Experimental and Clinical Endocrinology & Diabetes
@ 1999 Johann Ambrosius Barth
Telomerase activity in benign and malignant adrenal tumors
C. M. Bamberger1,2, T. Else1, A .- M. Bamberger3, A. Frilling4, F. U. Beil2, B. Allolio5, H. M. Schulte1
1IHF Institute for Hormone and Fertility Research, University of Hamburg; 2Dept. of Medicine, 3Institute for Pathology, University Clinic Eppendorf, Martinistr. 52, Hamburg; 4University Clinic Essen, Dept. of General Surgery and Transplantation, Essen; 5University Clinic Würzburg, Dept. of Endocrinology, Würzburg, Germany
Key words: Adrenal tumors, telomerase
Summary: Histological analysis of surgically removed adrenal masses often fails to differentiate between benign and malignant tumors. In normal cells, the telomeric ends of the chromosomes are shortened with each cell division, leading to chromosome destabil- ization and cellular senescence after a critical number of cell cycles. In tumor cells, telomere shortening is prevented by a specific DNA polymerase, called telomerase. In an effort to clarify the role of telomerase in the pathogenesis of adrenal tumors, and to test whether its activity could serve as marker of malignancy, we meas- ured telomerase activity in 41 human adrenal tissue samples that
were classified both by the clinical course and by histological exam- ination. Telomerase activity was dermined by TRAP ELISA and expressed as high (>50% of positive control telomerase activity), medium (31-50%), low (11-30%), very low (≤10%), or absent (0%). The 8 normal adrenal tissue samples showed very low levels of telomerase activity. Mean telomerase activity also very low in 3/3 incidentaloinas, 6/6 Cushing adenomas, 6/6 Conn adenomas, 7/7 adrenocortical carcinomas, 8/8 benign pheochromocytomas, and 2/3 malignant pheochromocytomas. In contrast, one malignant pheochromocytoma showed high telomerase activity. These data indicate that telomerase activity may not be a suitable marker for malignancy in the adrenal gland. Our results also challenge the current dogma of close correlation between cell dedifferentiation and telomerase activity.
Introduction
Adrenal tumors are very common in humans. How- ever, very little is known about the molecular patho- genesis underlying the development of adrenal neo- plasms. Furthermore, the histopathological differen- tiation between benign and malignant adrenal tumors is often difficult to achieve, and in many cases, this distinction can only be made by the clinical course. So far, there is no suitable marker that can distinguish benign from malignant adrenal lesions with sufficient specificity. Analysis of tumor clonality, for instance, revealed that most adrenal carcinomas are mono- clonal in origin. However, this is also true for a sub- stantial number of adrenal adenomas (Beuschlein et al., 1994; Gicquel et al., 1994). Staining for markers of proliferative activity, such as Ki67 or PCNA, were not strictly associated with the malignant state either (Goldblum et al., 1993). Marx et al. demonstrated that normal adrenocortical cells expressed the MHC class II antigen, whereas adrenal carcinomas were MHC class II-negative. Again, some adrenal ade- nomas did not show MHC class II staining either (Marx et al., 1996).
In normal cells, the stabilizing ends of the chromo- somes, termed telomeres, are shortened with each cell division, since DNA replication does not allow for completion of chromosome ends (Blackburn, 1991; Harley, 1991; Allsopp et al., 1995; Autexier and Gre- ider, 1996; Shay and Wright, 1996; Shay and Bac- chetti, 1997; Greider, 1998). It is believed, that after a defined number of divisions, telomere length has critically shortened and forces the cells to enter the state of senenescence (Blackburn, 1991; Harley, 1991; Allsopp et al., 1995). Malignant tumor cells have es- caped this ‘clock of senescence’ because they express telomerase, a ribonucleoprotein DNA polymerase, which can counteract telomer shortening by net telo- mere elongation (Yu et al., 1990; Rhyu, 1995; Aute- xier and Greider, 1996; Greider, 1996; Harley and Kim, 1996; Shay and Wright, 1996; Shay and Bacch- etti, 1997; Greider, 1998). Therefore, determination of telomerase activity has been proposed as a diagnostic and prognostic marker in cancer. Most malignant tu- mors show indeed increased telomerase activity as compared to the normal tissue they are derived from (Kim et al., 1994; Hiyama et al., 1995; Rhyu, 1995; Autexier and Greider, 1996; Harley and Kim, 1996; Nouso et al., 1996).
There are no studies analyzing telomerase activity in the human adrenal gland. The current study was, therefore, designed to determine telomerase activity in the normal and neoplastic human adrenal gland and to test whether it could serve as a marker of ma- lignancy in this tissue.
Materials and methods
Histopathological analysis
Normal adrenals and adrenal tumors were removed by abdominal surgery (nephrectomy for renal carci- noma, adrenalectomy for hormone-producing tu- mors, or adrenalectomy for tumors of suspicious structure and/or size as determined by adrenal CT or MRI). Part of the tissue was immediately frozen in liquid nitrogen and stored until measurement of telo- merase activity (see below). The remaining tissue was embedded in paraffine following standard protocols. Serial sections of 4-6 um were cut from the paraffin blocks and stained with hematoxylin-eosin. Tissue slides were analyzed independently by two patholo- gists. The diagnosis of malignancy was based both on the subsequent clinical course and on the results of the histopathological analysis. Telomerase activity was determined in a blinded fashion, i. e. without knowing the clinical and/or histopathological data. In total, 41 human adrenal tissue samples were analyzed (8 normal adrenals, 3 incidentalomas, 6 Cushing adenomas, 6 Conn adenomas, 7 adrenocortical carci- nomas, 8 benign pheochromocytomas, and 3 malig- nant pheochromocytomas).
Telomerase assays
Telomerase activity in adrenal tissue samples was de- termined by a modified telomeric repeat amplification protocol (TRAP) (Kim et al., 1994), following the in- structions of the manufacturer (Telomerase PCR EL- ISA, Boehringer Mannheim, Germany). Briefly, 50 cryostat sections from each frozen adrenal tissue were incubated for 30 min with lysis buffer, and cell debris was removed by centrifugation. Protein concen- trations were measured in all samples, and 5 µg pro- tein/tissue sample were used for the assay. In the first step of the assay, synthetic PI-TS-primers, to which any telomerase present in the sample should add tel- omeric repeats, were added to the lysate. The elong- ation product was amplified by PCR, using the pri- mers P1-TS and P2, which contains telomeric repeat- specific sequences. An aliquot of the PCR product was then denatured at 94℃ and hybridized at 50℃ to a digoxigenin-labeled, telomeric repeat-specific cDNA probe. The product was immobilized via the biotin-labeled primer sequences to a streptavidin-co- ated microtiter plate and incubated with a peroxidase- conjugated anti-digoxigenin antibody. Finally, the
probe was visualized by adding 3,3’,5,5’-tetramethyl benzidine (TMB), which is metabolized to a colored reaction product by peroxidase. Absorbance was de- termined in a microplate reader at 450 nm. All meas- urements were performed in duplicates. A standard curve was established using extracts from telomerase- positive Jurkat T lymphoma cells (0.1 ng, 1 ng. 10 ng). 10 ng of RNAse treated Jurkat cell lysate was used as a negative control. Telomerase activity was expressed as high (>50% of positive control telomer- ase activity), medium (31-50%), low (10-30%), very low (<10%), or absent (0%). Statistical analysis was performed using the unpaired t-test.
Results
The results of the telomerase assays are shown in Fig. 1 and Table 1. Telomerase activity in normal adrenal tissue samples was very low (1.3 ± 0.131% of maxi- mum activity, defined as telomerase activity in 10 ng Jurkat cell extract). Mean telomerase activity was not significantly different from normal tissue in 3/3 inci- dentalomas, 6/6 Cushing adenomas, 6/6 Conn aden- omas, 7/7 adrenocortical carcinomas, 8/8 benign pheochromocytomas, and 2/3 malignant pheochro- mocytomas. Only one malignant pheochromocytoma had high levels of telomerase activity. There was a tendency towards lower telomerase activity levels in Cushing adenomas and benign pheochromocytomas compared to normal adrenals. Furthermore, all but one adrenal carcinomas had higher telomerase activi- ties than the Cushing adenomas (Fig. 2). However, because of the small number of cases and the very low absolute levels of telomerase activity, we do not consider these differences significant at the present time, even though the pure statistical numbers would indicate so.
Discussion
In this study, we analyzed telomerase activity in nor- mal and neoplastic human adrenals. Telomerase ac- tivity was very low (yet detectable) in all but one tis- sue samples and did not distinguish benign from ma- lignant adrenal tissue. Cushing adenomas and benign pheochromocytomas displayed a tendency towards lower levels of telomerase activity than the other tis- sues, including normal adrenal.
As would be expected, telomerase activity in the normal human adrenal was very low. This is consist- ent with the telomerase hypothesis, which states that normal cells sustain a progressive shortening of their telomeres because they do not express significant amounts of this enzyme (Blackburn, 1991; Harley, 1991; Allsopp et al., 1995; Autexier and Greider, 1996; Shay and Wright, 1996; Shay and Bacchetti, 1997; Greider, 1998). Few exceptions to this rule have since been reported, and they basically include all
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Fig. 2 Comparison of telomerase activity in Cushing adenomas and adrenal carcinomas. All but one carcinoma had higher telo- merase activity than the adenomas
rapidly dividing cells, such as activated lymphocytes (Bodnar et al., 1996).
Surprisingly, telomerase activity was not signifi- cantly elevated in benign or malignant adrenal tu- mors. Adrenal tumors, thus, belong to the approx. 10% of neoplasm with low or undetectable telomerase activity (Autexier and Greider, 1996; Shay and Wright, 1996; Shay and Bacchetti, 1997; Greider, 1998). Since tumors consist of potentially immortal and indefinitely dividing cells, one has to assume some type of protective mechanism against telomere shortening in tissues with low levels of telomerase ac- tivity. It has indeed been shown that telomere length- ening does not necessarily depend on the presence of telomerase (Bryan et al., 1995; Blasco et al., 1997).
| Tissue | Telomerase activity (%) | SD (%) |
|---|---|---|
| Normal adrenals | 1.30 | 0.131 |
| Incidentalomas | 1.21 | 0.080 |
| Cushing adenomas | 1.12 | 0.028 |
| Conn adenomas | 1.22 | 0.058 |
| Adrenal carcinomas | 1.81 | 0.946 |
| Benign | 1.13 | 0.035 |
| pheochromocytomas | ||
| Malignant | 34.08 | 57.086 |
| pheochromocytomas |
The authors of the former study suggested that an alternative, possibly recombination-mediated mecha- nism might be operative in cells lacking telomerase activity. Alternatively, telomerase activity could be switched on late in tumor development, i.e. after the potential date of surgery. Consistent with this hypo- thesis, telomere length was reported to be reduced in many tumors. Blasco et al. reported that the telomer- ase RNA component was expressed in preneoplastic stages in a transgenic mouse model of multistage tumorigenesis, whereas telomerase activity was de- tected only in late tumor stages (Blasco et al., 1996). To further determine the functional role of telomerase in the normal and neoplastic adrenal gland, it will be crucial to analyze the expression of telomerase com- ponents (van Steensel and de Lange, 1997) as well as the actual telomere length in this tissue.
Telomerase activity has been proposed as a diag- nostic and prognostic marker for various human can- cers (Blackburn, 1991; Harley, 1991; Allsopp et al., 1995; Autexier and Greider, 1996; Shay and Wright, 1996; Shay and Bacchetti, 1997; Greider, 1998). It has been demonstrated that assessment of telomerase ac- tivity in exfoliated cells from urine or colonic washes
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could establish the diagnosis of bladder and colon cancer, respectively (Kinoshita et al., 1997; Yoshida et al., 1997; Yoshida et al., 1997). In these tumors, telomerase might also be useful to determine the stage of the disease (Yoshida et al., 1997; Yoshida et al., 1997). Prostate cancer can be distinguished from be- nign hyperplasia by analyzing telomerase activity (Sommerfeld et al., 1996). In the human adrenal gland, however, telomerase activity does not seem to represent a suitable marker for malignancy. Of the 33 tumors analyzed, only one sample, a malignant pheochromocytoma, showed high levels of telomerase activity. Kubota et al. previously reported elevated te- lomerase activity in 3/3 malignant pheochromocy- tomas (Kubota et al., 1998). In our study, 2/3 malig- nant pheochromocytomas displayed very low telo- merase activity. We, therefore, conclude that telomer- ase is not a reliable marker to distinguish benign from malignant pheochromocytomas either.
In summary, we have demonstrated very low telo- merase activity in all but one adrenal tissue samples analyzed. These data indicate that telomerase activity may not be a suitable marker for malignancy in the adrenal gland. Telomerase activity may distinguish Cushing adenomas from Cushing carcinomas, how- ever, a larger number of cases is required to confirm these findings. Finally, our results challenge the cur- rent dogma of close correlation between neoplastic growth and telomerase activity.
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Christoph M. Bamberger, M. D. IHF Institute for Hormone and Fertility Research University of Hamburg Grandweg 64 D-22529 Hamburg Tel. (+49) 40-561 908 88 Fax (+49) 40-561 908 64 e-mail: < bamberger@plexus.uke.uni-hamburg.de>