Coxsackie and adenovirus receptor is a critical regulator for the survival and growth of oral squamous carcinoma cells.

Coxsackie and adenovirus receptor (CAR) is essential for adenovirus infection to target cells, and its constitutive expression in various cancerous and normal tissues has been reported. Recently, the biological role of CAR in human cancers of several different origins has been investigated with respect to tumor progression, metastasis and tumorigenesis. However, its biological function in tumor cells remains controversial. Here we report the critical role of CAR in growth regulation of oral squamous cell carcinomas (SCCs) in vitro and in vivo via the specific interaction with Rho-associated protein kinase (ROCK). Loss of endogenous CAR expression by knockdown using specific small interfering RNA (siRNA) against CAR facilitates growth suppression of SCC cells due to cell dissociation, followed by apoptosis. The consequent morphological reaction was reminiscent of anoikis, rather than epithelial-mesenchymal transition, and the dissociation of oral SCC cells was triggered not by lack of contact with extracellular matrix, but by loss of cell-to-cell contact caused by abnormal translocation of E-cadherin from surface membrane to cytoplasm. Immunoprecipitation assays of the CAR-transfected oral SCC cell line, HSC-2, with or without ROCK inhibitor (Y-27632) revealed that CAR directly associates with ROCKI and ROCKII, which results in inhibition of ROCK activity and contributes to maintenance of cell-to-cell adhesion for their growth and survival. Based on these findings, in vivo behavior of CAR-downregulated HSC-2 cells from siRNA knockdown was compared with that of normally CAR-expressing cells in intraperitoneally xenografted mouse models. The mice engrafted with CAR siRNA-pretreated HSC-2 cells showed poor formation of metastatic foci in contrast to those implanted with the control siRNA-pretreated cells. Thus, CAR substantially has an impact on growth and survival of oral SCC cells as a negative regulator of ROCK in vitro and in vivo.

Expression pattern of REIC/Dkk-3 in mouse squamous epithelia.

The REIC/Dkk (reduced expression in immortalized cells/Dickkopf-3) gene was originally identified as a tumour-suppressor gene with reduced expression in immortalized cells, cancer-cell lines and tumour tissues. Of the four members of the Dkk family, the REIC/Dkk-3 protein is unique in terms of DNA sequence, expression profiles and biological functions. In this study, we investigated and compared the expression patterns of the REIC/Dkk-3 protein in mouse squamous epithelia. Expression of REIC/Dkk-3 in the back skin was localized to the upper layer of the interfollicular epidermis, and the inner root sheath of hair follicles. Expression of REIC/Dkk-3 was detected in the ear skin, oral mucosa, tongue, oesophagus, uterine cervix, footpad and tail skin, but not in the cornea. Interestingly, expression was localized to the upper layers of these epithelial tissues. The physiological function of REIC/Dkk-3 is still unclear, but our detailed observation highlight its unique expression pattern in squamous epithelia.

Degrading ability of oligocyclic aromates by Phanerochaete sordida selected via screening of white rot fungi.

Seventy-nine white rot strains were screened to determine if they had the potential for use in the degradation of oligocyclic aromates (PAHs) by measuring their dye-decoloration rate. Fourteen strains that were selected based on their dye-decoloration rate were then evaluated for the ability to tolerate various levels of PAHs spiked in agar medium. The ability of white rot fungi to degrade 3- or 4-ring PAHs (anthracene, phenanthrene, fluoranthene, pyrene) was determined. Two strains of Phanerochaete sordida (KUC8369, KUC8370) were possible PAHs degraders, degrading a significantly greater amount of phenanthrene and fluoranthene than the culture collection strain P. chrysosporium (a known PAHs degrader). The production of manganese peroxidase, the only extracellular ligninolytic enzyme detected during the cultivation, was evaluated.

Application of a thermo-reversible gelation polymer, mebiol gel, for stem cell culture and regenerative medicine.

Recent studies have revealed the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer, Mebiol Gel. It is a purely synthesized biocompatible copolymer composed of thermoresponsive polymer blocks [poly(N-isopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol [PEG]). Mebiol Gel is characterized by its temperature-dependent dynamic visccoelastic properties. Mebiol Gel is used as a biocompatible scaffold for three-dimensional culture without any toxicity. Representative biological scaffolds for three-dimensional culture, i.e. type I collagen and Matrigel, interact with cells and affect cellular functions, but Mebiol Gel hardly showed such effects. Because of its innertness, Mebiol Gel enables clonal expansion of single stem cells. Application of Mebiol Gel to tissue defects in animal models revealed that Mebiol Gel enhanced tissue regeneration with activation of stem cells and prevention of inflammation. Thus, Mebiol Gel is suitable for preparation of cells for transplantation and is useful for direct application to promote regeneration of damaged tissues in vivo.

REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer.

The overexpression of reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in human prostatic and testicular cancer cells. The aim of this study is to examine the potential of REIC/Dkk-3 as a therapeutic target against breast cancer. First, the in vitro apoptotic effect of Ad-REIC treatment was investigated in breast cancer cell lines and the adenovirus-mediated overexpression of REIC/Dkk-3 was thus found to lead to apoptotic cell death in a c-Jun-NH(2)-kinase (JNK) phosphorylaion-dependent manner. Moreover, an in vivo apoptotic effect and MCF/Wt tumor growth inhibition were observed in the mouse model after intratumoral Ad-REIC injection. As multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer, the in vitro effects of Ad-REIC treatment were investigated in terms of the sensitivity of multidrug-resistant MCF7/ADR cells to doxorubicin and of the P-glycoprotein expression. Ad-REIC treatment in MCF7/ADR cells also downregulated P-glycoprotein expresssion through JNK activation, and sensitized its drug resistance against doxorubicin. Therefore, not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using Ad-REIC. We suggest that REIC/Dkk-3 is a novel target for breast cancer treatment and that Ad-REIC might be an attractive agent against drug-resistant cancer in combination with conventional antineoplastic agents.

Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model.

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

Novel collagen sponge reinforced with polyglycolic acid fiber produces robust, normal hair in murine hair reconstitution model.

The hair reconstitution assay is a useful system for studying cell-cell and epithelial-mesenchymal interaction. The current method consists of transplantation of both epidermal and dermal cells, using a silicone chamber placed on an athymic nude mouse. However, because of leakage and tilting of the grafted cells, the rate and area of hair growth vary depending on the chamber. We modified this method by using a collagen sponge as a scaffold and compared two types of collagen sponges, each having different tensile strengths. A conventional collagen sponge disturbed normal hair follicle formation; in contrast, a collagen sponge containing polyglycolic acid (PGA) fiber supported proper restructuring of skin and hair follicles. These data suggested the usefulness of PGA fiber-containing collagen sponges for hair reconstitution in research and clinical applications.

Neuronal interactions are higher in the cortex than thalamus in the somatosensory pathway.

Previous studies have shown significant correlated discharges (noise correlation) and synergistic information coding among adjacent cortical neurons. In order to investigate whether such interactions are present at an earlier stage of sensory processing, we compared noise correlation and synergistic information transmission in the ventral posterolateral nucleus (VPLn) of thalamus and primary somatosensory cortex (SI) of anesthetized rats. A hind paw was stimulated electrically and responses of several neighboring neurons were recorded simultaneously with a tetrode. Analyses indicated that noise correlation in the SI was about four times higher than in the VPLn, and, interestingly, it was significantly reduced following sensory stimulation in both regions. Spike count distributions of individual VPLn units contained higher amounts of information about the delivery of external stimulation compared with those of SI units. When simultaneously recorded units were considered together, transmission of information was more interactive (synergistic or redundant) among SI than VPLn units. On average, information transmission was independent in the VPLn, but synergistic in the SI. The difference in synergistic information coding was largely attributable to different levels of noise correlation and their modulation by external sensory stimulation. These results indicate that neuronal interactions are relatively low at the thalamic level, but much enhanced at the cortical level along the somatosensory pathway. The enhanced neuronal interactions in the cortex may reflect the role of cortex in extracting higher features of sensory stimuli.

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k(1)), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k(1) value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k(1) value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k(1) values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.

Reactivation of liver-specific gene expression in an immortalized human hepatocyte cell line by introduction of the human HNF4alpha2 gene.

An immortalized human hepatocyte cell line (OUMS-29) was established from fetal liver by transfection with the SV-40 large T antigen gene that has certain liver-specific functions such as albumin production and enzyme activities of CYP1A1, 1A2, and 2E1. To make OUMS-29 cells express other liver-specific functions, the human hepatocyte nuclear factor 4alpha2 (HNF4alpha2) gene was introduced into the cells, because this gene was found to be markedly down-regulated. The transduced HNF4alpha2 was overexpressed in the nuclei of the transfected cells, and its DNA-binding activity was also detected. The liver-specific genes such as apolipoprotein AI, CII, CIII, blood coagulation factor X, alpha1-antitrypsin, and HNF1alpha were up-regulated. Thus, this cell line is expected to be a useful tool for studying the differentiated human hepatocyte functions.

Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin.

A novel mouse cDNA named hornerin was isolated by RNA differential display applied to developing mouse skin. Hornerin, which has 2,496 amino acids, comprises EF-hand domains at the N terminus followed by a spacer sequence and a large repetitive domain, indicating that hornerin is a novel member of the "fused gene"-type cornified envelope precursor protein family. The repetitive domain of hornerin was found to be rich in glycine, serine, and glutamine. Hornerin was expressed in the tongue, esophagus, forestomach, and skin among the adult mouse tissues examined, all of them cornifying stratified epithelium. In the embryonic mouse skin, hornerin mRNA was first detected on gestational day 15.5 in the epidermis coincidentally with the formation of a granular layer. In accordance with this, hornerin was detected in the granular and cornified layers of the mature epidermis. In the granular cells of the epidermis, the hornerin protein was detected in keratohyalin granules together with profilaggrin. Furthermore, Western blot analysis of the mouse skin showed that the hornerin protein was cleaved during the process of epidermal differentiation, indicating possible posttranslational proteolytic processing as is observed in profilaggrin. Differentiation of primary mouse epidermal keratinocytes with 0.12 mm Ca(2+) resulted in the induction of hornerin. These results indicate that hornerin is structurally as well as functionally most similar to profilaggrin among the family members and possibly plays pleiotropic roles, including a role in cornification.

Functional abrogation of p53 is required for T-Ag induced proliferation in cardiomyocytes.

Targeted expression of the SV40 large T-antigen oncoprotein (T-Ag) induces cardiomyocyte proliferation in the atria and ventricles of transgenic mice. Previous studies have identified the p53 tumor suppressor, p107 (a homologue of the retinoblastoma tumor suppressor), and p193 (a novel BH3 only proapoptosis protein) as prominent TAg binding proteins in cardiomyocyte cell lines derived from these transgenic mice. To further explore the significance of these protein-protein interactions in the regulation of cardiomyocyte proliferation, a transgene comprising the human atrial natriuretic factor (ANF) promoter and sequences encoding a mutant T-Ag lacking the p53 binding domain was generated. Repeated micro-injection of this DNA gave rise to genetically mosaic animals with minimal transgene content, suggesting that widespread cardiac expression of mutant T-Ag was deleterious. This notion was supported by the observation that the transgene was selectively lost from the cardiac myocytes (but not the cardiac fibroblasts) in the mosaic animals. Crosses between the mosaic mice and animals expressing a cardiac restricted dominant negative p53 resulted in transgene transmission with ensuing overt cardiac tumorigenesis. Transfection of the mutant T-Ag in embryonic stem (ES) cell-derived cardiomyocytes resulted in wide-spread cell death with characteristics typical of apoptosis. Co-transfection with a dominant negative p53 transgene rescued mutant TAg-induced cell death in the ES-derived cardiomyocyte cultures, resulting in a marked proliferative response similar to that seen in vivo with the rescued transgenic mouse study. These results indicate that T-Ag expression in the absence of p53 functional abrogation results in cardiomyocyte death.

PKCtheta II, a new isoform of protein kinase C specifically expressed in the seminiferous tubules of mouse testis.

Protein kinase C (PKC) theta, a Ca(2+)-independent isoform of PKC, has been known to be expressed in skeletal muscle and T cells. In the present study, we isolated and characterized a smaller transcript expressed in the mouse testis, the cDNA of which is referred hereafter as PKCthetaII and the original PKCtheta as PKCthetaI. The cDNA clone of PKCthetaII has 2184 base pairs and 464 amino acids in the possible open reading frame, consisting of the 5' unique sequence of 20 amino acids and the PKCthetaI sequence of 444 amino acids. Genomic DNA analysis revealed that transcription of PKCthetaII is initiated from the PKCthetaII-specific exon, which is located between exons 7 and 8 of the PKCtheta gene, indicating that alternative splicing is the mechanism by which PKCthetaII is generated. PKCthetaII is expressed exclusively in the testis in an age-dependent manner with sexual maturation. In situ hybridization and reverse transcription-polymerase chain reaction of microdissected tissues clearly demonstrated that PKCthetaII is expressed in the seminiferous tubules of the mouse testis. Consistent with its molecular structure lacking the C1 regulatory domain, PKCthetaII is constitutively active as determined by an in vitro kinase assay, being independent of PKC activators, e.g. phosphatidylserine and phorbol ester. PKCthetaII may play a crucial role in spermatogenesis or some related function of the testis.

Helicobacter pylori induces pepsinogen secretion by rat gastric cells in culture via a cAMP signal pathway.

Infection with Helicobater pylori (H. pylori) is associated with various stomach diseases such as chronic gastritis, peptic ulcer, and gastric carcinoma. In order to investigate the mechanisms of enhanced production of pepsinogen by H. pylori in cultured rat gastric cells that have the potential to produce pepsinogen, secretion and synthesis of pepsinogen in the cells exposed to H. pylori extract were determined by measuring the hydrolysis of hemoglobin. Various drugs were used to study the mechanisms of effects of H. pylori on the cells. Exposure of the gastric cells to H. pylori extract caused a significant increase in pepsinogen secretion into the culture medium within 30-180 min in a dose-dependent manner, accompanied by a significant increase in pepsinogen synthesis in the gastric cells after 60 min of incubation. Heat treatment of the H. pylori sonicate at 100 degrees C for 10 min completely abolished the stimulatory effect of H. pylori on pepsinogen secretion. 2',3'-Dideoxyadenosine (50 microM), a specific adenylate cyclase inhibitor, abolished the effect of H. pylori-induced pepsinogen secretion. Puromycin (10 microg/ml), a protein synthesis inhibitor, and nicorandil (0.1 mM), a specific intracellular calcium antagonist, reduced the H. pylori-induced pepsinogen secretion by 37% (p<0.01) and 25% (p<0.05), respectively. On the other hand, actinomycin D (1 microg/ml), an RNA synthesis inhibitor, did not affect the H. pylori-induced pepsinogen secretion. Consequently, dibutyryl cAMP potentially stimulated the pepsinogen secretion from gastric epithelial cells in a dose-dependent manner. H. pylori induces pepsinogen secretion and synthesis by gastric epithelial cells through an increase in the intracellular cAMP and mobilization of the intracellular calcium. In addition, H. pylori affects pepsinogen synthesis at the translational level.

Screening for genes involved in tissue invasion based on placenta formation and cancer cell lines with low and high metastatic potential.

During the formation of placenta, trophoblast cells vigorously invade maternal uterine tissues, sharing many features with the invasion of cancers. We applied RNA differential display to placenta tissues from 8.5 to 17.5 days post-coitus (dpc) ICR mice, and isolated 188 cDNA fragments expressed differentially. Among the 25 known cDNA fragments thus far analyzed, six cDNAs have been reported to be relevant to tumor invasion and/or metastasis. Furthermore, 11 of 20 unknown cDNAs isolated showed differential expression between the pairs of cancer cell lines with low and high metastatic potential, indicating potential usefulness of the present two-step approach.

Suppression of hair follicle development inhibits induction of sonic hedgehog, patched, and patched-2 in hair germs in mice.

Embryonic induction of hair follicles is a fascinating model of localized morphogenesis from a simple homogeneous epithelial cell sheet. Accumulating evidence indicates that Sonic hedgehog (Shh) signaling plays a central role in hair follicle formation. We quantitated the expression levels of Shh and its receptor genes, Patched (Ptc) and Patched-2 (Ptch2), in two distinct experimental systems in which the development of hair follicles was suppressed. Shh, Ptc, and Ptch2 were induced about six- to tenfold in normal embryonic hair germs in vivo as well as in developing skin tissue maintained in organ culture. This induction was almost completely inhibited both in the developing skin tissue of ICR mice cultured with 30ng/ml epidermal growth factor and in embryos of Tabby mutant mice (a model of hypohidrotic ectodermal dysplasia) at 14.5-15.5 days postcoitus. Expression of Shh, Ptc and Ptch2 was induced in the Tabby embryos at 16.5 days postcoitus, indicating that Shh signaling may be involved in the formation not only of the well-studied guard hair but also of the awl hair. The potential of the two biological systems for studying molecular mechanisms in hair follicle formation, particularly at an early phase including Shh signaling, is discussed.

A carcinoembryonic antigen family cDNA from mouse placenta encoding a protein with a rare domain composition.

The carcinoembryonic antigen (CEA) family comprises many members with pleiotropic functions. Among normal tissues, placenta is characterized by the abundant production of many different kinds of CEA-related proteins, which are apparently important for the maintenance of pregnancy. Using RNA differential display applied to mouse placentae at different gestational days, we have isolated a novel CEA-related cDNA designated as Ceacam11. Ceacam11 cDNA encodes 303 amino acids with a possible signal peptide and two immunoglobulin variable region-like domains. This domain composition is observed only in mouse Cea10/Ceacam10 among the many CEA family members thus far reported. The transcript of Ceacam11 was first detected in the placenta at 12.5 days post-coitus (dpc) and the level increased progressively towards 17.5 dpc. The expression of Ceacam11 appears to be reciprocal to that of Ceacam10, since the Ceacam10 transcripts were detected at 8.5 and 10.5 dpc, but not at 12.5 to 17.5 dpc. In situ hybridization showed that the expression of Ceacam11 was localized to the spongiotrophoblast of the placenta. Except for in the placenta, Ceacam11 transcripts were not detected in any adult tissues examined, including brain, lung, glandular stomach, small intestine, colon, liver, kidney and testis.

Cathepsin-6, a novel cysteine proteinase showing homology with and co-localized expression with cathepsin J/P in the labyrinthine layer of mouse placenta.

A novel cysteine proteinase, cathepsin-6, was isolated by RNA differential display from mouse placenta. Cathepsin-6 showed the highest homology with cathepsin J (same as P) and L. The structural features including the catalytic triad of the C1 proteinase family were well conserved in cathepsin-6. The expression of cathepsin-6 and cathepsin J/P was restricted in labyrinthine trophoblasts of the placenta.

Cholesterol sulfate, an activator of protein kinase C mediating squamous cell differentiation: a review.

Activity of protein kinase C (PKC) depends on the interaction with polar head-groups of two membrane lipids, i.e., phosphatidylserine and diacylglycerol. We demonstrated that cholesterol metabolism is directly involved in activation of the eta isoform of protein kinase C (PKCeta), which is predominantly expressed in epithelial tissues in close association with epithelial differentiation. We found that PKCeta was activated by cholesterol sulfate (CS), a metabolite of cholesterol formed during squamous cell differentiation. In the presence of CS, phorbol ester only weakly enhanced the activity of PKCeta. CS also activated PKCeta, PKCdelta and PKCepsilon in a dose-dependent manner, when assayed using purified recombinant materials. However, when partially purified materials were used from overexpressing normal human keratinocytes, only PKCeta was activated by CS among the isoforms examined. All the existing lines of evidence, mainly supplied from our laboratory, suggest that CS is involved in a signal transduction of squamous cell differentiation and thereby modifying squamous cell carcinogenesis.

A tetratricopeptide repeat-containing protein gene, tpis, whose expression is induced with differentiation of spermatogenic cells.

The tetratricopeptide repeat (TPR) is a degenerate 34-amino-acid sequence which forms scaffolds to mediate protein-protein interactions. We have isolated a cDNA named tpis from mouse embryonic skin and found that the deduced 529-amino-acid sequence contained 5 TPRs. In addition to skin, the transcript of tpis was detected in tissues with stratified squamous epithelium, e.g., tongue, esophagus, and forestomach. tpis was most strongly expressed in testis among adult tissues examined. The transcript of tpis from testis was longer, encoding 372 additional amino acid residues at the 5'-side with 3 more TPRs. In situ hybridization revealed specific expression of tpis at a distinct differentiation stage of spermatogenic cells, indicating involvement of tpis in spermatogenesis. Chromosomal localization of the tpis gene was determined as 18.10 cM of chromosome 15.