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.

Transactivation of the human cdc2 promoter by adenovirus E1A in cycling cells is mediated by induction of a 110-kDa CCAAT-box-binding factor.

Cyclin-dependent protein kinases (Cdks) are key regulatory proteins of the eukaryotic cell cycle. Cdc2 is expressed in late G1/S phase and functions in the G2 to M phase transition. Adenovirus E1A proteins are known to induce the expression of p34cdc2 and DNA synthesis in normal quiescent cells. In this study, mutational analysis of the human cdc2 promoter revealed that transactivation of the promoter by the E1A proteins in cycling cells is mediated through the two CCAAT box binding motifs. A 110-kDa protein (CBF/cdc2) was identified in nuclear extracts from monkey kidney (CV-1) cells stably expressing E1A as well as from adenovirus-transformed human 293 cells. Further, we show that this EIA-inducible CBF/cdc2 is related to the CBF which was shown to activate the heat shock protein 70 promoter. Analyses of the functional domain(s) of E1A required for the induction of the CBF and transactivation of the cdc2 promoter in these conditions revealed that E1A mutants which were defective in binding the pRB family of proteins or the cellular p300 protein were still active in assays measuring the induction of the CBF and transactivation of the cdc2 promoter, albeit with reduced efficiencies. But the E1A mutant which lost both functional domains was inactive in these assays. These results suggest that E1A has redundant functional domains for the induction of the 110-kDa CBF and activation of human cdc2 gene expression.

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.

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.

Acceleration of lipid peroxidation in human paraquat poisoning.

A 20-year-old man ingested approximately 15 to 20 mL of a 24% solution of paraquat. Acute renal and liver failure developed. The patient began undergoing hemodialysis and receiving vitamin E, but he died of pulmonary fibrosis the 27th day after the ingestion. During a two-week period of observation, evidence of lipid peroxidation in his serum was noted by determining malondialdehyde levels. Guinea pigs exposed to paraquat showed similar patterns of malondialdehyde presence in plasma. These results indicate that increased serum or plasma levels of lipid peroxide occurring in paraquat poisoning might reflect production of highly reactive oxygen free radicals and that, possibly, levels of lipid peroxides could serve as indicators of the efficacy of therapy directed toward scavenging free radicals.

Hepatitis C virus NS5A protein is phosphorylated in vitro by a stably bound protein kinase from HeLa cells and by cAMP-dependent protein kinase A-alpha catalytic subunit.

Hepatitis C virus (HCV) has a positive-strand RNA genome that codes for a polyprotein precursor, which is processed co- and post-translationally by cellular and viral proteinases into three structural and at least six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists in two phosphorylated forms of 56-kDa and 58-kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from HeLa cells and hepatocellular carcinoma (HepG2) cells by co-immunoprecipitation and by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein produced in E. coli. This protein kinase could phosphorylate in vitro the native NS5A on serine residues, (GST)-NS5A, histone H1, and casein as substrates. In addition, the GST-NS5A was also phosphorylated in vitro by the cAMP-dependent protein kinase A-alpha catalytic subunit.

Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain).

Sequences totalling 5472 nucleotides (nt) from four complementary DNA (cDNA) clones of the dengue virus type 2 (DEN-2) RNA (New Guinea strain, NGS-C) have been reported previously [Yaegashi et al., Gene 46 (1986) 257-267; Putnak et al., Virology 163 (1988) 93-103]. This report describes the complete nucleotide sequence, with the exception of about 7 nt at the 5'-noncoding region, of this RNA genome derived from several cDNA clones. It is 10,723 nt in length and contains a single long open reading frame of 10,173 nt, encoding a polyprotein of 3391 amino acids. The genomic organization is similar to that of other flaviviruses that have recently been reported. Among the three DEN-2 strains - the Jamaica genotype (DEN-2JAM), the DEN-2NGS-C, and the S1 candidate vaccine strain derived from Puerto Rico (PR)-159 isolate (DEN-2S1) - which have been sequenced to date, the amino acid sequences of the polyproteins bear 94%-99% similarity. When the amino acid sequences of DEN-2NGS-C are compared with those of the other two strains, the variations are greater in the DEN-2S1 than in the DEN-2JAM. When DEN-2 and DEN-4 are compared, the overall amino acid identities range from 30% to 80% in both the structural and nonstructural proteins; whereas between DEN-2 and DEN-1, they range from 68% to 79% in the region encoding the structural proteins and the nonstructural protein NS1.

Partial sequence analysis of cloned dengue virus type 2 genome.

Dengue virus (DEN) is a member of flaviviruses and contains a single, (+)-strand RNA of approx. 11 kb. Complementary DNA copy of the RNA was synthesized using reverse transcriptase and oligo(dT) as primer. The double-stranded DNA copy was cloned at the PstI site of pUC13'-1 vector and was used to transform Escherichia coli JM83. Eleven transfomants were found to contain DEN insert as screened by colony hybridization. Three clones were chosen for further characterization by nucleotide (nt), sequence analysis. Two of these clones overlapped by 470 bp. Sequences of these three clones totalling about 4.6 kb were obtained. Translation of this DNA in all possible reading frames revealed the presence of long open reading frames spanning the entire length of the cDNA clones. The putative polypeptides derived from the nt sequence are 885 and 643 amino acids in length and show homology to the region of polyprotein coded by the yellow fever virus genome corresponding to the non-structural proteins [Rice et al., Science 229 (1985) 726-733]. The significant homology between these two viruses in the regions coding for the non-structural proteins NS3 and NS5 suggests an important role for these two proteins in the life cycle of these viruses.

Characterization of the nuclear localization signal and subcellular distribution of hepatitis C virus nonstructural protein NS5A.

Hepatitis C virus (HCV) has a positive strand RNA genome that codes for a polyprotein that is processed co-translationally and post-translationally into three structural and at least seven nonstructural (NS) proteins. To investigate the function of NS5A, a recombinant vaccinia virus was constructed in which the NS5A gene was cloned under the control of T7 promoter and encephalomyocarditis virus 5'-untranslated region (EMCV-UTR) for cap-independent translation in mammalian cells. In addition, the NS5A gene was also cloned under the control of cytomegalovirus (CMV) early promoter. The NS5A expressed in monkey kidney (CV-1) cells was located predominantly in the cytoplasm. Using immunohistochemical analysis, the subcellular distribution of NS5A in liver biopsy samples from chronic HCV-infected patients was also found to be in the cytoplasm. However, the NS5A protein has a stretch of positively charged domain in the vicinity of proline and valine residues, (PPRKKRTVV), characteristic of a nuclear localization signal (NLS), in the COOH-terminal half of the protein. To investigate whether the putative NLS of NS5A is functional, chimeric expression plasmids were constructed in which regions containing the NLS were fused to the N-terminus of the E. coli beta-galactosidase (E. coli beta-Gal). The expression of the fusion proteins in CV-1 cells resulted in their nuclear localization, indicating that the putative NLS is functional in targeting the heterologous protein, E. coli beta-Gal, to the nucleus, although the native NS5A is retained in the cytoplasm.

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.

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.

Shortened microsatellite d(CA)21 sequence down-regulates promoter activity of matrix metalloproteinase 9 gene.

One characteristic elements in the promoter of the matrix metalloproteinase 9 (MMP-9) gene is the d(CA) repeat. To investigate whether this element regulates the transcription of the MMP-9 gene and its enzymatic activities, we sequenced the promoter region isolated from esophageal carcinoma cell lines. TE9 cells with low MMP-9 enzymatic activity had the number of d(CA) repeats shortened from 21 to 14 or 18. TE8, TE10 and TE11 cells with high MMP-9 activities had 21 or 23 d(CA) repeats. Luciferase assays using MMP-9 promoter containing 18, 14 or 0 d(CA) repeats showed transcriptional activities which were 50, 50 or 5%, respectively, of the level achieved with promoter containing 21 d(CA) repeats. Sequence analysis of the promoter of 223 Japanese subjects revealed that most had two alleles with 20, 21 or 22 d(CA) repeats, whereas six had one or two alleles with 14, 18 or 19 d(CA) repeats. We postulate that length alteration of the d(CA) repeat causes phenotypic differences among carcinoma cells and that microsatellite instability may contribute to the polymorphism of d(CA) repeat length.

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.

A product of growth arrest-specific gene 6 modulates scavenger receptor expression in human vascular smooth muscle cells.

Although Gas6 is identified as a growth factor for vascular smooth muscle cells (VSMCs), its roles in these cells have not been clearly elucidated. To examine the role of Gas6 in atherosclerosis, we examined the effects of Gas6 on scavenger receptor family expression in VSMCs. Scavenger receptor class A, one of the scavenger receptor family members, was upregulated in VSMCs by Gas6. Furthermore, the atherogenic lipoprotein, oxidized LDL, induced Gas6 production in these cells. These results indicate that Gas6 plays an important role in foam cell formation in human VSMCs.

Anti-proliferative effect of 2-methoxyestradiol on cultured smooth muscle cells from rabbit aorta.

The proliferation of cultured smooth muscle cells from rabbit aorta was inhibited markedly by 2-methoxyestradiol, considerably by 2-hydroxyestradiol, and only slightly by 17 beta-estradiol. The effects at doses from 1 to 40 nmol/ml were cytostatic rather than cytocidal. 2-Methoxyestradiol could be useful to suppress excessive proliferation of arterial smooth muscle cells, and thus to prevent the progression of atherosclerosis.

Production of tissue collagenase (matrix metalloproteinase 1) by human aortic smooth muscle cells in response to platelet-derived growth factor.

The production of the precursor of tissue collagenase/matrix metalloproteinase 1 (proMMP-1) by cultured human aortic medial smooth muscle cells (SMCs) was significantly enhanced by the treatment of the cells with platelet-derived growth factor (PDGF), interleukin 1 or 12-O-tetradecanoylphorbol-13-acetate (TPA). The response to PDGF of SMCs exhibited a tendency to be age-dependent: only SMCs obtained from older individuals (age: 54, 56, 72 and 74 years) responded to PDGF and synthesized proMMP-1, but not SMCs from young individuals (age: 10, 16 and 41 years), and weak responsiveness with a 19-year-old individual. On the other hand, induction of proMMP-1 synthesis in SMCs by TPA was not discriminated by age. The synthesis of two other related matrix metalloproteinases was also examined. Matrix metalloproteinase 2 was found to be constitutively expressed in zymogen form in SMCs and its synthesis was not affected by the treatments with PDGF, interleukin 1 or TPA. The synthesis of matrix metalloproteinase 3 (stromelysin) was not detected in SMCs from both young and old individuals even after the treatment with PDGF, interleukin-1, prostaglandin E2 or TPA. The ability of SMCs to synthesize and secrete proMMP-1 in response to PDGF suggests that this enzyme plays an important role in the migration of PDGF-stimulated SMCs from the media into the intima of aorta and the eventual formation of atherosclerotic plaques.

Atherogenic change in the thoracic aorta of the senescence-accelerated mouse.

Biochemical and histological changes in the thoracic aorta of the senescence-accelerated prone mouse were examined in comparison with those of the senescence-accelerated resistant mouse. At 3 and 5 months of age, the lipid peroxide level in the thoracic aorta was higher in the former than in the latter. At 5 months of age, levels of both total cholesterol and triglycerides in the aorta were higher in the former than in the latter, and vice versa for the level of aortic phospholipids. At 5 months of age, the prone mice showed macrophage invasion beneath the endothelial cells of the thoracic aorta, whereas the resistant ones did not.