Cancer-associated fibroblasts induce cancer cell apoptosis that regulates invasion mode of tumours.

In the process of cancer spreading, different modes of invasion exist. One is expansive invasion, in which a group of cancer cells gradually expands along with cancer cell proliferation. Invasion of cancer cells is also modified by their interaction with stromal cells including cancer-associated fibroblasts (CAFs). Cancer cells co-invade with CAFs, and invasion by CAFs frequently precede invasion by cancer cells, which indicates CAF-led cancer cell invasion. Here, we show that CAFs induce apoptosis in gastric cancer cells, which prevented expansive invasion by cancer cells and instead facilitated CAF-led invasion. Death receptor 4 and activation of caspase-8 in cancer cells mediated cancer cell apoptosis induced by CAFs, which was dependent on contact between cancer cells and CAFs. Apoptotic cancer cells in turn released apoptotic vesicles and stimulated invasion of CAFs. Accordingly, cancer cells followed the migrating CAFs. Treatment with a caspase inhibitor, ZVAD, or forced expression of a death domain fragment in cancer cells prevented cancer cell apoptosis induced by CAFs and increased expansive invasion by cancer cells in extracellular gel invasion assays, while the rate of cancer cell invasion led by CAFs was decreased. Death domain-fragment expression also prevented intramural invasion by gastric cancer cells in the stomach. Because CAF-led invasion is characterized by the movement of individual cancer cells away from the tumour, adequate cancer cell apoptosis may promote cancer dissemination.

Analysis of the NPHP genes in two Japanese patients with suspected sporadic juvenile or adolescent nephronophthisis.

BACKGROUND: Mutations in 3 genes (NPHP1, NPHP3 and NPHP4) have been identified in patients with juvenile or adolescent nephronophthisis (NPHP) without extrarenal involvement, mainly in patients from western countries. In this study, we analyzed mutations in the NPHP genes of 2 Japanese patients with suspected sporadic juvenile or adolescent NPHP without extrarenal involvement. METHODS: A renal biopsy was performed in the 2 patients. Genomic DNA was prepared from peripheral blood mononuclear cells of the patients and their family members. The above NPHP genes were examined by deletion analysis or direct automated sequencing of polymerase chain reaction-amplified DNA products. RESULTS: Histological findings in the patients were compatible with those of NPHP. In 1 patient, we identified a novel deletion mutation including about half of exons of the NPHP1 gene. In another patient, there was no mutation in the NPHP genes examined. CONCLUSIONS: We found a novel NPHP1 deletion in 1 patient. To our knowledge, this is the second Japanese NPHP case in which genetic diagnosis was made.

LPP inhibits collective cell migration during lung cancer dissemination.

Lipoma preferred partner (LPP) is a LIM domain protein, which has multiple functions as an actin-binding protein and a transcriptional coactivator, and it has been suggested that LPP has some roles in cell migration or invasion, however, its role in cancer cells remains to be elucidated. Here, we showed that LPP degraded N-cadherin in lung cancer, PC14PE6 cells via regulating the expression of matrix metalloproteinase 15 (MMP-15), and loss-of-LPP increases collective cell migration (CCM) and dissemination consequently. Knockdown of LPP and its functional partner, Etv5, markedly restores the full-length N-cadherin and increases cell-cell adhesion. We investigated the common target of LPP and Etv5, and found that MMP-15 is transcribed as their direct transcriptional target. Furthermore, MMP-15 could directly digest the N-cadherin extracellular domain. LPP knockdown in PC14PE6 cells increases N-cadherin-dependent CCM in the three-dimensional collagen gel invasion assays, and promoted the dissemination of cancer cells when they were orthotopically implanted in nude mice. Immunohistochemistry of lung adenocarcinoma specimens revealed the heterogeneity of LPP intensity and complementary expression of LPP and N-cadherin in the primary tumors. These findings suggest that loss-of-LPP, Etv5 or MMP-15 can be a prognostic marker of increasing malignancy.

Tks5 activation in mesothelial cells creates invasion front of peritoneal carcinomatosis.

Scirrhous gastric cancer is frequently associated with peritoneal dissemination, and the interaction of cancer cells with peritoneal mesothelial cells (PMCs) is crucial for the establishment of the metastasis in the peritoneum. Although cells derived from PMCs are detected within tumors of peritoneal carcinomatosis, how PMCs are incorporated into tumor architecture is not understood. The present study shows that PMCs create the invasion front of peritoneal carcinomatosis, which depends on activation of Tks5 in PMCs. In peritoneal tumor implants, PMCs represent majority of cells located at the invasive edge of the cancer tissue. Exogenously implanted PMCs and host PMCs aggressively invade into abdominal wall upon the peritoneal inoculation of cancer cells, and PMCs locate ahead of cancer cells in the direction of invasion. Tks5, a substrate of Src kinase, is predominantly expressed in the PMCs of cancer tissue, and promotes the invasion of PMCs and cancer cells. Expression and activation of Tks5 was induced in PMCs following their exposure to gastric cancer cells, and increased Tks5 expression was detected in PMCs located at the invasion front. Reduced Tks5 expression in PMCs blocked PMC invasion, which in turn prevents cancer cell invasion both in vitro and in vivo. The peritoneal dissemination of gastric cancer was significantly increased by mixing cancer cells and PMCs, and was suppressed by knockdown of Tks5 in PMCs. These results suggest that cancer-activated PMCs create invasion front by guiding cancer cells. Signaling leading to Tks5 activation in PMCs may be a suitable therapeutic target for prevention of peritoneal carcinomatosis.

Asporin activates coordinated invasion of scirrhous gastric cancer and cancer-associated fibroblasts.

Scirrhous gastric cancer, which has the worst prognosis among the various types of gastric cancer, is highly invasive and associated with abundant stromal fibroblasts. Although cancer-associated fibroblasts (CAFs) have been proposed to generate a tumor-supportive extracellular matrix that promotes the expansion of this type of cancer, the molecular mechanisms by which CAFs assist cancer cells are not yet fully understood. Here, we show for the first time that Asporin, a small leucine-rich proteoglycan (SLRP), is predominantly expressed in CAFs, and has essential roles in promoting co-invasion of CAFs and cancer cells. CAFs of scirrhous gastric cancer possess high potential for invasion, and invasion by CAFs frequently proceeded invasion by cancer cells, both in vitro and in vivo. Expression of Asporin was induced in fibroblasts by exposure to gastric cancer cells. Asporin secreted from CAFs activates Rac1 via an interaction with CD44 and promotes invasion by CAFs themselves. Moreover, Asporin promoted invasion by neighboring cancer cells, via paracrine effects mediated by activation of the CD44-Rac1 pathway. These results suggest that Asporin is a unique SLRP that promotes progression of scirrhous gastric cancer and is required for coordinated invasion by CAFs and cancer cells. Therefore, Asporin may represent a new therapeutic target molecule for the development of drugs aimed at manipulating the cancer microenvironment.

Biliary excretion of copper in LEC rat after introduction of copper transporting P-type ATPase, ATP7B.

Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of hepatic copper that results from reduced biliary copper excretion and disturbed incorporation of copper into ceruloplasmin. The ATP7B gene, responsible for the disease, encodes a copper transporting P-type ATPase. We previously demonstrated the involvement of ATP7B in hepatic copper secretion into plasma after the introduction of ATP7B into the Long-Evans Cinnamon (LEC) rat, a rodent model of Wilson's disease. In this study we found the increased copper contents of the hepatic lysosomal fractions and bile in the LEC rats after ATP7B introduction, indicating the participation of ATP7B in the biliary excretory pathway for copper.

Evaluation of transcatheter arterial embolization with epirubicin-lipiodol emulsion for hepatocellular carcinoma.

A total of 18 patients with hepatocellular carcinoma (HCC) were treated by transcatheter arterial embolization (TAE) with a 4'-epi-doxorubicin (EDX)-lipiodol emulsion. Infusion of the EDX-lipiodol emulsion (EDX-L) via the hepatic artery was followed by the injection of gelatin sponge in 12 cases. The response and survival of these 12 patients following EDX-L treatment were compared with those of 42 subjects treated with a doxorubicin-lipiodol emulsion (DX-L) and those of 23 patients treated by TAE with gelatin sponge (GS) only. In the group treated with EDX-L, nine cases were AFP-positive in sera and four showed a decrease in serum AFP values to less than 10% of the pretreatment level. Seven cases showed a partial response, and nine cases showed no change in the size of the tumor. In the group treated with EDX-L, nine cases are alive, and the oldest has survived for more than 431 days since the treatment. The half-year survival value was 57%, and the 1-year survival value was 49%. These values did not differ significantly from those calculated for the group treated with DX-L. The 1-year survival value determined for patients treated with a lipiodol emulsion (EDX-L or DX-L) followed by GS was 65%, and the 2-year survival value was 39%. These results rates are significantly better than those obtained in patients treated with GS only (1-year survival, 39%; 2-year survival, 13%.

The process of calcification during development of the rat tracheal cartilage characterized by distribution of alkaline phosphatase activity and immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans.

The rat tracheal cartilage was shown to calcify during development. The process of calcification was characterized in terms of distribution of alkaline phosphatase (ALP) activity and alterations to immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans during the development of the tracheal cartilage, in comparison with calcification of the epiphyseal growth plate cartilage. ALP activity was not identified in the tracheal cartilage in the course of calcification, which therefore differed from that in the growth plate. The tracheal cartilage matrix was not resorbed or invaded by type I collagen during calcification. This suggests that no osteogenesis is involved in calcification of the cartilage. Immunoreactivity for type II collagen became weaker in the central region of the tracheal cartilage during development. No net loss of proteoglycans was identified with Alcian blue staining after calcification of the tracheal cartilage. Immunoreactivity for chondroitin 4-sulphate increased in the calcified tracheal cartilage, while reactivity for chondroitin 6-sulphate was weaker in the calcified area than in the surrounding uncalcified region of the tracheal cartilage. The alteration of the extracellular matrices during development may be involved in the calcification of the rat tracheal cartilage.

A nonsense mutation (R220X) in the alpha-galactosidase A gene causes typical Fabry disease in both genders.

BACKGROUND: Fabry disease is an X-linked recessive disorder resulting from a deficiency of lysosomal alpha-galactosidase A (alpha-Gal A). Chronic renal failure is an important cause of death in patients with Fabry disease. We report on patients with Fabry disease (a hemizygous male and his mother) due to a nonsense mutation (R220X) in the alpha-Gal A gene. METHODS: The proband, a 41-year-old man, and his 71-year-old mother presented with renal and cardiac manifestations of Fabry disease. Histological examination and molecular analysis of the alpha-Gal A gene were performed. RESULTS: Typical histological findings of Fabry disease were observed in a renal biopsy specimen from the proband and in renal and myocardial necropsy specimens from the mother. Sequencing of a full-length alpha-Gal A cDNA from the proband indicated a C-T transition at codon 220, resulting in substitution of the predictable termination for arginine (R220X). Examination of genomic alpha-Gal A DNA revealed that the proband was a hemizygote and the mother was a heterozygous carrier for the mutation. CONCLUSION: This is the first detailed report of family members with Fabry disease due to a nonsense mutation (R220X) in the alpha-Gal A gene. Our study indicates that this mutation causes the typical disease in both genders.

Simultaneous detection of hepatitis B, C, and G viral genomes by multiplex PCR method.

We established a multiplex polymerase chain reaction (PCR) method for simultaneous detection of hepatitis B, C, and G viral genomes. The levels of concordance with the data obtained by conventional single PCR method were 100% for single infection, 98 to 100% for double infections, and 92% for triple infections. This method is not only suited to rapid, large-scale epidemiological screening and clinical diagnosis of those virus infections occurring alone or in combination, but is also time- and cost-effective.

Observation of an energetic-particle-driven instability in the wall-stabilized high-beta plasmas in the JT-60U tokamak.

We have observed a bursting mode in the high-beta plasmas above the ideal beta limit without a conducting wall. The mode frequency is chirping down as the mode amplitude increases, and its initial value is close to the precession frequency of the trapped energetic particle from the perpendicular neutral beams. The mode structure is radially extended with a peak around the q = 2 surface. This mode can finally trigger the resistive wall mode (RWM) despite enough plasma rotation for RWM stabilization. It is concluded that the mode is driven by trapped energetic particles. The mode is attributed to the interaction between the trapped energetic particles and a marginally stable mode in the wall-stabilized high-beta_{N} region.

Identification of a low plasma-rotation threshold for stabilization of the resistive-wall mode.

The plasma rotation necessary for stabilization of resistive-wall modes (RWMs) is investigated by controlling the toroidal plasma rotation with external momentum input by injection of tangential neutral beams. The observed threshold is 0.3% of the Alfvén velocity and much smaller than the previous experimental results obtained with magnetic braking. This low critical rotation has a very weak beta dependence as the ideal wall limit is approached. These results indicate that for large plasmas such as in future fusion reactors with low rotation, the requirement of the additional feedback control system for stabilizing RWM is much reduced.

Susceptibility of Oka varicella vaccine strain to antiviral drugs.

Susceptibility of Oka varicella vaccine virus to antiherpetic drugs was determined by the effective dose for 50% plaque reduction (ED50) using cell-free virus preparation. ED50 values were 3.02 microM for acyclovir, 3.72 microM for vidarabine, 0.0035 microM for sorivudine, and 4.67 microM for penciclovir. Oka varicella vaccine virus was as susceptible to these drugs as wild-type viruses. Sensitivity of thymidine kinase (TK)-deficient virus to penciclovir and of some DNA polymerase (DPase) mutants to sorivudine suggested that these drugs might be used for the treatment of vaccine recipients, even if Oka varicella vaccine became acyclovir-resistant by mutations in the TK or DPase genes, respectively. This result encourages the wider use of Oka varicella vaccine even for immunocompromised hosts because of its attenuation and susceptibility to chemotherapeutic drugs.

Hypomethylation of the c-myc oncogene in liver cirrhosis and chronic hepatitis.

In order to investigate how chronic liver diseases, including liver cirrhosis and chronic hepatitis, are associate with hepatocarcinogenesis in terms of gene alteration, the methylation states of the c-myc and c-Ki-ras genes were examined in 34 liver tissues from patients with chronic liver disease without hepatocellular carcinoma (HCC), 34 non-tumor liver tissues from patients with HCC, 18 HCC tissues and 31 control liver tissues. The methylation states were analyzed by the Southern hybridization method using the restriction endonuclease isoschizomers MspI and HpaII. The CCGG sites at the second exon of the c-myc gene tended to be more extensively hypomethylated both in chronic liver disease and in non-tumor tissues than in control livers. Whereas the CCGG sites of the c-Ki-ras, and the third exon of the c-myc gene tended to be hypomethylated only in HCC tissues in comparison with other tissue groups. These results suggest that chronic liver disease may be situated between normal liver and HCC based on the state of DNA methylation and associated with the development of HCC through hypomethylation of the c-myc and/or c-Ki-ras gene.

Production of 5-formyluracil from thymine in an in vitro active oxygen-generating system.

Thymine was placed in a model active oxygen-generating system containing ferrous sulfate, EDTA, and ascorbic acid. The oxidative products of thymine were separated by Sephadex LH-20 chromatography and a reversed phase high-performance liquid chromatography (HPLC) into at least five major components. One of them had a UV spectrum characteristic of 5-formyluracil and mass spectrometric analysis of this material also indicated this material to be 5-formyluracil.

Enhanced ATPase activity in liver cell nuclei induced by administration of mitomycin C to rats.

Intraperitoneal administration of mitomycin C (40 micrograms/100 g body weight) to male Wistar rats increased the ATPase activity in hypotonic extracts of liver cell nuclei for 4 days after injection. Partially purified ATPase, obtained by the DEAE-cellulose column chromatography of these extracts, showed a 14 times higher specific activity than that found in normal rat liver nuclei. The enzymatic activity was strongly enhanced by the addition of polynucleotides, especially poly A and poly I, to the assay mixture, but was inhibited by GTP, a chelating agent, heparin and thiol-group inhibitors. Quercetin and oligomycin were less effective, and ouabain showed no inhibitory effect. Mg2+ ions were essential, but neither Ca2+, Na+ nor K+ ions were required for the manifestation of the activity. These characteristic properties of the enzyme are similar to those of a nucleoside triphosphatase found in the nuclear matrix and envelope, suggesting that some energy-providing mechanisms involved in the repair processes of DNA damage or cellular injury are induced by mitomycin C administration.

Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay.

A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells.

Emergence and selection of HBV variants in an anti-HBe positive patient persistently infected with quasi-species.

BACKGROUND/AIMS: Infection with the hepatitis B e antigen (HBeAg) negative variant of hepatitis B virus (HBV) causes chronic liver disease characterised by occasional acute exacerbations. This virus exhibits a high prevalence of mutations in the core region. Our aim was to study the changes in the pre-core/core region of the virus in relation to exacerbations of the disease. METHODS/RESULTS: We performed direct sequencing on DNA amplified from 7 sequential sera taken over a 5-year period from a hepatitis B surface antigen (HBsAg) and anti-HBe positive Greek patient infected with the HBeAg negative variant. The patient had chronic hepatitis with several acute exacerbation episodes and underwent interferon therapy twice. We found significant variability in the core region at different time points. To determine whether these variants were present in the initial serum sample and subsequently selected under immune pressure or whether they arose de novo during the course of the disease, we cloned the pre-core/core region from 4 sera before and after episodes of acute exacerbation. Fifteen clones from each time point were sequenced. Fourteen nucleotide mutations in the pre-core/core region were recorded, 7 (50%) of which led to amino-acid substitutions. All the amino-acid changes occurred at recognised B- and CD4+ epitopes. The cloning results indicate the presence of quasi-species in all the samples investigated. Some of the variants present as a minor population in the first sample appear to have been selected and become dominant in subsequent sera. However, the emergence of novel variants, not present at a detectable level in earlier samples, during the course of the disease, was also established. The quasi-species nature of HBV only became apparent after the cloning experiments and was not obvious from the direct sequencing results. CONCLUSIONS: New dominant variants occurring during the course of the disease arose either by the selection of pre-existing mutants that were not detected by direct sequencing or by mutation of existing strains. All changes were located within B- and CD4+ epitopes. The continuous production and selection of variants may enable virus to evade elimination by the immune system, resulting in persistent infection.