Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre.

Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least 6 months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80-90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.

Antitumor activity of TMPyP4 interacting G-quadruplex in retinoblastoma cell lines.

To investigate the molecular mechanism of the antitumor activity of the cationic porphyrin 5, 10, 15, 20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4) in retinoblastoma cell lines, Y79 and WERI-Rb1 cells were treated with TMPyP4 for 0-72 h, after which growth inhibition, modulation of the cell cycle and the induction of apoptosis were examined. In addition, the effect of TMPyP4 on the susceptibility to irradiation was evaluated in Y79 and WERI-Rb1 cells. In vitro telomeric repeat amplification protocol assay showed TMPyP4 (10-100 microM) directly blocked telomerase elongation, suggesting that TMPyP4 can form stable guanine (G)-quadruplexes in extending telomere repeats in substrate oligonucleotides. The antiproliferative activities of TMPyP4 assessed with the MTS assay and expressed in terms of IC(50): Y79 cells, 60 microM; WERI-Rb1 cells, 45 microM. Treatment with TMPyP4 at doses of 10, 20, 50 or 100 microM for 48 or 72 h significantly inhibited the growth of Y79 and WERI-Rb1 cells. Apoptosis, as assessed with CaspACE FITC-VAD-FMK, was induced by TMPyP4 in a dose-dependent manner. Induction of apoptosis by TMPyP4 was associated with increased expression of phosphorylated DNA damage response factor H2AX (Ser139), phosphorylated p53 (Ser46) protein and activation of mitogen-activated protein kinases in Y79 and WERI-Rb1 cells. Moreover, TMPyP4 significantly enhanced the susceptibility to irradiation in both cell lines. This study provides insight into the molecular mechanism of the antitumor effects of TMPyP4. G-quadruplex structure may be a potential therapeutic target in retinoblastoma.

Adhesion to fibronectin induces megakaryocytic differentiation of JAS-REN cells.

BACKGROUND: Binding of integrins to the extracellular matrix elicits various responses. We have previously reported a megakaryocytic-erythroid cell line (JAS-R) that showed phenotypic changes after adhesion to plastic dishes. However, the matrix protein and the mechanism responsible for megakaryocytic differentiation still remain unknown. MATERIALS AND METHODS: JAS-REN (erythroid) cells were cultured on dishes coated with various proteins. The cells were treated with RGDS, a tetrapeptide ligand to integrins, or phorbol ester (12-o-tetradecanoylphorbol-13-acetate, TPA) for 48 hours and then were harvested. Subsequently, the cell surface markers were analyzed using flow cytometry and gene expression was studied by RT-PCR. RESULTS: The JAS-REN cells adhered to fibronectin-coated dishes, but showed poor adhesion to dishes coated with collagen, laminin or poly-D-lysine. The TPA-stimulated JAS-REN cells showed an increase in the expression of integrin alphaIIbbeta3 complex (CD41a) and integrin beta3 (CD61), while glycophorin A (CD235a) expression was decreased. JAS-REN cells that were adherent to fibronectin-coated dishes also showed a similar pattern of phenotype to TPA-treated cells, but the changes were not so prominent. RT-PCR revealed that TPA treatment altered the gene expression profile of JAS-REN cells, making it similar to that of JAS-RAD (megakaryocytic) cells. The RGDS-treated and fibronectin adherent JAS-REN cells also showed a mostly similar expression profile to JAS-RAD cells, but these two stimuli did not alter the gene expression profile as TPA stimulation did. Transcription factors, FLI1 and GFI1, were induced by all stimuli. CONCLUSION: Signals triggered by adhesion to fibronectin result in the induction of FLI1 that may play a pivotal role in the lineage shift of JAS-REN cells from erythroid to megakaryocytic.

Effects of prednisolone on specifically expressed genes in pediatric acute B-lymphoblastic leukemia.

Although glucocorticoid is essential in the treatment of pediatric acute lymphoblastic leukemia (ALL), their precise mechanisms of action remain unclear. We used DNA microarray to evaluate prednisolone-regulated genes in pre-B-ALL cells from 2 pediatric patients. We found up-regulation of 26 genes in ALL cells from both patients, compared with peripheral normal B lymphocytes before maintenance chemotherapy. Treatment with prednisolone for 48 hours induced down-regulation of 5 genes (terminal deoxynucleotidyl transferase, heparin-binding epidermal growth factorlike growth factor, pre-B-lymphocyte genes 1 and 3, and immunoglobulin lambda-like polypeptide) among 26 specifically expressed genes in pre-B-ALL cells from both patients.

Segregation of megakaryocytic or erythroid cells from a megakaryocytic leukemia cell line (JAS-R) by adhesion during culture.

Adhesion is one of the important biologic characteristics of leukemic cells. We previously reported a new megakaryocytic-erythroid cell line, JAS-R. In this study, JAS-R cells were segregated into two types by the differences of attachment to culture dishes. One type (designated as JAS-RAD cells) adhered to the substratum of the culture dishes, while the other (JAS-REN cells) grew as a single-cell suspension. Adhesion of JAS-RAD was inhibited by treatment with RGDS oligopeptide. Flow cytometric analysis revealed that JAS-RAD cells had high expression of CD41a and CD61 versus low CD235a expression, and JAS-REN showed low expression of CD41a, and CD61, and high CD235a. The two phenotypes were reciprocally exchangeable by selecting adherent or suspended cells from each type of culture. Microarray analysis and RT-PCR revealed that JAS-RAD cells expressed four major alpha-granule genes and JAS-REN cells expressed beta-globin. Interestingly, erythropoietin was only secreted by JAS-RAD cells. With regard to transcription factors, it was shown that GFI1, FLI1 and RUNX1 were strongly expressed in JAS-RAD cells while GATA1, FOG1 and NFE2 were equally expressed by both types. These findings indicate that adhesion via integrins is related to the phenotypic shift of JAS-R cells between megakaryocytic and erythroid lineages.

JAS-R, a new megakaryo-erythroid leukemic cell line that secretes erythropoietin.

BACKGROUND: The processes of leukemogenesis and differentiation of the megakaryo-erythroid lineage remain poorly understood. Leukemic cell lines derived from megakaryocytic leukemia are valuable reagents for studies on these events. MATERIALS AND METHODS: A new cell line, JAS-R, was established from a 64-year-old patient with acute megakaryocytic leukemia (AML M7). Its characteristics were studied by morphological, immunophenotypic and molecular biological analysis. RESULTS: Immunophenotyping showed that the JAS-R cells were positive for CD33, CD41 and CD61, as well as moderately to weakly positive for CD4, CD7, CD13 and glycophorin A. Chromosomal analysis revealed a composite karyotype, but no major translocation abnormalities were observed. Electron microscopy disclosed that the JAS-R cells had numerous surface blebs and some cells also had alpha-granules and demarcation membranes. The mRNAs of 4 major proteins (platelet factor 4, beta-thromboglobulin, selectin-P and thrombospondin 1) found in alpha-granules were all expressed by the JAS-R cells. In particular, expression of platelet factor 4 was high. To further characterize JAS-R cells, comparison with 4 other megakaryo-etythroid cell lines (CMK, MEG-01, K562 and KU812) was done by gene expression profiling using an oligo-DNA microarray. The results showed that JAS-R was a distinctive cell line. It was noteworthy that the JAS-R cells secreted erythropoietin and expressed erythropoietin receptor. A neutralizing antibody for erythropoietin partly inhibited the proliferation of the cells. CONCLUSION: JAS-R may be a useful cell line for investigating the differentiation and leukemogenesis of megakaryo-erythroid cells and for studying the influence of erythropoietin on these cells.

Novel drug delivery system using thermoreversible gelation polymer for malignant glioma.

Many approaches to local tumor treatment have been reported and their efficacy demonstrated in patients with malignant glioma. We studied thermoreversible gelation polymer (TGP) as a novel drug delivery system (DDS) for treating this type of tumor. TGP exhibits sol-gel transition i.e., is water-soluble in the sol phase below the chosen sol-gel transiting temperature and water-insoluble in the gel phase above this temperature. We conjugated doxorubicin with TGP to prepare doxorubicin-TGP (DXR-TGP), then studied the kinetics of doxorubicin release from TGP and the antitumor activity of DXR-TGP in vitro and in vivo. The diffusive speed of doxorubicin from TGP was 9.4x10(-7) cm(2)/s and doxorubicin was reliably released from TGP. DXR-TGP showed antitumor activity against the human glioma cell lines T98G and U87MG and in a subcutaneous tumor model in nude mice. Pathologically, detection of the proliferation marker Ki-67 was considerably lower in the DXR-TGP group than in the control group (30-40% vs. 60-70%, respectively). This is to the best of our knowledge the first report of TGP as a novel drug delivery system, and further we provide evidence that TGP exhibits potential for use as a novel DDS for malignant glioma.

Depsipeptide-resistant KU812 cells show reversible P-glycoprotein expression, hyper-acetylated histones, and modulated gene expression profile.

Depsipeptide (FK228), a histone deacetylase inhibitor, is a promising new anticancer agent. The mechanism of resistance to this agent was studied using KU812 cells. Depsipeptide-resistant KU812 cells expressed P-glycoprotein (P-gp) and their resistance was abolished by co-treatment with verapamil. P-gp expression returned to the parental cell level when resistant cells were cultured in depsipeptide-free medium, while resistant cells cultured in the medium containing 16 nM depsipeptide still showed hyper-acetylation of histones. Moreover, resistant cells showed erythroid differentiation. Microarray analysis revealed that 28 genes showed increased expression and three genes showed decreased expression in resistant cells compared with parental cells. These 31 genes had various functions relating to signal transduction, cell cycle, apoptosis, and control of cell morphology and differentiation. Among the 28 genes that were upregulated, 15 genes also showed an increased expression in parental cells treated with 4 nM depsipeptide for 48 h, while the other 13 genes including P-gp were different. Among the three genes with decreased expression, HEP27 was most dramatically downregulated. These findings suggest that continuous exposure to depsipeptide reversibly induces P-gp, which contributes to the onset of resistance, but the altered gene expression profile of resistant cells may also play a role.

Gene and protein expression profiling during differentiation of neuroblastoma cells triggered by 13-cis retinoic acid.

PURPOSE: The precise changes in RNA and protein expression that accompany neuroblastoma differentiation remain unknown. The authors used microarray technologies to screen molecules associated with the differentiation of neuroblastoma (NB) cells induced by 13-cis retinoic acid. METHODS: The authors quantified the expression of 2,061 RNA transcripts related to oncogenesis and of 380 proteins expressed in SK-N-SH and CHP-134 NB cell lines in the presence or absence of 13-cis retinoic acid. RESULTS: Hierarchical clustering captured gene expression altered during neuroblastoma differentiation induced by 13-cis retinoic acid. Several genes were further abstracted based on P values below 0.0017 or protein chips observed in both NB cell lines. The altered expressions of gene products revealed by both DNA and protein chips were in agreement. The expressions of N-myc, cyclin D3, and Wnt10B were downregulated, whereas those of retinoblastoma (RB) and related genes (p107, RB2/p130, p300/CBP, E2F-1, DP-1) as well as others were upregulated. CONCLUSIONS: These results suggest that microarray technology can screen for genes that are important in neuroblastoma differentiation.