Variable Expression of the Transcription Factors cAMP Response Element-Binding Protein and Inducible cAMP Early Repressor in the Normal Adrenal Cortex and in Adrenocortical Adenomas and Carcinomas
ALESSANDRO PERI, PAOLA LUCIANI, BARBARA CONFORTI, SILVANA BAGLIONI-PERI, FEDERICA CIOPPI, CLARA CRESCIOLI, PIETRO FERRUZZI, STEFANIA GELMINI, GIORGIO ARNALDI, GABRIELLA NESI, MARIO SERIO, FRANCO MANTERO, AND MASSIMO MANNELLI
Endocrine Unit (A.P., P.L., B.C., F.C., C.C., P.F., M.S., M.M.), Medical Genetics Unit (S.B .- P.), and Clinical Biochemistry Unit (S.G.), Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy; Department of Human Pathology and Oncology (G.N.), University of Florence, 50139 Florence, Italy; Department of Internal Medicine (G.A.), University of Ancona, 60131 Ancona, Italy; and Department of Medical and Surgical Sciences (F.M.), University of Padova, 35131 Padova, Italy
The molecular mechanisms leading to adrenocortical tumor- igenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP path- way, regulates both cell proliferation/differentiation and ste- roid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP respon- sive element modulator (CREM) and cAMP responsive ele- ment binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms a, B, y, and 72 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight
adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adre- nals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carci- nomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the car- cinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early re- pressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex. (J Clin Endocrinol Metab 86: 5443-5449, 2001)
I N THE ADRENAL cortex, the pituitary hormone ACTH acts as the major activator of the cAMP-dependent path- way. ACTH regulates cell differentiation, steroid synthesis, and, to a lesser extent, cell proliferation (1). However, the regulation of adrenocortical cells by ACTH-driven cAMP signaling appears to be complex, because an inhibitory role of ACTH on cell proliferation in vitro has also been observed (2,3). Accordingly, complex multiple molecular mechanisms are likely to be related to adrenocortical tumorigenesis, as it is suggested by different reports. LOH of the ACTH receptor (ACTH-R) gene and reduced levels of mRNA (4) have been observed, for instance, in adrenal cancer and have been as- sociated with cellular dedifferentiation. The observation that approximately one third of patients with MEN type 1 have adrenocortical tumors (5) prompted investigation of the as- sociation between mutations of the MEN 1 gene and adrenal neoplasia. LOH at 11q13 has been described in adrenal tu-
mors, although it is not completely clear whether it involves the menin locus or not (6, 7). Furthermore, the activation of the proto-oncogene K-ras (8), p53 mutations (9), or overex- pression of IGF II and IGF-binding protein-2 (10) or epider- mal growth factor receptor (11) have been observed with variable frequency. However, the molecular mechanisms leading to adrenocortical tumorigenesis appear to be only partially known and are yet to be clearly elucidated.
Most of the intracellular effects of cAMP are mediated by the activation of PKA. PKA activates, by phosphorylation at specific serine sites, the nuclear transcription factors cAMP response element modulator (CREM) and cAMP response element binding protein (CREB) (12). Phosphorylated CREB and CREM bind as dimers to palindromic cAMP response element sequences, thus modulating the expression of cAMP-dependent genes. A classical cAMP response element has been observed, for instance, in the promoter of 11ß- hydroxylase (CYP11B1) or aldosterone synthase (CYP11B2) genes (13). The peculiar aspect of CREB and CREM genes resides in the fact that they can encode different isoforms, either activating or inhibiting gene expression, by mecha-
Abbreviations: ACTH-R, ACTH receptor; CBP, CREB binding pro- tein; CREB, cAMP responsive element binding protein; CREM, cAMP responsive element modulator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICER, inducible cAMP early repressor.
nisms of alternative exon splicing, alternative promoter us- age, and autoregulation of promoters (12). In particular, an internal promoter of CREM gene directs the expression of the repressor isoforms named inducible cAMP early repressors (ICERs) in response to cAMP activation (12). Recently, in the human adrenocortical cancer cell line H295R, loss of expres- sion of CREB gene and overexpression of the activator CREM T, compared with the normal adrenal cortex, have been ob- served (14). It has been hypothesized that this pattern of expression could be linked to cellular transformation. To better define whether cAMP-dependent transcription factors play a role in adrenocortical tumorigenesis, we studied the expression of CREB, CREM, and ICER in normal human adrenal cortex (n = 3), in cortisol-secreting adrenocortical adenomas (n = 8), and in adrenocortical carcinomas (n = 8) by RT-PCR.
Materials and Methods
Patients
Nineteen patients were included in the study after obtaining in- formed consent. Three human normal adrenal glands (from patients undergoing nephrectomy), eight adrenocortical adenomas, and eight adrenal carcinomas were studied. The clinical characteristics of the pa- tients are reported in Table 1. Tissue specimens, obtained at surgery, were immediately frozen in liquid nitrogen and stored at -80 C until RNA extraction. Hematoxylin and eosin-stained sections were prepared, reviewed, and classified according to the criteria of Weiss (15).
ACTH receptor expression
The gene expression of the ACTH-R was evaluated by quantitative/ competitive RT-PCR, as described previously (16). Briefly, the following primers were used: 5’-ACTGTCCTCGTGTGGTTTTG-3’ and 5’-AGAT- GAAGACCCCGAGCAG-3’. A nonhomologous RNA competitor was constructed and used in RT-PCR experiments. There was a 66-bp dif- ference between the length of the competitor and the normal ACTH-R transcript. Five increasing amounts of competitor were mixed with fixed amounts of tumoral RNA after RNase-free DNase digestion. The bands corresponding to the competitor and the transcript products were re- solved by gel electrophoresis. The densitometric ratios were determined and were then plotted against the number of RNA competitor molecules added to each RT-PCR. The number of ACTH-R mRNA copies was extrapolated considering the value 1 of the competitor / target ratio as the
point in which the number of competitor molecules is equal to that of the target (tumoral ACTH-R).
Telomerase assay
Telomerase activity was measured as described previously (17). Briefly, tissue samples (~100 mg) were homogenized and centrifuged. The supernatants were frozen and stored at -80 C. Protein concentration was measured by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA). Protein (6 µg ) was used for telomerase assay. RNase (Roche Diagnostics, Monza, Italy; 0.5 µg) was used for each assay for 30 min at 37 C to inactivate telomerase. Each extract was assayed in 47.2 ul reaction mixture containing 10 mm Tris-HCI (pH 8.3), 50 mM KC1, 4.5 mM MgCl2, 1 mM dNTP, 20 PM TAG-U primer, and 0.5 ALM LT4 gene 32 protein (Roche Diagnostics). After 60 min at 30 C for telomerase-medi- ated extension of TAG-U primer, the reaction mixture was subjected to PCR. A 10 min at 72 C step followed PCR cycles after the addition of a second reaction mixture containing 20 PM CTA-R primer and 1.5 U Taq Gold (Perkin-Elmer Corp., Norwalk, CT). We diluted 10 ul each PCR product with 490 pl 10 mM Tris-HC1, 1 mM EDTA (pH 7.5), and then 500 ul ultrasensitive fluorescent dye PicoGreen (Molecular Probes, Inc., Eu- gene, OR). Fluorescence was measured in a spectrofluorometer RF-540 (Shimadzu) using standard wavelengths. DNA concentration was cal- culated for each sample on a calibration curve generated by dilutions of a control DNA (0-100 µg/liter). The final DNA concentration of each sample was determined by subtracting the DNA amount obtained in the same specimen after RNase treatment. Telomerase activity was calcu- lated as the mean of duplicates for each sample and expressed as nano- grams of DNA per microgram of protein. A negative control, obtained after pretreatment of the sample with RNase, was also assayed for each specimen.
CREB, CREM, and ICER RT-PCR
RT-PCR was performed on total RNAs (0.5 µg for each reaction) using CREB-, CREM-, and ICER-specific primers. In particular, CREB primers spanned sequences at the 5’- and 3’-end of CREB mRNA (18). CREM primers were able to generate two different amplified products (243 and 390 bp), corresponding to the isoforms a, B, y (repressors), and +2 (activator) and to the activators T, Ta, and 71, respectively, as described previously (19). ICER primers were designed to detect ICER I and ICER II isoforms, as described previously (20). The primers were synthesized by Roche Diagnostics. Preliminary experiments were performed to de- termine the PCR cycles corresponding to the exponential phase of am- plification. Thereafter, the PCR were always stopped in the exponential phase (35 cycles). Only in those cases in which no amplified signal was detectable did we further assess the negativity by extending the number of cycles. Each experiment was repeated three times to confirm the
| Patient (no.) | Age (yr) | Sex | Stage at surgery | Postsurgical outcome | Tumor size (cm) | Tumor secretion | ACTH-R (no. of copies*) | Telomerase activity (ng DNA/µg protein) |
|---|---|---|---|---|---|---|---|---|
| 4 | 56 | F | Cured (5 ** ) | 4.0 | C | 6.76 | n.d. | |
| 5 | 47 | F | Cured (6) | 2.0 | C | 6.50 | 12.7 | |
| 6 | 57 | F | Cured (5) | 4.7 | C | 5.96 | 2.3 | |
| 7 | 27 | F | Cured (6) | 2.0 | C | 6.41 | 0.6 | |
| 8 | 62 | F | Cured (7) | 5.5 | C | 6.51 | 7.6 | |
| 9 | 66 | M | Cured (5) | 4.0 | C | 6.70 | 5.0 | |
| 10 | 56 | F | Cured (2) | 4.0 | None | 6.38 | 7.0 | |
| 11 | 58 | F | Cured (3) | 1.8 | C | 6.87 | 2.7 | |
| 12 | 28 | F | III | Recurrence, mitotane | 22.0 | A | 4.91 | 27.6 |
| 13 | 33 | F | II | Recurrence, mitotane | 5.0 | A | 5.41 | 17.8 |
| 14 | 45 | F | IV | Metastasis, mitotane | 15.0 | None | 5.48 | 18.8 |
| 15 | 71 | F | IV | Metastasis, mitotane | 8.0 | None | 5.74 | 17.8 |
| 16 | 69 | F | III | Metastasis, died | 6.0 | A+M | 5.41 | 11.5 |
| 17 | 21 | F | IV | Metastasis, died | 12.0 | C | 5.73 | 15.8 |
| 18 | 50 | M | II | Remission | 9.0 | None | 5.06 | 15.2 |
| 19 | 61 | F | III | Recurrence, mitotane | 8.0 | None | 5.20 | 13.2 |
Histological diagnosis in patients 4-11, adenoma; in patients 12-19, carcinoma. C, Cortisol; A, androgens; M, mineralcorticoids.
*, Log (copies)/100 ng RNA; ** , years of follow-up; n.d., not determined.
results. The quality of RNAs was assessed by performing additional RT-PCR using primers specific for the glyceraldehyde-3-phosphate de- hydrogenase (GAPDH) gene, as described previously (21). Finally, in each RT-PCR experiment, a no RNA reaction was added as a negative control. RT-PCR products were subjected to agarose gel electrophoresis, Southern blot, hybridization to CREB-, CREM-, and ICER-specific probes and immunochemiluminescent detection, as described previ- ously (19, 20). The membranes were exposed to x-ray films. The time of exposure of the film was kept constant in each experiment (5 min).
Sequence analysis of ICER RT-PCR products
RT-PCR for the determination of ICER transcripts originated signals of different length. The specificity of the putative signals corresponding to ICER I and II isoforms was validated by additional sequence analysis, as described previously, using 32[P]-yATP-labeled primers (20). Ampli- fied products were electrophoresed on acrylamide gel (6%) in the pres- ence of 7 M urea. Then, the gels were blotted on Whatman 3 M filters (Whatman International Ltd., Maidstone, UK). After drying, the filters were transferred to an x-ray cassette and exposed to x-ray films for 12 h.
Statistical analysis
Statistical comparison between groups was performed using the t test. Differences were considered as statistically significant at the 0.05 level.
Results
Histology, ACTH receptor expression, and telomerase activity
Total RNA from normal adrenal glands (n = 3), adreno- cortical adenomas (n = 8), and adrenocortical carcinomas (n = 8) was subjected to RT-PCR. The histological features of the adrenal carcinomas, according to the criteria of Weiss (15), are shown in Table 2.
In addition to histological assessment and a prolonged follow-up (4.9 ± 0.6 yr; mean ± SE) of the patients with adenoma, further characterization was performed on the tissue samples. ACTH-R expression, which can be altered in adrenal carcinoma (4), was determined by competitive RT- PCR and was found to be significantly higher in adrenal adenomas than in carcinomas [6.51 ± 0.1 vs. 5.37 ± 0.1 log(no. copies)/100 ng RNA; mean ± SE; P = 0.000001] (Table 1). Telomerase activity, which has been shown to be a potential marker for differentiating benign from malignant adrenal tumors (22, 23), was also determined, and it was found to be higher in the carcinomas (adenomas, 5.41 + 1.54, vs. carci- nomas, 17.21 ± 1.72 ng DNA/µg protein; mean ± SE; P = 0.00023) (Table 1).
| Patient (no.) | Necrosis | Mitotic index (no./10 hpf) | Vascular invasion | Capsular invasion |
|---|---|---|---|---|
| 12 | + | 10 | + | + |
| 13 | + | 16 | - | + |
| 14 | + | 8 | + | + |
| 15 | + | 12 | + | + |
| 16 | + | 21 | + | + |
| 17 | + | 40 | + | + |
| 18 | + | 14 | - | + |
| 19 | + | 16 | + | + |
hpf, High-power fields.
Analysis of CREB transcripts
RT-PCR analysis of CREB transcripts revealed the pres- ence of a specific signal in all of the samples from normal adrenal gland and adrenal adenoma. The specificity of the 1026-bp signal, corresponding to the full-length transcript of CREB gene, was confirmed by Southern blotting and hy- bridization to a CREB-specific probe. The results, as obtained by chemiluminescent detection, are shown in Fig. 1A (no. 1-3, normal adrenal; 4-11, adenoma). Conversely, signals corresponding to CREB transcripts were detectable in only four of eight adrenocortical carcinomas (no. 12-15), whereas no amplified signal was in any case obtained (experiments were repeated three times) in the remaining samples (no. 16-19). In these cases, no signal was detectable, even after extending the number of PCR cycles (data not shown). The quality of RNAs was assessed by the analysis of GAPDH transcripts. A GAPDH-specific signal was readily detectable in all samples (Fig. 1B), thus excluding that the absence of CREB transcripts in some samples was due to RNA degra- dation. No significant difference in ACTH-R mRNA levels was observed between the carcinomas expressing and those not expressing CREB (5.38 ± 0.17 vs. 5.35 ± 0.15 log(no. copies)/100 ng RNA; mean ± SE, P = 0.88).
Analysis of CREM transcripts
CREM-specific primers, spanning sequences from exon B (sense primer) and from exon D (antisense primer) of CREM gene, were designed for RT-PCR experiments. These primers have been designed and used previously in our laboratory (19) and are able to generate two specific signals correspond- ing to different CREM isoforms (see Materials and Methods). The expected signal of 243 bp, corresponding to the CREM repressors a, B, and y and to the activator +2, was detected in all RNAs from normal adrenals (no. 1-3), adenomas (no. 4-11), and carcinomas (no. 12-18), with the exception of one case (no. 19). The specificity of the signal was validated by hybridization of RT-PCR products to a CREM-specific probe, and the results are shown in Fig. 2A. However, in no case was the presence of the 390-bp signal corresponding to the acti- vators T, Ta, and 1, which was readily detectable in other cell systems such as germ cells (19), observed in the adrenal cortex.
Analysis of ICER transcripts
For the detection of ICER transcripts by RT-PCR, primers were selected from the internal promoter and exon y (sense primer) and from exon Ib (antisense primer) of CREM gene. These primers were designed to detect ICER I (657 bp) and ICER II (257 bp) isoforms. Amplified products were hybrid- ized to a specific oligonucleotide probe. Signals correspond- ing to ICER I and ICER II transcripts were detected in all normal adrenal glands (Fig. 2B). In adrenocortical adenomas, ICER I and II transcripts were observed in all samples, with the exception of one case (no. 7). Conversely, in adrenal carcinomas, the presence of ICER I and II mRNA was de- tected in only three of eight cases (no. 12-14), whereas no detectable levels of expression were repeatedly found in the remaining cases (no. 15-19) (Fig. 2B). In those cases in which
A CREB
St N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
bp
1026 -
B GAPDH
St N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1
19
bp
193 -
no signal was detected, the negativity was confirmed even after extension of the number of PCR cycles (data not shown). Sequence analysis of the different amplified products, which was performed in samples from normal adrenals as well as from adenomas and carcinomas, confirmed that the 657- and 257-bp signals correspond to ICER I and ICER II, respectively (data not shown), as described previously in pituitary ade- nomas (20). The middle two amplified fragments, previously detected also in pituitary adenomas (20), contain partial se- quences of ICER I (data not shown) and might correspond to different, so far undescribed, ICER isoforms, which will need further characterization.
Discussion
ACTH, by activating the cAMP-dependent pathway, has a moderate effect on adrenal cell proliferation or even a mild anti-proliferative effect in vitro (2, 3), whereas it plays a pivotal role in regulating steroid hormone synthesis (1) and, hence, in maintaining a differentiated phenotype. LOH of the ACTH receptor gene and reduced expression of ACTH re- ceptor mRNA have been detected in a subset of adrenocor- tical carcinomas (4). Because LOH is a characteristic of many tumor types, it has been suggested that the ACTH-R gene may act as a tumor suppressor gene and that the LOH of this gene may result in loss of differentiation and in growth
advantage. The possible involvement of the cAMP-depen- dent pathway in neoplastic transformation of adrenocortical cells has been recently highlighted by the identification of the gene for Carney complex, a disease characterized by different clinical features, including the presence of pigmented adre- nocortical tumors. In fact, mutations of the gene encoding the protein kinase type I-a regulatory subunit, an apparent tu- mor suppressor gene, have been detected in a subset of patients with this disease (24).
In this report, we focused on two downstream targets of the cAMP-dependent pathway, i.e. CREM and CREB, and we investigated their expression in the normal adrenal gland as well as in adrenocortical adenomas and carcinomas. We de- tected CREM-specific transcripts corresponding to the tran- scriptional activator 72 and to the inhibitors a, ß, and y in RNAs from all normal adrenals, adenomas, and carcinomas, with only one exception in the last group. In no case were transcripts corresponding to CREM activators T, Ta, and 71 detectable. Therefore, an altered pattern of CREM expression does not appear to be a molecular feature associated with adrenal tumorigenesis. On the other hand, different patterns of expression of ICER isoforms, which derive from an inter- nal promoter of the CREM gene, were observed. In partic- ular, whereas ICER isoforms were readily detectable in all normal adrenal glands and adenomas (with the exception of
A CREM
1
2
3
4
5
7
8
9
10
11
12
13
14
15
16
17
St
N
6
19
bp
243 -
B
ICER
St
N
1
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7
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15
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bp
653 -
517 -
453 -
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298 -
one case), lack of expression was found in five of eight car- cinomas. Similarly, CREB transcripts were consistently ob- served in normal adrenal cortex and in adrenal adenomas, whereas detectable mRNA levels were not found in half of the adrenal cancers. The CREB transcript that we detected corresponds to the full-length transcript of CREB gene, which originates a transcriptional activator, upon phosphor- ylation at Ser-133. Conversely, CREB repressors originate by mechanisms of alternative exon splicing or by alternative start sites of translation of CREB gene. As a consequence, lack of transcription of one or more exons occurs, and the result- ing proteins cannot undergo phosphorylation-mediated ac- tivation. In keeping with our results, in a recent report the absence of CREB expression was observed in the human adrenocortical cancer cell line H295R (14). However, at vari- ance with our findings, in that study a compensatory over- expression of CREM T, which is usually absent or expressed at a very low level in the normal adrenal cortex, was detected in H295 cells (14). This apparent discrepancy might be due to the different cell system (i.e. adrenal tumoral tissues ob- tained at surgery vs. a single cultured cell line). In view of our results, it is noteworthy that both the promoter of CREB gene and the internal promoter of CREM gene that directs the expression of ICER isoforms, but not the upstream promoter, are autoregulated by cAMP. In fact, three CREs are present in the promoter region of CREB gene (25); two pairs of closely spaced CREs are contained in the internal promoter of CREM gene (26). As a result, activation of the cAMP pathway, by enhancing the levels of phosphorylated, hence activated, CREB, stimulates CREB and ICER expression. These consid-
erations, in conjunction with our experimental data, support the hypothesis that adrenal malignancies can be associated to an alteration of the cAMP-dependent signaling. In this scenario, the lack of expression of CREB (and/or ICER) in a consistent percentage of cases of adrenal carcinoma may be regarded as a marker of loss of cell differentiation. To clarify whether or not the absence of CREB/ICER mRNA was a consequence of a different molecular alteration such as re- duced ACTH-R expression, which may be a feature of ad- renal cancer (4), the levels of transcript of the ACTH-R gene were determined. The expression levels of this gene did not differ between the carcinomas expressing CREB/ICER and those showing loss of CREB/ICER expression, thus suggest- ing that the absence of CREB/ICER mRNA in a subset of adrenal carcinomas does not appear to be secondary to an upstream disregulation at the receptor level. Interestingly, CREB activation via Ser133-phosphorylation has been related to cell differentiation in different tissues, such as the brain (27) and the adipose tissue (28). In the blood system, phos- phorylated CREB has been shown to induce the differenti- ation of megakaryocytes (29). A CREB gain-of-function mutant, which induced high levels of constitutive Ser133- phosphorylation, has been found to promote cell differenti- ation in vitro (30). On the other hand, mice lacking CREB expression have been generated; the mutant mice invariably die from respiratory distress, associated to surfactant defi- ciency, immediately after birth (31). Significantly, a severe impairment in brain and T cell development has been ob- served. Furthermore, transgenic mice, in which expression of a dominant negative CREB isoform was targeted to the thy-
roid gland, exhibited severe growth retardation and primary hypothyroidism; histologically, the thyroid glands were characterized by poorly developed follicles (32).
CREB-mediated activation of transcription is a multifac- torial process that includes the involvement of different co- activators interacting with the transcriptional apparatus. CREB binding protein (CBP) and p300 are two factors con- necting with CREB only in its phosphorylated form (33, 34), thus participating in the molecular events resulting in the stimulation of gene expression. CBP and p300 take part in a variety of cellular processes, such as cell growth, differenti- ation, and apoptosis (35). It is noteworthy that alterations of the human CBP gene have been implicated in hematological malignancies, such as acute or chronic myeloid leukemia (36, 37). Similarly, inactivation of p300 gene has been related to leukemia (38) as well as to gastric and colorectal carcinomas (39), suggesting that these coactivators of CREB function may serve as tumor suppressor proteins. These data confirm that cAMP signaling is strongly involved in the control of cell growth and differentiation and that alterations disrupting the integrity of this pathway may result in neoplastic proliferation.
Because the number of cases of adrenocortical cancer that we examined so far is limited, it is not possible to conclu- sively establish whether there is a relationship between the presence or the absence of expression of CREB or ICER and the clinical features of the patients. In a recent report we observed that the high levels of telomerase activity in adrenal carcinomas were positively correlated with the tumor size (23). However, in the present study it may be noteworthy to observe that, among the eight patients with adrenal carci- noma, the two patients who died from metastatic disease did not show detectable levels of CREB and ICER expression. Therefore, although any conclusive statement will be possi- ble only after extending the number of cases examined, pre- liminary observations seem to suggest that the lack of ex- pression of CREB/ICER in cases of adrenal cancer might be linked to a more severe outcome.
In conclusion, in the present study we have demonstrated for the first time that, whereas cAMP-dependent transcrip- tion factors are consistently expressed in the normal adrenal gland and in adrenocortical adenomas, loss of expression of CREB and/or ICER may be a feature of adrenal cancer. Because cAMP signaling is involved in the processes leading to cell differentiation, it can be hypothesized that an alter- ation of the cAMP-dependent transcription factor machinery may be associated with impaired cell differentiation and with a transformed phenotype in the adrenal cortex.
Acknowledgments
We thank Dr. Marina Scarpelli (Department of Pathology, University of Ancona, Italy); Prof. Andrea Amorosi (Department of Pathology, University of Catanzaro, Italy); Prof. Marco Carini (Urology Unit, Os- pedale Santa Maria Annunziata, Florence, Italy); and Prof. Domenico Borrelli and Dr. Andrea Valeri (General Surgery Unit, Ospedale Careggi, Florence, Italy) for valuable contributions.
Received December 19, 2000. Accepted August 13, 2001.
Address all correspondence and requests for reprints to: Dr. Massimo Mannelli, Department of Clinical Physiopathology, Endocrine Unit, Uni-
versity of Florence, Viale Pieraccini, 6, 50139 Florence, Italy. E-mail: m.mannelli@dfc.unifi.it.
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