RT-PCR Analysis.
Total RNA was isolated by guanidinium thiocyanate, and 2.5 µg of total RNA was annealed with short random oligonucleotides (decamers) and retrotranscribed into cDNA using the first-strand synthesis kit RETROscript (Ambion Inc., Austin, TX). PCR reactions were performed using a fraction of the cDNA reaction. Both hTR and hTERT were amplified as described previously (6)
. The amplification of 18S rRNA was performed using a 3:7 mixture of 18S rRNA primer pairs and 18S rRNA Competimers (Ambion) for 23–25 cycles. All of the samples were assayed simultaneously to minimize experimental variation. Amplified products were resolved on a 6% PAGE and visualized by staining with ethidium bromide.
Microarray Fabrication.
A nonredundant set of 1500 prostate-derived cDNA clones were identified from the Prostate Expression Database (PEDB), a public sequence repository of expressed sequence tag (EST) data derived from human prostate cDNA libraries (26)
. Individual clone inserts were amplified by PCR as template and processed as previously described (27)
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Probe Construction and Microarray Hybridization.
Total RNA was isolated from P69 and M12 cells using TRIzol (Life Technologies, Inc.). Fluorescence-labeled probes were made from 1 µg of poly(A)+ RNA or 30 µg of total RNA in a reaction volume of 20 µl containing 1 µl of anchored oligo(dT) primer (Amersham-Pharmacia), 0.05 mM Cy3-dCTP (Amersham-Pharmacia), 0.05 mM dCTP, 0.1 mM each dGTP, dATP, dTTP, and 200 units Superscript II reverse transcriptase (Life Technologies, Inc.). Reactants were incubated at 42°C for 120 min followed by heating to 94°C for 3 min. Unlabeled RNA was hydrolyzed by the addition of 1 µl of 5 N NaOH and heating to 37°C for 10 min, followed by neutralization. After chromatography (Qiagen), 1 µg of dA/dT 12–18 (Amersham-Pharmacia) and 1 µg of human Cot1 DNA (Life Technologies, Inc.) were added to the probe, heat denatured, combined with an equal volume of 2xmicroarray hybridization solution (Amersham-Pharmacia) and prehybridized at 50°C for 1 h. The mixture was then placed onto a microarray slide with a coverslip and hybridized in a humid chamber at 52°C for 16 h. After washing, the slide was rinsed in distilled water to remove trace salts and dried.
Image Acquisition and Data Analysis.
Fluorescence intensities of the immobilized targets were measured using a laser confocal microscope (Molecular Dynamics). Intensity data were integrated at a pixel resolution of 10 µM using 20 pixels per spot, recorded at 16 bits. For each experiment, each cDNA was represented twice on each slide, and the experiments were performed in duplicate producing four data points per cDNA clone per hybridization probe. Intensity ratios for each cDNA clone hybridization with probes derived from P69 and M12 cells were calculated.
Protein Extraction and Immunoblotting.
Cells were harvested at 60–80% confluency using a modified radioimmuno protection assay buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1% aprotinin, and 100 mM DTT]. Lysates were clarified by centrifugation at 14,000 x g for 20 min at 4°C. Five to 25 µg of total cellular protein were subjected to 8–12% SDS-PAGE at 150 V for 1.5 h and transferred to nitrocellulose (Hybond; Amersham-Pharmacia, Buckinghamshire, England) at 100 V for 1 h. Membranes were blocked in PBS with 0.1% Tween 20 (PBS-T) and 5% milk for 1 h, followed by incubation in primary antibody for up to 2 h at room temperature. All of the chaperone antibodies are commercially available from Stressgen (Vancouver, British Columbia) and were used at the following dilutions in PBS-T with 5% milk: hsp90 (AC88, 1:5000); hsp70 (C92F3A-5, 1:5000); p23 (JJ3, 1:5000); hsp27 (G3.1, 1:2000); hsp40/ydj (Ab1, 1:10,000); hsf-1 (SPA-901, 1:10,000); and ß-actin (1:5,000; from Sigma Chemical Co.). Blots were washed and incubated with secondary antibody (1:5,000 for both mouse and rabbit) conjugated with horseradish peroxidase (Amersham) in PBS-T and 5% milk for 1 h at room temperature. After washing, the signal was detected using the SuperSignal Chemiluminescent Substrate kit (Pierce, Rockford, IL), followed by detection using both autoradiography and digital imaging with the ChemiImager 4400 (Alpha Innotech, San Leandro, CA). Because two antibodies can be used simultaneously to probe a given blot with the standard, ß-actin antibody, effective quantitation can be achieved by spot densitometry and determining the ratio of the ß-actin signal to that of the specific band of interest.
In Vitro Reconstitution.
To assemble telomerase, 5 µl of water-sonicated P69 extract was mixed with water, hsp90/p23, hsp90/p23/hsp70/p60/hsp40, or rabbit reticulocyte lysate in a 10-µl assembly assay and incubated at 30°C for 90 min. To assemble telomerase with purified cofactors, the P69 extract as above was mixed with 500 ng of p23, 750 ng of hsp90, 125 ng of hsp70, 25 ng of HOP (p60), and 25 ng of hsp40/ydj-1 in the presence of 10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, and 2 mM DTT (pH 7.5) in a total volume of 10-µl. Of the resulting reconstitution, 2-µl (corresponding to 4,000 cell equivalents) were subjected to the TRAPeze assay.
Immunohistochemistry.
Formalin-fixed, paraffin-embedded tissues from four cases of BPH and 14 cases of prostate carcinoma were immunohistochemically analyzed for hsp90 and p23 expression. Four-µm tissue sections were deparaffinized in xylene, rehydrated in graded alcohols, and washed in PBS. The tissue sections were microwaved in 10 mM citrate buffer solution for antigen retrieval at pH 6.0 in a thermoresistant container for 12 min and cooled for 20 min (28)
. Endogenous peroxidase activity was quenched in 0.3% hydrogen peroxide for 10 min. The sections were then incubated overnight at 4°C with either a p23 (1:3000 dilution), hsp90 (1:100 diluted; Ref. 17
), or cytomegalovirus (negative control) monoclonal antibody (MAb 8120; 1:1000 dilution; Chemicon International, Temecula, CA). Antigen-antibody complexes were visualized using a streptavidin-biotin staining technique (Vector Laboratories, Burlingame, CA) according to the manufacturer’s recommendations. Diaminobenzidine (DAB) was used as a chromogen, and hematoxylin as a counterstain.
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RESULTS |
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). Normal prostate epithelial cells taken from a patient after radical prostatectomy were transfected with SV40 large T antigen and selected on the basis of growth (immortalization; Ref. 22
). As summarized in Fig. 1
, the parental SV40 T antigen-immortalized prostate epithelial cell line, P69SV40Tag, is rarely tumorigenic, but after an extended latent interval, 2 mice of 17 that were given injections did form tumors (M1929 and M2205; 22
). From each of these sporadic tumors, cell lines were established in culture and subsequently reinjected into nude mice. One additional tumor-derived, nonmetastatic cell line was cultured (M2182) and again reinjected for an additional round of in vivo selection to obtain metastatic lines, where the tumorigenic and metastatic cell line, M12, was established (24)
. These cell lines, derived from a single immortalized cell line, provide a system from a defined genetic lineage in which to study the molecular changes that occur during prostate cancer progression.
Telomerase Activity and Telomerase Component Expression Levels in Tumorigenic Prostate Cells.
The levels of telomerase activity in the P69 cell line and its derived tumorigenic and metastatic lines were reproducibly elevated >10-fold in the tumorigenic cell lines in at least three independent experiments (Fig. 2A)
. This dramatic increase in telomerase further indicates the importance of telomerase activation in the transformation process (29)
. There is very little difference in the telomere lengths in these cells, with the P69 cell line having long and heterogeneous telomere lengths, whereas M12 and M2182 have shorter, more homogeneous lengths (data not shown).
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), suggesting a nontranscriptional mechanism for the telomerase increase in this prostate cancer system.
hsp90 Chaperones and Telomerase.
The hsp90 chaperone machinery has been shown to be required for functional assembly of human telomerase (17)
. During the course of microarray analysis of RNA from the P69 and M12 cell lines, the M12 mRNA for hsp90 bound 3-fold higher to hsp90 cDNA targets than that for the P69 mRNA (data not shown). Because there were no substantial differences in the levels of the telomerase components and the microarray data suggested an increase in hsp90 in the tumorigenic line M12, these findings prompted a study of the mechanisms for enhanced telomerase activity other than transcriptional regulation: an increase in the assembly of the telomerase holoenzyme. Consistent with the microarray data, Fig. 3
demonstrates a sizable increase in the expression levels of hsp90 and p23, known telomerase interactors. Relative amounts were determined by comparison with the ß-actin internal control and suggest a 5- to 10-fold increase in hsp90 and p23 levels as cells progress to a tumorigenic state (Fig. 3)
. In addition, we found by Northern analysis that hsp90 mRNA levels were elevated in the tumorigenic cells when compared with the P69 cells (data not shown). To determine the generality of this finding, we tested the DU145 prostate tumor-derived cell line for increased chaperone levels and also found a uniform increase in chaperone levels, consistent with the levels seen among tumorigenic cell lines from P69SV40Tag system (Fig. 3)
. In fact, it appears that the expression of a number of molecular chaperones was dramatically elevated as cell progress to a tumorigenic state (Fig. 4)
. In 2–3 independent experiments, the levels of hsp70 and hsp27, and to a lesser extent hsp40, are increased on the order of 2- to 4-fold in the M2182 and M12 tumorigenic cell lines when compared with the nontumorigenic line P69. In addition, hsf-1 was also substantially enhanced in the tumorigenic cell lines (Fig. 4B)
, suggesting a transcriptional mechanism for the chaperone increase during prostate cancer progression. At this point, it is critical to note the importance of the microarray data, which provided the key evidence to suggest the alternative to telomerase assembly/regulation and the role of chaperones in tumor progression.
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; nevertheless, telomerase activity was not enhanced by the addition of nonspecific protein (data not shown). This suggests that there is a significant amount of unfolded hTERT in the P69 cells, and that supplementing the extract with additional purified chaperones promotes the assembly of active telomerase activity. Taken together, these data indicate that enhanced telomerase assembly, rather than increased expression of telomerase components, is the cause of the elevated telomerase expression in tumorigenic cells.
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, top panels) compared with normal or BPH within the same section (Fig. 6
, bottom panels). For both the BPH and prostate carcinoma sections, control antibodies were negative for staining (data not shown). The antibodies for hsp90 and p23 are highly specific as evidenced by the lack of background observed after Western blotting (see Fig. 3
). Whereas secretory epithelial cells in the hyperplastic glands exhibit only weak cytoplasmic staining for p23, basal cells of the glands are consistently intensely stained (Fig. 6)
. Although telomerase is primarily nuclear and hsp90 and p23 are predominantly cytoplasmic, we observed specific, intense nuclear staining for both hsp90 and p23, which implies that either telomerase assembly occurs in the nucleus or that hsp90 and p23 remain associated with the functional telomerase enzyme. On the basis of our data, we suspect that contrary to other chaperone targets, hsp90 and p23 remain stably bound to active telomerase (30)
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DISCUSSION |
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The hsp90-related molecular chaperones have been shown to be elevated in a variety of cancers (reviewed in Ref. 31 ). We show that hsp90 levels were elevated in the tumorigenic M12 cells compared with the rarely tumorigenic P69 cells by microarray analysis. We also found that a number of chaperone-related proteins, including hsp70, p23, hsp40, hsp27, and hsf-1, are dramatically increased during prostate cancer progression, not only in this experimental system, but in an alternative prostate tumor-derived cell line and in primary prostate cancers as well. Therefore, these data suggested that increased telomerase assembly, rather than expression of telomerase components, occurs during tumorigenic conversion. This was confirmed by enhanced telomerase activity after the addition of purified chaperones in an in vitro P69 reconstitution system, which suggests that a significant portion of the telomerase is unfolded or unassembled because of inadequate chaperone expression. To our knowledge, this is the first report showing an increased chaperone-mediated assembly of telomerase as a mechanism that contributes to tumorigenic conversion.
The increased chaperone levels observed during prostate cancer progression may function not only to assemble telomerase but also to stabilize the hTERT protein and prevent degradation. With some chaperone proteins expressed in limiting amounts, certain other chaperones (specifically hsp70) are capable of targeting chaperone-associated proteins for ubiquitin-mediated degradation (32) . Importantly, we have shown that inactive hTERT, but not active telomerase, is associated with hsp70. Thus, in the absence of adequate hsp90 and/or p23, P69 hTERT may be targeted for hsp70-dependent degradation. Even so, our results indicate that there is a significant fraction of unfolded/inactive hTERT in P69 cells, given the fact that we observe increased telomerase activity after the addition of purified chaperone components using lysates from the low telomerase P69 cells. Together, these data suggest that there is substantial unfolded telomerase in extracted P69 cells and chaperones are the limiting components for increased telomerase assembly and activity.
It is important to distinguish our study from that of others regarding the hsp90, hsp70, and hsf-1 (33) . We show substantial differences in hsp90 and hsp70 expression between nontumorigenic and tumorigenic cells, in which Hoang et al. (33) compare tumorigenic-but-nonmetastatic to metastatic cells and find no changes in hsp90 or hsp70 levels. However, they do find that the transcription factor hsf-1 is elevated in metastatic PC-3 cells, whereas we find no enhancement of hsf-1 between M2182 (tumorigenic) and M12 (metastatic). The reason for this discrepancy in hsf-1 expression levels is unclear but may reflect differences in the culture conditions or the experimental systems themselves.
The hsp90 foldosome is minimally composed of hsp90, p23, hsp70, HOP, and hsp40/ydj (18 , 19) . For glucocorticoid receptors, effective ligand binding requires stabilization of the receptor, and purified chaperone proteins mediate the formation of active receptor, establishing sufficiency for a functional complex. Whereas hsp90 and p23 appear associated with the receptor, hsp70, HOP, and hsp40 remain essential for the ATP-dependent hsp90/p23-receptor association. Purified versions of each of the 5-foldosome proteins were able to mediate the assembly of functional telomerase in vitro (17) . Here, we show that increased assembly of human telomerase in extracts from the nontumorigenic P69 cells is mediated by the addition of purified chaperones. It is important to note that whereas expression of hTERT transcriptionally may not change, the association with chaperones may stabilize the protein and provide for an increased half-life of hTERT, allowing for the enhanced telomerase activity observed during progression. However, because we found that the addition of purified chaperones to a P69 extract enhanced activity, this suggests that chaperone-mediated hTERT stability only partially contributes to the telomerase enhancement observed, and increased assembly of telomerase is occurring.
The question still remains as to how chaperone-mediated enhancement of telomerase contributes to tumorigenesis. One possibility is that the tumorigenic cell lines shorten their telomeres more rapidly than the nontumorigenic cells and maintenance at such a short length (our tumorigenic cells have shorter telomeres than nontumorigenic cells) requires increased telomerase activity, providing cells with the ability to continuously proliferate. Alternatively, it may be possible that the telomerase increase is only an effect of the elevated chaperone levels, and that the chaperone increase contributes to the transformation process. Currently, each of these options is under investigation to understand the role of telomerase and chaperones in tumorigenic transformation and how this interaction contributes to the maintenance of a tumorigenic phenotype.
The mechanism for chaperone increase during prostate cancer transformation may be in part at the transcriptional level, as indicated by some of the data presented here. First, the M12 subline exhibits a reproducible increase in hsp90 mRNA expression using both microarray and Northern analyses. Second, the tumorigenic cells have elevated protein levels of hsf-1, which is known to have binding sites at the promoters of hsp90, hsp70, and hsp27 (34) . Thus, the increase in chaperones and telomerase assembly may be a novel mechanism for prostate cancer transformation that is ultimately mediated by hsf-1. In addition, progression of prostate cancer may require a ubiquitous increase in chaperone levels and protein folding. Because the hsp90 chaperone complex affects a number of cellular targets, it is likely that the overall up-regulation of chaperones during transformation will increase the activity and function of many other signaling and growth factor pathways. Even so, telomerase, as an hsp90 target, remains a critical component in the tumorigenic conversion process.
We have shown in an experimental model of cancer progression, and in prostate carcinoma tissues, that the hsp90-associated chaperone proteins are consistently up-regulated in malignant epithelial cells. Thus, we believe that detection of elevated levels of hsp90/p23/hsp70, as well as telomerase, may be useful for cancer diagnostics. In addition, increased chaperone levels and/or telomerase may have prognostic value in predicting prostate cancer or tumorigenic/metastatic potential in less aggressive disease. Further study into the Gleason’s score and tumor grade will be critical to our understanding of the chaperone/telomerase association during prostate cancer progression.
Hsp90 blocking agents, such as geldanamycin and radicicol, have been touted as anticancer compounds and are currently in clinical trials (35) . Geldanamycin is a benzoquinone ansamycin that blocks the association of p23 by associating with the hsp90 molecule in the ATP binding pocket. Although geldanamycin does not prevent hsp90 from associating with its targets, it does prevent the folding or assembly of those targets that would normally occur in the presence of ATP and p23. Without question, the hsp90 machinery (hsp90 and/or p23) is a potential target for gene- or chemotherapy, especially knowing that hsp90-associated chaperones are up-regulated as cancer cells progress. Because telomerase is a target for hsp90-mediated assembly of functional enzyme, inhibition of hsp90 function would likely inhibit telomerase and potentially prevent cancer progression. We have previously shown that geldanamycin blocks telomerase assembly in vitro and in vivo (17) . However, hsp90 has numerous targets within a given cell, and blanket treatment with hsp90 inhibitors may yield nonspecific effects on both normal and cancer cells. Therefore, inhibition of telomerase assembly as a method for adjuvant cancer therapy using hsp90 as a target needs to be a highly specific and efficient process. Consideration of this type of anti-telomerase therapy suggests that combinational treatments will be most effective and that further investigation into the mechanisms underlying the telomerase assembly process is required.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants RO1-CA 58126 (to J. L. W.) and RO1-DK 52683 (to J. L. W.) and grants from The Jeffress Memorial Trust (S. E. H.) and The Mary Kay Ash Charitable Foundation (to S. E. H.). S. E. H. is a V Foundation Scholar. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Massey Cancer Center, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298. E-mail: seholt{at}hsc.vcu.edu
3 The abbreviations used are: hTR, human telomerase RNA; hTERT, human telomerase reverse transcriptase; hsp, heat shock protein; HOP, hsp organizing protein; hsf, heat shock transcription factor; BPH, benign prostate hyperplasia; RT-PCR, reverse transcription-PCR.
Received 1/11/01. Accepted 4/ 6/01.
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