Exosomal miR-493 suppresses MAD2L1 and induces chemoresistance to intraperitoneal paclitaxel therapy in gastric cancer patients with peritoneal metastasis.

Intraperitoneal (IP) chemotherapy with paclitaxel (PTX) for gastric cancer (GC) with peritoneal metastasis (PM) is considered a promising treatment approach, however, there are no useful biomarkers to predict the efficacy of IP therapy. We examined the association between intra-peritoneal exosomes, particularly exosomal micro-RNAs (exo-miRNAs), and IP-chemo sensitivity. MKN45 cells that were cultured with intra-peritoneal exosomes from patients who did not respond to IP therapy with PTX (IPnon-respond group) exhibited resistance to PTX compared with exosomes from responding patients (IPrespond group) (p = 0.002). A comprehensive search for exo-miRNAs indicated that miR-493 was significantly up-regulated in exosomes from the IPnon-respond group compared with those collected from the IPrespond group. The expression of miR-493 in PTX-resistant MKN45 cells (MKN45PTX-res) was higher compared with that in MKN45. In addition, MKN45PTX-res cells exhibited lower MAD2L1 gene and protein expression compared with MKN45. Finally, miR-493 enhancement by transfection of miR-493 mimics significantly down-regulated MAD2L1 expression in MKN45 cells and reduced PTX sensitivity. Our results suggest that intra-peritoneal exo-miR-493 is involved in chemoresistance to PTX by downregulating MAD2L1 in GC with PM. Exo-miR-493 may be a biomarker for chemoresistance and prognosis of GC patients with PM and may also be a promising therapeutic target.

Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-ß-cyclodextrin (MßCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

Linderapyrone analogue LPD-01 as a cancer treatment agent by targeting importin7.

The Wnt/ß-catenin signaling pathway plays important roles in several cancer cells, including cell proliferation and development. We previously succeeded in synthesizing a small molecule compound inhibiting the Wnt/ß-catenin signaling pathway, named LPD-01 (1), and 1 inhibited the growth of human colorectal cancer (HT-29) cells. In this study, we revealed that 1 inhibits the growth of HT-29 cells stronger than that of another human colorectal cancer (SW480) cells. Therefore, we have attempted to identify the target proteins of 1 in HT-29 cells. Firstly, we investigated the effect on the expression levels of the Wnt/ß-catenin signaling pathway-related proteins. As a result, 1 inhibited the expression of target proteins of Wnt/ß-catenin signaling pathway (c-Myc and Survivin) and their genes, whereas the amount of transcriptional co-activator (ß-catenin) was not decreased, suggesting that 1 inhibited the Wnt/ß-catenin signaling pathway without affecting ß-catenin. Next, we investigated the target proteins of 1 using magnetic FG beads. Chemical pull-down assay combined with mass spectrometry suggested that 1 directly binds to importin7. As expected, 1 inhibited the nuclear translocation of importin7 cargoes such as Smad2 and Smad3 in TGF-ß-stimulated HT-29 cells. In addition, the knockdown of importin7 by siRNA reduced the expression of target genes of Wnt/ß-catenin signaling pathway. These results suggest that importin7 is one of the target proteins of 1 for inhibition of the Wnt/ß-catenin signaling pathway.

Eph signal inhibition potentiates the growth-inhibitory effects of PLK1 inhibition toward cancer cells.

Anti-mitotic drugs are clinically used as anti-cancer treatments. Polo-like kinase 1 (PLK1) is a promising target against cancer cell division due to its importance in the whole process of mitosis, and thus PLK1-targeting agents have been developed in the last few decades. Clinical trial studies show that several PLK1 inhibitors are generally well-tolerated. However, the response rates are limited; therefore, it is needed to improve the efficacy of those drugs. Here, we show that NVP-BHG712, an erythropoietin-producing human hepatocellular (Eph) signaling inhibitor, potentiates the growth-inhibitory effects of the PLK1 inhibitors BI2536 and BI6727 in cancer cells. This combination treatment strongly suppresses cancer spheroid formation. Moreover, the combination drastically arrests cells at mitosis by continuous activation of the spindle assembly checkpoint (SAC), thereby inducing apoptosis. SAC activation caused by the combination of NVP-BHG712 and BI2536 is due to the inhibition of centrosome maturation and separation. Although the inactivation level of the PLK1 kinase is comparable between BI2536 treatment alone and combination treatment, the combination treatment strongly inactivates MAPK signaling in mitosis. Since inhibition of MAPK signaling potentiates the efficacy of BI2536 treatment, inactivation of PLK1 kinase and MAPK signaling contributes to the strong inhibition of centrosome separation. These results suggest that Eph signal inhibition potentiates the effect of PLK1 inhibition, leading to strong mitotic arrest via SAC activation and the subsequent reduction of cancer cell survival. The combination of PLK1 inhibition and Eph signal inhibition will provide a new effective strategy for targeting cancer cell division.

Highly genomic instability of super-polyploid strains of Saccharomyces cerevisiae.

Polyploid (2n, 3n, and 4n) genomes are known to be unstable in Saccharomyces cerevisiae. Here, we attempted construction of super-polypoid strains (defined as having higher ploidy than tetraploidy) up to 32n by using the matα2-PBT method that we newly developed and investigated their genomic stability. It is known that cell size increases as ploidy increases up to tetraploid. However, unexpectedly, there was no change in the average cell size of the super-polyploid strains compared with tetraploid or pentaploid strains. Smaller sized cells were observed at a rather higher frequency in super-polyploid cell populations compared with those of diploid, triploid and tetraploid strains, suggesting that ploidy reduction in super-polyploid strains occurs quickly at a relatively high frequency. Assuming that ploidy reduction occurs through chromosome loss (or non-disjunction) during mitotic growth, we also estimated the frequency of chromosome loss (or non-disjunction) in various polyploid strains. Our results indicated that the frequency of chromosome loss (or non-disjunction) is drastically increased (10-2-10-3/cells plated) in super-polyploid strains compared with that (10-4-10-5/cells plated) of conventional polyploid (2n-4n) strains. This is the first attempt of construction of super-polyploid strains and investigation of their genomic stability in S. cerevisiae. We believe that the matα2-PBT method will be an invaluable tool for investigating a variety of interesting issues regarding polyploidy and their genomic characterization in eukaryotes.

The C-terminal tail of Rad17, iVERGE, binds the 9‒1‒1 complex independently of AAA+ ATPase domains to provide another clamp-loader interface.

The ATR pathway plays a crucial role in maintaining genome integrity as the major DNA damage checkpoint. It also attracts attention as a therapeutic target in cancer treatment. The Rad17-RFC2-5 complex loads the Rad9-Hus1-Rad1 (9-1-1) DNA clamp complex onto damaged chromatin to activate the ATR pathway. We previously reported that phosphorylation of a polyanionic C-terminal tail of human Rad17, iVERGE, is essential for the interaction between Rad17 and the 9-1-1 complex. However, the molecular mechanism has remained unclear. Here, we show that iVERGE directly interacts with the Hus1 subunit of the 9-1-1 complex through Rad17-S667 phosphorylation independently of the AAA+ ATPase domains. An exogenous iVERGE peptide interacted with the 9-1-1 complex in vivo. The binding conformation of the iVERGE peptide was analyzed by de novo modeling with docking simulation, simulated annealing-molecular dynamics simulation, and the fragment molecular orbital method. The in silico analyses predicted the association of the iVERGE peptide with the hydrophobic and basic patches on the Hus1 protein, and the corresponding Hus1 mutants were deficient in the interaction with the iVERGE peptide in vivo. The iVERGE peptide occupied the same position as the C-terminus of Saccharomyces cerevisiae RAD24 on MEC3. The interaction energy calculation suggested that the Rad17 KYxxL motif and the iVERGE peptide are the primary and secondary interaction surfaces between the Rad17-RFC2-5 and 9-1-1 complexes. Our data reveal a novel molecular interface, iVERGE, between the Rad17-RFC2-5 and 9-1-1 complexes in vertebrates and implicate that Rad17 utilizes two distinct molecular interfaces to regulate the 9-1-1 complex.

Phosphotyrosine proteomics in cells synchronized at monopolar cytokinesis reveals EphA2 as functioning in cytokinesis.

Cytokinesis is the final step of the cell division in which cellular components are separated into two daughter cells. This process is regulated through the phosphorylation of different classes of proteins by serine/threonine (Ser/Thr) kinases such as Aurora B and Polo-like kinase 1 (PLK1). Conversely, the role of phosphorylation at tyrosine residues during cytokinesis has not been studied in detail yet. In this study, we performed a phosphotyrosine proteomic analysis of cells undergoing monopolar cytokinesis synchronized by using the Eg5 inhibitor (+)-S-trityl-l-cysteine (STLC) and the CDK1 inhibitor RO-3306. Phosphotyrosine proteomics gave 362 tyrosine-phosphorylated peptides. Western blot analysis of proteins revealed tyrosine phosphorylation in mitogen-activated protein kinase 14 (MAPK14), vimentin, ephrin type-A receptor 2 (EphA2), and myelin protein zero-like protein 1 (MPZL1) during monopolar cytokinesis. Additionally, we demonstrated that EphA2, a protein with unknown function during cytokinesis, is involved in cytokinesis. EphA2 knockdown accelerated epithelial cell transforming 2 (Ect2) knockdown-induced multinucleation, suggesting that EphA2 plays a role in cytokinesis in a particular situation. The list also included many proteins previously reported to play roles during cytokinesis. These results evidence that the identified phosphopeptides facilitate the identification of novel tyrosine phosphorylation signaling involved in regulating cytokinesis.

v-Src delocalizes Aurora B by suppressing Aurora B kinase activity during monopolar cytokinesis.

c-Src tyrosine kinase plays roles in a wide range of signaling events and its increased activity is frequently observed in a variety of epithelial and non-epithelial cancers. v-Src, an oncogene first identified in the Rous sarcoma virus, is an oncogenic version of c-Src and has constitutively active tyrosine kinase activity. We previously showed that v-Src induces Aurora B delocalization, resulting in cytokinesis failure and binucleated cell formation. In the present study, we explored the mechanism underlying v-Src-induced Aurora B delocalization. Treatment with the Eg5 inhibitor (+)-S-trityl-L-cysteine (STLC) arrested cells in a prometaphase-like state with a monopolar spindle; upon further inhibition of cyclin-dependent kinase (CDK1) by RO-3306, cells underwent monopolar cytokinesis with bleb-like protrusions. Aurora B was localized to the protruding furrow region or the polarized plasma membrane 30 min after RO-3306 addition, whereas inducible v-Src expression caused Aurora B delocalization in cells undergoing monopolar cytokinesis. Delocalization was similarly observed in monopolar cytokinesis induced by inhibiting Mps1, instead of CDK1, in the STLC-arrested mitotic cells. Importantly, western blotting analysis and in vitro kinase assay revealed that v-Src decreased the levels of Aurora B autophosphorylation and its kinase activity. Furthermore, like v-Src, treatment with the Aurora B inhibitor ZM447439 also caused Aurora B delocalization at concentrations that partially inhibited Aurora B autophosphorylation. Given that phosphorylation of Aurora B by v-Src was not observed, these results suggest that v-Src causes Aurora B delocalization by indirectly suppressing Aurora B kinase activity.

Distinct effects of heat shock temperatures on mitotic progression by influencing the spindle assembly checkpoint.

Heat shock is a physiological and environmental stress that leads to the denaturation and inactivation of cellular proteins and is used in hyperthermia cancer therapy. Previously, we revealed that mild heat shock (42 °C) delays the mitotic progression by activating the spindle assembly checkpoint (SAC). However, it is unclear whether SAC activation is maintained at higher temperatures than 42 °C. Here, we demonstrated that a high temperature of 44 °C just before mitotic entry led to a prolonged mitotic delay in the early phase, which was shortened by the SAC inhibitor, AZ3146, indicating SAC activation. Interestingly, mitotic slippage was observed at 44 °C after a prolonged delay but not at 42 °C heat shock. Furthermore, the multinuclear cells were generated by mitotic slippage in 44 °C-treated cells. Immunofluorescence analysis revealed that heat shock at 44 °C reduces the kinetochore localization of MAD2, which is essential for mitotic checkpoint activation, in nocodazole-arrested mitotic cells. These results indicate that 44 °C heat shock causes SAC inactivation even after full activation of SAC and suggest that decreased localization of MAD2 at the kinetochore is involved in heat shock-induced mitotic slippage, resulting in multinucleation. Since mitotic slippage causes drug resistance and chromosomal instability, we propose that there may be a risk of cancer malignancy when the cells are exposed to high temperatures.

Epstein-Barr virus reactivation in peripheral B lymphocytes induces IgM-type thyrotropin receptor autoantibody production in patients with Graves' disease.

Epstein-Barr virus (EBV) is a human herpes virus that latently infects B lymphocytes. When EBV is reactivated, host B cells differentiate into plasma cells and produce IgM-dominant antibodies as well as many progeny virions. The aims of the present study were to confirm the IgM dominance of thyrotropin-receptor antibodies (TRAbs) produced by EBV reactivation and investigate the roles of TRAb-IgM in Graves' disease. Peripheral blood mononuclear cells (PBMCs) containing TRAb-producing cells were stimulated for EBV reactivation, and TRAb-IgM and TRAb-IgG were measured by ELISA. TRAb-IgM were purified and TSH-binding inhibitory activities were assessed using a radio-receptor assay. Porcine thyroid follicular epithelial cells were cultured with TRAb-IgM and/or complements to measure the intracellular levels of cAMP and the amount of LDH released. TRAb-IgM/TRAb-IgG (the MG ratio) was examined in sequential serum samples of Graves' disease and compared among groups of thyroid function. The results obtained showed that IgM-dominant TRAb production was induced by EBV reactivation. TRAb-IgM did not inhibit TSH binding to TSH receptors and did not transduce hormone-producing signals. However, it destroyed thyroid follicular epithelial cells with complements. The MG ratio was significantly higher in samples of hyperthyroidism or hypothyroidism than in those with normal function or in healthy controls. A close relationship was observed between TRAb-IgM produced by EBV reactivation and the development and exacerbation of Graves' disease. The present results provide novel insights for the development of prophylaxis and therapeutics for Graves' disease.

SH2D4A promotes centrosome maturation to support spindle microtubule formation and mitotic progression.

Mitotic progression requires the precise formation of spindle microtubules based on mature centrosomes. During the G2/M transition, centrosome maturation progresses, and associated microtubules bundle to form mitotic spindle fibers and capture the chromosomes for alignment at the cell equator. Mitotic kinases-induced phosphorylation signaling is necessary for these processes. Here, we identified SH2 domain-containing protein 4A (SH2D4A/PPP1R38) as a new mitotic regulator. SH2D4A knockdown delays mitotic progression. The time-lapse imaging analysis showed that SH2D4A specifically contributes to the alignment of chromosomes. The cold treatment assay and microtubule regrowth assay indicated that SH2D4A promotes microtubule nucleation to support kinetochore-microtubule attachment. This may be due to the centrosome maturation by SH2D4A via centrosomal recruitment of pericentriolar material (PCM) such as cep192, γ-tubulin, and PLK1. SH2D4A was found to be a negative regulator of PP1 phosphatase. Consistently, treatment with a PP1 inhibitor rescues SH2D4A-knockdown-induced phenotypes, including the microtubule nucleation and centrosomal recruitment of active PLK1. These results suggest that SH2D4A is involved in PCM recruitment to centrosomes and centrosome maturation through attenuation of PP1 phosphatases, accelerating the spindle formation and supporting mitotic progression.

Azaphilones produced by Penicillium maximae with their cell death-inducing activity on Adriamycin-treated cancer cell.

BACKGROUND: Heat shock proteins (Hsps) are overexpressed in several tumors and contribute to cell proliferation, metastasis, and anticancer drug resistance. Therefore, Hsp inhibitors have enhanced cytotoxicity as chemotherapeutic agents and may be effective with a reduced dosage for tumor therapy to avoid side effects. RESULTS: Four new azaphilones, maximazaphilones I-IV (1-4), and three known compounds (5-7) have been isolated from the airborne-derived fungus Penicillium maximae. Inhibitory effects of isolated compounds against induction of Hsp105 were evaluated by the luciferase assay system using Hsp105 promoter. In this assay, 2-4, 6, and 7 significantly inhibited hsp105 promoter activity without cytotoxicity. In addition, all isolated compounds except for 5 significantly induced the death of Adriamycin (ADR)-treated HeLa cells. Interestingly, 1-4, 6, and 7 didn't show anti-proliferative and cell death-inducing activity without ADR. CONCLUSION: This study revealed the chemical structures of maximazaphilones I-IV (1-4) and the potency of azaphilones may be useful for cancer treatment and reducing the dose of anticancer agents. In addition, one of the mechanisms of cell death-inducing activity for 2-4, 6, and 7 was suggested to be inhibitory effects of Hsp105 expression.

TYRO3 promotes chemoresistance via increased LC3 expression in pancreatic cancer.

Pancreatic cancer (PC) is an aggressive malignancy with few treatment options, and improved treatment strategies are urgently required. TYRO3, a member of the TAM receptor tyrosine kinase family, is a known oncogene; however, the relationship between TYRO3 expression and PC chemoresistance remains to be elucidated. We performed gain- and loss-of-function experiments on TYRO3 to examine whether it is involved in chemoresistance in PC cells. TYRO3 knockdown decreased cell viability and enhanced apoptosis following treatment of PC cells with gemcitabine and 5-fluorouracil (5-FU). In contrast, no such effects were observed in TYRO3-overexpressing PC cells. It is known that autophagy is associated with cancer chemoresistance. We then examined effects of TYRO3 on autophagy in PC cells. TYRO3 overexpression increased LC3 mRNA levels and induced LC3 puncta in PC cells. Inhibition of autophagy by chloroquine mitigated cell resistance to gemcitabine and 5-FU. In a xenograft mouse model, TYRO3 silencing significantly increased sensitivity of the cells to gemcitabine and 5-FU. To further investigate the involvement of autophagy in patients with PC, we immunohistochemically analyzed LC3 expression in the tissues of patients who underwent pancreatectomy and compared it with disease prognosis and TYRO3 expression. LC3 expression was negatively and positively correlated with prognosis and TYRO3 expression, respectively. Furthermore, LC3- and TYRO3-positive patients had a significantly worse prognosis among patients with PC who received chemotherapy after recurrence. These results indicated that the TYRO3-autophagy signaling pathway confers PC resistance to gemcitabine and 5-FU, and could be a novel therapeutic target to resolve PC chemoresistance.

GPX4 Regulates Tumor Cell Proliferation via Suppressing Ferroptosis and Exhibits Prognostic Significance in Gastric Cancer.

BACKGROUND/AIM: Gastric cancer (GC) is the fourth leading cause of cancer-related death worldwide. Glutathione peroxidase 4 (GPX4) is a glutathione-dependent antioxidant enzyme known to regulate ferroptosis, which is a non-apoptotic form of cell death accompanied by iron-dependent accumulation of reactive oxygen species (ROS). This study evaluated the expression and function of GPX4 in GC. MATERIALS AND METHODS: The expression of GPX4 was examined in five human GC cell lines (KATO-III, MKN-1, MKN-28, MKN-45, and MKN-74) using real-time quantitative PCR and western blotting. The role of GPX4 in GC was examined using small interference RNA and cell proliferation and ROS assays. Finally, we analyzed GPX4 expression in tumor tissues from 106 patients who underwent GC surgery using immunohistochemistry and evaluated the relationship between GPX4 levels and clinical outcomes of GC. RESULTS: GPX4 was expressed in all GC cell lines at various levels. GPX4 silencing and inhibition significantly reduced cell proliferation and increased ROS generation. Furthermore, the mRNA levels of prostaglandin-endoperoxide synthase 2, a known biomarker of ferroptosis, were increased after GPX4 silencing. GPX4 expression was found to be an independent prognostic factor for overall and disease-specific survival in GC patients. CONCLUSION: GPX4 can regulate cancer cell death via ferroptosis in GC cell lines and represents a significant risk factor for survival in patients with GC.

Rad17 Translocates to Nucleolus upon UV Irradiation through Nucleolar Localization Signal in the Central Basic Domain.

The nucleolus is a non-membranous structure in the nucleus and forms around ribosomal DNA repeats. It plays a major role in ribosomal biogenesis through the transcription of ribosomal DNA and regulates mRNA translation in response to cellular stress including DNA damage. Rad17 is one of the proteins that initiate and maintain the activation of the ATR pathway, one of the major DNA damage checkpoints. We have recently reported that the central basic domain of Rad17 contains a nuclear localization signal and that the nuclear translocation of Rad17 promotes its proteasomal degradation. Here, we show that the central basic domain contains the nucleolar localization signal as well as the nuclear localization signal. The nucleolar localization signal overlaps with the nuclear localization signal and is capable of transporting an exogenous protein into the nucleolus. Phosphomimetic mutations of the central basic domain inhibit nucleolar accumulation, suggesting that the post-translational modification sites regulate the nucleolar localization. Nucleolar accumulation of Rad17 is promoted by proteasome inhibition and UV irradiation. Our data show the nucleolar localization of Rad17 and suggest a possible role of Rad17 in the nucleolus upon UV irradiation.

Prognostication of early-onset endometrioid endometrial cancer based on genome-wide DNA methylation profiles.

OBJECTIVE: The aim of this study was to establish criteria that would indicate whether fertility preservation therapy would likely be safe for patients aged 40 years or less with endometrioid endometrial cancer based on their DNA methylation profile. METHODS: Forty-nine fresh-frozen tissue samples from patients with endometrial cancer from an initial cohort and 31 formalin-fixed paraffin-embedded tissue samples from a second cohort were subjected to genome-wide DNA methylation analysis using the Infinium MethylationEPIC BeadChip. RESULTS: Epigenomic clustering of early-onset endometrial cancer was correlated with the widely used recurrence risk classification. Genes showing differences in DNA methylation levels between the low-recurrence-risk category and intermediate- and high-risk categories were accumulated in pathways related to fibroblast growth factor and nuclear factor-κB signaling. DNA hypomethylation and overexpression of ZBTB38 were frequently observed in the low-risk category. Eight hundred thirty-one marker CpG probes showed area under the curve values of >0.7 on the receiver operating characteristic curve for discrimination of patients belonging to the low-risk category. By combining marker CpG sites, seven panels for placing patients into the low-risk category with 91.3% or more sensitivity and specificity in both the initial and second cohorts were established. CONCLUSIONS: DNA methylation diagnostics criteria using up to 6 of 8 CpG sites for LPP, FOXO1, RNF4, EXOC6B, CCPG1, RREB1 and ZBTB38 may be applicable to recurrence risk estimation for patients aged 40 years or less with endometrial cancer, regardless of tumor cell content, even if formalin-fixed paraffin-embedded biopsy or curettage materials are used.

Novel breeding method, matα2-PBT, to construct isogenic series of polyploid strains of Saccharomyces cerevisiae.

How ploidy is determined in organisms is an important issue in bioscience. Polyploidy is believed to be relevant to useful traits of domesticated plants and microorganisms. As such, polyploidy is central to many applications in biotechnology. However, studies of polyploidy are poorly advanced because no methodologies to construct desired polyploid have been developed for any organism. Herein we describe the development of a novel breeding technology, matα2-PBT, to generate polyploid strains of Saccharomyces cerevisiae. S. cerevisiae has two mating types, a and α, determined by MATa and MATα gene each of which consists of a1 and a2 and α1 and α2 cistrons. This novel technology exploits an interesting feature of a specific mutation, matα2-102, in the MATα2 gene. Unlike the MATα wild-type strain, which gives a non-mating phenotype when mated with MATa cells, the matα2-102 strain confers an α mating-type to a-type strains when mated with a-type strains. We constructed plasmid with the cloned matα2-102 mutant gene. An a-type cells harboring this plasmid displayed an α mating-type and mated with a-type cells. Because the resultant hybrid displays an α mating-type, it can mate again with a-type cells. By repeating this procedure, we have constructed an isogenic series of haploid to tetraploid of S. cerevisiae. Although whether even higher polyploid than tetraploid can be constructed by using this technology remains to be determined in the future, we believe that it became possible for the first time with matα2-PBT method to investigate whether higher polyploid than tetraploid can be constructed.

Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci.

Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.

Hsp70 promotes maturation of uromodulin mutants that cause familial juvenile hyperuricemic nephropathy and suppresses cellular damage.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD. Here we studied effects of genetic expression and pharmacological induction of Hsp70 on the UMOD mutants C112Y and C217G. METHODS: We expressed wild type (WT), C112Y and C217G in HEK293 cells and studied their maturation and cellular damage using western blot and flow cytometry. RESULTS: Expression of C112Y or C217G increased pro-apoptotic proteins, decreased anti-apoptotic proteins, and induced cellular apoptosis as examined by annexin V staining and flow cytometry. Overexpression of Hsp70 or administration of an Hsp70 inducer geranylgeranylacetone (GGA) promoted maturation of the mutant proteins, increased their secreted forms, normalized the levels of pro- and anti-apoptotic proteins and suppressed apoptosis. CONCLUSION: These findings indicated that Hsp70 enhanced maturation of C112Y and C217G and reduced cellular apoptosis, suggesting that Hsp70 induction might be of a therapeutic value for treatment of FJHN.