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ABSTRACT: Gankyrin (also called p28 or PSMD10) is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It consists of 7 ankyrin repeats and interacts with multiple proteins including Rb, Cdk4, MDM2 and NF-κB. To assess the oncogenic activity in vivo, we produced transgenic mice that overexpress gankyrin specifically in the hepatocytes. Unexpectedly, 5 of 7 F2 transgenic mice overexpressing hepatitis B virus X protein (HBX) promoter-driven gankyrin, and one of 3 founder mice overexpressing serum amyloid P component (SAP) promoter-driven gankyrin developed hepatic vascular neoplasms (hemangioma/hemangiosarcomas) whereas none of the wild-type mice did. Endothelial overgrowth was more frequent in the livers of diethylnitrosamine-treated transgenic mice than wild-type mice. Mouse hepatoma Hepa1-6 cells overexpressing gankyrin formed tumors with more vascularity than parental Hepa1-6 cells in the transplanted mouse skin. We found that gankyrin binds to and sequester factor inhibiting hypoxia-inducible factor-1 (FIH-1), which results in decreased interaction between FIH-1 and hypoxia-inducible factor-1α (HIF-1α) and increased activity of HIF-1 to promote VEGF production. The effects of gankyrin were more prominent under 3% O(2) than 1% or 20% O(2) conditions. Thus, the present study clarified, at least partly, mechanisms of vascular tumorigenesis, and suggests that gankyrin might play a physiological role in hypoxic responses besides its roles as an oncoprotein.
Biochemical and Biophysical Research Communications 01/2013; · 2.48 Impact Factor
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ABSTRACT: BACKGROUND: There are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5[prime] flanking region of the mouse cirp gene. RESULTS: By transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5[prime] flanking region octanucleotide 5[prime]-TCCCCGCC-3[prime] is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32[degree sign]C relative to 37[degree sign]C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32[degree sign]C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32[degree sign]C than 37[degree sign]C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5[prime] flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5[prime] flanking region abolished the effects of Sp1 on the reporter gene expression both at 37 [degree sign]C and 32[degree sign]C. CONCLUSIONS: Cold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.
BMC Biotechnology 10/2012; 12(1):72. · 2.35 Impact Factor
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Tomoko Masuda,
Katsuhiko Itoh,
Hiroaki Higashitsuji,
Hisako Higashitsuji,
Noa Nakazawa,
Toshiharu Sakurai, Yu Liu,
Hiromu Tokuchi,
Takanori Fujita,
Yan Zhao,
Hiroyuki Nishiyama,
Takashi Tanaka,
Manabu Fukumoto,
Masahito Ikawa,
Masaru Okabe,
Jun Fujita
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ABSTRACT: Cold-inducible RNA-binding protein (Cirp) was the first cold-shock protein identified in mammals. It is structurally quite
different from bacterial cold-shock proteins and is induced in response to mild, but not severe, hypothermia. To clarify the
physiological function of Cirp in vivo, we produced cirp-knockout mice. They showed neither gross abnormality nor defect in fertility, but the number of undifferentiated spermatogonia
was significantly reduced and the recovery of spermatogenesis was delayed after treatment with a cytotoxic agent, busulfan.
Cirp accelerated cell-cycle progression from G0 to G1 as well as from G1 to S phase in cultured mouse embryonic fibroblasts.
Cirp directly bound to dual-specificity tyrosine-phosphorylation–regulated kinase 1B (Dyrk1b, also called Mirk) and inhibited
its binding to p27, resulting in decreased phosphorylation and destabilization of p27. Cirp did not affect binding of Dyrk1b
to cyclin D1 but inhibited phosphorylation of cyclin D1 by Dyrk1b, resulting in cyclin D1 stabilization. In the spermatogonial
cell line GC-1spg, suppression of Cirp expression increased the protein level of p27, decreased that of cyclin D1, and decreased
the growth rate, which depended on Dyrk1b. Consistent changes in the protein levels of p27 and cyclin D1 as well as the percentage
of cells in G0 phase were observed in undifferentiated spermatogonia of cirp-knockout mice. In undifferentiated spermatogonia of wild-type mice, Cirp and Dyrk1b colocalized in the nucleus. Thus, our
study demonstrates that Cirp functions to fine-tune the proliferation of undifferentiated spermatogonia by interacting with
Dyrk1b.
Proceedings of the National Academy of Sciences 07/2012; 109(27):10885-10890. · 9.68 Impact Factor
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ABSTRACT: HSCO (hepatoma subtracted-cDNA library clone one, also called ETHE1) was originally identified by its frequent overexpression in hepatocellular carcinomas. HSCO inhibits function of NF-kappaB by binding to RelA and accelerating its export from the nucleus. We show here that HSCO exhibits anti-apoptotic activity in cells exposed to DNA-damaging agents by suppressing transcriptional activity of p53. Induction of pro-apoptotic genes, Noxa, Perp, PIG3, and Bax were suppressed in cells over-expressing HSCO. By increasing ubiquitylation and degradation of p53, HSCO reduces p53 protein levels. HSCO specifically associates with histone deacetylase 1 (HDAC1) independently of Mdm2 and facilitates deacetylation of p53 at Lys-373/382 by HDAC1. The metallo-beta-lactamase family consensus sequence in HSCO is important for its effect on p53 deacetylation. Co-immunoprecipitation and immunofluorescence studies suggested that HSCO, HDAC1, and p53 form a complex in the nucleus. Thus, HSCO is a cofactor that increases the deacetylase activity of HDAC1 toward p53, leading to suppression of apoptosis. Treatment of hepatocellular carcinomas that retain wild-type p53 and overexpress HSCO with anti-HSCO agents might re-establish the p53 response and revert chemoresistance.
Journal of Biological Chemistry 06/2007; 282(18):13716-25. · 4.77 Impact Factor
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ABSTRACT: Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.
Biochimica et Biophysica Acta 04/2006; 1763(3):290-5. · 4.66 Impact Factor
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ABSTRACT: Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we examined the effects of low temperature (32 degrees C) on cells exposed to a variety of stress in vitro. We found that 32 degrees C suppressed induction of apoptosis by cytotoxic stimuli such as adriamycin, etoposide, thapsigargin, NaCl, H(2)O(2), and anti-Fas antibody. In adriamycin-treated BALB/3T3 cells, the down-shift in temperature from 37 degrees C to 32 degrees C increased the Bcl-xL protein level and decreased the mRNA level of Puma and mitochondrial translocation of Bax, suppressing caspase-9-mediated apoptosis. Furthermore, the protein level and stability of p53 were decreased, and its nuclear export was increased concomitant with Mdm2 mRNA upregulation. The low temperature effect was not observed in p53(-/-)/Mdm2(-/-) mouse embryonic fibroblasts, suggesting that the effect is mediated by suppression of the p53 pathway. In contrast, while thapsigargin-induced apoptosis was suppressed by the low temperature, no effect on the p53 protein level was observed. Furthermore, the survival rate of p53(-/-)/Mdm2(-/-) cells exposed to thapsigargin was increased when cultured at 32 degrees C compared with 37 degrees C. In conclusion, mild hypothermia protects cells from a variety of stress by p53-dependent and p53-independent mechanisms.
Experimental Cell Research 11/2005; 309(2):264-72. · 3.58 Impact Factor
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ABSTRACT: Ubiquitin-dependent proteolysis mediates selective destruction of various cell cycle regulators, transcription factors and tumor suppressors. Gankyrin, a seven ankyrin-repeat protein, was originally identified as an oncoprotein commonly overexpressed in hepatocellular carcinomas and independently as a protein associated with the 19S regulatory complex of the 26S proteasome. Gankyrin also binds to CDK4 and the tumor suppressor RB, and accelerates phosphorylation and proteasomal degradation of RB. Recently, we have shown that gankyrin has an anti-apoptotic activity in cells exposed to DNA-damaging agents. Gankyrin binds to MDM2, a major E3 ubiquitin ligase for p53, and increases ubiquitylation and degradation of p53. Gankyrin increases activities of CDK4 and MDM2, and facilitates targeting of polyubiquitylated proteins to the 26S proteasome. Furthermore, inhibition of gankyrin induces apoptosis in cancer cells. Therefore, gankyrin is a promising target for potential anticancer therapeutic agents.
Cell cycle (Georgetown, Tex.) 11/2005; 4(10):1335-7. · 5.36 Impact Factor
