Elzbieta Kulig

p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers.

p27kip1 (p27) is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. p27 expression is regulated by cell contact inhibition and by specific growth factors, such as transforming growth factor (TGF)-beta. Since the cloning of the p27 gene in 1994, a host of other functions have been associated with this cell cycle protein. In addition to its role as a CDKI, p27 is a putative tumor suppressor gene, regulator of drug resistance in solid tumors, and promoter of apoptosis; acts as a safeguard against inflammatory injury; and has a role in cell differentiation. The level of p27 protein expression decreases during tumor development and progression in some epithelial, lymphoid, and endocrine tissues. This decrease occurs mainly at the post-translational level with protein degradation by the ubiquitin-proteasome pathway. A large number of studies have characterized p27 as an independent prognostic factor in various human cancers, including breast, colon, and prostate adenocarcinomas. Here we review the role of p27 in the regulation of the cell cycle and other cell functions and as a diagnostic and prognostic marker in human neoplasms. We also review studies indicating the increasingly important roles of p27, other CDKIs, and cyclins in endocrine cell hyperplasia and tumor development.

Leptin and leptin receptor expression in rat and mouse pituitary cells.

Leptin is a circulating hormone secreted mainly by adipose tissue. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues. Various reports have suggested that the anterior pituitary may have a role in the regulatory effects of leptin. We recently localized leptin in the human anterior pituitary, but analysis of leptin in rodent pituitary has not been previously reported. In this study we examined rat and mouse pituitary tissues and various cell lines for leptin by RT-PCR, immunohistochemistry, and Western blotting. Leptin receptor messenger RNA was also examined in these tissues by RT-PCR. Leptin was present in a small percentage of rat (4.8 ± 0.7%) and mouse (7 ± 2%) pituitary cells. Colocalization studies with leptin and pituitary hormones showed leptin expression mainly in TSH cells (24 ± 2% of TSH cells in the rat pituitary and 31 ± 1% of TSH cells in the mouse pituitary). A folliculo-stellate (FS) cell line, TtT/GF, also expressed leptin. Th...

DNA Methylation Regulates p27Kip1 Expression in Rodent Pituitary Cell Lines

We previously reported loss of expression of p27Kip1 (p27) protein in rat GH3 and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH3 and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH3 and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH3 and GHRH-CL1 cells. After treatment of GH3 and GHRH-CL1 cells with 10 micromol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%/o) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH3 cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH3 cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.

Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Transforming Growth Factor-β1-Induced Apoptosis in a Human Pituitary Adenoma Cell Line

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from hypothalamic tissues based on its ability to stimulate cAMP production in cultured anterior pituitary cells. Recent studies have suggested a functional role for PACAP in the apoptosis of brain cells. However, the role of PACAP in regulating apoptosis in human pituitary adenomas has not previously been examined. Analysis of the cultured human pituitary adenoma cell line HP75, which expresses all three major PACAP receptors, showed that both PACAP-38 and PACAP-27 inhibited TGF-beta1-induced apoptosis. Treatment with the PACAP receptor antagonists PACAP 6-38 (PACAP type I receptor antagonist) and (p-chloro-D-Phe(6), Leu(17))-VIP (PACAP type II receptor antagonist) blocked the effects of PACAP-38 on the inhibition of transforming growth factor-beta1 (TGF-beta1)-induced apoptosis, confirming the specificity of the role of PACAP. Treatment with forskolin but not phorbol 12-myristate 13-acetate (PMA) also inhibited TGF-beta1-induced apoptosis. TGF-beta1 treatment was associated with an increase in mitogen-activated protein kinase (MAP kinase) when analyzed by Western blotting, but PACAP inhibition of TGF-beta1-induced apoptosis was not associated with activation of MAP kinase. Immunocytochemical analysis of the cell cycle cyclin-dependent kinase inhibitor p27 showed that treatment with TGF-beta1, forskolin, PMA, and PACAP increased p27 expression in cultured HP75 cells. These results indicate that PACAP is a highly specific inhibitor of TGF-beta1-induced apoptosis in the HP75 human pituitary adenoma cell line and that PACAP, TGF-beta1, forskolin, and PMA all stimulate expression of the TGF-beta-regulated cell cycle protein p27 in the HP75 human pituitary adenoma cell line. The HP75 cell line can be used as a model to study the regulation of apoptosis in human pituitary cells.

A human pituitary adenoma cell line proliferates and maintains some differentiated functions following expression of SV40 large T-antigen

Human pituitary cells proliferate very slowly in vitro. Only a few cell lines have been established, and these have been used mainly for short-term studies. To obtain immortalized cell lines of human pituitary adenomas for in vitro studies, we infected adenoma cells with a replication-defective recombinant human adenovirus, which contains an SV40 early large T-antigen. One of the cell lines (HP75), which has been studied in culture during 60 passages, has been extensively characterized. It expressed the large T-antigen protein and its mRNA, as well as the genes for FSH-β, LH-β and α-subunit (α-SU) of gonadotropin hormone.The HP75 cell line also expressed the genes of various members of the chromogranin (Cg)/secretogranin (Sg) family, including CgA as well as the prohormone convertases PC1/3 and PC2. CgA was processed to pancreastatin in vitro, which was secreted into the culture medium. Treatment with phorbol 12-myristate 13-acetate (PMA), TGF-β1, and forskolin increased CgA expression in the cells and stimulated pancreastatin secretion into the medium while inhibiting cell growth. The HP75 cell line also expressed TGF-β mRNA isoforms (β1, β2, β3) and the mRNAs for the receptors for TGF-β (RI, RII, and RIII). The cells responded to TGF-β1 in vitro by increasing CgA protein expression and pancreastatin secretion. TGF-β-RII protein and mRNA expression were both increased by PMA.Ultrastructural studies showed that the HP75 cells had very few dense-core secretory granules and a poorly developed Golgi complex. After treatment with TGF-β1 and PMA, there was an increase in the development of rough endoplasmic reticulum and the Golgi complex.This is the first report of the development of an immortalized human pituitary cell line that retains some differentiated functions. HP75 can be used to study TGF-β and CgA functions in pituitary cells. Replication-defective recombinant human adenovirus with an SV40 large T-antigen insert can be used to generate other immortalized human pituitary cell lines for in vitro studies.

Distribution and regulation of proconvertases PC1 and PC2 in human pituitary adenomas.

Pituitary adenomas are members of the family of neuroendocrine cells and tumors which have secretory granules containing chromogranins/secretogranins and other proteins. Pituitary adenomas express the neuroendocrine specific proconvertases PC1 (also known as PC3) and PC2, which are important for the proteolytic processing of chromogranins/secretogranins molecules. We examined the distribution of PC1 and PC2 in primary cultures of 20 pituitary adenomas and analyzed the regulation of the proconvertase mRNAs and proteins by various secretagogues including hypothalamic hormones and phorbol ester to determine the role of PC1 and PC2 in CgA processing in pituitary adenomas. Although PC2 was present in all adenomas, there was a differential distribution of PC1 with PRL adenomas expressing lower levels of PC1 compared to other adenoma types by RT-PCR analysis, in situ hybridization and immunostaining. Treatment of primary cultures of pituitary adenomas with phorbol 12-myristrate 13-acetate (PMA) resulted in an increase in pancreastatin (PST) secretion in most pituitary adenomas and increased PC1 mRNA and protein expression in gonadotroph adenomas, but not in other types of adenomas. Analysis of a human pituitary adenoma cell line, immortalized by recombinant defective adenovirus (HP75), which expressed chromogranin A, FSH, PC1 and PC2 showed that PST was secreted by these immortalized cells. Treatment with TGFβ1 resulted in an increase in PST secretion and in PC1 mRNA and protein. These results indicate that a) there is a differential distribution of PC1 in human pituitary adenomas with PRL adenomas expressing very little PC1 mRNA and protein and b) that PC1 expression in gonadotropin hormone-producing adenomas is regulated by PMA and TGFβ1. These findings support the observation that chromogranin A is a substrate for the endoproteinase PC1 in human pituitary adenoma cells.

Remodeling of hyperplastic pituitaries in hypothyroid α-subunit knockout mice after thyroxine and 17β-estradiol treatment: Role of apoptosis

Hyperplasia of pituitary thyrotrophs is often associated with hypothyroidism. In this study, the effects of thyroxine and 17β-estradiol on thyrotroph hyperplasia was analyzed using a hypothyroid mouse model resulting from targeted disruption of the glycoprotein hormone α-subunit (αSU) gene, which leads to lack of functional thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and underdevelopment of the thyroid and gonads. Thyroxine replacement for 2 mo resulted in a decrease in the relative percent of thyrotrophs and an increase of lactotrophs and somatotrophs numbers to normal values. A twofold increase in the relative percent of gonadotrophs was observed compared to wild-type mouse pitutary. Treatment for 2 mo with 17β-estradiol led to an increase in lactotroph numbers to normal levels, but had no influence on thyrotroph hyperplasia. Rearrangement of the hyperplastic pituitary phenotype after hormonal replacement proceeded without any evidence of pituitary cell necrosis. A slight increase in apoptotic cell death was observed in hormone-treated pituitaries, and this was localized to TSH cells by double-labeling experiments. Chronic thyroxine treatment resulted in increased expression of Bcl-2 protein in hypertrophied pituitary cells, whereas 17β-estradiol increased expression of Bad protein in prolactin cells.These results suggest that apoptotic cell death is involved in reversal of thyrotroph hyperplasia in the presence of thyroid hormone. Thyroxine and 17β-estradiol may influence cell death in this model by regulating expression of the Bcl-2 protein family in a cell-type specific manner.