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Original Studies |
Departments of Histopathology (K.L., S.J., D.G.L.) and Endocrinology (M.K., Z.K., G.K., P.J.J., J.P.M., G.M.B., A.B.G.), St. Bartholomew’s Hospital, London EC1A 7BE; Centre for Cell Molecular Medicine (R.N.C.), University of Keele, Stoke-on-Trent ST4 7Q3; and Ludwig Institute for Cancer Research (X.L.), St. Mary’s Hospital, London W2 1PG, United Kingdom
Address correspondence and requests for reprints to: Professor Ashley B. Grossman, Department of Endocrinology, St. Bartholomew’s Hospital, West Smithfield, London EC1A 7B, United Kingdom, E-mail: A.B.Grossman{at}mds.qmw.ac.uk
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Abstract |
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500 cells were
analyzed. We also analyzed normal pituitaries using double-labeling for
p27 and each of the pituitary hormones to characterize the expression
of p27 in each cell type. p27 was expressed in normal pituitary cells;
in tumors expressing GH, prolactin, TSH, and FSH; and in aggressive
tumors, but markedly reduced expression of p27 was seen in corticotroph
tumors and pituitary carcinomas. In the normal pituitary, somatotroph,
lactotroph, and thyrotroph cells showed strong p27 staining, whereas
normal corticotroph cells showed a much lower level of p27 staining
(P < 0.001). Somatotroph, lactotroph, gonadotroph,
and thyrotroph adenomas showed a lower level of p27 expression compared
with normal somatotrophs (P = 0.02), lactotrophs
(P = 0.03), gonadotrophs (P =
0.01), and thyrotrophs, respectively, whereas the lower level of p27
expression present in normal corticotrophs virtually disappeared in
corticotroph adenomas (P = 0.001). We conclude that pituitary adenomas show a lower level of p27 protein expression than the normal cells from which they are derived, with malignant transformation leading to complete loss of p27 immunoreactivity. Corticotrophs are quite different to other pituitary cell types in terms of p27 immunoreactivity because both normal and tumorous corticotrophs have low p27 staining, and we speculate that this may relate to their inherent control mechanisms.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cell cycle is controlled at different stages by altered activities of specific cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs) (5, 6). Cyclins activate the CDKs with which they associate and then direct the activated kinase complexes to appropriate substrates, for example, the retinoblastoma protein (7). The resulting phosphorylation causes these substrates to execute key steps in cell proliferation; thus, retinoblastoma is inactivated by phosphorylation allowing cell cycle progression. The activity of cyclin-CDK complexes is regulated by two families of CDKI proteins that generally inhibit cell cycle progression. The INK4 group includes p16, p18, p15, and p19; p15 and p16 abnormalities have been described in various human malignancies (8). The kip/cip family comprises three structurally related proteins: p21, p27, and p57 are all capable of binding to and inhibiting most cyclin/CDK complexes, including cyclin D and cyclin E complexes (9, 10, 11, 12). Overexpression of these CDKIs causes cell cycle arrest, and thus, these agents act as negative regulators of the cell cycle. The p27 gene maps to chromosome band 12p13 (13, 14) and regulates progression from G1 into S phase by binding to and inhibiting the cyclin E/CDK and cyclin D/CDK complexes for entry into S phase (9, 12). p27 is present in large amounts in quiescent cells and declines when cells proliferate in response to mitogenic signals, such as growth factors and cytokines (15, 16, 17). In proliferating cells, p27 is expressed at a threshold level, much of it bound in a complex with cyclin D/CDK4 (9, 18, 19). As quiescent cells enter the cycle, the level of cyclin D complexes exceeds that of p27, countering its inhibitory effects. The levels of p27 in relation to cyclin D/CDK4 are crucial in determining whether the latter is enzymatically active or not (20).
It has been reported that mice with a genomic knockout of the p27 gene develop multiorgan hyperplasia, resulting in increased animal size, abnormalities in the thymus, retina, adrenal glands and gonadal organs, and pituitary tumors that develop from the pars intermedia and immunostain for ACTH (21, 22, 23). Recently, it has been shown that mice haplo-insufficient for the p27 gene also show increased animal size and the spontaneous development of intermediate lobe pituitary tumors, and are sensitive to irradiation- or chemical-induced carcinogenesis (24). It has been suggested that p27 could be important in the genesis of pituitary tumors.
Several groups, including our own, have previously investigated p27 messenger (m) RNA expression and the presence of gene mutations in human corticotroph PAs, concluding that abnormalities of p27 mRNA expression do not seem to be important in the pathogenesis of benign corticotroph tumor formation (25, 26, 27, 28). However, our preliminary studies of p27 immunostaining suggested decreased protein expression in pituitary carcinoma, but with no obvious change in benign corticotrophinomas. We have now sought to verify whether the amount of p27 protein product might be involved in the pathogenesis of human pituitary tumors, specifically those with an ACTH-secreting phenotype and aggressive forms of pituitary tumors, using a larger group of tissues and more precise quantitation.
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Materials and Methods |
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We studied 107 pituitary tissue samples removed at transsphenoidal surgery. These were classified histologically into 20 normal pituitaries, 73 PAs, 9 aggressive pituitary tumors, and 5 metastatic pituitary tumors. The normal pituitaries were sections cut from tissue removed at transsphenoidal surgery for presumptive tumors that proved on standard hematoxylin and eosin (H and E), immunohistochemical, and reticulin staining to consist of normal pituitary cells and architecture. The normal pituitaries included tissue from patients with the clinical diagnosis of Cushing’s disease (n = 14), prolactinoma (n = 3), acromegaly (n = 1), NFPA (n = 1), and arachnoid cyst (n = 1). Abnormal pituitary tissue was classified as adenoma, aggressive adenoma, or carcinoma. The 73 adenomas were categorized as GH-secreting tumors (n = 19), ACTH-secreting tumors (n = 21), prolactinomas (n = 7), 2 TSH-omas, and NFPAs (n = 18); we also looked at FSH-omas (n = 6), all of which showed markedly increased FSH secretion in in vitro culture (29) and clinical signs suggestive of increased FSH secretion in 2, although FSH immunostaining did not show uniform FSH positivity in these tumors. Aggressive pituitary tumors showed invasive growth and/or recurrence; this group included five NFPAs, two prolactinomas, and two somatotroph adenomas. The pituitary carcinoma group included three ACTH-secreting tumors and two prolactinomas; these patients had histologically verified extrapituitary metastases. All patients with the clinical diagnosis of Cushing’s disease routinely received 6–8 weeks of medical therapy with metyrapone and/or ketoconazole to normalize cortisol levels before surgery.
Immunohistochemistry
Tissue preparation.All tissues were collected at routine transsphenoidal surgery and prepared for pathological examination in a standard manner. Paraffin sections cut at 3 µm were air-dried, then placed in a 60 C oven overnight. Sections were de-waxed in xylene, followed by immersion into a solution of 750 µl 30% hydrogen peroxide and 50 ml methanol for 10 min to block endogenous peroxide. Sections were rehydrated to tap water, ready for antigen retrieval. Sections for p27 immunostaining required heat-mediated antigen retrieval treatment (30). Sections were "superheated" for 4 min in 0.01 M citrate buffer (pH 6.0), then placed into tap water immediately to avoid drying of sections. Sections were transferred to phosphate-buffered saline (PBS) before immunostaining.
Immunostaining–p27.Immunohistochemistry was performed using a standard avidin-biotin complex (ABC) method by an automated staining machine (Optimax Plus 1.5; BioGenex Laboratories, Inc. Finchampstead, Berkshire, UK). The primary antibody, antihuman p27 (31), was placed on sections at 1:50 dilution for 40 min at room temperature in a wet chamber. Sections were washed in PBS and then incubated in a biotinylated antimouse second layer for 30 min. Sections were again washed in PBS, then incubated in the avidin-biotin peroxidase complex (Vectastain Elite ABC peroxide kit PK6200; Vector Laboratories, Inc., Peterborough, UK) for 20 min. After three washes in PBS, sections were visualized with activated 3'3' diaminobenzidene-tetrahydrochloride solution (DAB) (Kentec DAB tablets 4170; Biostat, Stockport, UK) for 10 min; this resulted in a brown end-product. Sections were counterstained with Gill’s hematoxylin, dehydrated through graded alcohol before mounting in DPX (BDU, Poole, UK). In the tumor samples, consecutive sections were immunostained for the relevant pituitary hormone to ensure that the area counted for p27 was homogeneous for the hormone stain.
Immunostaining–pituitary hormones.Pituitary hormone stains were performed to establish the hormone phenotype of the tumor samples. GH, ACTH, TSH, LH, and FSH antibodies were supplied by DAKO Corp. (Oxford, UK). The prolactin antibody was purchased from Novacastra (Newcastle-upon-Tyne, UK). Sections were incubated overnight in the primary antibody at 4 C. The standard ABC protocol was followed thereafter. A normal human pituitary was used as a positive control; the hormone antibody was omitted and replaced by mouse immunoglobulin as a negative control.
Double-labeling.Seven normal pituitary samples underwent double-staining for p27 and each of the pituitary hormones (32). Sections were stained for p27 antibody using the avidin-biotin peroxidase method and visualized using DAB. Sections were then stained for pituitary hormones (ACTH, LH, FSH, prolactin, GH, and TSH) using the avidin-biotin alkaline phosphatase conjugate and the Fast Red TR/naphthol AS-MX detection system (product no F4648; Sigma, Dorset, UK). This permits the ratio of p27 expression in normal hormone-secreting cells to be determined. ACTH-, LH-, FSH-, prolactin-, GH-, and TSH-positive cells showed red cytoplasmic staining, whereas p27-positive cells showed brown nuclear staining. Cells that were negative for both stains had clear cytoplasm and blue nuclei.
H and E and reticulin staining.Sections from each pituitary sample were stained with H and E stain as a general stain to demonstrate the various tissue components. A reticulin stain was performed to provide an indication of tissue architecture.
Controls.Specificity of the p27 staining was assessed initially by preabsorption of the antibody with the protein used in its generation; this completely abolished p27 staining.
Positive controls: Human tonsil tissue was used as a positive control because this contains lymphoid tissue with variable proliferative activity. In the mantle zone the cells are predominately quiescent, demonstrating high levels of p27, whereas cells in the germinal centers are highly proliferative and express low levels of p27.
Negative controls: Negative controls were used in each staining batch in which the primary antibody was omitted and replaced by mouse immunoglobulin (negative control mouse immunoglobulin; Biogenex). The ABC protocol was followed thereafter. Positive and negative controls were run with every experimental tissue section. All sections were examined at x40 magnification using bright field microscopy.
Quantitation.Cells were assessed for the intensity of
staining, and cells with strong or moderate staining were counted as
positive, cells with no staining were counted as negative, whereas
cells with weak staining were scored separately (Fig. 1
). Sections were assessed by a single
observer blinded to the diagnosis. In each section, 500 cells were
analyzed. The percentage of p27-positive or -negative cells was then
expressed as a ratio of positive or negative cells to the total number
of cells counted. Photographs were taken with a Sony 3CCD Color video
camera and printed on Sony color video printer UP-5200MDP (Sony Corp., Japan).
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Statistical Analysis.The data were analyzed by nonparametric tests (Mann-Whitney U test and the Kruskal-Wallis test, as appropriate) using the Arcus Quickstat Biomedical version 1.2 (Buchan I; Addison Wesley Longman Ltd., Cambridge, UK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). However, the tumorous group was
inhomogeneous and showed significant variability in p27 staining
(P < 0.001 by Kruskal-Wallis test; Fig. 2). The
GH-secreting tumors, nonfunctioning tumors, prolactinomas, TSH-omas,
FSH-omas, and aggressive tumors showed slightly less p27 staining than
normal pituitary tissue; however, corticotroph adenomas and metastatic
tumors showed a significantly lower level of p27 expression
(P < 0.0001 compared with normal tissue;
P < 0.01 compared with the other tumor types;
Figs. 2, 3, and 4). The number of p27-negative cells
was also considerably higher in corticotroph and metastatic tumors
(P < 0.01; Fig. 4).
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). However, although the
pituitary carcinoma studied showed very little p27 immunostaining (78%
of the cells negative), its mRNA expression was not proportional to the
protein expression: the normal pituitary showed a p27 to GAPDH ratio of
0.31, whereas the carcinoma showed a slightly higher ratio (0.72; Fig. 5).
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and 7).
Around 40% of the GH-, prolactin-, TSH-, FSH-, and LH-secreting cells
showed positive p27 staining, whereas the corticotroph cells uniquely
showed a much lower level of p27 expression (3%) that was
significantly different to each of the other cell types (Kruskal-Wallis
test, P = 0.006; individual comparisons,
P < 0.05). The p27 staining for the normal tissue
from patients with the clinical diagnosis of Cushing’s disease was
similar to those with other pathology (p27 staining in normal tissue
from Cushing’s disease 57 ± 4% vs. normal tissue
from other tumors 55 ± 10%; P = 0.8).
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]. There
was a similar difference in the TSH-secreting cells compared with the
TSH-omas, although, due to the small sample size, this did not attain
statistical significance (P = 0.1; Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is increasing evidence for an association between cyclin-CDK complexes and CDKIs and cancer (33). There is aberrant expression of p27 in neoplasms with a marked decrease of p27 expression in some benign and malignant neoplasms compared with normal tissue (34). The 12p13 chromosomal band is known to be deleted in leukemias (13), although no mutations of p27 were found in 147 human primary solid tumors (14). In addition, a decrease or absence of p27 protein is a powerful negative prognostic marker in patients with breast (35, 36) and colorectal carcinomas (31, 37). High cyclin E and low p27 expression is associated with increased mortality among breast cancer patients (35). The correlation of p27 expression and the aggressiveness of gastric carcinomas shows that there is a significant difference between high p27 expression and low p27 expression in the survival of patients with gastric carcinomas (38). However, although certain of the CDKIs show frequent mutations in a variety of different malignancies (33, 39), no gene abnormalities were found in 28 PAs in one study (26) or in 18 nonfunctioning and somatotroph tumors in another, apart from the known polymorphism described at codon 109 (27). We were also unable to demonstrate any gene mutation in the exonic sequence of p27 in corticotrophinomas (25). Furthermore, p27 mRNA levels in human PAs were not different from normal tissue in several studies (25, 28), and we were also unable to show any loss of heterozygosity in the p27 region in a subset of corticotrophinomas investigated (25). However, it has been reported that there is less p27 protein expressed in PA tissue when 4 normal human pituitaries (removed at autopsy) were compared with 14 adenomatous pituitaries, with ACTH-secreting adenomas showing the lowest level of positivity (28, 34), although this was not confirmed in a more recent study investigating NFPAs and GH-secreting tumors (27). Our results, using a rigorously quantitative technique on a large number and variety of tumors and normal pituitaries, clearly suggest that adenomatous tissue shows less p27 compared with normal pituitaries. Because the normal pituitary contains a heterogeneous variety of cell types, we also carried out double-labeling studies to compare directly p27 immunopositivity within a given secreting cell, and again we noted a significant loss of p27 in tumorous cells of any class compared with the untransformed cells.
It is likely that p27 protein expression in PAs and pituitary carcinomas is regulated by translational and posttranslational mechanisms. Indeed, it has been found that p27 is degraded by the ubiquitin-proteasome pathway, that this process has been suggested to be more active in more malignant tumors, and that its activity is inversely correlated with the p27 protein content of the tissue (17, 37). Aggressive colorectal carcinomas show higher specific proteolytic activity and lower levels of p27 (37). Recently, another protein has been implicated in p27 protein regulation: Jab1 (Jun activation domain-binding protein) controls the activity of p27 by facilitating its degradation, possibly by assisting relocalization of p27 from the nucleus into the cytoplasm (40, 41). Unlike other cell cycle proteins, for which correlations have been found between the abundance of these proteins and changes in their mRNA content, a decline in p27 protein occurs in the presence of constant amounts of mRNA and protein synthesis (17). Here, we also demonstrate that a sample with very little p27 protein content showed a comparable amount of mRNA expressed in accordance with earlier more extensive studies of p27 mRNA expression (25, 28). In all types of pituitary tumors, p27 staining was significantly less than in the corresponding normal cell type, in accordance with data from nonendocrine tumors, suggesting that p27 is, indeed, an important regulator of cell proliferation. Finally, although only a few pituitary carcinomas were assessed, our data indicate that the progression from adenoma to carcinoma is also associated with a further loss of p27 protein expression. This seemed to be true for both ACTH- and prolactin-secreting tumors.
As corticotroph tumors showed a lower level of p27 staining
compared with other benign pituitary tumors, we speculated whether this
is a phenomenon characteristic of corticotroph adenomas or is present
in normal ACTH-producing cells, as well. Previous studies have
suggested that in normal rat pituitary more p27 is present in
prolactin, gonadotroph, and TSH cells, and less in GH and ACTH cells
(42); in normal human pituitaries, gonadotrophs and thyrotrophs show
the highest level of p27 staining, whereas ACTH cells show the lowest
level (28). Our results suggest that low p27 staining is solely a
characteristic of corticotrophs; all other cell types have populations
containing 20 times as many p27-positive cells as ACTH-containing
cells (see Fig. 7
). This may indicate that corticotroph cells are more
proliferative than other cell types, although there are few data in the
literature on the proliferative rate of different pituitary cell types
(43). However, in humans corticotroph tumors characteristically show
increased invasiveness and recurrence compared with other
hormone-secreting pituitary tumors (44, 45). It would, therefore, seem
that low p27 immunopositivity is a characteristic of corticotrophs that
may reflect their intrinsic proliferative rate, although there is no
direct evidence in support of this speculation. However, adenomatous
transformation of any cell type seems to lead to a reduction in p27
immunopositivity from the preceding basal level specific to that
cell.
In conclusion, in the normal pituitary there is variable expression of the CDKI p27, with relatively weaker staining in corticotrophs compared with other cell types. Tumorous transformation is associated with a reduction in p27 nuclear staining in each cell type, with a virtual absence of p27 in pituitary carcinomas. We suggest that these changes may reflect the proliferative activity of the cells and in turn implicate an abnormality of the pathway regulating p27 breakdown in pituitary oncogenesis.
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Footnotes |
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2 Present address: Institute of Endocrinology, Tashkent,
Uzbekistan.
Received February 3, 1999.
Revised April 2, 1999.
Accepted July 8, 1999.
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References |
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chain
of Gs and stimulate adenylyl cyclase in human pituitary tumors. Nature. 340:692–696.[CrossRef][Medline]
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