Shigeru Sugaya

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays.

Abstract Kita, K., Sugaya, S., Zhai, L., Wu, Y. P., Wano, C., Chigira, S., Nomura, J., Takahashi, S., Ichinose, M. and Suzuki, N. Involvement of LEU13 in Interferon-Induced Refractoriness of Human RSa Cells to Cell Killing by X Rays. Radiat. Res. 160, 302–308 (2003). Culture of human cells with human interferon α and β (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IFr and F-IFr cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UV^r-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14–18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CΘ, Src, Bax and μ-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated μ-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, μ-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells. Cell Death and Differentiation (2000) 7, 531–537

Suppression of okadaic acid-induced apoptosis by overexpression of calpastatin in human UVr-1 cells

Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UVr‐1 fibroblasts by transfection of its cDNA. The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5‐fluorouracil, mitomycin C and methotrexate. After treatment with OA, a typical apoptotic DNA ladder was observed in control vector‐transfected cells but not in calpastatin‐transfected cells. This indicates that OA‐induced apoptosis was suppressed by overexpression of calpastatin. Further immunoblot analysis showed that the OA‐induced hyperphosphorylation of c‐Jun was inhibited in calpastatin‐transfected cells. This might be involved in the resistance to OA‐induced cell death in calpastatin‐overproducing cells.

Downregulation of microRNA-431 by human interferon-β inhibits viability of medulloblastoma and glioblastoma cells via upregulation of SOCS6

miRNAs are small non-coding RNAs that inhibit gene expression by cleaving or hindering the translation of target mRNAs. In this study, we focused on miR-431, which mediated inhibition of cell viability by human interferon-β (HuIFN-β). We aimed to demonstrate an antineoplastic effect of HuIFN-β via miR-431 expression against medulloblastoma and glioblastoma, because HuIFN-β is frequently used in adjuvant therapy of these tumors. Addition of HuIFN-β to medulloblastoma and glioblastoma cells reduced viability, significantly decreased miR-431 expression, upregulated expression of SOCS6 (putative miR-431 target genes) and inhibited Janus kinase (JAK) 1 and signal transducer and activator of transcription (STAT) 2. The mitogen-activated protein kinase (MAPK) pathway, but not the phosphoinositide 3-kinase (PI3K)-Akt pathway, was downregulated in medulloblastoma cells, whereas the PI3K-Akt pathway, but not the MAPK pathway, was downregulated in glioblastoma cells. Addition of HuIFN-β and transient transfection with miR-431 to medulloblastoma and glioblastoma cells did not reduce viability, downregulated expression of SOCS6, and concomitantly activated the JAK1 and STAT2. We propose that, in medulloblastoma and glioblastoma cells, HuIFN-β decreases miR-431 expression and upregulates SOCS6 expression, and consequently inhibit cell proliferation by suppressing the JAK-STAT signaling pathway.

Inhibition of cell viability by human IFN-β is mediated by microRNA-431

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression by cleaving or hindering the translation of target mRNAs. We used microarray-based comparative transcriptome analysis to identify changes in miRNA expression and function between a human cell line, RSa, which is highly sensitive to HuIFN-β-mediated inhibition of cell viability, and its variant, the F-IFr cell line, which is relatively resistant to the cytokine. miR-431 expression was significantly higher in RSa cells compared with F-IFr cells. The addition of HuIFN-β to RSa cultures reduced cell viability, down-regulated expression of IGF1R and IRS2 (putative miR-431 target genes), and inhibited the PI3K-Akt and MAPK pathways. The survival of F-IFr cells was not reduced by HuIFN-β, but transient transfection with miR-431 precursors significantly decreased viability and concomitantly down-regulated IGF1R and IRS2 expression. In addition, the MAPK pathway, but not the PI3K-Akt pathway, was suppressed in F-IFr cells. Based on these results, we propose that, in RSa cells, HuIFN-β-induced miR-431 expression may down-regulate IGF1R and IRS2 expression, and consequently inhibit cell proliferation by suppressing the MAPK pathway.

EFFECTS OF 3-D CLINO-ROTATION ON GENE EXPRESSION IN HUMAN FIBROBLAST CELLS

Continuous variation in the direction of the gravity vector leads to various cellular responses including modulation of gene expression. Complementary DNA (cDNA) array analyses are available to observe the variation of gene expression under different conditions. In this study, expression levels of 588 representative genes were compared using the Atlas™ human cDNA expression array in human fibroblast cells with and without 3‐dimensional (3‐D) clinostat. Five upregulated and 8 downregulated genes were detected. Among these genes, upregulation of XRCC1, and downregulation of ERB‐B2 and p21Cip1/Waf1 were confirmed by RT‐PCR. These results suggested that the gene expression levels ofXRCC1, ERB‐B2 and p21Cip1/Waf1 were modulated by vector‐averaged microgravity induced by 3‐D clinostat in human fibroblast cells. Our findings may be a basis for the biological study of 3‐D culture systems.

Extracellular release of annexin II from pancreatic cancer cells and resistance to anticancer drug-induced apoptosis by supplementation of recombinant annexin II.

Objectives Extracellular microenvironment plays crucial roles in the development of cancers and chemoresistance. Pancreatic carcinoma is resistant to almost all chemotherapeutic agents. In this study, we identified annexin II in the medium from pancreatic cancer cells as a protein released into the extracellular environment. Methods Medium from 5-hour cultures of various cancer cells was collected. Proteins in the medium were detected by molecular mass analysis and immunoblotting. Anticancer drug sensitivity of cells preincubated with or without recombinant annexin II (rANX II) was measured using crystal violet assay and colony survival assay. Apoptosis-related molecules were analyzed by immunoblotting. Results Recombinant ANX II supplementation in the medium confers resistance to anticancer drugs, including cisplatin, 5-fluorouracil, and gemcitabine, in MiaPaCa-2 and AsPC-1 cells. In MiaPaCa-2 cells, rANX II supplementation resulted in suppression of caspase-3 activation associated with increased Bcl-2/Bax ratios. Suppression of cisplatin-induced cell death by rANX II supplementation was canceled by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase signal pathways. Conclusions The current study is the first report to demonstrate that supplementation of rANX II in the medium increased resistance to anticancer drugs in pancreatic cancer cells. Recombinant ANX II exerts cell death–suppressive function by antagonizing cisplatin-induced apoptosis.

Annexin II, a Novel HSP27‐interacted Protein, is Involved in Resistance to UVC‐induced Cell Death in Human APr‐1 Cells

Heat shock protein 27 (HSP27) is implicated in diverse biologic functions as a molecular chaperone. We found that HSP27 is involved in the protection of human cells against UVC lethality. To elucidate the molecular mechanisms underlying UVC resistance, we searched for HSP27‐interacted proteins related to resistance in UVC‐resistant human cells, APr‐1. Three candidates for HSP27‐interacted proteins were found from cell lysates using an affinity column coupled with GST‐fused HSP27 protein. Interaction between HSP27 and two candidates, annexin II and HSP70, was confirmed by immunoprecipitation analysis. After UVC irradiation, the amount of the complex of HSP27 and annexin II decreased in the postnuclear fraction, while it increased in the nuclear fraction. Cells transfected with annexin II–siRNA were more susceptible to UVC lethality. These results suggest that annexin II is a novel HSP27‐interacted protein which is involved in UVC resistance in human cells, at least those tested here.

Anti-proliferative and apoptosis-inducible activity of labdane and abietane diterpenoids from the pulp of Torreya nucifera in HeLa cells.

Two abietane and one labdane type diterpenoids were isolated from the methanol extracts of Torreya nucifera pulp and investigated for their ability to inhibit the growth of human cancer cells. Among the three compounds, the labdane compound kayadiol was found to have the most effective inhibitory effect against a wide variety of human cancer cells. Using the MTT assay, kayadiol was determined to have an IC50 (50% inhibition concentration) of 30 µM in HeLa cells, and also to exhibit anti-proliferative effects towards six other human cancer cell lines, with IC50 values of 30-50 µM. Kayadiol treatment of HeLa cells resulted in a dose-dependent generation of apoptotic events, including DNA laddering (≤100 µM). Moreover, kayadiol-treated HeLa cells showed activation of caspases-3 and -9, as well as an increase in the depolarization of mitochondrial membrane potential and the Bax/Bcl-2 ratio. These results indicate that a mitochondria-related apoptotic pathway is involved in the kayadiol-induced death of HeLa cells. Kayadiol is therefore a promising novel anti-proliferative agent and merits further investigation.

Down-regulation of Molecular Chaperone 78-kd Glucose-regulated Protein/immunoglobulin-binding Protein Expression Involved in Enhancement of Human Rs Cell Mutability

Objectives: Enhancement of cell mutability via extracellular materials of cancer cells is a crucial event leading to the development of cancers; however, the activation process of mutability is still not well understood. In this study, to identify the regulatory mechanism of cell mutability, we investigated mutability modulated in response to human pancreatic cancer cell-conditioned medium and identified the candidates for cellular molecules involved in the mutability modulation. Methods: To test the mutation-modulating effects of the conditioned medium, human RS cells were cultured with medium derived by culturing human pancreatic cancer KP-4 cells, followed by irradiation with UV (mainly 254 nm in wavelength). Mutations were detected by phenotypic ouabain resistance and genetic base substitution of K-ras codon 12. Messenger RNA differential display was used to identify genes that were differentially expressed between conditioned medium-treated and mock-treated RSa cells. The influence of 78-kd glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) expression on mutability was assessed by the down-regulation of GRP78/BiP using antisense oligonucleotides or antisense complementary DNA. Results: The UV-induced mutagenicity in RS cells was strengthened by preculture with KP-4 cell-conditioned medium. Messenger RNA differential display revealed that GRP78/BiP expression was suppressed in RS cells after treatment of the conditioned medium. Furthermore, the level of UV-induced mutations was elevated significantly in GRP78/BiP down-regulated cells. Conclusions: Culture of human RS cells with pancreatic cancer KP-4 cell-conditioned medium resulted in increased UV mutagenicity, possibly via the down-regulation of GRP78/BiP.Abbreviations: EMEM - Eagle minimal essential medium, DMEM/F12 - Dulbecco modified Eagle medium/nutrient mixture F12, OuaR - ouabain resistant, UV - far-ultraviolet ray, GAPDH - glyceraldehyde 3-phosphate dehydrogenase, PNA - peptide nucleic acid, GRP78 - 78-kd glucose-regulated protein, BiP - immunoglobulin-binding protein, ER - endoplasmic reticulum