Administration of Human-Derived Mesenchymal Stem Cells Activates Locally Stimulated Endogenous Neural Progenitors and Reduces Neurological Dysfunction in Mice after Ischemic Stroke.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin-GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP+ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry+ h-MSCs stimulated GFP+ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 104 cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

Targeted Suicide Gene Therapy with Retroviral Replicating Vectors for Experimental Canine Cancers.

Cancer in dogs has increased in recent years and is a leading cause of death. We have developed a retroviral replicating vector (RRV) that specifically targets cancer cells for infection and replication. RRV carrying a suicide gene induced synchronized killing of cancer cells when administered with a prodrug after infection. In this study, we evaluated two distinct RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) in canine tumor models both in vitro and in vivo. Despite low infection rates in normal canine cells, both RRVs efficiently infected and replicated within all the canine tumor cells tested. The efficient intratumoral spread of the RRVs after their intratumoral injection was also demonstrated in nude mouse models of subcutaneous canine tumor xenografts. When both RRVs encoded a yeast cytosine deaminase suicide gene, which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil, they caused tumor-cell-specific 5-FC-induced killing of the canine tumor cells in vitro. Furthermore, in the AZACF- and AZACH-cell subcutaneous tumor xenograft models, both RRVs exerted significant antitumor effects. These results suggest that RRV-mediated suicide gene therapy is a novel therapeutic approach to canine cancers.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

BACKGROUND/AIM: Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. MATERIALS AND METHODS: Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. RESULTS: Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. CONCLUSION: RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Retroviral Replicating Vectors Mediated Prodrug Activator Gene Therapy in a Gastric Cancer Model.

Retroviral replicating vectors (RRVs) selectively replicate and can specifically introduce prodrug-activating genes into tumor cells, whereby subsequent prodrug administration induces the death of the infected tumor cells. We assessed the ability of two distinct RRVs generated from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which infect cells via type-III sodium-dependent phosphate transporters, PiT-2 and PiT-1, respectively, to infect human gastric cancer (GC) cells. A quantitative RT-PCR showed that all tested GC cell lines had higher expression levels of PiT-2 than PiT-1. Accordingly, AMLV, encoding a green fluorescent protein gene, infected and replicated more efficiently than GALV in most GC cell lines, whereas both RRVs had a low infection rate in human fibroblasts. RRV encoding a cytosine deaminase prodrug activator gene, which converts the prodrug 5-flucytosine (5-FC) to the active drug 5-fluorouracil, showed that AMLV promoted superior 5-FC-induced cytotoxicity compared with GALV, which correlated with the viral receptor expression level and viral spread. In MKN-74 subcutaneous xenograft models, AMLV had significant antitumor effects compared with GALV. Furthermore, in the MKN-74 recurrent tumor model in which 5-FC was discontinued, the resumption of 5-FC administration reduced the tumor volume. Thus, RRV-mediated prodrug activator gene therapy might be beneficial for treating human GC.

Microglia Negatively Regulate the Proliferation and Neuronal Differentiation of Neural Stem/Progenitor Cells Isolated from Poststroke Mouse Brains.

We previously demonstrated that neural stem/progenitor cells (NSPCs) were induced within and around the ischemic areas in a mouse model of ischemic stroke. These injury/ischemia-induced NSPCs (iNSPCs) differentiated to electrophysiologically functional neurons in vitro, indicating the presence of a self-repair system following injury. However, during the healing process after stroke, ischemic areas were gradually occupied by inflammatory cells, mainly microglial cells/macrophages (MGs/MΦs), and neurogenesis rarely occurred within and around the ischemic areas. Therefore, to achieve neural regeneration by utilizing endogenous iNSPCs, regulation of MGs/MΦs after an ischemic stroke might be necessary. To test this hypothesis, we used iNSPCs isolated from the ischemic areas after a stroke in our mouse model to investigate the role of MGs/MΦs in iNSPC regulation. In coculture experiments, we show that the presence of MGs/MΦs significantly reduces not only the proliferation but also the differentiation of iNSPCs toward neuronal cells, thereby preventing neurogenesis. These effects, however, are mitigated by MG/MΦ depletion using clodronate encapsulated in liposomes. Additionally, gene ontology analysis reveals that proliferation and neuronal differentiation are negatively regulated in iNSPCs cocultured with MGs/MΦs. These results indicate that MGs/MΦs negatively impact neurogenesis via iNSPCs, suggesting that the regulation of MGs/MΦs is essential to achieve iNSPC-based neural regeneration following an ischemic stroke.

Transplantation of Human Brain-Derived Ischemia-Induced Multipotent Stem Cells Ameliorates Neurological Dysfunction in Mice After Stroke.

We recently demonstrated that injury/ischemia-induced multipotent stem cells (iSCs) develop within post-stroke human brains. Because iSCs are stem cells induced under pathological conditions, such as ischemic stroke, the use of human brain-derived iSCs (h-iSCs) may represent a novel therapy for stroke patients. We performed a preclinical study by transplanting h-iSCs transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion (MCAO). Compared with PBS-treated controls, h-iSC transplantation significantly improved neurological function. To identify the underlying mechanism, green fluorescent protein (GFP)-labeled h-iSCs were transplanted into post-stroke mouse brains. Immunohistochemistry revealed that GFP+ h-iSCs survived around the ischemic areas and some differentiated into mature neuronal cells. To determine the effect on endogenous neural stem/progenitor cells (NSPCs) by h-iSC transplantation, mCherry-labeled h-iSCs were administered to Nestin-GFP transgenic mice which were subjected to MCAO. As a result, many GFP+ NSPCs were observed around the injured sites compared with controls, indicating that mCherry+ h-iSCs activate GFP+ endogenous NSPCs. In support of these findings, coculture studies revealed that the presence of h-iSCs promotes the proliferation of endogenous NSPCs and increases neurogenesis. In addition, coculture experiments indicated neuronal network formation between h-iSC- and NSPC-derived neurons. These results suggest that h-iSCs exert positive effects on neural regeneration through not only neural replacement by grafted cells but also neurogenesis by activated endogenous NSPCs. Thus, h-iSCs have the potential to be a novel source of cell therapy for stroke patients.

Different Contacted Cell Types Contribute to Acquiring Different Properties in Brain Microglial Cells upon Intercellular Interaction.

Microglial cells (MGs), originally derived from progenitor cells in a yolk sac during early development, are glial cells located in a physiological and pathological brain. Since the brain contains various cell types, MGs could frequently interact with different cells, such as astrocytes (ACs), pericytes (PCs), and endothelial cells (ECs). However, how microglial traits are regulated via cell-cell interactions by ACs, PCs, or ECs and how they are different depending on the contacted cell types is unclear. This study aimed to clarify these questions by coculturing MGs with ACs, PCs, or ECs using mouse brain-derived cells, and microglial phenotypic changes were investigated under culture conditions that enabled direct cell-cell contact. Our results showed that ACs or PCs dose-dependently increased the number of MG, while ECs decreased it. Microarray and gene ontology analysis showed that cell fate-related genes (e.g., cell cycle, proliferation, growth, death, and apoptosis) of MGs were altered after a cell-cell contact with ACs, PCs, and ECs. Notably, microarray analysis showed that several genes, such as gap junction protein alpha 1 (Gja1), were prominently upregulated in MGs after coincubation with ACs, PCs, or ECs, regardless of cell types. Similarly, immunohistochemistry showed that an increased Gja1 expression was observed in MGs after coincubation with ACs, PCs, or ECs. Immunofluorescent and fluorescence-activated cell sorting analysis also showed that calcein-AM was transferred into MGs after coincubation with ACs, PCs, or ECs, confirming that intercellular interactions occurred between these cells. However, while Gja1 inhibition reduced the number of MGs after coincubation with ACs and PCs, this was increased after coincubation with ECs; this indicates that ACs and PCs positively regulate microglial numbers via Gja1, while ECs decrease it. Results show that ACs, PCs, or ECs exert both common and specific cell type-dependent effects on MGs through intercellular interactions. These findings also suggest that brain microglial phenotypes are different depending on their surrounding cell types, such as ACs, PCs, or ECs.

BAP1 depletion in human B-lymphoblast cells affects the production of innate immune cytokines and chemokines.

BRCA1 associated protein 1 (BAP1) is a ubiquitin C-terminal hydrolase that deubiquitinates histone H2AK119ub and other proteins and regulates the expression of multiple genes. The knockout of this tumor suppressor gene results in severe thymic atrophy, complete loss of the T cell lineage, and abnormal B cell development in mice. In the current study, we investigated in vitro effects of BAP1 knockout on cytokine and chemokine production using the human B-lymphoblast cell line TSCE5. We confirmed that knockout changed the production of innate immune-associated genes and their receptors. The CCL19, CCR7, CCL2, and CXCR5 genes associated with T and B cell migration were upregulated. Knockout cells producing high levels of CCL19 showed acceleration of actin polymerization, which is essential for cell migration. CD69, PTPRC, and TLR3 genes that activate inflammation were downregulated. The tumor necrosis factor ligand genes TNF, LTA, and TNFSF10 were downregulated by knockout. In knockout cells, TNFα production was strongly downregulated upon the addition of H2 O2 , but NF-κB in the basal condition and when TNFα was added was augmented, suggesting that these cells could respond to TNFα. These results indicated that BAP1 affects the expression of chemokines and cytokines, T and B cell migration, and activated inflammation associating with innate immunity.

Assessment of circulating microRNA specific for patients with familial adenomatous polyposis.

Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of diseases. However, usefulness of circulating miRNAs as biomarkers for hereditary gastrointestinal diseases have not been confirmed yet. We explored circulating miRNAs specific for patients with familial adenomatous polyposis (FAP) as a representative hereditary gastrointestinal disease. Next-generation sequencing (NGS) indicated that plasma miR-143-3p, miR-183-5p, and miR-885-5p were candidate biomarkers for five FAP patients compared to three healthy donors due to moderate copy number and significant difference. MiR-16-5p was considered as an internal control due to minimum difference in expression across FAP patients and healthy donors. Validation studies by real-time PCR showed that mean ratios of maximum expression and minimum expression were 2.2 for miR-143-3p/miR-16-5p, 3.4 for miR-143-3p/miR-103a-3p, 5.1 for miR-183-5p/miR-16-5p, and 4.9 for miR-885-5p/miR-16-5p by using the samples collected at different time points of eight FAP patients. MiR-143-3p/16-5p was further assessed using specimens from 16 FAP patients and 7 healthy donors. MiR-143-3p was upregulated in FAP patients compared to healthy donors (P = 0.04), but not significantly influenced by clinicopathological features. However, miR-143-3p expression in colonic tumors was rare for upregulation, although there was a significant difference by existence of desmoid tumors. MiR-143-3p transfection significantly inhibited colorectal cancer cell proliferation compared to control microRNA transfection. Our data suggested regulation of miR-143-3p expression differed by samples (plasma or colonic tumors) in most FAP patients. Upregulation of plasma miR-143-3p expression may be helpful for diagnosis of FAP, although suppressive effect on tumorigenesis seemed insufficient in FAP patients.

A patient-derived xenograft and a cell line derived from it form a useful preclinical model for small bowel adenocarcinoma.

Basic and clinical studies on small bowel adenocarcinoma (SBA) are limited due to the rare nature of this cancer. We established a patient-derived xenograft (PDX) model from the tumor tissue of an advanced SBA patient with liver and peritoneal metastasis, and a cell line from the PDX. In the PDX model, compared to the control group, 5-fluorouracil (5-FU) treatment resulted in statistically significant tumor growth inhibition (TGI), while oxaliplatin (OHP) and irinotecan had no significant inhibitory effects. In combination with 5-FU, OHP showed the highest rate of TGI. The IC50 for OHP was significantly lower than those for paclitaxel, gemcitabine, and trifluorothymidine in the PDX-derived cell line when compared to in HT29, a colon cancer cell line. Genetic analysis of the patient tumor, PDX tumor, and the cell line demonstrated consistency in the microsatellite status and mutations in TP53, APC, HRAS, CSF1R, FGFR3, FLT3, PDGFRA, and RET genes. However, the PDX tumor alone had additional mutations, indicating that the PDX-derived cell line may support the unstable genetic status of the PDX. Our findings confirmed the effectiveness of the combination of OHP and 5-FU, which is a common treatment for advanced SBA and advanced colorectal cancer, in a preclinical model. This preclinical model of SBA can help in further understanding the biology of SBA.

Splicing modulator FR901464 is a potential agent for colorectal cancer in combination therapy.

FR901464 (FR) was first described as an anticancer drug and later identified as a modulator of splicing factor 3B subunit 1 (SF3B1). Although the effectiveness of splicing modulators has been investigated in colorectal cancer (CRC) cells, their usefulness in animal experiments has not been confirmed. The association of SF3B1 with CRC progression and the influence of FR on transcriptional activity in CRC has not been fully elucidated. FR showed strong cytotoxicity against CRC cell lines, SF3B1-mutated cancer cell lines, and human fibroblasts with IC50 values less than 1 ng/ml. FR-resistant clones derived from HCT116, DLD1, Lovo, and CT26 cells showed IC50 values greater than 100 ng/ml. SF3B1 sequencing demonstrated low frequencies of SF3B1 mutations in CRC and mutations in codon 1074 of exon 22 in all FR-resistant clones. Unlike hematological malignancies, SF3B1 expression was not associated with CRC progression. Although FR showed significant growth inhibition in a xenograft model of RKO cells, severe toxicity was also induced. These data indicated CRC might be a suitable target of FR unless toxicity occurs. Microarray analysis and real-time quantitative PCR demonstrated downregulation of genes associated with Fanconi anemia (BRCA1 and BRCA2) and 28 driver oncogenes. These data suggested combination treatment of FR with other anticancer drugs whose sensitivity is associated with genes affected by FR treatment. Combination treatment with PARP1 inhibitor olaparib, whose sensitivity was enhanced by BRCA 1/2 deficiency, showed synergistic effects in CRC cells. Our data indicates the potential of FR in combination therapy rather than monotherapy for CRC treatment.

Dual-vector prodrug activator gene therapy using retroviral replicating vectors.

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.

Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors.

Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression.

Cytotoxicity of replication-competent adenoviruses powered by an exogenous regulatory region is not linearly correlated with the viral infectivity/gene expression or with the E1A-activating ability but is associated with the p53 genotypes.

BACKGROUND: Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. METHODS: We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5' regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. RESULTS: We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. CONCLUSIONS: Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression.

Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model.

Cancer vaccine application is limited to specific cancer types because few cancer-associated antigens are known to induce tumor rejection. Accordingly, we assessed the utility of Ad881, an oncolytic adenovirus in which viral replication was strictly regulated by the cancer-specific midkine promoter, as a cancer vaccine in a murine colorectal cancer model lacking specific cancer-associated antigens. In CT26 and CMT93 cells, Ad881 (multiplicity of infection: 100 or 1,000) showed stronger cytotoxicity and oncolysis in vitro than its equivalent replication-defective adenovirus, Ad884. CT26 cells (1 × 104) infected with Ad881 (multiplicity of infection: 1,000) for 24 hours were suitable as vaccine antigens without tumor formation in our model. Repeated vaccinations, but not single vaccination, induced a greater prophylactic immune response. The percentage of mice that rejected the tumor challenge was 0, 4, and 38% after no vaccination, single vaccination, and repeated vaccinations, respectively. Immunogenic cell death marker high-mobility group box 1 protein (HMGB1) and adenosine triphosphate in culture medium were higher after Ad881 infection (24.3 ng/ml and 48.2 nmol/l, respectively) than after Ad884 infection (8.6 ng/ml and 15.4 nmol/l, respectively) or oxaliplatin treatment (3.7 ng/ml and 1.8 nmol/l, respectively). These results indicate that repeated whole cell vaccination using an oncolytic adenovirus may be a potent approach to evoke immunogenic cell death.

Replication-competent adenoviruses with the type 35-derived fiber-knob region achieve reactive oxygen species-dependent cytotoxicity and produce greater toxicity than those with the type 5-derived region in pancreatic carcinoma.

Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad.

Combinatorial anti-angiogenic gene therapy in a human malignant mesothelioma model.

Anti-angiogenic gene therapy represents a promising strategy for cancer; however, it has rarely been tested in malignant mesothelioma, a highly aggressive tumor associated with asbestos with poor prognosis. In the present study, we investigated whether anti-angiogenic factors such as angiostatin, endostatin and the soluble form of vascular endothelial growth factor receptor 2 (sFlk1) were able to inhibit endothelial cell proliferation via lentivirus-mediated gene transfer into malignant mesothelioma cells in culture. We also assessed whether a dual-agent strategy had greater therapeutic benefit. Human malignant pleural mesothelioma MSTO-211H cells were transduced using lentiviral vectors that individually expressed angiostatin, endostatin and sFlk1 and linked to enhanced green fluorescent protein (EGFP) marker gene expression via an internal ribosome entry site. The lentivirus expressing EGFP alone was used as a control. The resultant cells designated as MSTO-A, MSTO-E, MSTO-F and MSTO-C were confirmed by western blot analysis and fluorescence microscopy to stably express the corresponding proteins. No differences were observed in the in vitro growth rates between any of these cells. However, co-culture of MSTO-A, MSTO-E and MSTO-F showed significant suppression of human umbilical endothelial cell growth in vitro compared with that of MSTO-C. Furthermore, a combination of any two among MSTO-A, MSTO-E and MSTO-F significantly enhanced efficacy. These results suggest that combinatorial anti-angiogenic gene therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy than that of a single-agent regimen.

Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells.

Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.

Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway.

The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor. FIR is alternatively spliced to lack the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. FIR and FIRΔexon2 form homo- or heterodimers that complex with SAP155. SAP155, a subunit of the essential splicing factor 3b subcomplex in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1. In contrast, FIR was co-immunoprecipitated with Ku86 and DNA-PKcs. siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, whereas siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression. Thus the mechanical interaction of FIR/FIRΔexon2/SAP155 bridges c-myc and P27Kip1 expression, potentially integrates cell-cycle progression and c-myc transcription in cell. Bleomycin(BLM) is an anticancer agent that introduces DNA breaks. Because DNA breaks generate the recruitment of Ku86/Ku70 to bind to the broken DNA ends, the possible involvement of FIR and Ku86/Ku70 interaction in the BLM-induced DNA damage repair response was investigated in this study. First, BLM treatment reduced SAP155 expression and increased FIR and FIRΔexon2 mRNA expression as well as the ratio of FIRΔexon2:FIR in hepatoblastoma cells (HLE and HLF). Second, FIR or FIRΔexon2 adenovirus vectors (Ad-FIR or Ad-FIRΔexon2) increased Ku86/Ku70 and P27Kip1 expression in vitro. Third, BLM decreased P27Kip1 protein expression, whereas increased P27Kip1 and γH2AX expression with Ad-FIRΔexon2. Together, the interaction of FIR/SAP155 modulates FIR splicing and involves in cell-cycle control or cell fate via P27Kip1 and c-myc in BLM-induced DNA damage pathway. This novel function of FIR splicing will contribute to clinical studies of cancer management through elucidating the mechanical interaction of FIR/FIRΔexon2/SAP155 as a potential target for cancer treatment.