PUMA facilitates EMI1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells.

Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration, while preventing cell transformation after damage. Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation (IR), while without promoting tumorigenesis in the long-term survivors. This finding suggests that PUMA (p53 upregulated modulator of apoptosis) may have a function other than regulates apoptosis. Here, we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells (PSCs) and immortalized hematopoietic progenitor cells (HPCs) after IR. We found that PUMA-deficient PSCs and HPCs exhibited a significant higher double-strand break (DSB) DNA repair activity via Rad51-mediated homologous recombination (HR). This is because PUMA can be associated with early mitotic inhibitor 1 (EMI1) and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation, thereby inhibiting Rad51 nuclear translocation and HR DNA repair. Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.

Double-blind randomized controlled trial on efficacy of cupulolith repositioning maneuver for treatment of apogeotropic horizontal canal benign paroxysmal positional vertigo.

Backgrounds: Although various therapeutic maneuvers have been proposed, it is still unclear which maneuver is better to treat apogeotropic horizontal canal benign paroxysmal positional vertigo (HC-BPPV).Objectives: This study aimed to assess the therapeutic efficacy of the cupulolith repositioning maneuver (CuRM) in apogeotropic HC-BPPV in comparison with the therapeutic head-shaking maneuver and modified Lempert maneuver.Materials and Method: This is double-blind randomized prospective study. Forty-nine consecutive patients diagnosed with apogeotropic HC-BPPV were allocated randomly to CuRM (n = 18), therapeutic head-shaking (n = 16), or modified Lempert maneuver (n = 15). The presence of nystagmus and vertigo on positional testing were evaluated at 30 min, on 1 day, and 1 week after treatment.Results: There were no significant differences in any clinical characteristics between the three groups at randomization. After a single trial of therapeutic maneuvers on the initial visit day, the CuRM (38.9%) and therapeutic head shaking maneuver (12.5%) did not show differences compared to modified Lempert maneuver (33.3%). The therapeutic effects on the 2nd day and at 1 week after treatment also did not differ between the three groups.Conclusions: Although the CuRM is theoretically considered to be a better therapeutic method, the therapeutic efficacy of CuRM was not statistically different compared to the other two maneuvers.

Polyethylene glycol-decorated doxorubicin/carboxymethyl chitosan/gold nanocomplex for reducing drug efflux in cancer cells and extending circulation in blood stream.

Polyethylene glycol (PEG)-decorated doxorubicin (Dox)/carboxymethyl chitosan (CMC)/gold nanoparticles (AuNPs) have been developed for cancer therapy. CMC was used as a reducing and stabilizing agent for the fabrication of AuNPs and Dox was loaded onto AuNPs as a chemotherapeutic agent. Dox-loaded CMC-stabilized AuNPs (Dox/CMC-AuNPs) with a mean diameter of 104.0 nm, zeta potential of -48.32 mV, and drug loading efficiency of 60.14% were prepared. PEG was attached to CMC-AuNPs for enhancing systemic drug exposure and prolonging the circulation in blood stream. Compared with Dox/CMC-AuNPs, Dox-loaded PEGylated CMC-AuNPs (Dox/CMC-AuNPs-PEG) showed a reduced hydrodynamic size (71.2 nm), less negative zeta potential (-12.83 mV), and an enhanced Dox loading efficiency (73.14%). Dox/CMC-AuNPs and Dox/CMC-AuNPs-PEG exhibited sustained and pH-dependent drug release profiles and exhibited antiproliferation effects against the A549 cells. In a bi-directional transport study of Caco-2 cell monolayers, AuNPs reduced the efflux ratio, which indicated that the P-glycoprotein-mediated multidrug resistance (MDR) was overcome. Dox/CMC-AuNPs-PEG resulted in reduced drug clearance (CL) and improved half-life (t1/2), compared with Dox/CMC-AuNPs, in rats after intravenous administration. These results suggest that Dox/CMC-AuNPs-PEG could be a promising nanotherapeutic approach to overcome MDR in cancer and prolong their circulation in the blood stream.

Data mining of micrornas in breast carcinogenesis which may be a potential target for cancer prevention.

The microRNAs (miRNAs) negatively regulate the stability and translation of target messenger RNAs by selectively binding. It has been implicated in diverse processes such as cellular differentiation, cell-cycle control, apoptosis, and carcinogenesis. Examination of tumor-specific miRNA expression profiles has revealed wide spread dysregulation of these molecules in diverse cancers. The available genomic bulk evidences were extracted from The Cancer Genome Atlas by using IluminaGA_miRNASeq platform in human breast cancer samples. After mining collected data, group of each miRNA ID was analyzed through five D/Bs (mirWalk, miranda, mirDB, RNA22, and TargetScan) on predicted and validated miRNA targets. Oncogenes known to have a high correlation with breast cancer (C-myc, HER2, cyclin D-1, N-RAS, FGF-4, FGF-3, BRCA1, and BRCA2) are subject in this study to select their relevant miRNAs. Function of miRNA regulation will be essential to achieve a complete understanding of carcinogenesis and these miRNAs would be potential target for breast cancer prevention.

Biodistribution and in vivo efficacy of genetically modified human mesenchymal stem cells systemically transplanted into a mouse bone fracture model.

Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical application due to their ability to undergo multi-lineage differentiation. Recently, ex vivo genetic modification of hMSCs was attempted to increase their differentiation potential. The present study was conducted to evaluate the biodistribution and in vivo efficacy of genetically modified hMSCs. To accomplish this, Runx2, which is a key transcription factor associated with osteoblast differentiation, was transduced into hMSCs using lentiviral vectors expressing green fluorescent protein (GFP) or luciferase. Here, we developed an experimental fracture in mice femur to investigate the effects of Runx2-transduced hMSCs on bone healing and migration into injury site. We conducted bio-luminescence imaging (BLI) using luciferase-tagged vector and quantitative real-time PCR using GFP probe to investigate the biodistribution of Runx2-transduced hMSCs in the fracture model. The biodistribution of hMSC cells in the fractured femur was observed at 14 days post-transplantation upon both BLI imaging and real-time PCR. Moreover, the fractured mice transplanted with Runx2-transduced hMSCs showed superior bone healing when compared to mock-transduced hMSC and MRC5 fibroblasts which were used as control. These data suggested that transplanted genetically modified hMSCs systemically migrate to the fractured femur, where they contribute to bone formation in vivo.

Hematopoietic stem cell regeneration enhanced by ectopic expression of ROS-detoxifying enzymes in transplant mice.

High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo.

Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells.

Previous studies have demonstrated that a small subset of cancer cells is capable of tumor initiation. The existence of tumor initiating cancer stem cells (CSCs) has several implications in terms of future cancer treatment and therapies. However, recently, several researchers proposed that differentiated cancer cells (non-CSCs) can convert to stem-like cells to maintain equilibrium. These results imply that removing CSCs may prompt non-CSCs in the tumor to convert into stem cells to maintain the equilibrium. Interleukin-6 (IL-6) has been found to play an important role in the inducible formation of CSCs and their dynamic equilibrium with non-stem cells. In this study, we used CSC-like human breast cancer cells and their alternate subset non-CSCs to investigate how IL-6 regulates the conversion of non-CSCs to CSCs. MDA-MB-231 and MDA-MB-453 CSC-like cells formed mammospheres well, whereas most of non-stem cells died by anoikis and only part of the remaining non-stem cells produced viable mammospheres. Similar results were observed in xenograft tumor formation. Data from cytokine array assay show that IL-6 was secreted from non-CSCs when cells were cultured in ultra-low attachment plates. IL-6 regulates CSC-associated OCT-4 gene expression through the IL-6-JAK1-STAT3 signal transduction pathway in non-CSCs. Inhibiting this pathway by treatment with anti-IL-6 antibody (1 µg/ml) or niclosamide (0.5-2 µM)/LLL12 (5-10 µM) effectively prevented OCT-4 gene expression. These results suggest that the IL-6-JAK1-STAT3 signal transduction pathway plays an important role in the conversion of non-CSCs into CSCs through regulation of OCT-4 gene expression.

Transferability of a modified embryonic stem cell test using a new endpoint for developmental neurotoxicity.

We developed and analyzed a new surrogate endpoint of the mouse embryonic stem cell test (EST) for developmental neurotoxicity. To determine the sensitivity, specificity, and transferability of the new endpoint, a pre-validation team from three independent laboratories optimized and standardized the protocol for neuronal differentiation of mouse embryonic stem cells (mESCs) by measuring the neuronal differentiation rates of mESCs under different culture conditions, such as the presence or absence of basic fibroblast growth factor (bFGF) in the growth media and varying lengths of culture. In addition, a component ratio of neuronal cells was measured by using flow cytometry analysis of ß-III tubulin (Tuj1)-positive cells and real-time polymerase chain reaction analysis of microtubule-associated protein 2 (MAP2) mRNA. Our results showed that the best growth was achieved by culturing mESCs for 12 d in N2B27 medium without bFGF or ascorbic acid. Lead (II) acetate and aroclor 1254 were used to test the usefulness of the new endpoint. When we used the known ID(50) values for lead (II) acetate in the EST model, it was classified as non-embryotoxic; however, when we used the new ID(50) values that we determined in this study, it was classified as weakly embryotoxic. Aroclor 1254 and penicillin G were also classified as weakly embryotoxic and non-embryotoxic compounds, respectively, when cardiac and neuronal differentiation ID(50) values were used. Therefore, our new surrogate endpoint for developmental neurotoxicity is not only sensitive and specific but also transferable among laboratories.

Embryotoxicity assessment of developmental neurotoxicants using a neuronal endpoint in the embryonic stem cell test.

The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj-1 ID50) using flow cytometry analysis of Tuj-1-positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj-1 ID50 in the EST instead of cardiac ID50, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj-1 ID50) , we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID50), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj-1 ID50 can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals.

Embryotoxicity of lead (II) acetate and aroclor 1254 using a new end point of the embryonic stem cell test.

We developed a new end point of the mouse stem cell test (EST) for developmental neurotoxicity. We tested 2 developmental neurotoxicants, namely, lead (II) acetate and Aroclor 1254, using this EST. Our results showed that lead (II) acetate is nonembryotoxic, and Aroclor 1254 is weakly embryotoxic. To identify a new end point for developmental neurotoxicity, we used the default method of neuronal differentiation for D3 mouse embryonic stem cells with basic fibroblast growth factor (bFGF) and ascorbic acid. Flow cytometry and real-time polymerase chain reaction were used to quantify the inhibition of neuronal differentiation. Our results showed that both lead (II) acetate and Aroclor 1254 reduced the percentage of microtubule-associated protein 2 (MAP-2)-positive cells and the messenger RNA (mRNA) expression level of MAP-2 in a dose-dependent manner. These results suggested that these methods can be used to develop an additional end point of the EST for developmental neurotoxicity using default differentiation of mouse embryonic stem cells.

Telomerase reverse transcriptase related with telomerase activity regulates tumorigenic potential of mouse embryonic stem cells.

Embryonic stem cell (ESC) research gave rise to the possibility that stem cell therapy could be used in the treatment of incurable diseases such as neurodegenerative disorders. However, problems related to the tumorigenicity of undifferentiated ESCs must be resolved before such cells can be used in the application of cell replacement therapies. In the present study, we attempted to determine biomarkers that predicted tumor formation of undifferentiated ESCs in vivo. We differentiated mouse ESCs (R1 cell line) into neural lineage using a 5-step method, and evaluated the expression of oncogenes (p53, Bax, c-myc, Bcl2, K-ras), telomerase-related genes (TERT, TRF), and telomerase activity and telomere length during differentiation of ESCs. The expression of oncogenes did not show a significant change during differentiation steps, but the expression of telomerase reverse transcriptase (TERT) and telomerase activity correlated with mouse ESCs differentiation. To investigate the possibility of mouse TERT (mTERT) as a biomarker of tumorigenicity of undifferentiated ESCs, we established mTERT knockdown ESCs using the shRNA lentivirus vector and evaluated its tumorigenicity in vivo using nude mice. Tumor volumes significantly decreased, and appearances of tumor formation in mice were delayed in the TERT-knockdown ESC treated group compared with the undifferentiated ESC treated group. Altogether, these results suggested that mTERT might be potentially beneficial as a biomarker, rather than oncogenes of somatic cells, for the assessment of ESCs tumorigenicity.

Biological characterization of long-term cultured human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical applications. The reason is that they may have the plasticity needed to differentiate into multiple lineages and the ability to expand ex vivo. For the therapeutic applications of hMSCs to be of practical use, it is crucial to assess the efficacy and safety of hMSCs in long-term ex vivo expansion. In this study, we cultured hMSCs by population doubling (PD) 60, and investigated their growth, osteogenic and adipogenic differential abilities, change of surface markers, telomerase activity, telomere length, and gene expression related to tumorigenesis. An in vivo tumorigenesis assay was also carried out. In long-term expanded hMSCs, the cells became aged above PD 30 and their adipogenic and osteogenic differentiation potential decreased. Telomerase activity unchanged whereas telomere length decreased and karyotypes were not changed. Gene expressions related to tumorigenesis decreased in proportion as the PD of hMSCs increased. In vivo transplantation of long-term cultured hMSCs to nude mice did not result in tumor formation. These findings suggest that diverse tests for cellular therapy should be considered during the ex vivo culture of hMSCs, particularly when a prolonged and extended propagation period is required.

The effects of cyclin-dependent kinase inhibitors on adipogenic differentiation of human mesenchymal stem cells.

The molecular mechanisms that couple growth arrest and cell differentiation were examined during adipogenesis. Here, to understand the cyclin-dependent kinase inhibitor (CKI) genes involved in the progression of adipogenic differentiation, we examined changes in the protein and mRNA expression levels of CKI genes in vitro. During the onset of growth arrest associated with adipogenic differentiation, two independent families of CKI genes, p27Kip1 and p18INK4c, were significantly increased. The expressions of p27Kip1 and p18INK4c, regulated at the level of protein and mRNA accumulation, were directly coupled to adipogenic differentiation. This finding was supported by the inhibition of adipogenic differentiation caused by short interfering RNA (siRNA). In this study, we investigated the regulatory effects of transforming growth factor beta-1 (TGFbeta-1) on CKI genes involved in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Only the up-regulation of p18INK4c during adipogenic differentiation, and not that of the p27Kip1 gene was prevented by treatment with TGFbeta-1, one of the factors that inhibit adipogenesis in vitro. This finding indicates a close correlation between adipogenic differentiation and p18INK4c induction in hMSCs. Thus, these data demonstrate a role for the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors in regulating adipogenic differentiation, thereby providing a molecular mechanism that couples growth arrest and differentiation.

The haploinsufficient tumor suppressor p18 upregulates p53 via interactions with ATM/ATR.

p18 was first identified as a factor associated with a macromolecular tRNA synthetase complex. Here we describe the mouse p18 loss-of-function phenotype and a role for p18 in the DNA damage response. Inactivation of both p18 alleles caused embryonic lethality, while heterozygous mice showed high susceptibility to spontaneous tumors. p18 was induced and translocated to the nucleus in response to DNA damage. Expression of p18 resulted in elevated p53 levels, while p18 depletion blocked p53 induction. p18 directly interacted with ATM/ATR in response to DNA damage. The activity of ATM was dependent on the level of p18, suggesting the requirement of p18 for the activation of ATM. Low p18 expression was frequently observed in different human cancer cell lines and tissues. These results suggest that p18 is a haploinsufficient tumor suppressor and a key factor for ATM/ATR-mediated p53 activation.

In vitro development of resistance to a novel fluoroquinolone, DW286, in methicillin-resistant Staphylococcus aureus clinical isolates.

In vitro development of resistance to a novel fluoroquinolone, DW286, as well as to ciprofloxacin, gemifloxacin, sparfloxacin and trovafloxacin, was investigated in eight methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. The strains were subcultured in subinhibitory concentrations of each agent during a 50 day period. Subculturing in most agents led to the selection of 37 mutants with increased MICs. The DW286 MICs were increased from 0.004-0.031 to 0.125-0.5 mg/L in five strains after 13-47 passages, and were not increased in three strains. The ciprofloxacin, gemifloxacin, sparfloxacin and trovafloxacin-selected mutants showed relatively weak cross-resistance to DW286. DNA sequencing analyses of all of the selected mutants revealed a few point mutations responsible for the high level of resistance, but actually these variations did not confer high resistance to fluoroquinolones. In the presence of reserpine, an inhibitor of the Gram-positive efflux pump, of 36 mutants 22 had two- to 16-fold lower ciprofloxacin MICs, and 20 had two- to 16-fold lower gemifloxacin MICs. However, sparfloxacin, trovafloxacin and DW286 were not good substrates for efflux pumps.

In vitro and in vivo antibacterial activities of DW286, a new fluoronaphthyridone antibiotic.

The in vitro and in vivo activities of DW286, a novel fluoronaphthyridone with potent antibacterial activity, were compared with those of ciprofloxacin, gemifloxacin, sparfloxacin, and trovafloxacin. Against gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis, the in vitro activity of DW286 was stronger than that of any other reference antibiotic. Against gram-negative bacteria, the activity of DW286 was similar to those of trovafloxacin and gemifloxacin but was weaker than that of ciprofloxacin. In a mouse systemic infection caused by three S. aureus strains, including methicillin-resistant S. aureus and quinolone-resistant S. aureus (QRSA), DW286 demonstrated the most potent activity, as found in vitro. Specially, DW286 is >or=8-fold more active against QRSA than the other fluoroquinolones. And the 50% protective doses for DW286 were correspondent with the in vitro activities.