Mutation and expression analysis of the p27/kip1 gene in corticotrophin-secreting tumours.

The molecular mechanisms leading to Cushing's disease are unclear. Inhibitors of cyclin-cyclin dependent kinase (CDK) complexes are regulators of the cell cycle and may function as tumour suppressor genes, many of which have been involved in the pathogenesis of several human malignancies. A member of this family, the p27/kip1 gene, maps to chromosome 12p13 and encodes an inhibitor of several cyclin-CDK complexes; these control the progression of the cell cycle from G1 to S-phase. Complete lack of p27/kip1 function, as occurs in the p27/kip1 'knockout' mouse, produces a complex phenotype associated with the development of pituitary tumours, specifically those of the intermediate lobe corticotrophs. We therefore investigated whether structural and functional abnormalities of the p27/kip1 gene and loss at the chromosome 12p13 region were present in human corticotrophin (ACTH)-secreting pituitary tumours. We studied 21 pituitary tumours, of which 20 were ACTH-secreting (two of these had biochemical and histological features of 'intermediate-lobe' tumours and one was malignant) while the remaining tumour was a prolactinoma; three ectopic secretors of ACTH (two bronchial and one thymic carcinoid); and a non-secretory thymic carcinoid. The whole coding region of the p27/ kip1 gene was screened for mutations by PCR-SSCP analysis and/or direct sequencing, while tumour mRNA expression was analysed by means of a semi-quantitative duplex PCR. Three polymorphic microsatellite markers of the 12p13 region were used to assess loss of heterozygosity (LOH) in 12 samples. Finally, tumour p27/kip1 protein expression was assessed by immunohistochemistry using a monoclonal antibody in 12 samples suitable for analysis. No sequence abnormalities were found in any of the samples other than a previously-described polymorphism. No LOH was observed in the tumours analysed. p27/kip1 mRNA expression was similar in tumour samples in comparison with normal pituitaries. Seven of the eight corticotroph tumours analysed by immunohistochemistry stained positive for p27/kip1, including the intermediate lobe. The only malignant pituitary tumour in the original series showed an absence of staining for p27/kip1. In addition, the three carcinoid tumours studied were negative on immunohistochemistry. Of a further three malignant pituitary tumours assessed, two (including a prolactinoma) were essentially negative, while the third was moderately positive. We conclude that mutations of the p27/kip1 gene, deletions of the 12p13 area or changes in expression, are not a general feature of corticotroph tumours, even those with intermediate lobe characteristics. However, other mechanisms of p27/kip1 inactivation, such as an abnormality at the post-translational level, may be related to more aggressive histological subtypes of ACTH-secreting and possibly other pituitary tumours

Expression of the growth hormone secretagogue receptor in pituitary adenomas and other neuroendocrine tumors.

Synthetic GH secretagogues (GHSs; GH-releasing peptides and their nonpeptide mimetics) stimulate GH release, activate the hypothalamo-pituitary-adrenal axis, and release PRL in vivo. Patients with acromegaly show an exuberant GH response to GHSs, whereas patients with pituitary-dependent ACTH-secreting tumors show an exaggerated rise in ACTH and cortisol. We, therefore, studied the presence of GHS receptor (GHS-R) messenger ribonucleic acid (RNA) in 38 human pituitary tumors of different cell types, 3 ectopic ACTH-secreting tumors, a pancreatic gastrinoma, 3 insulinomas, and a non-secreting thymic carcinoid as well as in 7 normal pituitary glands. Certain pituitary tumors were also studied by in vitro cell culture with measurement of secreted GH, ACTH, PRL, FSH, LH, alpha-subunit, and TSH. RNA was extracted from tissue samples and, after RT, a duplex PCR reaction with primers for the GHS-R gene and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was performed, allowing semiquantitation of GHS-R expression. All the somatotroph adenomas (n = 8) showed a 2-10 times higher expression of the GHS-R gene compared to normal pituitaries. Higher than normal expression was shown in 5 of 18 tumors from patients with ACTH-secreting pituitary adenomas and in 1 of 3 ectopic ACTH-secreting carcinoid tumors. Two of the pituitary ACTH-secreting adenoma samples showed completely absent expression of the GHS-R, 8 showed expression similar to that of normal pituitary tissue, and 3 of the corticotroph adenoma tissue samples and 2 ectopic ACTH-secreting tumors showed a very low level of expression. One of 4 prolactinoma samples showed a high level of expression, 1 showed expression similar to that of normal pituitary, and 2 samples showed a very low level of expression. Nonfunctioning pituitary adenoma samples showed either absent or very low level expression of the GHS-R. The pancreatic gastrinoma sample showed expression similar to that of normal pituitary tissue, whereas 3 insulinomas showed low level expression of the GHS-R gene; a nonsecreting thymic carcinoid tumor showed no detectable expression. In summary, although GHS-R messenger RNA is abundant in human somatotroph adenomas, it is also present in other pituitary adenomas, particularly ACTH-secreting tumors. These findings may explain the in vivo responses to GHSs in patients harboring such tumors. It also appears from our study that GHS-R may be expressed in other neuroendocrine tumors

?-d-Galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages

Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of 125 I labelled Evonymus europaea and Griffonia simplicifolia I-B 4 lectins. Treatment of the stimulated macrophages with coffee bean α-galactosidase abolished binding of the GS I-B 4 isolectin and changed the binding pattern of the Evonymus lectin. The affinity ( K a ) of Evonymus lectin for α-galactosidase-treated macrophages decreased approximately 23-fold, from 1.25×10 8 M −1 to 5.5×10 6 M −1 . Subsequent digestion of α-galactosidase-treated macrophages with α- l -fucosidase from Trichomonas foetus , further reduced binding of Evonymus lectin. Resident macrophages showed the same pattern of Evonymus lectin binding, with the same affinity, as α-galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of the Evonymus lectin which, in the absence of α- d -galactosyl groups, requires α- l -fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal α- l -fucosyl residues. It is also concluded that during macrophage stimulation/activation α- d -galactosyl residues are added to this glycoconjugate and that they form part of the receptor for Evonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains α- d -galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound to Corynaebacterium parvum activated macrophages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45796/1/10719_2004_Article_BF00731705.pd