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Original Studies |
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
Address all correspondence and requests for reprints to: Massimo Mannelli, M.D., Department of Clinical Pathophysiology, Endocrine Unit, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. E-mail: m.mannelli{at}dfc.unifi.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Telomerase is a ribonucleoprotein complex (3) that catalyzes the addition of telomeric repeats to the 3'-end of chromosome DNA, thereby preventing the loss of telomeric sequences 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 demonstrated in most human cancer tissues (6). Therefore, an increasing number of reports has recently been published on telomerase activity in different human cancers (7, 8, 9, 10, 11, 12, 13, 14) to evaluate whether it might constitute a good index of malignancy.
The differentiation between benign and malignant adrenocortical 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 characteristics, 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, 24, 25) telomerase activity has been measured in adrenocortical tumors, including several adrenal malignancies, where it was found to be positive. Nevertheless, in these studies the method employed was the traditional semiquantitative TRAP (telomerase repeat amplification 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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). Telomerase 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 variations were 12.3% and 14.5%,
respectively.
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-32P]deoxy-CTP (10 mCi/µL; 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 activity was
considered present when a 6-bp ladder pattern of bands, each
representing the addition of a hexanucleotide telomeric repeat by
telomerase, was observed after 24-h exposure at -80 C. Statistical comparison between groups was performed using the Wilcoxon signed rank test. Correlation was calculated by Pearson’s correlation coefficient.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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
repeats, was observed (Fig. 1). In
cortical adenomas, median telomerase 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 (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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Very recently, telomerase activity has been detected in different kinds of malignant tumors (6, 7, 8, 9, 10, 11, 12, 13, 14), including adrenal tumors (23, 24, 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 selectively 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 established on macro- and microscopic histological criteria as well as on patient follow-up, which, for patients affected by benign adenomas, lasted for at least 2 yr.
The results of our study indicate that adrenocortical carcinomas 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 mentioning that in carcinomas telomerase activity was significantly correlated to tumor diameter. Although tumor size at surgery 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 adrenocortical tumors is necessary to increase the number of observations, paying special attention to those tumors whose grade of malignancy is graded as intermediate by the conventional macro- 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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Revised September 10, 1999.
Accepted September 17, 1999.
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) and carcinomas (•).