Namiko Aiba

Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA.

Wilson’s disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver. WND (ATP7B) gene, which encodes a putative copper transporting P-type ATPase, is defective in the patients. To investigate the in vivo function of WND protein as well as its intracellular localization, WNDcDNA was introduced to the Long-Evans Cinnamon rat, known as a rodent model for Wilson’s disease, by recombinant adenovirus-mediated gene delivery. An immunofluorescent study and a subcellular fractionation study revealed the transgene expression in liver and its localization to the Golgi apparatus. Moreover, since the synthesis of holoceruloplasmin is disturbed in the Long-Evans Cinnamon rat, the plasma level of holoceruloplasmin, oxidase-active and copper-bound form, was examined to evaluate the function of WND protein with respect to the copper transport. Consequently, the appearance of holoceruloplasmin in plasma was confirmed by Western blot analysis and plasma measurements for the oxidase activity and the copper content. These findings indicate that introduced WND protein may function in the copper transport coupled with the synthesis of ceruloplasmin and that the Golgi apparatus is the likely site for WND protein to manifest its function.

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

Wilsons 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 Wilsons 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.

Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells.

The liver is believed to contain stem cells that can differentiate into either hepatocytes or biliary epithelial cells. In the present study, we established a nonhepatocytic epithelial cell line from the normal livers of adult rats. The established cells, designated HSL cells, were immunoreactive against alpha-fetoprotein, but neither albumin nor cytokeratin 19. To demonstrate the differentiation potential of HSL cells in vitro, the cells were cocultured with hepatic stellate cells as a mixture or separately using insert wells. Consequently, although coculture with hepatic stellate cells rendered HSL cells able to produce albumin, the mixed coculture system mimicking the hepatic environment elicited this phenomenon more effectively than the separated coculture system. In conclusion, HSL cells have immature properties and the potential to differentiate into mature cells. Not only the extracellular matrices but also soluble factors, which are produced by hepatic stellate cells, induce this maturation, demonstrating the importance of the hepatic environment for hepatocyte differentiation.

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.

Nm23-H1 regulates contact inhibition of locomotion, which is affected by ephrin-B1

Summary Contact inhibition of locomotion (CIL) is the process by which cells stop the continual migration in the same direction after collision with another cell. Highly invasive malignant cells exhibit diminished CIL when they contact stromal cells, which allows invasion of the tissue by tumors. We show that Nm23-H1 is essential for the suppression of Rac1 through inactivation of Tiam1 at the sites of cell–cell contact, which plays a pivotal role in CIL. U87MG cells show CIL when they contact normal glia. In spheroid confrontation assays U87MG cells showed only limited invasion of the glial population, but reduction of Nm23-H1 expression in U87MG cells abrogated CIL resulting in invasion. In U87MG cells, Nm23-H1 is translocated to the sites of contact with glia through association with &agr;-catenin and N-cadherin. Mutants of Nm23-H1, which lacked the binding ability with Tiam1, or &agr;-catenin did not restore CIL. Moreover, the expression of ephrin-B1 in tumor cells disrupted CIL and promoted invasion. As one mechanism, ephrin-B1 inhibits the association of Nm23-H1 with Tiam1, which contributes for activation of Rac1. These results indicate a novel function of Nm23-H1 to control CIL, and its negative regulation by ephrin-B1.

Monitoring the Expression Profiles of Doxorubicin-Resistant K562 Human Leukemia Cells by Serial Analysis of Gene Expression

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software.We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells.There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin β, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin ε 1, N-myristoyltransferase 1, hemoglobin α 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin β1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicinresistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis.

Gene Expression Profiling of Human Erythroid Progenitors by Micro-Serial analysis of Gene Expression

We compared the expression profiles of highly purified human CD34+ cells and erythroid progenitor cells by micro-serial analysis of gene expression (microSAGE). Human CD34+ cells were purified from granulocyte colony-stimulating factor-mobilized blood stem cells, and erythroid progenitors were obtained by cultivating these cells in the presence of stem cell factor, interleukin 3, and erythropoietin. Our 10,202 SAGE tags allowed us to identify 1354 different transcripts appearing more than once. Erythroid progenitor cells showed increased expression of LRBA, EEF1A1, HSPCA, PILRB, RANBP1, NACA, and SMURF. Overexpression of HSPCA was confirmed by real-time polymerase chain reaction analysis.MicroSAGE revealed an unexpected preferential expression of several genes in erythroid progenitor cells in addition to the known functional genes, including hemoglobins. Our results provide reference data for future studies of gene expression in various hematopoietic disorders, including myelodysplastic syndrome and leukemia.

Agr2 Mediates Paracrine Effects on Stromal Fibroblasts That Promote Invasion by Gastric Signet-Ring Carcinoma Cells

Agr2 is a disulfide isomerase residing in the endoplasmic reticulum (ER), which physiologically regulates protein folding and mediates resistance to ER stress. Agr2 is overexpressed in adenocarcinomas of various organs, where it participates in neoplastic transformation and metastasis, therefore acts as a pro-oncogenic protein. Besides its normal localization in the ER, Agr2 is also found in the serum and urine of cancer patients, although the physiological significance of extracellular Agr2 is poorly understood. In this study, we demonstrated that extracellular Agr2 can activate stromal fibroblasts and promote fibroblast-associated cancer invasion in gastric signet-ring cell carcinoma (SRCC), where Agr2 is highly expressed. Agr2 secreted from SRCC cells was incorporated by the surrounding gastric fibroblasts and promoted invasion by these cells. In turn, activated fibroblasts coordinated the invasive behavior of fibroblasts and cancer cells. Our findings suggested that Agr2 drives progression of gastric SRCC by exerting paracrine effects on fibroblasts in the tumor microenvironment, acting also to increase the growth and resistance of SRCC cells to oxidative and hypoxic stress as cell autonomous effects.

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.

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.