P53 protein and its messenger ribonucleic acid in human adrenal tumors
V. Adleff*, K. Rácz*, M. Tóth*, I. Varga*, A. Bezzegh ** , and E. Gláz
*Gastroenterological and Endocrinological Research Unit, Second Department of Medicine, Semmelweis University Medical School, ** Byk Gulden Reference Laboratory, National Institute of Oncology, Budapest, Hungary
ABSTRACT. The role of p53 tumor suppressor gene in the pathomechanism of adrenal tumors was investigated by measuring p53 protein and its messenger ribonucleic acid (mRNA) in 12 normal human adrenals as well as in 56 adrenal tumors (7 aldosterone-producing adenomas, 5 adrenocortical adenomas causing Cushing’s syndrome, 19 non- hyperfunctioning adrenocortical adenomas, 5 adrenocortical carcinomas, 12 pheochromocy- tomas, 3 myelolipomas, 4 ganglioneuromas and 1 hemangioma). The p53 protein concentration was significantly increased in aldosterone-producing adenomas (394±36 pg/mg cytosolic protein, mean±SE, vs 266±18 in normal human adrenals), whereas the concentration of this protein in Cushing’s adenomas, non-hyperfunctioning adre- nocortical adenomas, pheochromocytomas, and in
all but one adrenocortical carcinomas was similar to that measured in normal human adrenal tissues. One adrenocortical carcinoma tissue showed very high p53 protein content (3000 pg/mg cytosolic protein). By contrast, myelolipomas (23±20) gan- glioneuromas (43±15) and a hemangioma (11 pg/mg cytosolic protein) had very low p53 protlein content. Northern blot analysis revealed the pres- ence of p53 mRNA in each adrenal tissue exam- ined with highest levels in aldosterone-producing and Cushing’s adenomas. It is possible that the dif- ferences in p53 protein and/or mRNA contents re- flect corresponding differences in the pathogenetic importance of p53 alterations in these types of adrenal tumors.
(J. Endocrinol. Invest. 21: 753-757, 1998) @1998, Editrice Kurtis
INTRODUCTION
The p53 tumor suppressor gene, located on the short arm of the human chromosome 17, encodes a 53-kilodalton nuclear phosphoprotein that func- tions as a key regulator of cell proliferation (1, 2). This phosphoprotein also is recognized as an im- portant constituent of a pathway responsible for re- pairing DNA damage through mechanisms involv- ing growth arrest and apoptosis (3). It has been shown that alterations of the structure and/or ex- pression of the p53 gene are involed in the process of tumoral growth. Loss of function of the p53 pro- tein, mainly due to inactivating mutations of the p53 gene, occurs in several human tumors, includ-
ing adrenocortical adenomas and carcinomas, and pheochromocytomas (4-10).
With the aim of determining the potential role of p53 protein in the pathomechanism of adrenal tu- mors, in the present study we used a luminometric immunoassay to measure p53 protein levels in nor- mal human adrenals and different types of adrenal tumors. As shown in earlier studies, this method of- fered an excellent approach to evaluate the accu- mulation of p53 protein in tumoral tissues (11). This method allowed us to analyze, for the first time, quantitative differences in p53 protein content in a large series of normal and tumorous adrenals in- cluding aldosterone-producing and Cushing’s ade- nomas, non-hyperfunctioning adenomas, adreno- cortical carcinomas, pheochromocytomas, myelo- lipomas, ganglioneuromas, and a hemangioma. In addition, we wanted to examine whether differ- ences in p53 protein levels, if exist, could be due to respective differences in p53 messenger ribo- nucleic acid (mRNA) contents. For this reason we performed Northern blot hybridization studies to detect p53 mRNA in these adrenal tissues.
Key-words: p53 protein, p53 expression, luminometric immunoassay, adre- nal tumors, adrenal pathophysiology.
Correspondence: Károly Rácz, M.D., D.Sc., 2nd Dept. of Medicine, Sem- melweis University, 1088 Budapest, Szentkirályi u 46, Hungary. Accepted July 16, 1998.
MATERIALS AND METHODS
Patients and adrenal tissues
Aldosterone-producing adenomas were removed from 7 patients, who had typical symptoms of pri- mary aldosteronism, including severe hypokalemia, hypertension, polyuria, increased plasma aldosterone and 18-hydroxycorticosterone levels, and suppressed plasma renin activity. In all patients surgical removal of the adrenal adenomas resulted in a normalization of both clinical and biochemical symptoms. Adreno- cortical adenomas causing Cushing’s syndrome were obtained from 5 patients. In all five cases, the clinical and hormonal signs of cortisol overproduction were unequivocally confirmed by endocrinological evalu- ation and adrenal surgery resulted in a disappearance of all signs of Cushing’s syndrome. Non-hyperfunc- tioning adrenocortical adenomas were removed from 19 patients, whose adrenal tumors were discovered unexpectedly during abdominal ultrasound or com- puted tomography. In these patients, the diagnosis of non-hyperfunctioning adrenocortical tumor was es- tablished by detailed endocrine evaluation, which ex- cluded primary aldosteronism, Cushing’s syndrome, hyperandrogenism and pheochromocytoma. Adre- nocortical carcinomas were obtained from 5 patients with (2 cases) or without (3 cases) clinical and hormo- nal symptoms of hyperandrogenism and Cushing’s syndrome. Adrenal pheochromocytomas were ob- tained from 12 patients who presented with sustained or paroxysmal hypertension. In all cases, the diagno- sis was confirmed by preoperative endocrine testing which included 24-h urinary vanillylmandelic acid de- terminations, MIBG scintigraphy, CT scanning and examination of T2-weighted images on MRI. Myelo- lipomas (3 cases), ganglioneuromas (4 cases) and a hemangioma (1 case) were obtained from patients without any symptoms of adrenal disorders. These adrenal lesions were discovered incidentally during abdominal ultrasound or computed tomography and endocrine evaluation of the patients proved normal hormone values. Immediately after surgical removal, all adrenal tumors were quickly freed from adjacent tissues and pieces of the tumors were immediately frozen, then stored at -80 C. Samples from all tumoral specimens underwent histological evaluation which confirmed the clinical diagnosis.
Normal human adrenal tissues were obtained from 12 patients who underwent unilateral nephrectomy and adrenalectomy for treatment of non-metastatic kidney tumors (4 cases) or who were subjected to uni- lateral adrenalectomy for adrenal cysts (8 cases). The patients had neither clinical symptoms of adrenocor- tical hormone overproduction nor increased plasma cortisol, aldosterone or androgen hormone levels. Immediately after removal, macroscopically normal
adrenal tissues were dissected, frozen in liquid nitro- gen, and kept at -80 C until processing.
Determination of p53 protein
Frozen adrenal tissues were homogenized in 10 mmol/l TRIS buffer (pH 7.4) containing 1.5 mmol/l EDTA, 10 mmol/l sodium molybdate and 1.0 mmol/l monothioglycerol, and cytosolic preparations were obtained by centrifugation using a method described for steroid receptor analysis (12). Cytosolic p53 con- tent was determined with a monoclonal two-site single incubation luminometric immunoassay kit (Sangtec Medical, Bromma, Sweden). The assay was performed on a LIA-mat S300 analyzer (Byk- Sangtec, Dietzenbach, Germany). The monoclonal antibody was a DO1 type which detected both wildtype and mutant p53. The protein concentra- tions of the cytosolic extracts were measured by a standard method, and the p53 values were ex- pressed as pg/mg cytosolic protein. Statistical anal- ysis was undertaken using ANOVA.
Determination of p53 mRNA
Frozen adrenal tissues were weighed, and total RNA was isolated by the guanidinium thiocyanate method (13) using a single step extraction (14). RNA (30 µg) from each tissue was denatured in a mixture of gly- oxal and dimethylsulfoxide-containing sodium phos- phate. RNA samples were electrophoresed on formal- dehyde-containing agarose gels and transferred to Hybond N filters (Amersham International, Buskin- ghamshire, United Kingdom). The filters were expo- sed to UV light, baked for 1 h at 80 C, then prehybri- dized for 16 h at 42 C as previously described (15). Hybridization was carried out for 24 h at 42 C with 32P-labelled p53 probe. The hybridization probe was prepared from human wild-type p53 cDNA (pC53- SN3) (16) using [a-32P]dCTP (Isotope Institute, Buda- pest, Hungary) and a megaprime DNA-labelling sys- tem (Amersham International). After hybridization, the filters were washed and autoradiographed at -80 C with intensifying screens (DuPont GmbH, Dreieich, Germany) and Kodak X-OMAT films. After autora- diography, the radioactive probe was completely re- moved by two washes, and the filters were reautogra- phed to exclude the presence of residual radioac- tivity. Washed filters were prehybridized and then re- hybridized with actin cDNA (Oncor, Gaithersbury, MD) radiolabelled probe. The signal intensities of mRNA transscripts were quantitated by means of a densitometric camera scanner (Stratagene) and stan- dardization of hybridized p53 transscripts was valida- ted by densitometric scanning of actin transcripts. Hy- bridization signals were expressed in arbitrary units (mean±SEM).
RESULTS
To determine and compare the p53 protein con- tent in normal and tumorous adrenal tissues, cy- tosolic preparations were obtained and then used for the luminometric assay. The results of these studies showed detectable amounts of p53 protein in all normal and tumorous adrenal tissues. As shown in Figure 1, the concentration of p53 in nor- mal human adrenals was 266±18 pg per mg of cy- tosolic protein (mean±SE). Compared to normal adrenals, the p53 content was significantly increa- sed in aldosterone-producing adenomas (304+36 pg/mg cytosolic protein) whereas Cushing’s ade- nomas, non-hyperfunctioning adenomas and pheo- chromocytomas showed p53 concentrations simi- lar to those measured in normal adrenal tissues. Of the 5 adrenocortical carcinomas examined one had very high p53 content (3000 pg/mg cytosolic pro- tein) while the p53 concentrations in the remaining four carcinomas were not increased. Interestingly, myelolipomas, ganglioneuromas and a heman- gioma exhibited very low p53 contents (23±20, 43±15 and 11 pg/mg cytosolic protein, respec- tively) (Fig 1).
Figure 2 illustrates that in agreement with the pres- ence of p53 protein, these adrenal tissues expres-
pg p53/mg cystolic protein
600
*
400
T
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200
*
*
T
4
Normal
adrenals
Aldo-prod.
adenoma
Cushing’s
adenoma
Non-Hyper-
func. adenoma
Adrenocort.
carcinoma
chromocytoma
Myelolipoma
Pheo-
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neuroma
Hemangioma
sed p53 mRNA. The autoradiogram of a Northern blot shown in the middle part of Figure 2 demon- strates the presence of a single mRNA at 2.2 kilo- bases (kb), which corresponds to the known size of human p53 mRNA. When normalized to actin mRNA (bottom panel of Fig. 2), the intensity of mRNA sig- nals indicated high amounts of p53 mRNA in aldos- terone-producing and Cushing’s adenomas whereas other adrenal tissues had less p53 mRNA content. The top panel of Figure 2 shows that high p53 mRNA contents were not always accompanied with
p53 protein
pg/mg cytosolic protein
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400
200
p 53 mRNA
2.2 kb -
Actin RNA
2.2 kb -
1
2 3 4 5 6 7
increased p53 protein contents and that the relative intensities of the p53 mRNA signals failed to parallel the amounts of p53 protein in these adrenal tissues. This finding was confirmed in subsequent studies showing the absence of correlation between p53 pro- tein and its mRNA in a large number of tissues in- cluding 12 normal and 56 tumorous adrenals. These studies revealed again that aldosterone-producing and Cushing’s adenomas had significantly greater p53 mRNA content (p<0.05) than the other groups.
DISCUSSION
The present study indicates that p53 and its mRNA is present in normal human adrenals and in a large num- ber of different adrenal tumors, including aldos- terone-producing and Cushing’s adenomas, non-hy- perfunctioning adenomas, adrenocortical carcinomas, pheochromocytomas, myelolipomas, ganglioneuro- mas and a hemangioma. Among these tissues, the highest p53 protein content was found in an adreno- cortical carcinoma in which p53 protein concentra- tion was more than 10 times higher than that mea- sured in normal adrenal tissues. However, the p53 protein content in four other adrenocortical carcino- mas was not increased. These observations are in agreement with a few earlier studies showing im- munohistochemical accumulation of the p53 protein in some cases of sporadic adrenocortical carcinomas (5, 6, 17, 18). It has been proposed that mutations of the p53 gene, which are reportedly present in some cases of adrenocortical carcinomas (5, 6), may result in an increased half-life of the mutated p53 protein and may, therefore, be responsible for the increased p53 protein content of these adrenal tissues.
Our study showed that aldosterone-producing and Cushing’s adenomas had significantly increased p53 protein and/or mRNA contents compared to that measured in human normal adrenal tissues. Whether increased p53 protein and/or mRNA contents in these tumors represent underlying gene mutations or are the results of enhanced stability of wild-type p53 protein by other mechanisms remains unknown. p53 gene mutation in aldosterone-producing and Cushing’s adenomas has been the subject of a few previous reports but the results have not been en- tirely concordant. Lin et al. (7) reported a high fre- quency of mutations located outside the 4 known “hot spots” in exons 5-8 of the p53 gene in about 60% of adrenocortical adenomas, including aldos- terone-producing and Cushing’s adenomas. Howe- ver, other studies showed that mutations in the p53 gene, including those reported by Lin et al. occur in- frequently or are absent in these tissues (5, 6, 10) and, therefore, may not be responsible for the increased
p53 protein content. It is possible that the increased p53 protein in these tissues may result from either an increased expression or an abnormal stabilization of the wild-type protein leading to an increase in its half- life, as already proposed (8).
Our observation that non-hyperfunctioning adreno- cortical adenomas and pheochromocytomas failed to show increased p53 protein and/or mRNA con- tents are in agreement with earlier reports which ex- amined p53 protein accumulation by means of im- munochemistry and found an infrequent or absent p53 protein accumulation in these tissues (8, 10).
More interestingly, our study showed that myeloli- pomas, ganglioneuromas and a hemangioma had markedly reduced p53 protein content compared to that measured in normal adrenals or in other types of tumorous adrenal tissues. The meaning of reduced p53 protein content is not understood as p53 has never been investigated in these types of adrenal tu- mors. However, it cannot be ruled out that the low p53 protein content represent inherent differences in the cellular origin of these tumors or, alternatively, it may be linked to the tumoral process. It is also not known whether the markedly reduced p53 content could facilitate the growth rate of these tumors.
In conclusion, the present experiments showed that p53 protein and its mRNA is present in normal hu- man adrenals and in a large number of different types of adrenal tumors. Of the 5 adrenocortical carcino- mas examined, one had very high concentration of the p53 protein suggesting that marked p53 accu- mulation occurs in at least some proportion of adre- nocortical carcinoma tissues. In addition, we found that aldosterone-producing and Cushing’s adeno- mas, but not non-hyperfunctioning adenomas and pheochromocytomas had significantly increased p53 protein and/or mRNA content. By contrast, mye- lolipomas, ganglioneuromas and a hemangioma showed very low p53 content. It is possible that these differences in p53 protein and/or mRNA contents may be implicated in the pathophysiology of these types of adrenal tumors.
ACKNOWLEDGMENTS
We wish to thank Dr. Bert Vogelstein (The Johns Hopkins Oncology Center, Baltimore) for providing the p53 cDNA. This work was supported by grants T-023833 and T-013193 from the National Scientific Research Foundation of Hungary and by a grant ETT 468/1996 from the Ministry of Welfare, Hungary.
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