Aberrant DNA methylation of cyclin D2 and p27 genes in rodent pituitary tumor cell lines correlates with specific gene expression

We previously reported that increased DNA methylation was an important mechanism of silencing the p27 gene in some pituitary tumor cell lines [1]. DNA methylation correlated inversely with p27 gene expression. The p27 and cyclin D2 genes are located in the same region of mouse chromosome 6, rat chromosome 4, and human chromosome 12p13. Because both genes are located in the same gene cluster, we investigated whether methylation was a principal mechanism regulating cyclin D2 as well as p27 expression in rodent pituitary cell lines. Bisulfite genomic sequencing showed that the normally unmethylated cytosines of the p27 gene in normal pituitary (NP) were extensively methylated in GH3 and GHRH-CL1 cells, but not in AtT 20, αT₃-1 and LβT₂ cells; but cyclin D2 was extensively inactivated in various pituitary tumor cell lines by increased DNA methylation. These abnormalities of methylation in p27 and cyclin D2 genes occurred with different frequencies in five pituitary tumor cell lines with 100% (5/5) methylation of the cyclin D2 gene and 40% (2/5) methylation of the p27 gene. Treatment with the methyl transferase inhibitor 5′-aza-2′-deoxycytidine (AZAdC) increased expression of cyclin D2 and p27 in GH3 and GHRH-CL1 pituitary tumor cells. There was a correlation between hypermethylation and gene expression. GH₃ tumors implanted into Wistar-Furth rats in vivo did not change the methylation status of the p27 and cyclin D2 genes. These data indicate a coordinately reduced expression of these two linked genes in most rodent pituitary tumor cell lines and suggest that methylation of cyclin D2 and p27 might occur in a “hot spot” in this gene-rich cluster. Supported in part by NIH CA 37231 and CA 42951     

Regulation of estrogen receptor mRNA in normal and neoplastic rat pituitary tissues

The effects of estradiol 17β (E₂) on the regulation of estrogen receptor (ER) mRNA amounts in normal and neoplastic rat pituitary tissues were analyzed by in situ hybridization with an oligonucleotide probe. E₂ treatment produced a significant increase in ER mRNA amounts in two transplantable pituitary tumors (MtT/Wl5 and MtT/F4) and in the GH₃ cell line. ER mRNA amounts were also increased In normal pituitaries after 6 weeks of E₂ treatment, but the differences were not significant. Biochemical assay of ER proteins confirmed the presence of ER protein in MtT/Wl 5 and MtT/F4 tumors. “Cytoplasmic” ER proteins were decreased by E₂ in the MtT/Wl5 tumor.These results indicate that ER mRNA is present in normal pituitaries and in pituitary tumors and can be regulated by estrogen treatment in vivo and in vitro. The inhibitory effects of high estrogen concentration on proliferation of transplantable pituitary tumors in vivo and GH₃ cells in vitro is not explained by the absence of ER mRNAs in these tumors.     

DNA Methylation Regulates p27Kip1 Expression in Rodent Pituitary Cell Lines

We previously reported loss of expression of p27^Kip1 (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 μmol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%) 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.     

Plasma 1,25 Dihydroxy Vitamin D<Subscript>3</Subscript> Level and Expression of Vitamin D Receptor and Cathelicidin in Pulmonary Tuberculosis

Introduction  Vitamin D₃, which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D₃ levels and increased risk of tuberculosis have been reported. Study Subjects and Methods  Plasma 1,25 dihydroxy vitamin D₃ levels (1,25(OH)₂ D₃) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D₃. VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. Results  1,25(OH)₂ D₃ were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)₂ D₃ levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D₃-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). Conclusions  The present study suggests that PTB patients may have increased 1,25(OH)₂ D₃ levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)₂ D₃ might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.     

TNF-α mRNA Expression Correlates with TGF-β mRNA Expression <Emphasis Type="Italic">In Vivo</Emphasis>

TNF-α is postulated to play a significant role in regulating TGF-β₁ expression. In lung fibroblasts, for example, TNF-α is supposed to induce TGF-β₁ via AP-1 activation. TNF-α receptor, knock-out mice are resistant to induced fibrosis and over-expression of TNF-α causes increased TGF-β₁ production in mice. Therefore, we investigated whether TNF-α mRNA levels are associated with the TGF-β₁ mRNA levels of blood leucocytes in humans. Quantitative real-time PCR of TNF-α and TGF-β₁ was performed in 118 Germans. Calculations of expression were made with the 2^−ΔΔCT method. When the investigated population was divided in two groups (TNF-α low and TNF-α high) by the median of the determined TNF-α expression, highly significant (p < 0.0001) differences of TGF-β1 mRNA expression were revealed. Additionally, dividing the investigated population into quartiles of the determined TNF-α expression showed significantly different TGF-β1 mRNA expressions. Comparing the determined CT-values of TNF-α in context with these of TGF-β1, a coefficient of determination R ² = 0.4635 was calculated. In this study we demonstrated in vivo a significant association of the relative TNF-α/B2M mRNA expression and the relative TGF-β₁/B2M mRNA expression in 118 Germans.     

Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K<Superscript>+</Superscript> channels

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca²⁺ levels has been shown to rely on G_q/11 and PLC activation, the molecular mechanisms involved in the regulation of ERG K⁺ channels by TRH are still partially unknown. We have analysed the effects of βγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH₃ cells β-ARK/CT, Gα_t, and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the βγ scavengers affected the TRH-induced shift in V _1/2. Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH₃ cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH₃ cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca²⁺ and ERG responses in GH₃ cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH₃ and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.     

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.     

Cell Cycle Regulatory Proteins p27(kip), Cyclins Dl and E and Proliferative Activity in Oncocytic (Hurthle Cell) Lesions of the Thyroid

Cyclins are prime cell-cycle regulators central to the control of cell proliferation in eukaryotic cells. The formation of cyclin/cyclin-dependent kinases (CDK) complexes activates the kinases and initiates a cascade of events, which directs cells through the cell cycle. CDK inhibitors (CDKIs) such as p27^kip1 inhibit cyclin-CDK complexes and function as negative regulators of the cell cycle. Previous studies have shown that p27^kip1 is decreased in malignant relative to benign thyroid tumors, but its role and interaction with other cell cycle regulatory proteins have not been well established in oncocytic lesions of the thyroid. We studied the expression of p27^kip1, cyclins D1 and E, and Ki67 in 20 cases of oncocytic adenoma (AD), 6 cases of oncocytic carcinoma (CA), 8 cases of Hashimoto’s thyroiditis (HT), and 9 cases of nodular goiter with oncocytic change (NG) by immunohistochemistry. In the latter two lesions only oncocytic cells were evaluated. The positive staining was stratified into four groups. Statistical analysis was done using the Kruskal-Wallis one-way analysis of variance test, and, when significant, the Dunn multiple-comparisons procedure was used to determine pairwise differences. All 20 AD were p27^kip1 positive, 10 were 4+, 2 were 3+, and the remaining 8 were 1+. In contrast all 6 CA showed 4+ p27^kip1 staining, of the 8 HT, 2 were 4+, two 3+, three 1+, and 1 was negative. All 9 NG were p27 positive, 7 showed 4+, one 3+, and one 1+ staining. On pairwise comparison differences in p27^kip1 staining between AD and CA and between HT and CA were statistically significant (p=0.0243 and p=0.0142, respectively). In all but one case Ki67 expression was either very low (<3%) or negative. No significant differences were seen in the expression of cyclin D1 or cyclin E among the groups observed. In conclusion, the increased p27^kip1 expression in malignant oncocytic tumors relative to benign oncocytic lesions is unlike any other malignant progression reported in the thyroid and other organ systems in the body. This may reflect on the biologic nature of the oncocytic cells of the thyroid and the significance of this finding remains to be established. Proliferative activity as studied by Ki67 immunostaining was not helpful in distinguishing benign from malignant oncocytic tumors.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

Mycophenolate mofetil and roscovitine decrease cyclin expression and increase p27(kip1) expression in anti Thy1 mesangial proliferative nephritis

The response of mesangial cells to a phlogistic challenge includes cell proliferation and mesangial matrix expansion. Cell proliferation is a highly regulated process which includes enhancing factors such as cyclins, cyclin dependent kinases, and inhibitory proteins, such as p27(kip1). The aim of the study was to evaluate the effects of Mycophenolate mofetil (MMF), and roscovitine (R), on the cell cycle regulatory system when administered in the florid phase of the experimental model of mesangial proliferative nephritis induced by the anti Thy-1 antigen monoclonal antibody. Three days after nephritis induction, different groups were given MMF and R. Rats treated with MMF or R showed a slight decrease in mesangial proliferation and matrix expansion. Samples of cortical tissue were tested by 'real time' RT-PCR in order to study gene expression of cyclins B, D1, D2, D3, E, and the cyclin inhibitor p27(kip1). Localization of mRNA was evaluated by in situ hybridization. Real time RT-PCR analysis showed a significant decrease in cyclins B, D1, D2, and D3 in rats treated with either MMF or R as compared to controls. Both MMF and R treatment induced a significant increase in p27(kip1) mRNA expression. In situ hybridization showed a mesangial-endothelial expression pattern in glomeruli. The number of labelled cells per glomerulus, the number of positive glomeruli in each examined slide as well as cyclin D2 and D3 signal intensity was significantly lower in rats treated with MMF or R as compared to controls, whereas MMF or R treatment up-regulated p27(kip1) mRNA expression. Immunohistochemical evaluation of p27(kip1) aimed to examine the influence of MMF or R on protein expression confirmed up-regulation.     

A catalog of 106 single-nucleotide polymorphisms (SNPs) and 11 other types of variations in genes for transforming growth factor-beta1 (TGF-beta1) and its signaling pathway

 Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that is produced in the platelet, bone, placenta, and other tissues. It acts as a growth inhibitor in many types of cells, and also mediates extracellular matrix production and immunosuppression. Mutations in the specific domain of its gene (TGFB1) cause Camurati-Engelmann disease, a bone-sclerosing disorder, and those in other domains may be associated with osteoporosis. We identified 106 single-nucleotide polymorphisms and 11 other types of variations in TGFB1 and six other genes. These genes were TGF-β type I receptor gene (TGFBR1), TGF-β type II receptor gene (TGFBR2), SMAD2 gene (SMAD2), SMAD3 gene (SMAD3), SMAD4 gene (SMAD4), and SMAD7 gene (SMAD7), all of which compose the TGF-β1 signaling pathway. We also estimated allele frequencies of these DNA polymorphisms among 48 Japanese individuals. Our data will provide a useful resource for the study of disease susceptibility. Received: May 9, 2002 / Accepted: June 4, 2002     

Tissue-engineered skin using aggregates of normal human skin fibroblasts and biodegradable material

Higher-density inoculation of fibroblasts into a three-dimensional scaffold should accelerate wound healing after skin implantation. This study attempted to develop tissue-engineered skin with a higher density of fibroblasts. We first attempted to fabricate three-dimensional high-cell-density aggregates (spheroids) of normal human fibroblasts for application to tissue-engineered skin. Our method consisted of rotational shaking with nontreated dishes, decreasing fibroblast-meterial interactions, and augmenting cell-cell interaction. To prompt aggregate formation, the medium was supplemented with insulin, dexamethasone, ascorbic acid, and basic fibroblast growth factors that potentiate secretion of extracellular matrices. Under such improved conditions, fibroblasts were able to form spheroidal aggregates within 24 to 36h of rotational culture. Although the formed aggregates were irregular in shape and were composed of only several cells after 12h, they became almost spheroidal after 24h. The aggregates grew even more round after 36h, and their surfaces became smooth. After 36h of rotational culture, the fibroblast aggregates were collected and reinoculated onto a biodegradable mesh composed of polyglycolic acid coated with collagen. The aggregates were trapped in the material and became attached after 24h. Finally, because transforming growth factor-β₃ (TGF-β₃) is known to accelerate wound healing, we conducted a semiquantitative analysis of TGF-β₃ mRNA in both the fibroblast monolayers (two-dimensional culture) and the aggregates (three-dimensional culture). Analysis of TGF-β₃ mRNA expression showed that mRNA expression was greater in the fibroblasts of aggregates than in a monolayer. Therefore, our newly developed dermal graft is expected to accelerate wound healing faster than conventional grafts.     

Transforming growth factor-β stimulates IL-1β-induced monocyte chemoattractant protein-1 expression in human synovial cells via the ERK/AP-1 pathway

Objective and Design: Transforming growth factor- β (TGF-β) has not only a fibrogenic role, but also monocyte/ macrophage chemotactic properties in a synovial joint. However, little is known about the effects of TGF-β on monocyte chemoattractant protein-1 (MCP-1) expression in human synovial cells under inflammatory status. The aim of this study was to determine whether TGF-modulates MCP-1 production under the chronic inflammation, and to elucidate the cell signaling mechanism involved. Materials and methods: Human synovial cells were exposed to IL-1β, which mimics the environment of chronic inflammation. Production of MCP-1 protein and expression of MCP-1 mRNA were determined by ELISA and real-time PCR. Results: TGF-β upregulated the expression of MCP-1 mRNA and protein with or without IL-1β. TGF-β and IL-1β synergistically enhanced MCP-1 gene expression, and an AP-1 binding site was involved in the signal transduction. In addition, MEK inhibitor completely suppressed TGF-β-induced MCP-1 production. Conclusions: TGF-β and IL-1β synergistically enhance MCP-1 gene expression through the activation of the MEK/ERK1/2 pathways, which leads to AP-1 activation. The impairment of MCP-1 regulation by TGF-β in resident synovial cells might represent an important mechanism of chronic inflammation and tissue fibrosis in a synovial joint. MCP-1 should be considered a valid target for therapeutic intervention. Received 22 September 2005; returned for revision 3 February 2006; accepted by J. Skotnicki 29 May 2006     

Immunohistochemical analysis of p27 (Kip1) in human pituitary glands and in various types of pituitary adenomas

p27 (Kip1) plays regulatory roles in the cell cycle by inhibiting the activity of cyclin dependent kinases (CDKs). This immunohistochemical study is aimed at elucidating the expression of p27 in human pituitary and in various types of pituitary adenomas in order to clarify its role in the regulation of proliferation. Sixteen normal pituitary glands and 179 human pituitary adenomas were used for immunohistochemical studies. The tissues were fixed in 10% formalin and embedded in paraffin. Indirect peroxidase method was performed after heat-induced antigen retrieval using a monoclonal antibody against p27 protein. p27 protein was expressed in the nuclei of all 16 normal human pituitary glands. p27 protein was also expressed in 128 of 179 cases of pituitary adenomas (71.5%). A marked decrease of p27 expression was noted in ACTH-secreting adenomas, 8/20 (40.0%), compared with other types of pituitary adenomas—GH-secreting adenomas, 35/46 (76.1%); PRL-secreting adenomas, 22/33 (66.7%); TSH-secreting adenomas, 8/11 (72.7%); and nonfunctioning adenomas, 55/69 (79.7%). These results suggest that p27 may play some role in the regulation of proliferation in all types of pituitary adenomas. The lower levels of p27 in ACTH-secreting adenoma is of particular interest with respect to the intermediate lobe-derived pituitary tumor developed in p27 knockout mice.     

Overexpression of p21, cyclin E and decreased expression of p27 in DMBA (7, 12-dimethylbenzanthracene)-induced rat ovarian carcinogenesis

Ovarian cancer is a common gynecological malignancy and a leading cause of death in women. Inactivation of the tumor suppressor gene and deregulation of cyclin E are frequent in human ovarian cancer. The objective of this study was to investigate the expressions and roles of cyclin E, p21 and p27 in 7, 12-dimethylbenzanthracene (DMBA)-induced ovarian tumors in rats. The expressions of cyclin E, p21 and p27 were evaluated by immunohistochemistry and western blot analysis. The expressions of cyclin E and p21 in ovarian tumors was higher than that in normal ovarian surface epithelium. In contrast, the expression of p27 in ovarian tumors was lower than that in normal ovarian surface epithelium. But there were no differences among the cancer types. Positive correlation was present between cyclin E and p21. p27 was negatively correlated with cyclin E and p21. These results suggest that the increased expression of cyclin E and p21, and the decreased expression of p27, occur in DMBA-induced rat ovarian carcinogenesis and result in tumor progression.     

Evidence That Folliculo-Stellate Cells Mediate the Inhibitory Effect of Japanese Kampo Medicine,Unkei-To,on Growth Hormone Secretion in Rat Anterior Pituitary Cell Cultures

PROBLEM: Cytokine-induced neutrophil chemoattractant (CINC) was reported to influence anterior pituitary hormone release. We recently found that Unkei-to, one of the Japanese Kampo medicines, stimulated CINC secretion by rat anterior pituitary cells and the pituitary folliculo-stellate (FS)-like cell line (TtT/GF). Therefore, the effect of Unkei-to on growth hormone (GH) secretion by rat anterior pituitary cells was investigated. METHOD OF STUDY: Dispersed normal anterior pituitary cells, the folliculo-stellate-like cell line TtT/GF, and the GH₃ cell were used to test the effect of Unkei-to on GH secretion. In reconstitutive coculture experiments, TtT/GF cells were mixed with GH₃ cells at a ratio (TtT/GF cells: GH₃ cells) of 1:99. From this mixture, cells were seeded onto plates at a density of 10⁴ cells/well and were cultured for 5 days. The cells were then used in the experiments. RESULTS: Unkei-to at 20 μg/ml significantly inhibited GH secretion by normal anterior ptuitary cells within 12 hr of incubation. In contrast Unkei-to stimulated GH secretion by GH₃ cells in a time- and dose-dependent manner, suggesting that an accessory cell type was involved. To assess the contribution of CINC as a paracrine factor, an experiment using a reconstitutive coculture system was performed, and Unkei-to was found to inhibit GH secretion when GH₃ cells were cocultured with TtT/GF cells. The addition of anti-CINC antibody to the reconstitutive coculture system antagonized Unkei-to-inhibited GH secretion. CONCLUSION: CINC, which was secreted from FS cells by Unkei-to, may be responsible for mediating the inhibitory effect of Unkei-to on GH secretion by rat anterior pituitary cells.     

CD<Subscript>4</Subscript><Superscript>+</Superscript>CD<Subscript>25</Subscript><Superscript>+</Superscript> Regulatory T Cells Decreased the Antitumor Activity of Cytokine-Induced Killer (CIK) Cells of Lung Cancer Patients

CD₄ ⁺CD₂₅ ⁺ regulatory T cells (Tregs) have been shown to inhibit cytotoxic lymphocytes-mediated immune responses. Cytokine-induced killer (CIK) cells exert high impact on adoptive immunotherapeutic approaches. Therefore, the purpose of this report was to determine the effect of Tregs on CIK cell growth and CIK-induced cytotoxicity for inhibition of tumor growth in vivo as well as in vitro. After depletion of CD₄ ⁺CD₂₅ ⁺ cells before culture, the proliferation and cytotoxicity of CIK cells, which indicated in bromodeoxyuridine (BrdU) and lactic dehydrogenase (LDH) assays, were significantly increased. Depletion of CD₄ ⁺CD₂₅ ⁺ cells preculture also enhanced the suppression effect on the lung cancer cells inoculated in experimental animals. Blockage of glucocorticoid-induced tumor necrosis factor receptor (GITR) and transforming growth factor β1 (TGF-β1) by antibodies partially abrogated the suppressive effect of CD₄ ⁺CD₂₅ ⁺ cells on CIK. These results indicated that Tregs could inhibit the antitumor activity of CIK cells. The molecules TGF-β and GITR may contribute to the suppressive function of CD₄ ⁺CD₂₅ ⁺ cells.     

Cyclin-dependent kinase inhibitor p27Kipl expression and interaction with other cell cycle-associated proteins in mammary carcinoma

p27, cyclin D1, and retinoblastoma (Rb) protein have been demonstrated using immunohistochemistry in 189 cases of primary breast carcinoma with long-term follow-up. There was a statistically significant association between the expression of p27 and both cyclin D1 and the retinoblastoma gene product (pRb), corresponding to their close interactions in regulating the G₁/S transition in the cell cycle. Low levels of p27 were seen in high-grade, rapidly proliferating, oestrogen receptor-negative tumours. In univariate analysis, low p27 expression was associated with a reduced relapse-free and overall survival. In multivariate analysis, p27 was not an independent predictor of survival when either histological grade or proliferative activity (S-phase fraction) was included in the model. When the combined expression of p27 and cyclin D1 was related to survival, patients with high levels of p27, regardless of their cyclin D1 status, did well, whilst those with low p27 had a poor outcome. The only exception, in the latter group, was patients with tumours expressing high levels of cyclin D1, who did as well as the high p27 group. We have shown that in clinical material p27 expression is associated with proliferative activity and while univariate analysis shows it to be a significant indicator of prognosis, this significance is lost in multivariate analysis when traditional prognostic factors are included in the model. The interest in p27 expression in mammary carcinoma lies in its behaviour when examined in combination with other G₁ cell cycle regulators. Copyright © 1999 John Wiley & Sons, Ltd.