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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4280-4286
Copyright © 2003 by The Endocrine Society

Expression Profile of the Telomeric Complex Discriminates between Benign and Malignant Pheochromocytoma

Carsten Boltze, Jochen Mundschenk, Nicole Unger, Regine Schneider-Stock, Brigitte Peters, Christian Mawrin, Cuong Hoang-Vu, Albert Roessner and Hendrik Lehnert

Departments of Pathology (C.B., R.S.-S., A.R.), Endocrinology and Metabolism (J.M., N.U., H.L.), Biometrics (B.P.), and Neuropathology (C.M.), Otto von Guericke University, D-39120 Magdeburg, Germany; and Clinic of General Surgery, Martin Luther University (C.H.-V.), D-06097 Halle-Wittenberg, Germany

Address all correspondence and requests for reprints to: Carsten Boltze, M.D., Department of Pathology, Otto von Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany. E-mail: carsten.boltze{at}medizin.uni-magdeburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Telomerase, a ribonucleoprotein complex that includes the telomerase RNA component, the telomerase-associated protein (TP1), the telomerase catalytic subunit (hTERT), and the heat shock protein 90 (HSP90), is closely related to the malignant potential of human tumors. In pheochromocytomas (PC) it is very difficult to predict malignant potential by conventional histology or immunohistochemical and molecular markers. To test whether the expression of telomerase subunits is reflected in the malignant transition of PCs, we determined their mRNA and/or protein expression in 28 benign and nine malignant PCs and compared the results with telomerase activity. RT-PCR analysis revealed that TP1 was ubiquitously expressed. The telomerase RNA component was found in all malignant (100%) and in 13 of 28 (46%) benign PCs. In contrast, hTERT was clearly associated with aggressive biological behavior. All of the malignant (100%), but only two of 28 benign (7%) PCs expressed hTERT. HSP90 was increased in malignant PCs, but was also expressed at a lower level in benign tumors. High telomerase activity was measurable in hTERT-positive tissues only. Our data indicate that hTERT, HSP90, and telomerase activity are up-regulated in malignant cells of the adrenal medulla. The common expression of hTERT and telomerase activity thus represents an additional prognostic marker that may identify more aggressive tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DISCRIMINATION BETWEEN benign and malignant pheochromocytoma (PC) and, in particular, prediction of malignancy remain highly difficult tasks. With the exception of the demonstration of distant metastases, there are no clear-cut criteria that distinguish between benign and malignant tumors in the adrenal medulla. Both capsular invasion and focal vascular invasion are insufficient criteria, because both may be observed in benign PCs (BP) as well. According to Linnoila et al. (1), the following features are more frequent in malignant PC (MP): male predominance, extraadrenal location, greater tumor weight, confluent tumor necrosis, and the presence of vascular invasion and/or extensive local invasion. Hyaline cytoplasmic globules were found in 59% of BP and 32% of MP. Logistic regression analysis parameters revealed that extraadrenal location, coarse nodularity of the primary tumor, confluent tumor necrosis, and absence of cytoplasmic hyaline globules were most predictive of malignancy. Similarly, employing immunohistochemical or molecular markers still remains an inconclusive means of predicting malignant behavior. To date, only the Ki-67 labeling index has been proven important (2). The expression of cathepsin B, cathepsin D, type IV collagenase, basic fibroblast growth factor, or Bcl-2 was not informative (2). There is some evidence that the expression of cyclooxygenase (COX) may be of relevance; a malignant PC (MP) showed enhanced COX2 expression (3). Thus, the diagnostic dilemma of determining the nature of PC still needs to be resolved. This is particularly important, because approximately 10–15% of PCs are probably malignant (4).

Complex mechanisms regulating cellular life span have evolved in mammalian cells. Normal cells demonstrate a strictly limited growth potential and senescence after a defined number of cell divisions. In contrast, tumor cells often exhibit an apparently unlimited proliferation potential and are termed immortalized. It has been proposed that the progressive shortening of the ends of the eukaryotic chromosomes, the telomeres, is an important component of senescence and is involved in the control of the cell cycle. Telomeres are specialized nucleoprotein structures that maintain chromosomal stability by protecting ends from exonuclease and ligase digestion or illegitimate recombination. In addition, they ensure the complete chromosomal replication and proper segregation (5). Telomeres contain telomere-binding proteins and short guanine-rich sequences. In humans, the highly evolutionary conserved telomeric DNA is composed of the hexanucleotide tandem repeat 5'-TTAGGG-3' (6). The conventional DNA replication machinery cannot copy extreme terminal sequences of the lagging strand during replication of linear chromosomes; thus, 50–200 bp of telomeric DNA will be lost during each cell division. This problem is remedied by the production of telomerase, a ribonucleoprotein enzyme complex that synthesizes and maintains telomeric DNA by adding the hexanucleotide repeat units on the 3' ends of the single-stranded DNA (7).

Telomerases consist of several protein components and an RNA subunit. The intrinsic RNA component of telomerase complex (hTR) with a length of 400 nucleotides has been cloned from several species, including humans (8). The hTRs display low sequence homology, but share a conserved predicted secondary structure, consisting of a stem, a pseudoknot, and a set of stem-loop structures. The expression of hTR is no predictor of telomerase activity, because hTR is expressed in all cells (9).

The catalytic subunit of the telomerase complex is the human homolog of the yeast Saccharomyces cerevisiae gene EST2, a specialized reverse transcriptase that relies on an associated RNA to provide a template for the synthesis of DNA repeats. Accordingly, it is designated human telomerase reverse transcriptase (hTERT), also known as hTRT, hTCS1, TP2, and hEST2 (10). Human TERTs are large proteins (103–134 kDa). In contrast to conventional reverse transcriptases, which can copy long stretches of DNA or RNA, the polymerization activity of hTERTs is somehow restricted to copying a template of short RNA repeats. The heat shock protein 90 (HSP90) has been demonstrated to bind to hTERT and is considered a telomerase subunit (11). Geldanamycin, an HSP90 inhibitor, has been found to reduce the activity of reconstituted telomerase in cell extracts, demonstrating the role of HSP90 in the holoenzyme complex (12).

Besides hTERT and hTR, which are apparently both necessary and sufficient to form the catalytic core of telomerase, several other proteins that form a complex with the core enzyme have been identified (13). Unlike the hTERTs, which are phylogenetically conserved, the telomerase-associated proteins appear to lack structural and primary sequence conservation. One telomerase-associated protein (TP1), which is presumably the human homolog of the Tetrahymena telomerase p80 gene, is a large, ubiquitously expressed protein (230–240 kDa) that binds to hTR (14). The function of telomerase-associated proteins is not known, but they possibly regulate telomerase activity. Telomerase is expressed by most malignant cells and is normally inactive in most somatic cells, with the exception of proliferative stem cells, male germ cells, and activated lymphocytes (15). The activation of telomerase in malignant neoplasms seems to be an important step in tumorigenesis to gain the ability of indefinite proliferation and to become immortal.

There is a steadily growing interest in the potential application of telomerase as a diagnostic and prognostic marker. For this reason, the aim of the present investigation was to analyze the dynamics of various genes (TP1, hTR, hTERT, and HSP90) of the telomerase ribonucleoprotein enzyme complex and to study whether the expression of these genes and telomerase activity could serve as a diagnostic marker distinguishing benign from malignant neoplasms and as a marker of dedifferentiation, invasiveness, or aggressive behavior in PCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ethical approval

The studies involving the use of human tissues described in this paper were approved by the local ethical committees, and all patients gave written consent.

Pheochromocytoma tissues

Pheochromocytoma tissue was obtained from 37 patients undergoing surgery between 1994 and 2000. There were two clinically defined subject groups: 28 benign and nine malignant intramedullar PCs. All tumors showed the classical histology and the typical immunohistochemical pattern (positive immunoreactivity for vimentin, chromogranin, synaptophysin, and S100). The benign group included tumor samples obtained from eight females and 20 males (median, 52.2 yr; range, 27–72 yr; follow-up: median, 44.8 months; range, 24–72 months). The group of MP included three females and six males (median, 60.4 yr; range, 47–67 yr; tumor-free survival time: median, 17.8 months; range, 0–42 months; Tables 1Go and 2Go).


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  		TABLE 1. Clinical and tumor-specific data, as well as expression of telomerase-related genes (TP1, hTR, hTERT, and HSP90) and telomerase activity in benign and malignant pheochromocytoma

 

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  		TABLE 2. Uni- and multivariate analysis of the demographical, tumor-specific, and experimental data

 
RNA isolation and RT-PCR

RNA was isolated from frozen pheochromocytoma tissues using the TRIzol reagent (Life Technologies, Inc., Munich, Germany) following the manufacturer's recommendation. cDNA was synthesized from total RNAs using the Superscript II Kit (Life Technologies, Inc.).

PCR primers were as follows: TP1, 5'-TCAAGCCAAACCT-GAATCTGAG-3' (sense) and 5'-CCCGAGTGAATCTTTCTACGC-3' (antisense), amplicon 264 bp; hTR, 5'-CCTAAC-TGAGAAGGGCGTAGGC-3' (sense) and 5'-CTAGAATGAACGGTGGAAGGCG-3' (antisense), amplicon 273 bp; hTERT, 5'-CGGAAGAGTGTCTGGAGCAA-3' (sense) and 5'-GGATGAAGCGGAGTCTGGA-3' (antisense), amplicon 145 bp; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CAT-CACCATCTTCCAGGAGCG-3' (sense) and 5'-TGACCTTGCCCACAGCCTTG-3' (antisense), amplicon 443 bp.

A negative control without cDNA was included in each experiment. PCR products were resolved on ultrathin native polyacrylamide gels cross-linked with piperazine diacrylamide, baked on GelBond Pag (FMC Bioproducts, Rockland, ME), and visualized by silver staining. Bands were directly quantified by laser densitometry VDS (Amersham Pharmacia Biotech, Denver, CO). Expression was evaluated by estimating the intensity ratio between the TP1, hTR, and hTERT signal and the corresponding GAPDH signal. Normal placenta tissue was used as a negative control, and its expression level was defined as normal. In comparison with the placenta, 3-fold or more intensified signals were regarded as increased expression. The expression levels were classified as follows: -, no expression; +, low expression (3- to 10-fold); and 2+, high expression (>10-fold). Tissue from an expanded endometrioid ovarian carcinoma with 3-fold (TP1), 3-fold (hTR), and 4-fold (hTERT) increased expression was used as a positive control. To ensure consistency, each PCR reaction was carried out twice.

Quantitative real-time RT-PCR for expression of hTERT

Total RNA was isolated from fresh-frozen tissue by the TRISOLV system (Biozol, Eching, Germany) in accordance with the manufacturer’s protocol. RNA was treated with deoxyribonuclease (Eurogentec, Seraing, Belgium) and purified with the RNeasy Mini Prep Kit (Qiagen, Hilden, Germany). cDNA was synthesized from approximately 2 µg RNA using the FirstStrand Synthesis Kit (Amersham Pharmacia Biotech, Freiburg, Germany) with deoxythymidine18 primers. Relative concentrations of cDNA samples were evaluated by quantitative RT-PCR of GAPDH performed on the LightCycler (Roche, Mannheim, Germany), followed by analysis of the gene expression of hTERT using the same procedure. To amplify the cDNA, 5-µl aliquots of reverse transcribed cDNA (dilution, 1:5) were subjected to PCR amplification in 20 µl containing a final concentration of 2 mmol/liter MgCl2, 0.5 µmol/liter of the primer (see RT-PCR), and 2 µl of Ready-to-Use LightCycler DNA Master SYBRGreen I (Roche). For hot-start PCR, 0.16 µl/sample of TaqStart antibody (Clontech, Heidelberg, Germany) was added to the amplification mixture before the addition of primers and template cDNA. The reaction conditions were initial denaturation at 95 C for 2 min, followed by 37 cycles of denaturation at 95 C for 1 sec, annealing at 60 C for 5 sec, and extension at 72 C for 8 sec. Quantitative analysis was performed using the LightCycler Software (Roche) and a real-time fluorogenic detection system for a kinetic, rather than end-point, approach as on conventional polyacrylamide gels. The generation of quantitative data was based on different PCR kinetics of samples with different levels of target gene expression. We used a relative quantification in which the expression levels of pheochromocytoma samples were compared with the data from the colon adenocarcinoma telomerase-positive cell line SW-480 (ATCC CLL-228, American Type Culture Collection, Manassas, VA) (13) in a geometric dilution series. For analysis, the quantitative amounts of hTERT gene expression standardized on GAPDH expression were grouped as follows: negative = 0; less than 1:64 dilution of positive cell line SW-480 = 1; 1:64 to 1:32 dilution = 2; 1:32 to 1:16 dilution = 3; 1:16 to 1:8 dilution = 4; 1:8 to 1:4 dilution = 5; 1:4 to 1:2 dilution = 6; and more than 1:2 dilution of positive cell line SW-480 = 7.

hTERT immunohistochemistry

For immunohistochemical evaluation of hTERT on paraffin-embedded sections a new staining procedure was developed, because the antibody used has only been applied to frozen tissue. A microwave pretreatment (5 min at 600 watts and 15 min at 450 watts) in Glycabuffer (pH 3.0, BioGenex Laboratories, San Ramon, CA), followed after deparaffinization and rehydration of the 3-µm thin sections. Endogenous peroxidase activity was inhibited with a 3% solution of H2O2 in PBS, pH 7.5, for 15 min. Normal horse serum diluted 1:20 with PBS and containing 1% BSA was incubated with the samples for 20 min to suppress nonspecific binding. Tissue sections were incubated with the polyclonal antibody TRT (H-231, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:100 dilution for 1 h at 37 C. After washing in PBS, the samples were incubated with a 1:200 dilution of biotinylated antigoat secondary antibody (Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature. The detection of the bound antibody was accomplished by the avidin-biotin complex method (Vectastain Elite ABC Kit, Vector Laboratories, Inc.). A 0.1% solution of 3,3'-diaminobenzidine (5 min) (Sigma-Aldrich Corp., St. Louis, MO) was used as chromogen. The specificity of the immunostaining was checked by the omission of single steps in the immunohistochemical protocol and replacement of the primary antibody with nonimmune serum. Positively stained nuclei were counted and represented as a percentage.

HSP90 immunohistochemistry

Immunohistochemical studies for HSP90 also employed the avidin-biotin complex method. The 3-µm thin paraffin-embedded sections were rehydrated step by step with descending concentrations of ethanol before the staining procedure. Endogenous peroxidase was blocked by incubation in methanol containing 0.3% H2O2 for 35 min. After washing in 0.01 M PBS, the slides were incubated with 10% normal goat serum for 60 min to block nonspecific binding sites. Normal goat serum was removed, followed by incubation with the specific polyclonal antibody HSP90 (H-114, Santa Cruz Biotechnology, Inc.) at a 1:100 dilution overnight at room temperature, with subsequent staining as described for hTERT. Immunostaining was repeated on at least one occasion, and the results were assessed by considering both the scoring intensity of staining and the proportion of maximally stained cells as recommended by Remmele et al. (16). Maximally stained cells were proportionally distributed as follows: missing = 0; less than 10% = 1; 11–50% = 2; 51–80% = 3; and greater than 80% = 4. Staining intensity was classified as negative = 0; slight = 1; abundant = 2; or strong = 3. The product of staining intensity and the percentage of positive cells describes the score (0–12). A score of 0–2 was regarded as negative or low, 3–6 as moderate, and more than 6 as strong.

Telomerase assay

For telomerase activity analysis, the commercially available Telomerase PCR ELISA-Kit (Roche) was used. This is a photometric enzyme immunoassay using the telomeric repeat amplification protocol (TRAP) assay with nonradioactive detection, which is an extension of the original method described by Kim and Wu (17). The assay procedures were performed as described in the kit manual. All determinations were performed in triplicate.

Proliferation study

To detect the proliferation rate, an immunohistochemical study for the Ki-67 antigen was performed on representative paraffin sections with the use of appropriate positive and negative controls throughout. The monoclonal antibody anti-Ki-67 (Mib-1, 1:50; Dianova, Hamburg, Germany) was used. The alkaline phosphatase antialkaline phosphatase method was carried out on 4-µm thick tissue sections previously deparaffinized with xylene for 15 min and treated in a microwave oven using 0.01 mol/liter citrate buffer (pH 6.0) for 30 min. Incubation was performed with the primary antibody at 37 C. After washing, the sections were subsequently incubated for 30 min at room temperature with a biotinylated horse antimouse antibody and an avidin-biotin peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories, Inc.). The final reaction product was shown by incubation with 3,3'-diaminobenzidine (0.1% solution, 5 min; Sigma-Aldrich Corp.), and the nuclei were counterstained with Gui’s hematoxylin. Positively stained nuclei were counted and expressed as a percentage.

Statistical analysis

Telomerase activity, the expression of the different telomerase genes, and the clinical parameters, including pathological data, were tested for statistical significance using the proportional t test and uni- and multivariate analysis. P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparing demographic with clinical data, we found the following pattern: the mean age of the BP group was 54.3. The patients of the MP group with poorer clinical prognosis were, on the average, older (mean age, 60.4 yr; P = 0.067; Table 2Go). The MP group consisted of 20 males and eight females, whereas six males and three females constituted the BP group (P = 0.786; Table 2Go). Of 28 patients with BP, 24 were still alive, and four died of another disease after a 44.8-month follow-up (range, 24–72 months). Neither recurrences nor metastases were detectable during this time. In the MP group, eight of nine patients had tumor metastasis after a tumor-free time of 17.8 months (ranging from 0–42 months). Only one patient (no. 34, Table 1Go) was tumor free after 37 months. Two patients (no. 36 and 37) had metastases (lymph nodes and liver) at the time of surgery. Four of nine MP patients died of disease.

Proliferation study

The mean tumor weight in BP (80.5 g) and MP (94.5 g) showed no difference (P = 0.12). In contrast, MP had a substantially higher proliferation rate than BP (10.8% vs. 1.3%; P < 0.001). Considering MP and BP as one group, there was no significant correlation between the proliferation rate and tumor weight (P = 0.09). In MP alone, however, the proliferation rate correlates significantly with tumor weight (P = 0.02).

Telomerase genes and activity in pheochromocytomas

TP1. The mRNA coding for telomerase-associated protein was ubiquitously and equally expressed in all BP and MP (Fig. 1Go and Table 1Go).



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  		FIG. 1. Examples for the expression of telomerase-related genes and GAPDH as well as telomerase activity in pheochromocytoma tissue of five patients. -, No telomerase activity; +, positive telomerase activity. Patients 24, 25, and 26 had benign pheochromocytoma (B); patients 29 and 30 had malignant pheochromocytoma (M). For further clinical data, see Table 1Go.

 
hTR. The intrinsic RNA component of telomerase complex showed a different expression pattern. The RT-PCR revealed that hTR was expressed in 13 of 28 BP (46%) and in all nine MP (100%; Fig. 1Go and Table 1Go). No statistically significant correlation was observed between hTR expression and age (P = 0.54), sex (P = 0.78), tumor size (P = 0.67), or proliferation index (P = 0.11) in BP.

hTERT. The expression of mRNA for the catalytic subunit, human telomerase reverse transcriptase, was associated with malignancy. Human TERT was expressed in only two of 28 BP and in all of the nine MP (P < 0.01; Fig. 1Go and Table 1Go). In the two hTERT-positive BPs (no. 27 and 28, Table 1Go), there was a strong infiltration of lymphocytes, suggesting chronic inflammation. These two tumors were also found among the largest PC in BP and derived from the oldest patients. Also, the proliferation index was elevated. No statistically relevant correlation was seen between the data of quantitative real-time RT-PCR and age (P = 0.77), sex (P = 0.18), tumor size (P = 0.08), or proliferation index (P = 0.21). Complete agreement was found between mRNA and protein expression. Protein expression was only detectable in mRNA-positive cases. In these cases, the share of stained nuclei was 28.1% for BP and 72.1% for MP (P < 0.001; Fig. 2Go and Table 1Go). A close relationship between the number of stained nuclei (protein) and the level of quantitative real-time PCR (mRNA) was not seen (P = 0.09).



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  		FIG. 2. Detection of hTERT and HSP90 protein expression in pheochromocytomas. A, Ninety-three percent of BPs were immunonegative; only two cases showed positivity (inset, patient 28). In contrast, all MPs were immunopositive for hTERT with strongly stained nuclei (B). All BPs expressed HSP90 at a similarly low level (C). Also, patient 28 with positive hTERT expression showed low HSP90 expression (inset; Remmele score, 4). MPs had very strong immunopositivity for HSP90 (D; Remmele score, 9–12).

 
HSP90. All of the BP expressed the HSP90 protein in the cytoplasm and, sporadically, in the nuclei at a similarly low level. The Remmele score ranged between 3–6 (mean, 4.50). Patients 27 and 28 with positive hTERT expression also showed a low HSP90 expression (Remmele score, 4; Fig. 2Go and Table 1Go). In contrast, MP were very strongly immunopositive for HSP90 (Remmele score, 8–12; mean, 10.22; P < 0.01; Fig. 2Go and Table 2Go).

Telomerase activity. All 28 BPs were negative for telomerase activity. In contrast, telomerase activity was detectable in all MPs (P < 0.001; Fig. 3Go and Tables 1Go and 2Go). T activity was only measurable in tissues that expressed the catalytic subunit hTERT (P < 0.001). In BP, the expression of hTERT does not lead to activation of telomerase.



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  		FIG. 3. Nonradioactive TRAP assay in BP (B) and MP (M). Benign pheochromocytomas (cases 25–28) without telomerase activity only showed the 50-bp increment and the 36-bp internal control, whereas the high positive MPs (cases 36 and 37) showed a ladder-like pattern of PCR products. BPs 27 and 28 were immunopositive for hTERT, but were telomerase negative.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most PCs can be cured by surgical resection. In view of the unfavorable prognosis for surgical therapy in cases of late tumor detection and malignant tumors, clinicians are required to find early typical signs and symptoms of MP vs. BP. In a recent study by Glodny and colleagues (18), arterial hypertension, headaches, sweating, tachycardia, and pallor were found more often in patients with BPs, whereas abdominal pain and dorsalgia occurred more frequently in MP patients. Also, unusually short histories and extraadrenal tumor localization arouse suspicion of malignancy (18). Several clinical parameters, such as high preoperative 24-h urinary dopamine levels, high tumor weight, elevated tumor dopamine concentration, and persistent postoperative hypertension are suggestive of malignant disease, but are not sufficiently accurate to be considered diagnostic (19). Laboratory-based tests, including elevated serum chromogranin levels or the presence of multiploidy in tumor cell DNA, have also been employed to help distinguish malignant from BPs. Other approaches point to the expression or absence of various molecules or gene products as a means of predicting malignancy. Among these studies, the lack of neuropeptide Y expression (20), the overexpression of p53 and Bcl-2 (21), the presence of RET protooncogene point mutations (22), and elevated telomerase activity (23, 24, 25) have suggested a malignant potential in PCs.

In the last few years telomerase has attracted considerable interest as a discrimination marker and a possible target for therapeutic intervention of malignant tumors. The apparent absence of measurable telomerase activity in most normal tissues and the fact that more than 85% of tumor samples show telomerase activity (26) suggest that different telomerase genes play a key role in the growth of most tumors, and that telomerase inhibitors might have a place in cancer therapy. Telomerase activity is present in germ cells and is repressed in most somatic cells, but the mechanisms of repression are not yet known. In colorectal and gastric carcinomas (27, 28), breast tumors (29), thyroid carcinomas (30), and liposarcomas (31), telomerase activity has been described as a marker that correlated significantly with malignancy and progression of the clinical tumor stages. However, recent studies reported that telomerase activity was also expressed in normal nonneoplastic tissues as well in nonneoplastic hyperproliferative lesions (29, 32).

The function of telomerase clearly relates to cellular senescence and immortalization of normal cells. It is known that telomerase plays a role in the maintenance of telomeric integrity. Analysis of the components of the human telomerase complex indicates that their expression often does not correlate with telomerase activity. This applies to the RNA component hTR, which is expressed regardless of the immortalization status (8), and TP1, which is ubiquitously expressed (14). However, the newly cloned putative telomerase subunit hTERT may indeed be a marker for immortalization (33, 34). Human TERT was shown to be expressed in tumor cell lines and in a number of tumors. In contrast, no expression was detected in normal fibroblast-derived mortal cell lines or in normal tissues, such as heart, brain, placenta, liver, skeletal muscle, breast, ovary, and prostate (9).

As an accurate method for the diagnosis of MP does not exist, telomerase activity, which indicates the presence of immortal cells, and the catalytic subunit of telomerase might represent useful diagnostic tools. This study is the first for which a sufficiently large number of samples was available. It demonstrates that hTERT was clearly and highly significantly associated with differentiation, whereas the telomerase RNA component (hTR) was found in all malignant and in almost half of BPs. In addition, telomerase activity was measurable in the hTERT-positive tissues only, indicating that these markers represent an important addition to prognostic markers in PC.

Our data now clearly demonstrate that telomerase is expressed neither in cells of the normal adrenal medulla nor in those of BP. It is well known that in fetal adrenal glands neuroblasts increase in number and size until wk 14 and 20 of gestation and then regress. Moreover, Hiyama et al. (35) reported that the adrenal glands of fetuses at wk 16 and 18 of gestation had telomerase activity, whereas no activity was detected in the normal adrenal glands of a newborn, a 2-month-old infant, and a 5-yr-old boy. Thus, it appears plausible that the expression of telomerase in a PC should indicate the malignant behavior of the component cells.

To date, only telomerase activity has been investigated in a few studies, and the results achieved were equivocal. In one study a low mean telomerase activity was found in eight of eight BPs and in two of three MPs. Telomerase activity was high in only one MP (23). Two other studies yielded divergent results, showing that, based on a highly sensitive PCR detection method, telomerase activity was detected in one of 13 adrenal cortical tumors and in two of seven PCs; these telomerase-positive tumors were found to exhibit pathological features suggesting a malignant potential (24). Similarly, in another study telomerase activity was detected neither in normal adrenal medulla nor in BPs, whereas all three MPs showed elevated telomerase activity (25). Thus, our data clearly exceed the data, reported in previous studies, demonstrating the importance of both telomerase activity and hTERT expression. From these data, it now appears to be clear that the study of multiple components of the telomeric complex is necessary to discriminate between these two types of PCs.

Our data confirm that TP1 is ubiquitously expressed in benign and malignant tumors (14) and also in BP and MP. The expression of hTR showed a very different picture. hTR expression does not correlate with telomerase activity. Interestingly, telomerase activity is also measurable without hTR expression, and hTR is also expressed in telomerase-negative cells, an observation that has been described previously in other cell systems (8, 9). In these cases, the restoration of telomerase activity through ectopic expression of hTERT leads to telomere lengthening, with a significant extension of life span. This definitively excludes TP1 and hTR as markers distinguishing between BP and MP.

The expression of hTERT was associated with malignancy. Only two benign neoplasms (7%) showed positivity. However, these cases had no telomerase activity in the TRAP assay. At first, we could not explain this phenomenon. The critical reanalysis of these positive cases revealed that they contained extensive lymphoid infiltrates with germinal centers, which was also observed in other studies (36, 37). Recent studies investigated other binding proteins (i.e. molecular chaperone p23 and HSP90) of the telomerase complex (38). HSP90 has been demonstrated to bind to hTERT and contribute to telomerase activity (11). To clarify this question in PCs, we performed an HSP90 protein expression study. Although HSP90 was detectable in all tissues, significant up-regulation was found in the telomerase-positive MPs. The two hTERT-positive BP and all of the other BP showed only low protein expression.

HSP90 and p23 have been identified as components of progesterone and glucocorticoid receptor complexes. Subsequently, it was found that the presence of both molecules is required to maintain these receptors in a ligand-binding state (39). These observations led to the concept of a molecular chaperone machinery or foldosome that mediates the assembly of a biologically active protein complex. Similarly, HSP90 and p23 in the telomerase complex may also serve this foldosome function in assembling the active holoenzyme. This mechanistic model and our results indicate that not only hTERT, but also HSP90, overexpression is strongly associated with the activation of telomerase. Only the nine MPs were telomerase positive in the TRAP assay. These tumors expressed hTERT and overexpressed HSP90. In addition, hTR and TP1 were detectable in the MPs. We conclude that hTERT is a regulatable subunit of the telomeric complex, whereas the other components are more constantly expressed. We hypothesize that once hTERT is expressed, all of the other telomerase subunits can be assembled to form a highly active holoenzyme.

In summary, our data, obtained from a representative number of BPs and MPs, show that telomerase activity is up-regulated in neoplastic cells. They also show that the concomitant expression of hTERT and telomerase activity is a potential prognostic marker for the identification of invasive tumors, but may also be used as an additional diagnostic marker for the classification of pheochromocytomas.


   Acknowledgments
 
The technical assistance of Carola Kügler, Claudia Miethke, Nadine Wiest, and Kathrin Hammje is gratefully acknowledged.


   Footnotes
 
This work was supported by NOVARTIS, Germany.

Abbreviations: BP, Benign pheochromocytoma; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSP90, heat shock protein 90; hTERT, telomerase catalytic subunit; hTR, telomerase RNA component; MP, malignant pheochromocytoma; PC, pheochromocytoma; TP1, telomerase-associated protein; TRAP, telomeric repeat amplification protocol.

Received August 14, 2002.

Accepted May 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Linnoila RI, Keiser HR, Steinberg SM, Lack EE 1990 Histopathology of benign versus malignant sympathoadrenal paragangliomas. Clinicopathologic study of 120 cases including unusual histological features. Hum Pathol 21:1168–1180[CrossRef][Medline]
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