The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3690-3693
Copyright © 2003 by The Endocrine Society
Diagnostic and Prognostic Value of Fas and Telomeric-Repeat Binding Factor-1 Genes in Adrenal Tumors
Hajime Kanauchi,
Nobuyuki Wada,
David G. Ginzinger,
Mamie Yu,
Mariwil G. Wong,
Orlo H. Clark and
Quan-Yang Duh
Endocrine Surgical Oncology Fellows (H.K., N.W.), Department of Surgery, University of California San Francisco (UCSF), UCSF/Cancer Center (D.G.G., M.Y.) and UCSF/Mount Zion Medical Center (M.G.W., O.H.C.), San Francisco, California 94143; Surgical Service, Veterans Affairs Medical Center (Q.-Y.D.), San Francisco, California 94121; Department of Surgery, The University of Tokyo (H.K.), Tokyo 113, Japan; and First Department of Surgery, Yokohama City University, School of Medicine (N.W.), Yokohama 236, Japan
Address all correspondence and requests for reprints to: Quan-Yang Duh, M.D., Surgical Service 112, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: quan-yang.duh{at}med.va.gov.
 |
Abstract
|
|---|
It is often difficult to distinguish histologically between an adrenal cortical cancer and a benign adenoma, or to predict the prognosis of patients with adrenal cortical cancers. In this investigation, we examined whether apoptosis-regulating genes, bcl-xL and fas, and a telomere-related gene, telomeric-repeat binding factor-1 (TRF-1), differ between adrenal cortical cancers and benign adrenal tumors. Tissues from 4 adrenal cortical cancers were compared with 7 normal adrenal tissues, 17 cortical adenomas, 4 cortical hyperplasias, and 20 pheochromocytomas for expressions of bcl-xL and fas by RT-PCR, and for expressions of TRF-1 by real-time quantitative RT-PCR. All benign adrenal tissues expressed both the antiapoptosis gene, bcl-xL, and proapoptosis gene, fas, but the adrenal cortical cancers expressed only bcl-xL and not fas. TRF-1 increased by more than 30-fold in the adrenal cortical cancers, compared with benign adrenal tissues, and inversely correlated with the prognosis of patients with the adrenal cortical cancers. This lack of expression of fas in adrenal cortical cancer may help to distinguish it from benign adrenal tumors. The level of TRF-1 expression may be helpful prognostically for patients with adrenal cortical cancers.
|
Introduction
|
|---|
ADRENAL CORTICAL CANCER is an aggressive cancer that is often resistant to surgical and medical treatment. Patients with adrenal cortical cancer often have a poor prognosis (1). It is sometimes difficult to determine whether a small (<4 cm) adrenal cortical neoplasm is benign or malignant histologically (2). The prognosis of patients with adrenal cortical cancer depends mainly on the stage of cancer at diagnosis (3). Several studies have shown genetic abnormalities, including overexpression of IGF-2, loss of heterozygosity at several sites, and abnormalities of several oncogenes in adrenal cortical carcinoma (4, 5).
In a previous investigation, we reported that benign adrenal tissues express both the antiapoptosis gene bcl-2 and the proapoptosis gene bax. Adrenal cortical cancers, in contrast, express only bcl-2 but not bax. We also found that there was increased expression of the telomere-related gene, human telomerase reverse transcriptase (hTERT), in adrenal cortical cancers when compared with benign adrenal tumors. hTERT expression also seemed to correlate with the prognosis of patients with adrenal cortical cancer (6).
To further confirm whether the difference in expression of apoptosis-regulating genes and telomere-related genes would help to distinguish between benign and malignant adrenal cortical tumors or to predict the prognosis of patients with adrenal cortical cancers, we examined the expression of other apoptosis-regulating genes, bcl-xL and fas, and a telomere-related gene, telomeric-repeat binding factor-1 (TRF-1).
|
Materials and Methods
|
|---|
Materials
We analyzed 52 adrenal surgical specimens from patients who underwent adrenalectomy for primary adrenal tumors at the University of California San Francisco between 1995 and 2000. After resection, the specimens were frozen and stored at -80 C until RNA extraction. These adrenal specimens include 7 normal tissues adjacent to adrenal tumors (from 2 patients with aldosterone-secreting adenomas, 3 patients with nonfunctioning adrenal adenomas, and 2 patients with benign pheochromocytomas), 17 cortical adenomas (from 10 patients with aldosterone-secreting tumors and 7 patients with cortisol-secreting tumors), 4 cortical hyperplasias (from 3 patients with Cushing’s disease and 1 patient with ectopic ACTH syndrome), 20 benign pheochromocytomas, and 4 cortical cancers. The 20 pheochromocytomas were presumed to be benign, because long-term follow-up has not detected any metastasis. All 4 adrenal cortical cancers secreted cortisol (Table 1
). This investigation was approved by the Committee on Human Research, University of California San Francisco. We measured bcl-xL and fas gene expressions by RT-PCR, and TRF-1 gene expression by real-time quantitative RT-PCR.
Total RNA extraction and cDNA synthesis
RNA was extracted from adrenal specimens by the acid-phenol-guanidinium method. The quality of RNA samples was measured by the intensity of the 18S and 28S RNA bands, under UV light, after electrophoresis and staining with ethidium bromide. RNA was transcribed into first-strand cDNA using oligo dt primer for RT-PCR or using hexamers for real-time quantitative RT-PCR.
RT-PCR
Table 2 shows the primers for bcl-xL and fas (7). The PCR condition for bcl-xL and fas was as follows: one cycle of 94 C (2 min) and 92 C (2 min), 40 cycles of 55 C (30 sec), 72 C (90 sec), and 92 C (45 sec). PCR was conducted with 50 µl reaction volumes of 1x PCR buffer II (Applied Biosystems, Foster City, CA), 2.5 mM MgCl2, 200 nM each primer, 200 µM of each deoxynucleotide triphosphate, and 0.025 U/µl Taq polymerase with 2 µg cDNA. Fifteen microliters of the RT-PCR products were electrophoresed through 1.5% agarose gels and stained with ethidium bromide and examined under UV light. The following PCR products were detected: bcl-xL (379 bp) and fas (413 bp).
Real-time quantitative RT-PCR
Primers, a probe, and PCR conditions to measure the human ß-glucuronidase gene (used as an endogenous RNA control) expression have been previously described (6). The sequences of the oligonucleotide primers and the hybridization fluorescent probe for TRF-1 gene were designed and synthesized by Biosearch Technologies, Inc. (Novato, CA) (Table 2
). PCR for TRF-1 was conducted, in triplicate, with 50 µl reaction volumes of 1x PCR buffer A (Applied Biosystems), 6.5 mM MgCl2, 900 nM each of primers, 200 nM each of deoxynucleotide triphosphates, 100 nM probe, 0.025 U/µl Taq Gold (Applied Biosystems), and 50 ng cDNA, using ABI 7700 Prism (Applied Biosystems). The cycling conditions for TRF-1 were as follows: 1 cycle of 95 C (12 min), 45 cycles of 95 C (15 sec) and 60 C (1 min). Relative TRF-1 gene expression was determined by the 
- threshold cycle method (6, 8). Sequence Detection (version 1.7, Applied Biosystems) was used for analysis. The threshold cycle values for each set of three reactions were averaged for all subsequent calculations (6, 9).
Statistical analysis
Relative TRF-1 gene expression among the various adrenal tissues was compared using the Mann-Whitney U test. Differences were considered significant at P < 0.05.
|
Results
|
|---|
RT-PCR
Figure 1 shows representative bcl-xL (379 bp) and fas (413 bp) gene expression in various adrenal tissues. All adrenal tissues expressed bcl-xL gene. Normal adrenal tissues (7 of 7), cortical adenomas (17 of 17), cortical hyperplasias (4 of 4) and pheochromocytomas (20 of 20) expressed the fas gene, but cortical cancers (0 of 4) did not. Thus, all adrenal tissues, benign or malignant, expressed the antiapoptosis gene, bcl-xL. In contrast, all the benign adrenal tumors, but none of the cortical cancers, expressed the proapoptosis gene, fas.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 1. Representative expressions of bcl-xL and fas genes in adrenal tumors. RT-PCR products from adrenal tissues were electrophoresed on 1.5% agarose gel stained with ethidium bromide. Expression of ß-actin gene was used as an internal control. Normal tissue, Normal adrenocortical tissues adjacent to adrenal tumors (lanes 1 and 2); Adenoma, aldosterone-secreting (lane 3) and cortisol-secreting (lane 4) adrenal cortical adenomas, Hyperplasia, Cushing’s disease (lane 5) and ectopic ACTH-syndrome (lane 6); Pheochromocytoma, lanes 7 and 8; Cortical cancer, adrenal cortical cancer (lanes 9–12).
|
|
Real-time quantitative RT-PCR
Figure 2 shows, and Table 3 summarizes, the amount of relative TRF-1 gene expression in the adrenal tumors studied. TRF-1 gene expression was significantly higher in adrenal cortical cancers than in normal adrenal tissues, cortical adenomas, cortical hyperplasias, and pheochromocytomas. The three patients (cases 1–3) who had a very high TRF-1 gene expression died 5–24 months after adrenalectomy. The one patient (case 4) who did not have an increased TRF-1 gene expression is still alive 5 yr, after adrenalectomy, with distant metastasis.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2. Relationship between relative expression of TRF-1 gene and prognosis of adrenal cortical cancer. Relative gene expressions of adrenal tissues were measured by real-time quantitative RT-PCR. Normal tissue, Normal tissues adjacent to adrenal tumors; Adenoma, adrenal cortical adenoma; Hyperplasia, adrenal cortical hyperplasia; Cortical cancer, adrenal cortical cancer; Survival, survival period after adrenalectomy.
|
|
|
Discussion
|
|---|
In this investigation, we documented that all benign and malignant adrenal tissues that were evaluated expressed the antiapoptosis gene, bcl-xL, but only adrenal cortical cancers failed to express the proapoptosis gene, fas. bcl-xL inhibits apoptosis, whereas fas induces apoptosis (10, 11). Increased bcl-xL and decreased fas expression have been observed in other cancers (12, 13). The decreased expression of fas in adrenal cortical cancers may help them bypass the control of programmed cell death. Determination of antiapoptosis and proapoptosis gene expression may also help to differentiate between benign and malignant adrenal tumors, because none of our four adrenal cortical cancers expressed fas.
bcl-xL is one of the bcl gene family members, and it inhibits apoptosis independent of bcl-2 (14). It has become increasingly clear that abnormality in the regulation of programmed cell death is a critical component in multistep tumorigenesis (15, 16). Increased expression of bcl-xL could be involved in malignant behavior, because it inhibits apoptosis and increases tumor growth (12). We previously reported that adrenal cortical cancers failed to express bax; bax and fas are both proapoptosis genes (6, 17). Bax-induced apoptosis is mitochondria-dependent, whereas fas-induced apoptosis is mitochondria-independent (11). Fas expression has been reported to be suppressed in thyroid cancers and also adrenal cancers (13, 18).
Telomeres, telomerase, and hTERT are related to tumor growth and proliferation (19, 20, 21, 22). Telomeres are a repeating sequence of six bases, (TTAGGG)n at the ends of chromosomes. Telomeres shorten at each cell division. When telomere length is less than 4–6 kbp, normal cells can no longer proliferate. Telomerase can synthesize telomeric DNA repeats and compensate for such shortening. Many cancers have elevated telomerase activity that enables them to proliferate (23). hTERT elongates telomere length through activation of telomerase (24). TRF-1 binds to chromosome ends and limits telomere elongation (25).
Telomere length and telomerase activity are not always concordant. For example, thyroid cancer, in spite of having elevated telomerase activity, often has shorter telomeres than adjacent normal tissue (26, 27). We hypothesized that adrenal cortical cancers that had elevated hTERT expression also had increased TRF-1 expression. Telomere length is controlled by mechanisms that involve telomerase and TRF-1 (19, 28). TRF-1 binds to double-strand telomeric DNA and regulates telomere length without affecting the enzymatic activity of telomerase (25). Telomere length is shorter in some cancers, compared with adjacent normal tissue, despite higher telomerase activity in cancers (27, 29). This suggests a regulatory mechanism that limits telomere elongation by genes such as TRF-1, which is overexpressed in some cancers. hTERT gene expression examined in our previous investigation correlated with TRF-1 gene expression in adrenal cortical tumors (r = 0.475; P < 0.05, Pearson’s correlation coefficient) but not in pheochromocytomas (r = 0.009) (6). This finding suggests that the feedback mechanism of TRF-1 to check telomere length is maintained in adrenal cortical tumors; thus TRF-1 expression increases with increased hTERT expression. Parallel to the level of expression of hTERT, the increased expression of TRF-1 may correlate with malignancy.
Increased hTERT gene expression activates cell proliferation in cancers. Prognosis of patients with breast cancer was reported to be inversely proportional to hTERT gene expression (30). Our data indicates that TRF-1 gene expression correlates directly with hTERT gene expression in adrenal cortical tumors. The one patient who expressed lowest TRF-1 expression was the only one who survived more than 5 yr after adrenalectomy and was alive with distant metastasis. In our previous study, this tumor also had the lowest expression of hTERT gene, compared with the other three adrenal cortical cancers (6). Thus, TRF-1 gene expression, in addition to hTERT gene expression, may also help to predict tumor behavior in patients with adrenal cortical cancer.
In conclusion, adrenal cortical cancers, in contrast with benign adrenal tumors, fail to express fas gene. In addition, the level of TRF-1 gene expression seems to correlate with adrenal cortical cancer aggressiveness. These observations should be confirmed by a larger study that includes more adrenal cortical cancers, but these findings may help to select patients for adrenalectomy and guide postsurgical treatment.
|
Footnotes
|
|---|
This work was supported in part by Mt. Zion/ Health Systems, The James Martin Foundation, The Heller Family Foundation, and The Friends of Endocrine Surgery. Part of this study was presented at the 93rd Annual Meeting of The American Association for Cancer Research, San Francisco, California, April 6–10, 2002; and the 84th Annual Meeting of The Endocrine Society, San Francisco, California, June 19–22, 2002.
Abbreviations: hTERT, Human telomerase reverse transcriptase; TRF-1, telomeric-repeat binding factor-1.
Received June 20, 2002.
Accepted April 10, 2003.
|
References
|
|---|
- Vassilopoulou-Sellin R, Schultz PN 2001 Adrenocortical carcinoma. Clinical outcome at the end of the 20th century. Cancer 92:1113–1121[CrossRef][Medline]
- Barnett Jr CC, Varma DG, El-Naggar AK, Dackiw APB, Porter GA, Pearson AS, Kudelka AP, Gagel RF, Evans DB, Lee JE 2000 Limitations of size as a criterion in the evaluation of adrenal tumors. Surgery 128:973–983[CrossRef][Medline]
- Weiss LM, Medeiros LJ, Vickery Jr AL 1989 Pathologic features of prognostic significance in adrenocortical carcinoma. Am J Surg Pathol 13:202–206[Medline]
- Gicquel C, Bertherat J, Le Bouc Y, Bertagna X 2000 Pathogenesis of adrenocortical incidentalomas and genetic syndromes associated with adrenocortical neoplasms. Endocrinol Metab Clin North Am 29:1–13[CrossRef][Medline]
- Stratakis CA, Chrousos GP 2000. Adrenal cancer. Endocrinol Metab Clin North Am 29:15–25
- Kanauchi H, Wada N, Clark OH, Duh Q-Y 2002 Apoptosis regulating genes, bcl-2 and bax, and human telomerase reverse transcriptase (hTERT) mRNA expression in adrenal tumors: possible diagnostic and prognostic importance. Surgery 132:1021–1026[CrossRef][Medline]
- Agarwal R, Talati M, Lambert W, Clark AF, Wilson SE, Agarwal N, Wordinger RJ 1999 Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells. Exp Eye Res 68:583–590[CrossRef][Medline]
- Heid CA, Stevens J, Lival KJ, Willams PM 1996 Real time quantitative PCR. Genome Res 6:986–994[Abstract/Free Full Text]
- Ginzinger DG, Godfrey TE, Nigro J, Moore II DH, Suzuki S, Pallavicini MG, Gray JW, Jensen RH 2000 Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis. Cancer Res 60:5405–5409[Abstract/Free Full Text]
- Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB 1993 bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597–608[CrossRef][Medline]
- Nagata S, Golstein P 1995 The Fas death factor. Science 267:1449–1456[Abstract/Free Full Text]
- Leiter U, Schmid RM, Kaskel P, Peter RU, Krahn G 2000 Antiapoptotic bcl-2 and bcl-xL in advanced malignant melanoma. Arch Dermatol Res 292:225–232[CrossRef][Medline]
- Basolo F, Fiore L, Baldanzi A, Giannini R, Dell’Omodarme M, Fontanini G, Pacini F, Danesi R, Miccoli P, Toniolo A 2000 Suppression of Fas expression and down-regulation of Fas ligand in highly aggressive human thyroid carcinoma. Lab Invest 80:1413–1419[Medline]
- Minn AJ, Rudin CM, Boise LH, Thompson CB 1995 Expression of bcl-xL can confer a multidrug resistance phenotype. Blood 86:1903–1910[Abstract/Free Full Text]
- Wang DG, Johnston CF, Marley JJ, Phenix KV, Atkinson AB, Russell CF, Buchanan KD 1997 Expression of the apoptosis-suppressing gene BCL-2 in pheochromocytoma is associated with the expression of C-MYC. J Clin Endocrinol Metab 82:1949–1952[Abstract/Free Full Text]
- Bargou RC, Daniel PT, Mapara MY, Bommert K, Wagener C, Kallinich B, Royer HD, Dorken B 1995 Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer 60:854–859[Medline]
- Oltvai ZN, Milliman CL, Korsmeyer SJ 1993 Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619[CrossRef][Medline]
- Wolkersdorfer GW, Marx C, Lohmann T, Brauer S, Schroder S, Brown J, Mitsiades N, Chrousos GP, Bornstein SR 1998 The mercy of adrenocortical tumor cells on lymphocytes. Endocr Res 24:711–716[Medline]
- Levy MZ, Allsopp RC, Futcher AB, Greider CW, Harley CB 1992 Telomere end-replication problem and cell aging. J Mol Biol 225:951–960[CrossRef][Medline]
- Zeiger MA, Smallridge RC, Clark DP, Liang CK, Carty SE, Watson CG, Udelsman R, Saji M 1999 Human telomerase reverse transcriptase (hTERT) gene expression in FNA samples from thyroid neoplasms. Surgery 126:1195–1198[CrossRef][Medline]
- Brousset P, Chaouche N, Leprat F, Branet-Brousset F, Trouette H, Zenou RC, Merlio JP, Delsol G 1997 Telomerase activity in human thyroid carcinomas originating from the follicular cells. J Clin Endocrinol Metab 82:4214–4216[Abstract/Free Full Text]
- Mannelli M, Gelmini S, Arnaldi G, Becherini L, Bemporad D, Crescioli C, Pazzagli M, Mantero F, Serio M, Orlando C 2000 Telomerase activity is significantly enhanced in malignant adrenocortical tumors in comparison to benign adrenocortical adenomas. J Clin Endocrinol Metab 85:468–470[Abstract/Free Full Text]
- Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW 1994 Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015[Abstract/Free Full Text]
- Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR 1998 Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts. Cancer Res 58:4168–4172[Abstract/Free Full Text]
- Smogorzewska A, van Steensel B, Bianchi A, Oelmann S, Schaefer MR, Schnapp G, de Lange T 2000 Control of human telomere length by TRF1 and TRF2. Mol Cell Biol 20:1659–1668[Abstract/Free Full Text]
- Kammori M, Nakamura KI, Kawahara M, Mimura Y, Kaminishi M, Takubo K 2002 Telomere shortening with aging in human thyroid and parathyroid tissue. Exp Gerontol 37:513–521[CrossRef][Medline]
- Kammori M, Takubo K, Nakamura K, Furugouri E, Endo H, Kanauchi H, Mimura Y, Kaminishi M 2000 Telomerase activity and telomere length in benign and malignant human thyroid tissues. Cancer Lett 159:175–181[CrossRef][Medline]
- van Steensel B, de Lange T 1997 Control of telomere length by the human telomeric protein TRF1. Nature 385:740–743[CrossRef][Medline]
- Nakamura K, Furugori E, Esaki Y, Arai T, Sawabe M, Okayasu I, Fujiwara M, Kammori M, Mafune K, Kato M, Oshimura M, Sasajima K, Takubo K 2000 Correlation of telomere lengths in normal and cancers tissue in the large bowel. Cancer Lett 158:179–184[CrossRef][Medline]
- Bieche I, Nogues C, Paradis V, Olivi M, Bedossa P, Lidereau R, Vidaud M 2000 Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay. Clin Cancer Res 6:452–459[Abstract/Free Full Text]
Top
Abstract
Introduction
