COMMENTS
Telomerase Activity Is Significantly Enhanced in Malignant Adrenocortical Tumors in Comparison to Benign Adrenocortical Adenomas
MASSIMO MANNELLI, STEFANIA GELMINI, GIORGIO ARNALDI, LUCIA BECHERINI, DONATELLA BEMPORAD, CLARA CRESCIOLI, MARIO PAZZAGLI, FRANCO MANTERO, MARIO SERIO, AND CLAUDIO ORLANDO
Department of Clinical Pathophysiology, Endocrine Unit (M.M., L.B., D.B., C.C., M.S.) and Clinical Biochemical Unit (S.G., M.P., C.O.), University of Florence, 50139 Florence; and the Division of Endocrinology, Institute of Internal Medicine, University of Ancona (G.A., F.M.), Ancona, 60100 Italy
ABSTRACT
Telomerase is an enzyme that causes short repeated sequence addition to the ends of chromosomes, thereby preventing their short- ening during cell division and counteracting cell senescence. Telom- erase activity is generally absent in adult differentiated cells, whereas it has been demonstrated in tumor cells, suggesting that its presence might be considered an index of malignancy. To evaluate whether telomerase might be considered a good predictive index of malignancy in adrenocortical tumors, we measured telomerase activity in 11 ad- renal adenomas and 7 carcinomas obtained at surgery, using an
original quantitative method. Telomerase activity was significantly higher (P < 0.001) in carcinomas than in adenomas (median, 15.2 ng DNA/µg protein; range, 9.0-27.6 vs. 2.0; range, 0-8.3), and no overlap was observed between the 2 groups. In carcinomas, telomerase ac- tivity was significantly correlated with tumor diameter (r = 0.939; P < 0.0001), whereas in adenomas it was not. The results of this study suggest that quantitative telomerase measurement may represent a useful tool to differentiate malignant from benign adrenocortical tu- mors. (J Clin Endocrinol Metab 85: 468-470, 2000)
T ELOMERES (1) are repeated sequences at the ends of chromosomes that undergo shortening on continuous cell proliferation. In fact, the DNA polymerase complex does not replicate the very end of chromosomes during each cycle of cell proliferation. Telomeres stabilize chromosomes and prevent DNA degradation as well as provide a signal of cellular senescence. In fact, when telomere length is reduced to a critical point, normal somatic cells exit the cell cycle and become senescent (2).
Telomerase is a ribonucleoprotein complex (3) that cata- lyzes the addition of telomeric repeats to the 3’-end of chro- mosome DNA, thereby preventing the loss of telomeric se- quences at each cell division. Telomerase activity is variously distributed in adult somatic cells (4); it is clearly present in germinal cells of the testis and stem cells of regenerative tissues, whereas low activity has been demonstrated in some normal differentiated tissues. On the contrary, it is clearly detectable in cancer cell lines in vitro (5), and it has been recently demon- strated in most human cancer tissues (6). Therefore, an increas- ing number of reports has recently been published on telom- erase activity in different human cancers (7-14) to evaluate whether it might constitute a good index of malignancy.
The differentiation between benign and malignant adre- nocortical tumors is often difficult (15) on either clinical or morphological ground. Several criteria have been proposed in the past to distinguish between benign and malignant tumors (16). More recently, several cytological characteris- tics, such as the expression of the proliferating cell antigen (17), of the adrenal 4 binding protein (18), of the c-Myc protein (19), of the insulin-like growth factor II gene (20), or of the p53 protein (21) or the DNA index (19, 22), have been evaluated as indexes of malignancy, but to date none of them predicts malignancy in the single tumor with accuracy. In some recent reports (23-25) telomerase activity has been measured in adrenocortical tumors, including several adre- nal malignancies, where it was found to be positive. Nev- ertheless, in these studies the method employed was the traditional semiquantitative TRAP (telomerase repeat am- plification protocol) assay, and the series of adrenocortical cancers was limited to 1-2 specimens. In the present study we measured telomerase activity in 11 benign adrenocortical adenomas and 7 malignant adrenocortical carcinomas using an original assay (26), allowing a quantitative determination of telomerase activity.
Materials and Methods
Eighteen adrenocortical tumors (11 adenomas and 7 carcinomas) were studied. The clinical characteristics of each patient are reported in Table 1 as are the characteristics of each tumor. Tissue specimens were obtained at surgery, immediately frozen in liquid nitrogen, and stored at -80 C until extraction. The method we used to measure telomerase
| Patient no. | Age (yr) | Sex | Postsurgical outcome | Tumor size (cm) | Tumor secretion | Telomerase activity (ng DNA/µg protein) |
|---|---|---|---|---|---|---|
| 1 | 66 | M | Cured | 4.0 | C | 5.0 |
| 2 | 46 | F | Cured | 5.0 | None | 5.5 |
| 3 | 51 | F | Cured | 4.5 | C | ND |
| 4 | 38 | F | Cured | 2.0 | C | 8.3 |
| 5 | 57 | F | Cured | 4.7 | C | 2.0 |
| 6 | 27 | F | Cured | 2.0 | C | 0.6 |
| 7 | 21 | F | Cured | 8.0 | A | 3.5 |
| 8 | 21 | F | Cured | 5.5 | A | 2.7 |
| 9 | 24 | F | Cured | 3.0 | C | 0.6 |
| 10 | 62 | F | Dieda | 7.0 | C | ND |
| 11 | 44 | F | Cured | 4.0 | None | 5.5 |
| 12 | 69 | F | Metastasis, died | 6.0 | A + M | 11.5 |
| 13 | 45 | F | Recurrence, mitotane | 9.0 | C + A | 9.0 |
| 14 | 21 | F | Metastasis, died | 12.0 | C | 15.8 |
| 15 | 50 | M | Cured | 9.0 | None | 15.2 |
| 16 | 61 | F | Recurrence, mitotane | 8.0 | None | 13.2 |
| 17 | 28 | F | Recurrence, mitotane | 22.0 | A | 27.6 |
| 18 | 45 | F | Metastasis, mitotane | 15.0 | None | 18.8 |
Histological diagnosis in patients 1-11, adenomas; in patients 12-18, carcinomas. C, Cortisol; A, androgens; M, mineralcorticoids; ND, not detectable.
” Patient with Cushing’s syndrome due to bilateral adrenal adenomas, died from cardiac arrest 4 days after right adrenalectomy performed by laparoscopy.
activity has been reported previously (24). Each sample was assayed for telomerase activity in duplicate, starting from protein extracts of the tissue. A negative control, obtained after pretreatment of the sample with ribonuclease, was also assayed for each specimen. The protein concentration was measured in each extract by the Bio-Rad Laboratories, Inc., protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Telom- erase activity was calculated as the mean of duplicates and expressed as nanograms of DNA per µg proteins. In each assay we also evaluated a protein extract of a cell line (LNCaP) and a protein extract of a gastric tumor sample as positive controls. Human placental DNA was used as a negative control. The within- and between-assay coefficients of vari- ations were 12.3% and 14.5%, respectively.
In some tumor samples the presence of telomerase activity was also tested using the conventional TRAP assay (5) with autoradiographic revelation of radiolabeled PCR products. In this assay protocol, telom- erase reaction and amplification were performed with CX and TS prim- ers (5). For radiolabeling of PCR products 0.2 p&L [@-32P]deoxy-CTP (10 mCi/pL; 3000 Ci/mmol) were added to the reaction mix. We performed 35 PCR cycles of 95 C for 30 s, 50 C for 30 s, and 72 C for 60 s. The PCR products were resolved by electrophoresis on a 10% nondenaturing polyacrylamide gel, which was then autoradiographed. Telomerase ac- tivity was considered present when a 6-bp ladder pattern of bands, each representing the addition of a hexanucleotide telomeric repeat by te- lomerase, was observed after 24-h exposure at -80 C.
Statistical comparison between groups was performed using the Wil- coxon signed rank test. Correlation was calculated by Pearson’s corre- lation coefficient.
Results
Telomerase activity was detectable in all carcinomas ex- amined and in 9 of 11 adenomas (Table 1). No activity was detectable in 3 samples of normal adrenocortical tissue. To control for whether the results of our assay were related to telomerase activity, conventional TRAP assay based on 32P labeling was performed in 3 adenomas and 4 carcinomas. In each of the assayed adenomas and carcinomas, a typical 6-bp ladder pattern, representing the addition of telomeric re- peats, was observed (Fig. 1). In cortical adenomas, median te- lomerase activity was 2.0 ng DNA/µg protein (range, 0-8.3), whereas in cortical carcinomas, it was 15.2, (range, 9.0-27.6). Telomerase activity in the two groups was statistically different
Adenomas
Carcinomas
1
2
3
4
5
6
7
(P < 0.001). In carcinomas telomerase activity was significantly correlated with tumor diameter (r = 0.939; P < 0.0001), whereas in adenomas it was not (r = 0.213; Fig. 2).
Discussion
Although metastasizing or very large adrenocortical tu- mors are easily labeled as malignant, for most adrenal tu- mors it is difficult to predict malignant potential. In fact, although a multifactorial analysis has been proposed by Weiss (16) to discriminate between metastasizing and non- metastasizing adrenal tumors, the method is not easy, and sometimes it has been reported to give false results. For this
25
r = 0.213
20
r = 0.939
Tumor Size (cm)
15
10
5
0
0
5
10
15
20
25
30
Telomerase Activity (ng DNA/µg protein)
reason, many other indexes have been evaluated in the past (15, 17-22, 27), but none of them predicts malignancy with accuracy in the single tumor.
Very recently, telomerase activity has been detected in different kinds of malignant tumors (6-14), including adre- nal tumors (23-25), using a method that is conventionally called TRAP. The main advantage of this technique is the high sensitivity, which allows the revelation of telomerase activity even in small samples of cancer tissues or cultured cells. On the other hand, this approach does not provide quantitative information on the activity of the enzyme.
In this study we applied a modification of the TRAP assay (26), based on the use of a sensitive fluorochrome that se- lectively binds double strand DNA (28). As the TRAP assay uses a reaction that generates double strand DNA starting from cellular extracts, and the amount of DNA generated is proportional to the telomerase activity in the initial sample, the estimated DNA concentrations in post-PCR samples are quantitatively related to telomerase activity.
The above-mentioned quantitative method was applied to specimens obtained from 18 patients with adrenocortical tumor. The presence or absence of malignancy was estab- lished on macro- and microscopic histological criteria as well as on patient follow-up, which, for patients affected by be- nign adenomas, lasted for at least 2 yr.
The results of our study indicate that adrenocortical car- cinomas possess a higher telomerase activity than benign adenomas. In our series no overlap was observed between the two groups, although the difference between sample 4 (adenoma) and sample 13 (carcinoma) is rather small. The levels of telomerase activity we found in adrenal carcinomas are comparable to those previously detected in gastric and endometrial cancers and lower than those measured in breast and ovarian carcinomas (26). Moreover, it is worth mention- ing that in carcinomas telomerase activity was significantly correlated to tumor diameter. Although tumor size at sur- gery depends not only on cell proliferative potential but also on time of diagnosis, the correlation we found might suggest
that telomerase activity is a good index of tumor growth capacity. To draw final conclusions on the sensitivity and prognostic value of telomerase measurement in adrenocor- tical tumors is necessary to increase the number of observa- tions, paying special attention to those tumors whose grade of malignancy is graded as intermediate by the conventional mac- ro- and microscopic histological criteria. Should our data be confirmed in larger series, the quantitative measurement of telomerase activity might become one of the best available indexes to distinguish benign from malignant adrenocortical tumors.
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