Inactivation of the p16 gene in human pituitary nonfunctioning tumors by hypermethylation is more common in null cell adenomas.

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A/MTS1 (p16) gene is associated with loss of expression of p16 protein in pituitary tumors. We analyzed a series of 21 pituitary adenomas and three normal pituitaries along with a human pituitary cell line (HP75) for methylation of exon 1 by methylation-specific PCR, immunohistochemistry, and Western blotting. PCR analysis showed that 5/7 (71%) of null cell adenomas, but only 2/7 (29%) gonadotroph tumors were hypermethylated. In addition,1 of 2 ACTH tumors but no GH (n = 4) or PRL (n = 1) adenoma examined were hypermethylated. Immunostaining and Western blot analysis of protein expression supported the methylation-specific PCR analyses. These results show that p16 gene silencing by hypermethylation is more common in null cell adenomas compared to other nonfunctioning adenomas such as gonadotroph tumors and that the role of p16 in the pathogenesis of pituitary adenomas is restricted to specific tumor subtypes.

Analysis of beta-catenin mutations and alpha-, beta-, and gamma-catenin expression in normal and neoplastic human pituitary tissues.

The cadherin-catenin system mediates Ca(2+)-dependent cell-cell adhesion, and genetic alterations in these molecules play a significant role in multistage carcinogenesis. Mutations in the beta-catenin gene, mostly affecting exon 3, have been detected in malignant cell lines and in primary tumors. Immunohistochemical abnormalities in alpha-, beta-, and gamma-catenin have been reported in malignant and benign tumors, and nuclear localization of beta-catenin has been associated with mutations in exon 3 of this gene. Mutational analysis of exon 3 of the beta-catenin gene was undertaken by polymerase chain reaction (PCR) and sequencing using genomic DNA extracted from frozen tissues, including 4 normal pituitaries, 22 pituitary adenomas, and one pituitary carcinoma. Frozen sections from these cases were used for immunohistochemical detection of beta-catenin. We also analyzed immunohistochemical expression of alpha-, beta-, and gamma-catenin by paraffin sections from 154 pituitary tumors, including 148 adenomas and 6 carcinomas. Genomic DNA was extracted from paraffin sections of 2 gonadotroph tumors showing nuclear staining for beta-catenin and was used for PCR and sequencing of exon 3 of the beta-catenin gene. No mutations in exon 3 of the beta-catenin gene were found in any of the 23 cases analyzed by PCR and sequencing. In addition, the 2 cases studied by paraffin section immunohistochemistry, with nuclear staining for beta-catenin, were negative for mutations in this exon. Normal pituitary expressed all three catenin proteins. Immunostaining usually showed a membranous pattern of reactivity and was generally stronger in normal pituitary than in the adjacent adenomas. Stains for alpha-catenin were positive in fewer tumors than for beta-catenin. The lowest frequency immunopositive tumors and the weakest immunostaining was for gamma-catenin. All medically treated prolactinomas were negative for gamma-catenin, whereas treated growth hormone adenomas were less often positive for both alpha- and gamma-catenin than for untreated tumors. The percentage of positive cases for beta-catenin was the same in these two groups. Most pituitary carcinomas were negative for both alpha- and gamma-catenin but were beta-catenin positive. These results indicate that (i) mutations in exon 3 of the beta-catenin gene are uncommon in pituitary tumors, and (ii) expression of alpha-, beta-, and gamma-catenin is decreased in pituitary adenomas compared to normal pituitary tissues.

Analysis of homogeneous populations of anterior pituitary folliculostellate cells by laser capture microdissection and reverse transcription-polymerase chain reaction.

Pituitary folliculostellate (FS) cells are usually located between the secretory cells in the anterior pituitary, and they produce many peptides that exert a paracrine effect on hormone-producing pituitary cells. Previous approaches have been unsuccessful in obtaining homogeneous populations of FS cells. We used a combination of immunostaining with S100 protein followed by laser capture microdissection (Immuno-LCM) to obtain purified populations of rat FS cells. These cells were analyzed along with a mouse FS cell line (TtT/GF) by RT-PCR for gene expression. RT-PCR analyses showed that both FS cell populations expressed the mRNAs for glial fibrillary acidic protein, S100 protein, transforming growth factor-beta1 (TGFbeta1), TGFbeta receptor, interleukin-6, leptin, leptin receptor, pituitary adenylate cyclase-activating polypeptide (PACAP), and PACAP receptors. Both FS cell populations were negative for PRL, GH, and POMC, supporting the homogeneity of the rat FS cell population. TGFbeta1, but not PACAP-38, treatment stimulated cell proliferation in both FS cell populations. TGFbeta1 increased leptin, but not interleukin-6, mRNA expression in rat FS cells. However, TGFbeta1 inhibited leptin RNA expression in the TtT/GF cell line, as shown by RT-PCR and Northern blot analysis. These results indicate that 1) homogeneous populations of FS cells can be prepared by Immuno-LCM; 2) TGFbeta1 stimulates the proliferation of normal rat FS cells and the TtT/GF cell line; and 3) the effects of TGFbeta1 to stimulate leptin mRNA expression in rat FS cells but inhibit leptin mRNA expression in TtT/GF cells probably reflect alterations in signal transduction in the TtT/GF cell line.