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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 3 1220-1225
Copyright © 2000 by The Endocrine Society

Original Studies

Expression of Corticotropin-Releasing Hormone Messenger Ribonucleic Acid in Human Pituitary Corticotroph Adenomas Associated with Proliferative Potential

Bing Xu, Toshiaki Sano, Shozo Yamada, Chiun C. Li and Mitsuyoshi Hirokawa

Department of Pathology, University of Tokushima School of Medicine (B.X., T.S., C.C.L., M.H.), 770-8503 Tokushima; and Department of Neurosurgery, Toranomon Hospital (S.Y.), 105-0001 Tokyo, Japan

Address all correspondence and requests for reprints to: Dr. Bing Xu, Department of Pathology, University of Tokushima School of Medicine, 770-8503 Tokushima, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Among the factors that promote the growth of human pituitary corticotroph adenomas (hPCAs), the proliferative potential of CRH secreted by hPCAs on these tumors is not well known. In this study, the CRH messenger ribonucleic acid (mRNA) transcripts were demonstrated on paraffin sections using the quantitative in situ hybridization method in 37 of 43 hPCAs, including 17 of 22 microadenomas, 15 of 15 macroadenomas, and 5 of 6 locally invasive adenomas according to Hardy’s classification of pituitary adenomas. The more important findings were that CRH mRNA signal intensity in pituitary corticotroph adenoma cells was linearly correlated with Ki-67 tumor growth fractions (r = 0.802; P < 0.0001), and in macroadenoma and locally invasive adenoma cells it was significantly higher than in microadenoma cells (P = 0.035). On the other hand, CRH mRNA transcript accumulation was absent or negligible in 10 normal pituitary glands (P = 0.005).

This is the first report of the frequent expression of CRH mRNA localized in human pituitary corticotroph adenoma cells. These results indicate that CRH from a local source of corticotroph adenoma cells not only has autocrine/paracrine functions in corticotroph adenomatous tissue, but also is an important factor associated with a proliferative potential of hPCAs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HISTORICAL CONTROVERSY about the pathogenesis and prognosis of human pituitary corticotroph adenomas (hPCAs) has two conceptually opposing theories: that hPCAs arise as a primary defect at the pituitary level or as a result of dysfunctional regulation by hypothalamic and peripheral agents. The generally high cure rate of hPCAs (1, 2), the absence of identifiable corticotroph hyperplasia surrounding the tumor (3), and the monoclonality of the origin of the majority of hPCAs by X-chromosomal inactivation analysis are important evidence to support that an intrinsic pituitary defect may be the tumorigenesis of hPCAs (4, 5, 6). However, there are a number of associated neuroendocrine abnormalities observed in many hPCAs with Cushing’s syndrome (2, 7). The most obvious candidates are protooncogenes and tumor suppressor genes, which have been tested for abnormalities in hPCAs leading to tumorigenesis. Clear evidence of a defect in these genes has not been found, such as RAS, c-ERB2/neu, c-MYC, PKC, RET protooncogenes, and p53, Rb1 tumor suppressor genes (8, 9, 10, 11, 12), although a small fraction of hPCAs have activating Gs {alpha}-subunit mutations, 11q13 allelic loss, and p27 inactivation (13, 14, 15, 16).

During the past 2 decades, hyperplasia of corticotrophs, but without hPCA formation, caused by ectopic CRH oversecretion from prostate small cell carcinoma, bronchial carcinoid, and gangliocytoma has been reported (17, 18, 19, 20, 21). Similarly, the study of CRH transgenic mice found that high levels of CRH could produce corticotroph hyperplasia despite the fact that no corticotroph adenoma was seen (22). In accordance with these observations, CRH may have an important role in sustaining tumor development. Moreover, after the expression of CRH, GHRH, TRH, and GnRH messenger ribonucleic acids (mRNAs) in the nontumorous pituitary gland and pituitary adenomas has been demonstrated (23, 24, 25, 26), and the overexpression of the GHRH gene in pituitary somatotroph adenomas with acromegaly has been shown to be associated the neoplastic progression and clinical aggressiveness (27), the role of the hypothalamic hormones expressed by pituitary tumor cells themselves is attached importance. By analogy, expression of the CRH gene in hPCAs might be variable to promote tumor progression. Furthermore, not only has the CRH gene been shown to be expressed in mouse corticotropic tumor cell (28), but a study of hypothalamic hormone gene expression in pituitary tumors has demonstrated cell clusters containing a high level of CRH transcripts in 17% of 132 pituitary adenomas (23). These results make it possible to demonstrate this hypothesis. Here we report on the effects of CRH expression in hPCAs on tumor proliferation, which also supports this prediction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor specimens and control tissues

Forty-three consecutive hPCAs from 34 female and 9 male patients, whose mean age was 46.9 yr (range, 12–75 yr), were obtained during surgery at the University Hospital of Tokushima (Tokushima, Japan) and Toranomon Hospital (Tokyo, Japan) between 1990 and 1998. Included were 26 functioning corticotroph adenomas (FCAs) with Cushing’s diseases and 17 silent corticotroph adenomas (SCAs) in the light of functional classification of hPCAs, in which 1 of the FCAs and 4 of the SCAs were recurrent. In addition, according to Hardy’s classification of pituitary adenomas, which is based on tumor size and degree of local invasion (29), 22 cases of grade I hPCAs (microadenomas) containing 3 recurrent cases, 15 cases of grade II hPCAs (macroadenomas), and 6 cases of grade III hPCAs (locally invasive adenomas) including 2 recurrent cases were included in this series. These tissues were formalin fixed and paraffin embedded at the time of surgery, pathologically characterized by a single pathologist (T. Sano), and constituted the primary study set upon which quantitative in situ hybridization (ISH) analysis, CRH and proliferative marker MIB-1 immunostains, clinicopathological correlations, and statistical modeling were performed.

As control tissues, formalin-fixed and paraffin-embedded specimens of 10 normal autopsy pituitary glands were used, all of which were obtained from patients who died of nonendocrine causes and were retrieved within 4 h of death.

CRH probe

To detect specific nucleotide sequences of CRH mRNA, an oligonucleotide probe (Central Laboratory, Nippon Flour Mills, Atsugi, Japan) of 47 nucleotides was used for ISH. This probe (5'-GAG AGC CGC GGG GCT GTC GAG CGA GCG CCG AGG CAG CAG CAG CTG CT-3') derived from nucleotides 1668–1714 of the human CRH gene and was labeled at the 3'-end with biotin.

ISH protocol

ISH was performed on 5-µm slide-mounted sections of formalin-fixed and paraffin-embedded tissues, which were fixed on slides at 42 C overnight. After being deparaffinized in xylene, rehydrated through graded ethanol, and rinsed in 2 x SSC (standard sodium citrate), sections were treated with 0.2 N HCl at room temperature. They were then treated with 10 µg/mL proteinase K (DAKO Corp., Kyoto, Japan) in phosphate-buffered saline (PBS) for 10 min each at 25 C, followed by treatment with 2 mg/mL glycine in PBS for 10 min to inactivate the enzyme. As prehybridization, sections were treated with 50% formamide/2 x SSC at 37 C in a hybridization bath. For the hybridization step, sections were covered with 30 µL hybridization solution (Wako, Osaka, Japan) consisting of 1 µg/mL labeled probe in 50% deionized formamide, 2 x SSC, 1 µg/µL yeast transfer ribonucleic acid, 1 µg/µL sonicated salmon sperm deoxyribonucleic acid, 1 µg/µL BSA, and 10% dextran sulfate during incubation and hybridized for 20 h at 37 C in a humidified chamber. To remove the unbound probe, the sections were washed in 50% formamide/2 x SSC twice, 20 µg/mL ribonuclease A solution (Wako) in sodium tris-aminomethane buffer containing ethylenediaminetetraacetic acid once, and 0.1 x SSC twice at 25 C in a hybridization bath for 20 min each.

All of the immunologic detection steps of the hybridized probe were performed at room temperature, using part of the catalyzed signal amplification system (DAKO Corp.) composed of streptavidin-biotin-peroxidase complex to bind to the biotinylated probe via its streptavidin and bound peroxidase to catalyze precipitation onto the specimen of a biotinylated phenol, resulting in amplification of the number of biotin molecules available for binding to the next reagent, streptavidin-peroxidase. The final reaction product was visualized with 3,3'-diaminobenzidine, and slides of hPCAs and normal pituitary glands were stained with hematoxylin and 1% methyl green for nuclear detection, respectively.

To control the nonspecific binding of the probe, some sections were hybridized with an unrelated probe or with hybridization mixture without a probe. To confirm the specific hybridization of the probe to the target mRNA, some sections were treated with 100 µg/mL ribonuclease A and incubated in a humidified chamber for 2 h at 37 C after proteinase K digestion. Control sections were tested in parallel without ribonuclease A.

CRH mRNA signal intensity

To quantify the CRH mRNA signal intensity in hPCAs, the percentage of positive CRH mRNA signals within the cytoplasm of tumor cells vs. the total tumor cells in each tumor, five color microphotos at x200 magnification were taken from the unoverlapped representative fields, although among larger specimens a greater number of fields were required to fully screen the sectional area. An average of approximately 1000 cells were counted in each specimen. The CRH mRNA signal intensity was determined, expressed as the percentage of positive cells of ISH.

To quantify the CRH mRNA signal intensity in normal autopsy pituitary glands, serial sections were used for ISH and ACTH immunostaining. The percentage of positive CRH mRNA signals vs. the positive cells of ACTH immunostaining is regarded as the CRH mRNA signal intensity in normal control pituitary glands.

Immunostaining of MIB-1 and CRH

MIB-1 and CRH immunostains were performed on 5-µm sections of formalin-fixed and paraffin-embedded tissue. After routine deparaffinization, rehydration, and blockade of endogenous peroxidase activity, they were placed in a glass box filled with 10 mmol/L citrate buffer, pH 6.0, and antigenetically retrieved for 15 min at 800 watts in a microwave oven. Sections were incubated at 4 C overnight in the MIB-1 monoclonal antibody (1:50 dilution; Immunotech, Marseilles, France) or for 48 h in CRH polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); then the former was incubated in biotinylated rabbit antimouse IgG and avidin-biotinylated horseradish peroxidase complex (Vector Laboratories, Inc. Burlingame, CA) for 1 h each at room temperature, and the latter was incubated in biotinylated rabbit antimouse Igs and catalyzed signal amplification system. Sections were washed thoroughly in PBS buffer between each of the immunostaining procedures. Antigen-antibody complexes were detected with the 3,3'-diaminobenzidine/H2O2 reaction, and then sections were stained with 1% methyl green and coverslipped.

For negative controls, primary MIB-1 monoclonal antibody and CRH polyclonal antibody were substituted with PBS in duplicate sections. To confirm the specific binding of CRH polyclonal antibody to the target antigen, inhibition of immunohistochemical staining in positive samples by preincubating the antibody with 0.6 µg active peptide of human CRH containing 41 amino acids (Peptide Institute, Inc., Osaka, Japan) was performed at 4 C overnight, representing an approximately 100-fold excess of peptide over antibody.

Ki-67 antigen labeling index

The proliferating cell index was calculated from each slide according to the intensity of immunopositive nuclei. All definite nuclear immunostaining was considered positive; vascular components and nontumorous adenohypophysial cells were excluded. To obtain the percentage (labeling indexes) of Ki-67-immunoreactive nuclei vs. the total neoplastic nuclei in each tumor, five color microphotos at x200 magnification were taken from the unoverlapped representative fields, although among larger specimens a greater number of fields was required to fully screen the sectional area. An average of approximately 1000 nuclei were counted in each specimen. A Ki-67 antigen labeling index was determined and expressed as the percentage of Ki-67-immunoreactive nuclei.

Statistical analysis

Statistical correlation between two independent variables was computed by means of the Spearman rank-correlation coefficient, which does not require normal distribution. The Mann-Whitney U test was used to determine the difference in variability level between the distributions of two independent groups. P < 0.05 is considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CRH mRNA signal intensity in hPCAs and normal control pituitary tissues

The CRH mRNA signals were detected in 24 of 26 FCAs (92.3%) with Cushing’s disease, 13 of 17 SCAs (76.5%), and 6 of 10 normal pituitary glands (60%) by ISH (Fig. 1Go, a and b). Between FCAs (mean ± SEM, 12.66 ± 2.42%) and SCAs (6.02 ± 1.43%), the difference in CRH mRNA signal intensity did not reach statistical significance (P = 0.086). However, compared with 10 normal pituitary glands, the signals were variably positive. Between hPCAs (10.48 ± 1.66%) and normal pituitary glands (1.12 ± 0.34%), a significant difference in CRH mRNA signal intensity was noted (P = 0.005; Fig. 2Go). In other words, the CRH mRNA signal was absent or negligibly weak in nontumorous control pituitary tissues.



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  		Figure 1. Expression of CRH mRNA in a hPCA cell line and normal autopsy pituitary corticotrophs by ISH. The hybridization signals detected with biotin-labeled probe diffusely present in hPCAs (hematoxylin counterstain; a) and spottily present in normal autopsy pituitary glands (methyl green counterstain; b) are shown. A negative control experiment, using pretreatment with ribonuclease A digestion, shows no signal for CRH mRNA (hematoxylin counterstain; c). Magnification, x200.

 


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  		Figure 2. CRH mRNA signal intensity in normal autopsy pituitary glands (N), FCAs, and SCAs. The mean CRH mRNA signal intensity of hPCAs, either FCAs or SCAs, was significantly higher than that of normal autopsy pituitary glands (P = 0.005); however, between FCAs and SCAs, there was no significant difference.

 
Moreover, the CRH mRNA signals were positive in 17 of 22 grade I hPCAs (77.3%), 15 of 15 grade II hPCAs (100%), and 5 of 6 grade III hPCAs (83.3%), according to Hardy’s classification of pituitary adenomas. Between 22 grade I hPCAs (7.30 ± 1.53%) and 21 grade II III hPCAs (13.81 ± 2.86%), a statistical difference in the CRH mRNA signal intensity was found (P = 0.035; Fig. 3Go).



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  		Figure 3. CRH mRNA signal intensity in hPCAs of Hardy’s classification. The mean CRH mRNA signal intensity of grade II and III hPCAs was statistically higher than that of grade I hPCAs (P = 0.035).

 
Correlation between CRH mRNA signal intensity and Ki-67 labeling index

A Ki-67 labeling index for 43 hPCAs (1.96 ± 0.35%), ranging from 0–12.08%, was determined using the MIB-1 antibody (Fig. 4Go). Comparing CRH mRNA signal intensity with Ki-67 labeling index, a significant positive linear correlation was observed (r = 0.802; P < 0.0001; Fig. 5Go), although there was no difference in the Ki-67 labeling index (P = 0.619) between FCAs (1.95 ± 0.35%) and SCAs (1.36 ± 0.33%).



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  		Figure 4. Expression of Ki-67 antigen in the nuclei of the hPCA cells by MIB-1 immunostaining (methyl green counterstain). Magnification, x200.

 


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  		Figure 5. Correlation of CRH mRNA signal intensity vs. Ki-67 labeling index. The Spearman rank-correlation coefficient analysis reveals that a significant positive linear correlation exists between these two variables (r = 0.802; P < 0.0001).

 
CRH immunohistochemical analysis

In 5 of 26 FCAs and 4 of 17 SCAs, all of which expressed high levels of CRH mRNA, conclusive cellular localization of CRH protein could be demonstrated. These CRH-immunopositive hPCAs contained 5 recurrent cases, in which only 2 locally invasive macroadenomas showed strong positive CRH immunostaining (Fig. 6aGo). However, in 10 normal pituitary glands in which CRH mRNA signal was absent or at low levels, CRH immunoreactivity could not be demonstrated.



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  		Figure 6. Accumulation of CRH protein within the neoplastic cytoplasm of hPCAs by immunohistochemistry using CRH polyclonal antibody (a; methyl green counterstain). Specific binding of CRH polyclonal antibody to the target antigen, preincubated with the active peptide of human CRH, shows no signal for CRH antigen in hPCAs (b; methyl green counterstain). Magnification, x200.

 
CRH mRNA signal intensity in FCAs and preoperative and postoperative plasma ACTH and cortisol levels

In all corticotroph adenoma patients basal determinations of preoperative and 2-week postoperative plasma ACTH and cortisol levels (normal: ACTH, <60 pg/mL; cortisol, <19 µg/dL) and of ACTH responses to CRH were made. The higher preoperative plasma ACTH and cortisol levels of the FCA patients with Cushing’s disease were used for comparison with CRH mRNA signal intensity. A lack of significant linear correlation was observed not only between CRH mRNA signal intensity and preoperative plasma ACTH levels (r = 0.158; P = 0.458), but also between CRH mRNA signal intensity and preoperative plasma cortisol levels (r = 0.143; P = 0.513). In addition, in two patients 2-week postoperative plasma ACTH levels did not remit to normal. However, a month later their plasma ACTH levels returned to normal during follow-up.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that most human hPCAs, including FCAs and SCAs, are considered curable lesions, although FCAs pathologically hypersecrete ACTH and are usually associated with Cushing’s disease or Nelson’s syndrome (1, 2). However, rarely hPCAs are more aggressive, showing signs of local invasiveness and infrequent recurrences after curative resections by experienced neurosurgeons, even if their histological characters are benign (30). Furthermore, invasive hPCAs recur more frequently than noninvasive tumor (31). Underlying the tendencies of some hPCAs toward proliferative, invasive, or recurrent growth, there are presumably specific variabilities that promote tumor progression.

To find out whether CRH in the pituitary plays a role in the proliferation of hPCAs, in this study we applied the ISH method to detect CRH mRNA in formalin-fixed and paraffin-embedded human pituitary tissue and determined that accumulation of CRH mRNA transcripts is an important factor associated with the proliferative potential of hPCAs. Using this technique, CRH mRNA signals were either absent in 4 of 10 normal nontumorous pituitary glands or present at very low levels in 6 of 10 normal nontumorous pituitary glands. In contrast, they were obviously positive in 37 of 43 hPCAs, including 24 of 26 FCAs and 13 of 17 SCAs. The difference in CRH mRNA intensity between hPCAs and normal pituitary glands was statistically significant (P = 0.005) that CRH mRNA signals in normal pituitary glands could be neglected.

On the other hand, the MIB-1 monoclonal antibody reacts with the Ki-67 nuclear antigens expressed in G1, S, G2, and M phases, but not in G0 phase of the cell cycle. It has been shown to correlate with proliferative activity, intrinsic aggressiveness, and invasiveness of various tumor types as well as pituitary tumors (32, 33, 34). In this study, a Ki-67 labeling index of 43 hPCAs (1.96 ± 0.35%), ranging from 0–12.08%, was determined using the MIB-1 antibody. The more important finding was the significant positive linear correlation (r = 0.802; P < 0.0001) between CRH mRNA signal intensity and Ki-67 labeling index. In addition, to a certain extent it appears that proliferation and invasion of hPCAs are related to tumor size and type.

Macroadenomas are more often invasive than microadenomas, and SCA is one of the most commonly invasive pituitary adenomas (35, 36). In this study, CRH mRNA signal intensity in macroadenomas and locally invasive adenomas was significantly higher than that in microadenomas (P = 0.035). Finally, two locally invasive macroadenomas could be demonstrated to be strongly positive for CRH immunostaining. These results indicate that the CRH gene expressed by tumor cells themselves is an important factor associated with the proliferative potential of hPCAs. This is supported by the reports that ectopic CRH oversecretion from prostate small cell carcinoma, bronchial carcinoid, and gangliocytoma may cause hyperplasia of corticotropic cells (17, 18, 19, 20, 21), and experiments showing that CRH is capable of inducing a significant increase in corticotrophs in the anterior pituitaries of CRH transgenic mice (37, 38, 39). Moreover, the finding that human CRH receptor type I was cloned from a human corticotroph tumor strongly suggests that CRH-mediated autocrine and/or paracrine stimulation may be associated with tumor proliferative potential (40).

In human pituitary, although part of the hypothalamic-pituitary-adrenal axis, CRH is a potent secretagogue that stimulates not only ACTH synthesis (41, 42) but also ACTH release by corticotrophs and corticotroph adenoma cells via the cAMP-protein kinase A pathway (43, 44). ACTH secretion and growth of corticotrophs and corticotroph adenoma cells are also regulated by other stimulatory agents, such as vasopressin, pituitary leukemia inhibitory factor, and their specific receptors (45, 46, 47, 48), as well as inhibitory factors, such as corticotropin-releasing inhibitory factor and glucocorticoid and its receptor (49, 50, 51). Therefore, ACTH secretion is determined by a complex balance of these agents. In this study, the CRH mRNA signals were detected in both FCA and SCA cells, and there was no significant difference in CRH mRNA signal intensity between two groups (P = 0.086). Furthermore, there were no significant linear correlations between higher preoperative plasma ACTH and cortisol levels in the 26 FCA patients with Cushing’s disease and CRH mRNA signal intensity in adenoma cells. These results show that the hypercortisolemic state secondary to excess or dysregulated ACTH secretion caused by FCA might not only be determined by CRH from FCA cells themselves, and that plasma ACTH and cortisol levels leading to Cushing’s syndrome correlate with a complex balance of several agents.

In conclusion, the presence of CRH mRNA from a local source of corticotroph adenoma cells and the significant linear correlation between CRH mRNA signal intensity and tumor progression suggest that CRH-mediated autocrine and/or paracrine stimulation may be a mechanism associated with tumor proliferative potential.

Received July 20, 1999.

Revised December 1, 1999.

Accepted December 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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