The combination of strong expression of ZNF143 and high MIB-1 labelling index independently predicts shorter disease-specific survival in lung adenocarcinoma.

BACKGROUND: The transcription factor, zinc finger protein 143 (ZNF143), positively regulates many cell-cycle-related genes. The ZNF143 would show high expression of multiple solid tumours related closely to cancer cell growth, similar to the widely accepted Ki67 (MIB-1) protein, but the underlying mechanisms for ZNF143 remain unclear. We investigated the association of ZNF143 expression with clinicopathological features and prognoses of patients with lung adenocarcinoma. METHODS: Expressions of ZNF143 and MIB-1 were immunohistochemically analysed in 183 paraffin-embedded tumour samples of patients with lung adenocarcinoma. The ZNF143 expression was considered to be strong when >30% of the cancer cells demonstrated positive staining. RESULTS: Strong ZNF143+ expression showed a significantly close relationship to pathologically moderate to poor differentiation and highly invasive characteristics. The ZNF143 positivity potentially induced cell growth of lung adenocarcinoma, correlated significantly with high MIB-1 labelling index (⩾10%). Univariate and multivariate analyses demonstrated that both strong ZNF143+ and the high MIB-1 index group have only and significantly worse survival rates. CONCLUSIONS: The combination of strong ZNF143 expression and high MIB-1 index potentially predicts high proliferating activity and poor prognosis in patients with lung adenocarcinoma, and may offer a therapeutic target against ZNF143.

Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas.

BACKGROUND: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). METHODS: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. RESULTS: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low ß-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. CONCLUSION: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.

Polypeptide N-acetylgalactosaminyltransferase 6 expression in pancreatic cancer is an independent prognostic factor indicating better overall survival.

BACKGROUND: The family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) is responsible for the altered glycosylation in cancer. The purpose of our study was to investigate the clinical significance of two isoforms, GalNAc-T6 and -T3, and their correlation with the prognosis of pancreatic cancer. METHODS: Immunohistochemistry was used to analyse GalNAc-T6 and -T3 expressions in 70 clinicopathologically characterised pancreatic cancer cases. RESULTS: Positive expressions of GalNAc-T6 and -T3 were immunohistochemically identified in 51% (36 of 70) and in 77% (54 of 70) of patients, respectively. A close relationship was noted between GalNAc-T6 positive expression and pathological well/moderate differentiated type (P=0.001), small tumour size (P=0.044), absence of vascular invasion (P=0.009), and low stage of the American Joint Committee on Cancer systems (P=0.043). The expression of GalNAc-T3 significantly correlated with good differentiation (P=0.001), but not with other clinicopathologic features. Furthermore, univariate and multivariate analyses revealed that GalNAc-T6 expression was an independent prognosis indicator for the disease, whereas GalNAc-T3 expression had no impact on clinical outcome, even though 33 of 36 GalNAc-T6-positive cases also had a positive expression of GalNAc-T3 (P=0.001, r=0.356). CONCLUSION: Both GalNAc-T6 and -T3 expressions correlated significantly with tumour differentiation, whereas only GalNAc-T6 expression predicted prognosis in pancreatic cancer.

Clock and ATF4 transcription system regulates drug resistance in human cancer cell lines.

The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.

Rheumatoid synovial endothelial cells produce macrophage colony-stimulating factor leading to osteoclastogenesis in rheumatoid arthritis.

OBJECTIVES: Periarticular osteoporosis and joint destruction are major complications in rheumatoid arthritis (RA), caused by osteoclast-mediated bone resorption. However, the mechanisms of monocyte/osteoclast maturation and role of RA endothelial cells (RAECs) in the control of osteoclastogenesis remain unclear. The present study was designed to determine the most important factors that influence monocyte accumulation and osteoclast formation among the many factors produced by RAEC. METHODS: We analysed the expression profiles of various genes in human endothelial cells from various organs (RA synovium, umbilical vein, skin, liver sinusoid, renal glomerulus and brain) using oligonucleotide microarrays. Specifically, up-regulated gene in RAECs was assessed by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunostaining of RA synovia. Migration of monocytes was assessed by the chemotactic chamber EZ-TAXIScan. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation was observed by microscopy. RESULTS: Among many epithelial-expressed factors, macrophage colony-stimulating factor (M-CSF) gene was abundantly expressed specifically in RAECs. Genes of fibroblast growth factor-2, interleukin-6 and osteoprotegerin were also overexpressed in RAECs. Migration of monocytes and osteoclast formation in co-cultures promoted by culture supernatants of RAECs were inhibited by M-CSF neutralizing antibody. CONCLUSIONS: M-CSF produced by RAECs is involved in osteoclastogenesis from monocytes, migration and TRAP-positive MNC formation.

Expression of Y-box-binding protein dbpC/contrin, a potentially new cancer/testis antigen.

Y-box-binding proteins are members of the human cold-shock domain protein superfamily, which includes dbpA, dbpB/YB-1, and dbpC/contrin. dbpC/contrin is a germ cell-specific Y-box-binding protein and is suggested to function as a nuclear transcription factor and RNA-binding protein in the cytoplasm. Whereas ubiquitous dbpB/YB-1 expression has been well studied in various types of human carcinomas as a prognostic or predictive marker, the dbpC/contrin expression in human tumour cells has not been reported. In this report, we provide the first evidence showing that dbpC was highly expressed in human testicular seminoma and ovarian dysgerminomas, and in carcinomas in other tissues and that its expression in normal tissues is nearly restricted to germ cells and placental trophoblasts. These results indicate that dbpC/contrin would be a potentially novel cancer/testis antigen.

Transactivation of human cdc2 promoter by adenovirus E1A.

Expression of the adenovirus oncoprotein E1A 12S induces the heterotrimeric transcription factor, NF-Y. NF-Y binds to the two CCAAT motifs upstream of the transcriptional start site of the human cdc2 promoter and is required for activation of the promoter by E1A 12S in cycling cells. The observations that a number of eukaryotic cell cycle regulatory genes also contain the CCAAT motifs and NF-Y binds to them support the notion that E1A 12S could play an important role in deregulated expression of these genes through activation of NF-Y gene in cycling cells.

An immunohistochemical and ultrastructural study of syringocystadenoma papilliferum.

BACKGROUND: Syringocystadenoma papilliferum is a benign hamartomatous tumour of the skin. The histogenesis of this tumour is still controversial. There have been few reports regarding immunohistochemical investigations using only a limited range of antibodies and ultrastructural studies on this rare tumour. OBJECTIVES: To elucidate the immunohistochemical and ultrastructural properties of this tumour. METHODS: We investigated the immunohistological patterns of 12 different anticytokeratin (CK) antibodies and several other markers in five cases of this tumour, comparing them with the patterns in adult sweat glands. One of these cases was also evaluated ultrastructurally. RESULTS: The luminal columnar cells of the tumour were mostly positive for CK7 and more than 70% were positive for CK19. These cells showed the heterogeneous expression of CK1/5/10/14, CK14 and CK5/8. These patterns were also observed in the luminal cells in the secretory or the ductal portion of the adult sweat glands. The basal cuboidal cells of the tumour almost constantly expressed CK1/5/10/14, CK5/8, CK14 and CK7 (except for one case), similar to the patterns of basal cells in the transitional portion and myoepithelial cells in the sweat glands. However, the basal tumour cells expressed CK19 and vimentin heterogeneously, and alpha-smooth muscle actin focally (three cases). Ultrastructurally, the constituent epithelial cells were mainly divided into three types: luminal cells, basal cells and clear cells. The luminal tumour cells bore features of the secretory or ductal luminal cells of sweat glands, although they were somewhat immature in appearance. The basal tumour cells were fundamentally basaloid in nature. The clear cells were undifferentiated or primitive in appearance, suggesting stem or progenitor cell properties. Transitional forms between the clear cells and the other two cell types were also identified. CONCLUSIONS: The tumour epithelium was composed of several cell types demonstrating various developmental stages from the primitive clear cells to the basal cells demonstrating a tendency to differentiate toward basal cells in the apocrine transitional portion or myoepithelial lineage, or luminal cells toward the ductal or secretory epithelium. These results support the classical concept that syringocystadenoma papilliferum is a hamartomatous tumour that arises from pluripotent cells.

Histamine increases the expression of LOX-1 via H2 receptor in human monocytic THP-1 cells.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a member of the scavenger receptor family, and is known to be expressed in monocytes/macrophages. We investigated the effect of histamine on the expression of LOX-1 in cells of the human monocytic leukemia cell line THP-1. Histamine as well as forskolin and dibutyryl cyclic AMP (Bt2-cAMP) stimulated the THP-1 monocytes to express the LOX-1 gene at the transcription level. This histamine effect on LOX-1 gene expression, via the histamine H2 receptor-mediated cAMP signal transduction pathway, was reduced after differentiation of the cells into macrophages, even though forskolin and Bt2-cAMP still enhanced the gene expression. The alteration of the responsiveness of LOX-1 expression to histamine was related to suppressed expression of the H2 receptor in THP-1 macrophages. The switch of the predominant class of histamine receptors between H1 and H2 would modulate the effects of histamine on LOX-1 gene expression in monocytes and macrophages, and therefore, would play a certain role in the inflammatory aspects of atherogenesis.

Phosphatidylinositol 3-OH kinase-Akt/protein kinase B pathway mediates Gas6 induction of scavenger receptor a in immortalized human vascular smooth muscle cell line.

The growth arrest-specific gene 6 encodes a secreted protein, Gas6, which was originally identified as the ligand of a receptor, Axl, with tyrosine kinase activity. The class A scavenger receptor (SRA) mediates lipid uptake into cells, leading to the formation of foam cells, an important step in atherogenesis. Although Gas6 induces SRA expression, the underlying mechanism is not clear. In this report, we show that the Gas6-induced expression of SRA was mediated by the phosphatidylinositol 3-OH kinase (PI3-kinase)-serine/threonine kinase (Akt/protein kinase B [PKB]) pathway involving Akt phosphorylation. This pathway was activated by exposure to Gas6. Furthermore, the effect of Gas6 was abrogated by wortmannin, a specific inhibitor of PI3-kinase. We also demonstrated that the constitutively active form of Akt enhanced activity of the SRA promoter but that the dominant-negative mutant of Akt completely abolished the expression of SRA after treatment with Gas6. These results show that the PI3-kinase-Akt/PKB pathway participates in Gas6-induced SRA expression and suggests that the activation of Akt/PKB plays an important role in Gas6-induced atherosclerosis and foam cell formation in human vascular smooth muscle cells.

Effects of histamine and interleukin-4 synthesized in arterial intima on phagocytosis by monocytes/macrophages in relation to atherosclerosis.

We investigated the localization of histidine decarboxylase (HDC), which is the rate-limiting enzyme that generates histamine from histidine, in human aorta/coronary artery. RT-PCR and immunohistochemical staining revealed that the HDC gene was expressed in monocytes/macrophages and T cells in the arterial intima but not in smooth muscle cells in either the arterial intima or the media. A luciferase promoter assay with U937 and Jurkat cells demonstrated that interleukin-4 (IL-4) inhibited the expression of the HDC gene. In contrast, among a scavenger receptor family, IL-4 as well as histamine up-regulated U937 cells to express the LOX-1 gene but not the SR-A gene, which genes encode receptors that scavenge oxidized lipids. These findings suggest that histamine synthesized in the arterial wall participates in the initiation and progression of atherosclerosis and that IL-4 can act as an important inhibitory and/or stimulatory factor in the function of monocytes/macrophages modulated by histamine in relation to the process of atherosclerosis.

Expression of HDL receptor, CLA-1 in human smooth-muscle cells and effect of interferon-gamma on its regulation.

High-density lipoprotein (HDL) exerts antiatherogenic effects by various mechanisms. The protective effect of HDL is thought to involve the reverse transport of cholesterol from cells in the arterial wall to the liver for disposal. We previously identified human scavenger receptor BI (hSR-BI/CLA-1) as a receptor for human HDL, but did not examine the expression of hSR-BI/CLA-1 in smooth-muscle cells. In this present study, a human aortic intima smooth-muscle cell line immortalized with SV 40 DNA was established, and the expression of hSR-BI/CLA-1 in this cell line analyzed by Western blot and RT-PCR. HSR-BI/CLA-1 mRNA and protein were detected in both this cell line and primary human aortic smooth-muscle cells. A cytokine, interferon-gamma (IFN-gamma) inhibited the hSR-BI/CLA-1 protein expression, but not mRNA expression. This observation confirmed that selective cholesterol ester uptake from HDL was inhibited by IFN-gamma. These results indicated that hSR-BI/CLA-1 may be expressed in human smooth-muscle cells, and the expression may be modulated by IFN-gamma. HSR-BI/CLA-1 on smooth-muscle cells could play an important role in atherogenesis.

Induction of human matrix metalloproteinase-12 gene transcriptional activity by GM-CSF requires the AP-1 binding site in human U937 monocytic cells.

Matrix metalloproteinase-12 (MMP-12) is critical for the migration of monocytes/macrophages into inflammatory sites through the basement membranes. We previously reported that MMP-12 expression was initially induced by granulocyte macrophage colony-stimulating factor (GM-CSF) in human peripheral blood monocytes and U937 monocytic cells. To further elucidate the molecular mechanism for the regulation of MMP-12 expression by GM-CSF in monocytes, we determined the sequence requirements for the MMP-12 gene transcriptional response of U937 monocytic cells to GM-CSF by using luciferase reporter and electrophoretic mobility shift assays. A series of 5'-deletion and site-directed mutation of the human MMP-12 promoter demonstrated that an AP-1 site spanning the -81 to -75-bp region is critical for the induction of MMP-12 promoter activity by GM-CSF. The electrophoretic mobility shift assay revealed that AP-1 binding activity was increased by GM-CSF treatment and that the AP-1 complex induced by GM-CSF consisted of multiple Jun and Fos isoforms. These results indicate that MMP-12 expression in U937 monocytes was initially induced by GM-CSF through the AP-1 binding activity.

Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.

Hepatitis C virus nonstructural protein, NS5A, is a phosphoprotein produced from the processing of the viral polyprotein precursor. NS5A associates with several cellular proteins in mammalian cells, and the biological consequences of this interaction are currently unknown. To this end, five stable NS5A-expressing murine and human cell lines were established. Tetracycline-regulated NIH3T3 cells and rat liver epithelial cells as well as the constitutive, NS5A-expressing, human Chang liver, HeLa, and NIH3T3 cells all exhibited cell growth retardation compared with the control cells. Cell cycle analysis by flow cytometry indicated that the NS5A-expressing human epitheloid tumor cells had a reduced S phase and an increase in the G(2)/M phase, which could be explained by a p53-dependent induction of p21(Waf1/Cip1) protein and mRNA levels. NS5A interacts with Cdk1 in vivo and in vitro, and a significant portion of the p21(Waf1/Cip1) was found to be in a complex with Cdk2 in the NS5A-expressing human hepatic cell line. Cdk1 and cyclin B1 proteins were also reduced in human Chang liver cells consistent with the increase in G(2)/M phase. Our results suggest that the NS5A protein causes growth inhibition and cell cycle perturbations by targeting the Cdk1/2-cyclin complexes.

Comparison of three in vitro assay systems used for assessing cytotoxic effect of heavy metals on cultured human keratinocytes.

The cell viability assay using cultured cells is of great advantage to elucidate the biological effect of potentially toxic substances. Recently, a novel assay system, Tetracolor One cell proliferation assay (Seikagaku Co., Tokyo, Japan), has been developed. In this report, we compare the results of the Tetracolor One assay regarding the cytotoxic effect of three heavy metal salts on cultured adult keratinocytes to those of the neutral red dye uptake assay and the MTT eluted stain assay. In this study, these three methods showed almost similar results. Compared to the other two methods, however, the Tetracolor One assay, which requires only one-step procedure before spectrophotometric measurement, is easier to use, and errors in measurement, which may be produced through the multistep procedure, are much less in this assay. Therefore, we believe that the Tetracolor One assay system is useful for assessing the cytotoxic effect of heavy metals on cultured human keratinocytes.

Teratoid carcinosarcoma of the ovary with prominent neuroectodermal differentiation.

We present what we believe to be only the second report of ovarian teratoid carcinosarcoma. The patient, a 59-year-old woman, was admitted to hospital complaining of a pelvic mass and of abdominal fullness. Advanced ovarian cancer was diagnosed, and a tumorectomy was done. The tumor occupied the pelvis, and metastasis was found in the liver and spleen. The solid tumor was composed of chondrosarcoma, squamous cell carcinoma, adenocarcinoma and malignant neuroectodermal components, which contained ganglioneuroblastoma-like and medulloepithelioma-like areas. Immunohistochemically, the neuroectodermal cells were positive for both neural and epithelial markers. This ovarian tumor consisted of frankly malignant components, with prominent neuroectodermal elements mixed with epithelial and mesenchymal elements in an organoid fashion; a quite rare tumor.

Role of apoptosis in the closure of neonatal ductus arteriosus.

Apoptosis regulates the remodeling of tissue during embryonic development by eliminating unwanted cells and structures. The present study investigated smooth muscle cell (SMC) proliferation and apoptosis in the neonatal ductus arteriosus (DA) during closure. In the DA of 39 swine neonates and 5 autopsy human neonates, apoptosis was detected using in situ end-labeling and electron microscopy, and proliferation was evaluated using proliferating cell nuclear antigen. In swine, apoptosis of SMC was first observed at 24h after birth. After 48h, both apoptosis and proliferation quickly increased and became most prominent at 3 days, mainly in the intima and inner media. From 5 days, both apoptosis and proliferation quickly disappeared, and were present to a minor extent at the 2 weeks after birth. During these processes, there was no sign of inflammation or necrosis. In humans, apoptosis was found in tissue specimens obtained from 2 term neonates who died at 1 and 5 days after birth. These findings suggest that SMC contribute to the functional closure of the DA by active constriction, and soon after, they switch to proliferation and apoptosis, which may contribute to the anatomical closure of the DA.

Ovarian yolk sac tumor with virilization during pregnancy: immunohistochemical demonstration of Leydig cells as functioning stroma.

A case is reported of yolk sac tumor occurring in the left ovary and complicated by pregnancy. The 22-year-old patient presented at 28 weeks gestation with virilization and elevated serum levels of testosterone and alpha-fetoprotein. The tumor showed the typical features of yolk sac tumor with a mixture of islands of Leydig cells. The accumulations of Leydig cells were well demarcated from the cellular components of the yolk sac tumor and were distributed throughout the tumor, although with predominant localization at the periphery. By immunohistochemistry the Leydig cells were intensely positive for vimentin and negative for cytokeratins, allowing clear distinction from the cell components of the yolk sac tumor, which were positive for cytokeratins and negative for vimentin. Testosterone was also identified in the cytoplasm of the Leydig cells. After tumor resection the testosterone and alpha-fetoprotein levels declined simultaneously; this, together with the immunohistochemical demonstration of testosterone, indicates that the Leydig cells were responsible for the endocrine manifestations. Furthermore, antibodies against inhibin alpha-subunit and calretinin could be used to detect the Leydig cells. The present case, a combination of yolk sac tumor and Leydig cells acting as a functioning stroma and causing virilization during pregnancy, is very rare.

Matrix metalloproteinases in patients with osteoarthritis of the hip.

In 78 patients undergoing a total hip replacement we examined the production of matrix metalloproteinases (MMP) by the fibroblasts from the connective tissue of the acetabulum; we then correlated these findings with clinical and radiological characteristics of the same patients. In 53 patients only MMP-2 was produced; in 15 cases MMP-1, -2 and -3 were present; and in 10 cases not only MMP-1, -2, -3 but also MMP-9. Significant differences among the clinical and radiological parameters were found in the 3 subsets. A positive correlation between the production of MMP-9 and a rapid destruction of the hip joint was found.

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages.

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.