Hiroyoshi Iseki

Isolation of a novel mouse gene, mSVS-1/SUSD2, reversing tumorigenic phenotypes of cancer cells in vitro

We report isolation of a novel tumor‐reversing gene, tentatively named SVS‐1, encoding a protein of 820 amino acids with localization on the plasma membrane as a type I transmembrane protein. The gene was found among those downregulated in the activated oncogene‐v‐K‐ras‐transformed NIH3T3 cells, Ki3T3, with tumorigenic phenotype. SVS‐1 protein harbors several functional domains inherent to adhesion molecules. Histochemical staining of mouse tissues using antibody raised against the protein showed the expression of the protein in restricted regions and cells, for example, strongly positive in apical membranes of epithelial cells in renal tubules and bronchial tubes. The protein inducibly expressed in human fibrosarcoma HT1080 cells and cervical carcinoma HeLa cells was found to be localized primarily on the plasma membrane, as stained with antibodies against FLAG tag in the N‐terminus and against the C‐terminal peptide of the protein. Expression of the protein in cells induced a variety of biological effects on cancer cells: detachment from the substratum and aggregation of cells and growth inhibition in HeLa cells, but no inhibition in non‐tumorigenic mouse NIH3T3 cells. Inhibition of clonogenicity, anchorage‐independent growth, migration and invasion through Matrigel was also observed. Taken together these results suggest that the SVS‐1 gene is a possible tumor‐reversing gene. (Cancer Sci 2007; 98: 900–908)

Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes

BackgroundSenescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated.ResultsTo identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis.ConclusionsOur data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains.

Parvovirus Nonstructural Proteins Induce an Epigenetic Modification through Histone Acetylation in Host Genes and Revert Tumor Malignancy to Benignancy

ABSTRACT Several malignant tumor cells become apoptotic and revert to the benign phenotype upon parvovirus infection. Recently, we demonstrated that the rat parvovirus RPV/UT also induces apoptosis in the rat thymic lymphoma cell line C58(NT)D. However, a minority of cells that escaped apoptosis showed properties different from the parental cells, such as resistance to apoptosis, enhanced cell adherence, and suppressed tumorigenicity. The present study was performed to determine the molecular mechanism of parvovirus-induced phenotypic modification, including oncosuppression. We demonstrated that the nonstructural (NS) proteins of RPV/UT induced apoptosis in C58(NT)D cells and suppressed tumor growth in vivo. Interestingly, NS proteins induced the expression of ciliary neurotrophic factor receptor alpha, which is up-regulated in revertant cell clones, and enhanced histone acetylation of its gene. These results indicate that parvoviral NS regulate host gene expression through histone acetylation, suggesting a possible mechanism of oncosuppression.

Sirt1, p53, and p38MAPK Are Crucial Regulators of Detrimental Phenotypes of Embryonic Stem Cells with Max Expression Ablation†‡§

c‐Myc participates in diverse cellular processes including cell cycle control, tumorigenic transformation, and reprogramming of somatic cells to induced pluripotent cells. c‐Myc is also an important regulator of self‐renewal and pluripotency of embryonic stem cells (ESCs). We recently demonstrated that loss of the Max gene, encoding the best characterized partner for all Myc family proteins, causes loss of the pluripotent state and extensive cell death in ESCs strictly in this order. However, the mechanisms and molecules that are responsible for these phenotypes remain largely obscure. Here, we show that Sirt1, p53, and p38MAPK are crucially involved in the detrimental phenotype of Max‐null ESCs. Moreover, our analyses revealed that these proteins are involved at varying levels to one another in the hierarchy of the pathway leading to cell death in Max‐null ESCs. STEM CELLS2012;30:1634–1644

Propagation of Rat Parvovirus in Thymic Lymphoma Cell Line C58(NT)D and Subsequent Appearance of a Resistant Cell Clone after Lytic Infection

ABSTRACT Rat parvovirus (RPV) is nonpathogenic in rats but causes persistent lymphocytotropic infection. We found that RPV was propagated in rat thymic lymphoma cell line C58(NT)D and induced apoptosis. Interestingly, a resistant subclone, C58(NT)D/R, from surviving cells after lytic infection had differentiated phenotypic modifications, such as increased cell adherence, resistance to apoptosis, and suppressed tumorigenicity.

Human Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) gene is transcriptionally regulated by CREB and Wnt/β‐catenin signaling

The aberrant activation of Wnt signaling is a key process in colorectal tumorigenesis. Canonical Wnt signaling controls transcription of target genes via β‐catenin and T‐cell factor/lymphoid enhancer factor family transcription factor complex. Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) is a novel member of the Armadillo family which has two Armadillo repeats as opposed to more than six repeats in the classical Armadillo family members. Here we examine cis‐regulatory elements and trans‐acting factors involved in the transcriptional regulation of the ALEX1 gene. Site‐directed mutations of a cyclic AMP response element (CRE) and an E‐box impaired the basal activity of human ALEX1 promoter in colorectal and pancreatic cancer cell lines. Moreover, overexpression of CRE‐binding protein (CREB) increased the ALEX1 promoter activity in these cell lines, whereas knockdown of CREB expression decreased the expression level of ALEX1 mRNA. Interestingly, luciferase reporter analysis and quantitative real‐time RT‐PCR demonstrated that the ALEX1 promoter was up‐regulated in a CRE‐dependent manner by continuous activation of Wnt/β‐catenin signaling induced by a glycogen synthase kinase‐3 inhibitor and overexpression of β‐catenin. These results indicate that the CRE and E‐box sites are essential cis‐regulatory elements for ALEX1 promoter activity, and ALEX1 expression is regulated by CREB and Wntk/β‐catenin signaling. (Cancer Sci 2010)

von Willebrand factor type D domain mutant of SVS‐1/SUSD2, vWDm, induces apoptosis in HeLa cells

SVS‐1/SUSD2 is a novel gene, which inhibits growth and reverses tumorigenic phenotypes of cancer cells in vitro. Here we report identification of a mutant of SVS‐1, designated SVS‐1‐vWDm, in which conserved amino acids GLLG at positions 591–594 in von Willebrand factor type D (vWD) domain are replaced by AAAA. As observed by laser confocal microscope, intracellular localization of the mutant protein has changed such that both the N‐terminus and the C‐terminus of SVS‐1‐vWDm were localized in the inner surface of the plasma membrane, whereas the N‐terminus of SVS‐1 was localized in the outer surface of the plasma membrane. Additionally, SVS‐1‐vWDm was processed much less efficiently and in a slightly different manner. In in vitro studies, adenovirus‐mediated transduction of the SVS‐1‐vWDmgene induced growth suppression of HeLa cells in a dose‐dependent manner, as the wild‐type gene and inhibition of anchorage‐independent growth. Of great interest is the finding that the mutant protein, vWDm, but not the wild‐type one induced apoptosis, as observed by nuclear as well as DNA fragmentation. Activation of caspase‐3 and ‐9, but not caspase‐8 or ‐12, was also demonstrated in vWDm‐expressing cells. An inhibition of Akt phosphorylation, a major survival signaling component, also occurred in vWDm‐expressing HeLa cells. Together these data suggest that vWDm induces apoptosis by inactivation of survival signaling component Akt and activation of caspase cascade (mitochondrial pathway) in HeLa cells. We propose SVS‐1‐vWDmas an alternative gene for use in developing new therapeutic strategies for the treatment of cancer. (Cancer Sci 2007; 98: 909–915)

ALEX1 suppresses colony formation ability of human colorectal carcinoma cell lines

Arm protein lost in epithelial cancers, on chromosome X (ALEX; also known as armadillo repeat containing, X‐linked [ARMCX]) is a novel subgroup within the armadillo (ARM) family, which has several ARM repeat domains. The biological function of classical ARM family members such as β‐catenin is well understood, but that of the ALEX/ARMCX family members is largely unknown. Here we evaluate the effects of ALEX1 overexpression on in vitro colony formation ability and expression of ALEX1 mRNA in human colorectal tumor. Overexpression of ALEX1 suppressed the anchorage‐dependent and ‐independent colony formation of human colorectal carcinoma cell lines by the study of stable clones of HCT116 cells expressing ALEX1 protein. Bisulfite genomic sequencing revealed that the promoter region of ALEX1 gene was highly methylated in both HCT116 and SW480 cells in comparison with PANC‐1 and MCF‐7 cells, which express endogenous ALEX1 mRNA, indicating the capability of promoter methylation to silence ALEX1 gene in HCT116 and SW480 cells. Our current findings suggest that overexpression of ALEX1 play a negative role in human colorectal tumorigenesis. (Cancer Sci 2012; 103: 1267–1271)

Combined Overexpression of JARID2, PRDM14, ESRRB, and SALL4A Dramatically Improves Efficiency and Kinetics of Reprogramming to Induced Pluripotent Stem Cells

Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N‐terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2‐associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2‐dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2‐associated protein network contributes to overcoming reprogramming barriers. Stem Cells 2016;34:322–333

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1em1 (cre) Utr strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1em1 (cre) Utr) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1em1 (cre) Utr is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.