Expression of KL-6 mucin, a human MUC1 mucin, in intrahepatic cholangiocarcinoma and its potential involvement in tumor cell adhesion and invasion.

AIMS: Aberrant expressions of KL-6 mucin were proved to be associated with worse tumor behaviors of many carcinomas. This study was to evaluate the expression KL-6 mucin, a human MUC1 mucin, in intrahepatic cholangiocarcinoma (CC) and its significance in tumor progression. MAIN METHODS: KL-6 mucin expressions in 21 patients with CC, 12 with combined hepatocellular and cholangiocarcinoma (cHCC-CC), and 78 with hepatocellular carcinoma (HCC) were detected by immunohistochemical staining. The effects of two glycosylation inhibitors (tunicamycin and benzyl-alpha-N-acetylgalactosamine (BAG)) on CC cell proliferations were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. KL-6 mucin expressions were detected by immunocytochemical staining and western blotting after tunicamycin or BAG treatment. Cell adhesive and invasive properties were evaluated by adhesion tests and transwell chamber assays after tunicamycin or BAG treatment. KEY FINDINGS: Positive KL-6 mucin staining was observed in all CC tissues and CC areas of cHCC-CC tissues. Immunocytochemical staining and western blotting showed that KL-6 mucin expressions were significantly reduced after both inhibitors treatment. Cell adhesive properties were significantly decreased after both inhibitors treatment, while cell invasive abilities were significantly decreased after BAG but not tunicamycin treatment. SIGNIFICANCE: This study indicated that KL-6 mucin might be a specific tumor target for CC. Therapeutic strategies that target glycosylation of KL-6 mucin may be useful to control aggressive behaviors of CC.

Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sisters.

Cerebrotendinous xanthomatosis (CTX) is a hereditary disorder, which is inherited as an autosomally recessive disease, causing production of cholesterol and cholestanol xanthomas and mental retardation. The disease is caused by mutations in the gene for sterol 27-hydroxylase (CYP27A1). The only CTX patients diagnosed in Scandinavia are two Norwegian sisters from a consanguineous marriage. Here we have characterized the mutation and its functional consequences for the enzyme. Analysis of genomic DNA from cultured fibroblasts identified a base exchange C > T in position 1441, causing arginine at amino acid position 441 to be replaced by tryptophan. The same mutation was introduced by mutagenesis in the complimentary DNA (cDNA) for CYP27, ligated into the expression vector pcDNA4/HisMax and transfected into HEK293 cells. The mutated enzyme had less than 5% of the enzyme activity compared with the native enzyme. No abnormal catalytic products could be identified in the cell culture medium. Probably the mutation affects the haem binding within the holoenzyme. The mutation has also previously been reported in a Japanese family. This is the second example of a CTX-causing mutation that has been recognized in more than one population.

Matrix metalloproteinase-2 and -9 in bile as a marker of liver metastasis in colorectal cancer.

Matrix metallproteinases (MMP)-2 and -9 are associated with cancer invasion and metastasis. MMP-2 and MMP-9 activities have never been assayed in bile. In the present study we investigated whether MMP-2 and -9 activities in the bile could be a marker for evaluation of liver metastasis in colorectal cancer. Fifty-three patients underwent colorectal resection for histologically verified adenocarcinoma. Twenty-six patients had colorectal cancer without liver metastasis and 27 patients had metastatic liver tumor. Six patients were studied as carcinoma-free control. MMP-2 and MMP-9 activities were assayed in bile using gelatin zymography and quantitated. Active MMP-2 activity of colorectal cancer with liver metastasis group (24.1 +/- 2.5 pixel count) was significantly higher than that of colorectal cancer without liver metastasis group (11.4 +/- 1.3 pixel count) (P < 0.001) or of control group (6.4 +/- 1.0 pixel count) (P < 0.001). Active MMP-9 was not detected in bile. ProMMP-9 activity of colorectal cancer with liver metastasis group (530.3 +/- 127.5 pixel count) was significantly higher than that of colorectal cancer without liver metastasis group (213.9 +/- 33.2 pixel count) (P = 0.008). This is the first report showing that the levels of active MMP-2 and proMMP-9 in bile were significantly higher in liver metastasis of colorectal cancer than in metastasis-free colorectal cancer. The results suggest that activities of active MMP-2 and proMMP-9 in the bile may be useful markers for predicting liver metastasis in colorectal cancer.

Age- and gender-related changes in ligament components.

The age- and gender-related changes in extracellular matrix components (elastin, elastin cross-links, fibrillin, collagen and glycoprotein) and mineral components (calcium, Ca; phosphorus, P) in human lumbar yellow ligaments were investigated using samples obtained from surgical specimens. The mineral (Ca and P) contents increased with ageing (r = 0.703 and r = 0.772, respectively), whereas the contents of matrix components tended to decrease with ageing (elastin r = -0.261, elastin cross-links r = -0.213, fibrillin r = 0.494; collagen r = -0.322 and glycoprotein r = -0.143). Comparison of the male and female groups revealed that the ligament elastin content and elastin cross-links decreased in the male group, whereas the ligament collagen content decreased in the female group significantly in an age-dependent manner (r = -0.788, r = -0.753 and r = -0.721, respectively). These findings demonstrate age- and gender-related changes in mineral and matrix components (especially elastin and collagen) in the lumbar yellow ligaments in the Japanese population. It is suggested that elastin and collagen metabolism in ligaments changes both with age and according to gender.

Effects of fukinolic acid and cimicifugic acids from Cimicifuga species on collagenolytic activity.

The inhibitory collagenolytic activity (47-64% inhibition in 0.22-0.24 microM) of fukinolic acid and cimicifugic acids A, B, and C, which are esters of fukiic acid (3',4'-dihydroxybenzyl tartaric acid) was more potent than that (20-37% inhibition in 0.23-0.24 microM) of cimicifugic acids D, E, F, which are esters of pscidic acid (4'-hydroxybenzyl tartaric acid). Since fukiic acid showed weaker inhibition, and caffeic acid, ferulic acid, isoferulic acid, and p-coumaric acid showed far weaker activities, the entire structures of fukinolic acid and cimicifugic acids A, B, and C proved to be responsible for the inhibitory activities. Trypsin and pronase E hydrolyzed collagen nonselectively alone or in addition to collagenase. These collagenolytic activities were also inhibited by fukinolic acid. These results show that fukinolic acid may inhibit either the collagenolytic activities specific to collagenase or nonspecific to other emzymes. The present studies suggest the potential effect of fukinolic acid and cimicifugic acids of Cimicifuga rhizomes in preventing collagen degradation by collagenases or collagenolytic enzymes under pathological conditions, wound healing, or inflammation.

Frontal lobe dementia with abnormal cholesterol metabolism and heterozygous mutation in sterol 27-hydroxylase gene (CYP27).

Of the primary dementing disorders that cause frontotemporal dementia, the best-known is Pick disease. We report on a 44-year-old woman with progressive frontal lobe dementia and spastic paraplegia. Examination revealed increased serum levels of cholestanol with abnormal cholesterol metabolism and a heterozygous mutation of the sterol 27-hydroxylase gene (CYP27). Biochemical findings were compatible with cerebrotendinous xanthomatosis (CTX); however, the clinical manifestations were very dissimilar. To our knowledge, a symptomatic carrier of this mutation among CTX patients has not been reported. We speculate that the present patient has a previously undescribed neurodegenerative disease related to abnormal cholesterol metabolism with this heterozygous mutation.

Anti-alpha2 integrin antibody induces secretion and activation of 72-kDa progelatinase by human fibroblasts.

We examined the effects of the extracellular matrix proteins fibronectin, laminin, and matrigel on 72-kDa progelatinase secretion by human lip fibroblasts (KD) and skin fibroblasts (MRC9 and HSF). By gelatin zymography, 1.7 +/- 0.2-fold, 1.7 +/- 0.2-fold, and 1.8 +/- 0.3-fold increases were observed in 72-kDa progelatinase secretion from KD cells treated with fibronectin, laminin, and matrigel, respectively. Laminin and matrigel, but not fibronectin, stimulated 72-kDa progelatinase secretion from HSF cells. Fibronectin, laminin, and matrigel did not stimulate 72-kDa progelatinase secretion by MRC9 cells. Anti-alpha2 integrin antibody-stimulated 72-kDa progelatinase secretion and induced the 62-kDa activated form of 72-kDa progelatinase by KD cells. Activated p42 MAP kinase (MAPK) expression was down-regulated by anti-alpha2 integrin antibody. Anti-alpha2 integrin antibody stimulated 72-kDa progelatinase secretion by HSF cells without inducing the 62-kDa activated form. The data suggest that interaction between fibroblasts and extracellular matrix components via alpha2 integrin plays an important role in regulating secretion and activation of 72-kDa gelatinase and that down-regulation of activated p42 MAPK may be involved in the 72-kDa progelatinase activation mechanism.

Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum.

1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.

Nitric oxide stimulates elastin expression in chick aortic smooth muscle cells.

Nitric oxide (NO), an endothelium-dependent relaxing factor, regulates relaxation, proliferation, and migration of smooth muscle cells (SMCs) and most likely attenuates developing vascular disease such as atherosclerosis. We investigated whether or not NO is associated with regulation of aortic elasticity. S-Nitrosoglutathione (GSNO), a NO donor, stimulated tropoelastin synthesis in cultured SMCs during both the quiescent and proliferating phases. The stimulation of tropoelastin synthesis was dose-dependent within 1-100 nM. Maximum stimulation was detected by treatment with 100 nM GSNO for 24 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), an exogenous cyclic GMP analog, also upregulated tropoelastin synthesis. Tropoelastin and lysyl oxidase mRNA expression, as assessed by Northern blot analysis, was also stimulated by GSNO. Administration of KT5823, a cyclic GMP-dependent protein kinase inhibitor, inhibited the GSNO-induced tropoelastin synthesis. These results indicate that the stimulatory effects of GSNO are due to cyclic GMP dependent protein kinase (PKG) activation by NO. In conclusion, NO seems to enhance aortic elasticity via tropoelastin and lysyl oxidase upregulation.

Calreticulin is directly involved in anti-alpha3 integrin antibody-mediated secretion and activation of matrix metalloprotease-2.

Matrix metalloprotease-2 (MMP-2) plays a pivotal role in cancer invasion and metastasis. Invasive human rhabdomyosarcoma cells (RD) secrete proMMP-2. We recently reported that anti-alpha3 integrin antibody induced the activated form of MMP-2 and enhanced proMMP-2 secretion by RD cells with concomitant enhancement of RD cell invasion. Since recent studies showed that calreticulin interacts with integrin alpha subunit, we hypothesized that calreticulin may be involved in signal transduction of anti-alpha3 integrin antibody-mediated MMP-2 secretion and activation. Here we demonstrate that anti-alpha3 integrin antibody induced a transient enhanced interaction of calreticulin with alpha3 integrin. Transfection of antisense oligonucleotides of calreticulin in RD cells abrogated the interaction between calreticulin and alpha3 integrin, and completely suppressed activation of MMP-2 and enhanced secretion of proMMP-2 induced by anti-alpha3 integrin antibody. Transient overexpression of calreticulin cDNA in RD cells significantly increased secretion of proMMP-2. The results demonstrate for the first time that calreticulin is directly involved in MMP-2 secretion and activation.

Genetic analysis of phytosterolaemia.

Two women with multiple xanthomas, intermittent arthritis and thrombocytopenia were diagnosed as phytosterolaemia, an autosomal-recessive lipid storage disease, based on their increased serum concentrations of beta-sitosterol, campesterol and sitostanol. The gene responsible for this disease is located within a distance of 18 cM between microsatellite markers of D2S 1788 and D2S1352 at chromosome 2p21. We genotyped the patients and their family members with 16 microsatellite markers around this locus. The results from the homozygosity mapping of one family suggested that the gene was located within the distance of 12.6 cM between D2S2328 and D2S1352. We have shortened the genetic distance by 5.4 cM.

[Indication and outcome of extended right hemihepatectomy combined with pancreatoduodenectomy for bile duct carcinoma].

During the past 10 years, we have performed extended right hemihepatectomy combined with pancreatoduodenectomy (rtHPD) in eight patients with bile duct carcinoma. We compared the results in these patients with those in 43 bile duct carcinoma patients who underwent extrahepatic bile duct resection with more extensive hepatectomy than hemihepatectomy. Our indication for rtHPD is bile duct carcinoma of the diffuse type involving the intrapancreatic bile duct. For patients with obstructive jaundice, biliary drainage was performed preferentially in the part of the liver to be preserved. Portal vein embolization was performed before extended right hemihepatectomy or left trisectorectomy. Complete external drainage of pancreatic juice followed by second-stage pancreatojejunostomy was performed in five rtHPD patients. There were no hospital deaths or hepatic failures. There were four 5-year survivors after rtHPD. There was no significant difference between the cumulative 5-year survival rates after rtHPD (71%) and non-HPD (42%). Patients with bile duct carcinoma whose prognosis can be improved only by rtHPD exist and should be treated by rtHPD. However, considering the reported high mortality rate after this procedure, rtHPD should not be performed in an institution where its safety cannot be guaranteed.

p53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.

Novel acyl-CoA synthetase in adrenoleukodystrophy target tissues.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by demyelination of white matter. The X-ALD gene product adrenoleukodystrophy protein (ALDP) is expressed broadly among various tissues. However, deficiency of functional ALDP exclusively impairs brain, adrenal gland, and testis. Thus, loss of ALDP function is assumed to involve inactivation of a putative mediating factor that functions in a tissue-specific manner. Here we cloned a mouse cDNA encoding a novel protein, Lipidosin, that possesses long-chain acyl-CoA synthetase (LCAS) activity. Lipidosin is expressed exclusively in mouse brain, adrenal gland, and testis, which are affected by X-ALD. LCAS activity of Lipidosin was diminished by mutation of conserved amino acids within the AMP-binding domain. Mutation of the Drosophila homologue of Lipidosin has been reported to cause neuronal degeneration. Thus, Lipidosin may mediate the link between ALDP dysfunction and the impairment of fatty acid metabolism in X-ALD.

Effects of para-nonylphenol on 92 kDa gelatinase secretion by human peripheral lymphocytes and U937 cells in vitro.

Much attention has focused on environmental estrogenic chemicals such as para-nonylphenol which disrupt various tissues via the estrogen receptor. We studied effects of para-nonylphenol on gelatinase secretion by human lymphocytes in vitro. para-Nonylphenol (0.05-50 microM) dose dependently suppressed 92 kDa gelatinase secretion. The suppressive effect of 25 and 50 microM para-nonylphenol was completely blocked by tamoxifen. We also studied the effects of para-nonylphenol (0.05-50 microM) on 92 kDa gelatinase secretion by human leukemia U937 cells. para-Nonylphenol suppressed 92 kDa gelatinase secretion in a dose-dependent manner. The suppressive effect of 50 microM para-nonylphenol was completely blocked by tamoxifen. Estradiol did not significantly suppress 92 kDa gelatinase secretion. Our results suggest that para-nonylphenol suppressed 92 kDa gelatinase secretion via the estrogen receptor, however, para-nonylphenol interacts with the estrogen receptor in a manner distinct from estradiol. As this assay system is simple and rapid, it may prove useful to evaluate toxic effects of para-nonylphenol on human blood cells.

Overexpression of vacuolar ATPase 16-kDa subunit in 10T1/2 fibroblasts enhances invasion with concomitant induction of matrix metalloproteinase-2.

Recent studies show that the vacuolar-type H(+)-ATPase (V-ATPase) 16 kDa subunit is expressed on plasma membrane of cancer cells. We hypothesized that V-ATPase 16 kDa subunit is directly involved in cell invasion. In the present study we established transfectants overexpressing V-ATPase 16 kDa subunit at the mRNA level, and found that these transfectants showed an enhanced invasiveness through matrigel with a concomitant increases in secretion of matrix metalloproteinase-2. Moreover, antisense oligonucleotides of the V-ATPase 16 kDa subunit suppressed invasive human A549 cell invasion with concomitant decreases in secretion of matrix metalloproteinase-2. The results suggest that the V-ATPase 16 kDa subunit is directly involved in cell invasion and that matrix metalloproteinase-2 is responsible for promoting the invasion by the V-ATPase 16 kDa subunit.

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A.

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.