PDIA1 acts as master organizer of NOX1/NOX4 balance and phenotype response in vascular smooth muscle.

Changes in vascular smooth muscle cell (VSMC) phenotype underlie disease pathophysiology and are strongly regulated by NOX NADPH oxidases, with NOX1 favoring synthetic proliferative phenotype and NOX4 supporting differentiation. Growth factor-triggered NOX1 expression/activity strictly depends on the chaperone oxidoreductase protein disulfide isomerase-A1 (PDIA1). Intracellular PDIA1 is required for VSMC migration and cytoskeleton organization, while extracellular PDIA1 fine-tunes cytoskeletal mechanoadaptation and vascular remodeling. We hypothesized that PDIA1 orchestrates NOX1/NOX4 balance and VSMC phenotype. Using an inducible PDIA1 overexpression model in VSMC, we showed that early PDIA1 overexpression (for 24-48 h) increased NOX1 expression, hydrogen peroxide steady-state levels and spontaneous VSMC migration distances. Sustained PDIA1 overexpression for 72 h and 96 h supported high NOX1 levels while also increasing NOX4 expression and, remarkably, switched VSMC phenotype to differentiation. Differentiation was preceded by increased nuclear myocardin and serum response factor-response element activation, with no change in cell viability. Both NOX1 and hydrogen peroxide were necessary for later PDIA1-induced VSMC differentiation. In primary VSMC, PDIA1 knockdown decreased nuclear myocardin and increased the proliferating cell nuclear antigen expression. Newly-developed PDIA1-overexpressing mice (TgPDIA1) exhibited normal general and cardiovascular baseline phenotypes. However, in TgPDIA1 carotids, NOX1 was decreased while NOX4 and calponin expressions were enhanced, indicating overdifferentiation vs. normal carotids. Moreover, in a rabbit overdistension injury model during late vascular repair, PDIA1 silencing impaired VSMC redifferentiation and NOX1/NOX4 balance. Our results suggest a model in which PDIA1 acts as an upstream organizer of NOX1/NOX4 balance and related VSMC phenotype, accounting for baseline differentiation setpoint.

Proteasome and heat shock protein 70 (HSP70) inhibitors as therapeutic alternative in multiple myeloma.

HSP70 connects multiple signaling pathways that work synergistically to protect tumor cells from death by proteotoxic stress and represents a possible target to establish a new approach for multiple myeloma treatment. Therefore, bioluminescent cell lines RPMI8226-LUC-PURO and U266-LUC-PURO were treated with HSP70 (VER155008) and/or proteasome (bortezomib) inhibitors and immunodeficient mice were used for subcutaneous xenograft models to evaluate tumor growth reduction and tumor growth inhibition after treatment. Bioluminescence imaging was used to follow tumor response. Treatment with bortezomib showed ∼60% of late apoptosis in RPMI8226-LUC-PURO (without additional benefit of VER155008 in this cell line). However, U266-LUC-PURO showed ∼60% of cell death after treatment with VER155008 (alone or with bortezomib). RPMI8226-LUC-PURO xenograft presented tumor reduction by bioluminescence imaging after treatment with bortezomib, VER155008 or drug combination compared to controls. Treatment with bortezomib, alone or combined with VER155008, showed inhibition of tumor growth assessed by bioluminescence imaging after one week in both RPMI8226-LUC-PURO and U266-LUC-PURO cell lines when compared to controls. In conclusion, our study shows that the combination of proteasome and HSP70 inhibitors induced cell death in tumor cells in vitro (late apoptosis induction) and in vivo (inhibition of tumor growth) with special benefit in U266-LUC-PURO, bearing 17p deletion.

TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) stable silencing increases late apoptosis by upregulation of caspase 9 and APAF1 in RPMI8226 multiple myeloma cell line.

BACKGROUND: TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) modulates apoptotic pathways preventing the formation of the apoptosome complex. Our group previous study showed that 90% of patients' multiple myeloma (MM) marrow-derived plasma cells present TRIAP1 overexpression as compared to normal plasma cells. Due to high prevalence and lack of information on TRIAP1's role in MM biology, we decided to explore the impact of TRAIP1 through stable gene silencing in MM cell lines and its effect on cell cycle and apoptosis. METHODS: TRIAP1 expression was examined in MM cell lines by quantitative real time PCR. Cell lines were submitted to transduction with lentiviral vector encoding a TRIAP1-specific short hairpin RNA (shRNA) and, as control, encoding a non-targeting shRNA (scramble). Apoptosis was assessed by flow cytometry with annexin V and propidium iodide staining (the later also used for cell cycle), APAF1 and Caspase 9 apoptosome related genes expression and Caspase 9 and Caspase 3/7 activity. RESULTS: RPMI8226 and U266 cell lines were chosen for transduction experiments since they present higher levels of TRIAP1 expression. Inhibition of TRIAP1 in RPMI8226 cells increased the percentage of apoptotic cells, accompanied by increased expression of APAF1 and Caspase 9, and Caspase 9 and Caspase 3/7 activity. Transduced U266 cell line did not show sustained inhibition of TRIAP1 expression nor apoptosis induction. CONCLUSION: Stable silencing of TRIAP1 induces late apoptosis through APAF1/Caspase 9 pathway at least in RPMI8226 cell line, suggesting that it could be exploited as a potential target at least for a subgroup of MM patients. GENERAL SIGNIFICANCE: In the present study, we demonstrated effects of TRIAP1 silencing on RPMI8226 MM cell line and established its mechanism mediated through APAF1 and Caspase 9. No relevant effect was found after gene silencing in U266 cell line.

Genetic barcode sequencing for screening altered population dynamics of hematopoietic stem cells transduced with lentivirus.

Insertional mutagenesis has been associated with malignant cell transformation in gene therapy protocols, leading to discussions about vector security. Therefore, clonal analysis is important for the assessment of vector safety and its impact on patient health. Here, we report a unique approach to assess dynamic changes in clonality of lentivirus transduced cells upon Sanger sequence analysis of a specially designed genetic barcode. In our approach, changes in the electropherogram peaks are measured and compared between successive time points, revealing alteration in the cell population. After in vitro validation, barcoded lentiviral libraries carrying IL2RG or LMO2 transgenes, or empty vector were used to transduce mouse hematopoietic (ckit+) stem cells, which were subsequently transplanted in recipient mice. We found that neither the empty nor IL2RG encoding vector had an effect on cell dynamics. In sharp contrast, the LMO2 oncogene was associated with altered cell dynamics even though hematologic counts remained unchanged, suggesting that the barcode could reveal changes in cell populations not observed by the frontline clinical assay. We describe a simple and sensitive method for the analysis of clonality, which could be easily used by any laboratory for the assessment of cellular behavior upon lentiviral transduction.

Bone marrow stem cells in facial nerve regeneration from isolated stumps.

INTRODUCTION: Severe lesions in the facial nerve may have extensive axonal loss and leave isolated stumps that impose technical difficulties for nerve grafting. METHODS: We evaluated bone marrow stem cells (BMSC) in a silicone conduit for rat facial nerve regeneration from isolated stumps. Group A utilized empty silicone tubes; in groups B-D, the tube was filled with acellular gel; and, in groups C and D, undifferentiated BMSC (uBMSC) or Schwann-like cells differentiated from BMSC (dBMSC) were added, respectively. Compound muscle action potentials (CMAPs) were measured, and histology was evaluated. RESULTS: Groups C and D had the highest CMAP amplitudes. Group C had shorter CMAP durations than groups A, B, and D. Distal axonal number and density were increased in group C compared with groups A and B. CONCLUSIONS: Regeneration of the facial nerve was improved by both uBMSC and dBMSC in rats, yet uBMSC was associated with superior functional results.

Mesenchymal bone marrow stem cells within polyglycolic acid tube observed in vivo after six weeks enhance facial nerve regeneration.

Autografting is the gold-standard method for facial nerve repair with tissue loss. Its association with high-quality scaffolds and cell implants has disclosed distinct experimental outcomes. The aim of this study was to evaluate the functional and histological effects of bone marrow stem cells (BMSC) combined with polyglycolic acid tube (PGAt) in autografted rat facial nerves. After neurotmesis of the mandibular branch of the rat facial nerve, surgical repair consisted of nerve autografting (groups A-E) contained in pGAT (groups B-E), filled with basement membrane matrix (groups C-E) with undifferentiated BMSC (group D) or Schwann-like cells that had differentiated from BMSC (group E). Axon morphometrics and an objective compound muscle action potentials (CMAP) analysis were conducted. Immunofluorescence assays were carried out with Schwann cell marker S100 and anti-ß-galactosidase to label exogenous cells. Six weeks after surgery, animals from either cell-containing group had mean CMAP amplitudes significantly higher than control groups. Differently from other groups, facial nerves with Schwann-like cell implants had mean axonal densities within reference values. This same group had the highest mean axonal diameter in distal segments. We observed expression of the reporter gene lacZ in nerve cells in the graft and distally from it in groups D and E. Group-E cells had lacZ coexpressed with S100. In conclusion, regeneration of the facial nerve was improved by BMSC within PGAt in rats, yet Schwann-like cells were associated with superior effects. Accordingly, groups D and E had BMSC integrated in neural tissue with maintenance of former cell phenotype for six weeks.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6.

BACKGROUND: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. METHODS: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. RESULTS: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. CONCLUSIONS: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.

Evaluation of economically important traits from sixteen parental strains of the silkworm Bombyx mori L (Lepidoptera: Bombycidae).

The classification and characterization of silkworm strains are important for sericulture, which is supported by the constant development of new hybrids. In this study, 16 parental strains of Bombyx mori L from the germplasm banks of the Universidade Estadual de Maringá--UEM, and Associação dos Criadores de Bicho-da-Seda de Nova Esperança e Regiões Sericícolas do Paraná--ACESP, were evaluated regarding biological and productive traits economically important. The Chinese C122-B and C121-A, and the Japanese HA-A and HA-B strains yielded the highest cocoon weight, which is related to the raw silk percentage. Our data will be useful in breeding programs for the production of superior silkworm strains and hybrids.

Evaluation of economically important traits from sixteen parental strains of the silkworm Bombyx mori L (Lepidoptera: Bombycidae) / Avaliação de características economicamente importantes de dezesseis linhagens do bicho-da-seda Bombyx mori L (Lepidoptera: Bombycidae)

The classification and characterization of silkworm strains are important for sericulture, which is supported by the constant development of new hybrids. In this study, 16 parental strains of Bombyx mori L from the germplasm banks of the Universidade Estadual de Maringá - UEM, and Associação dos Criadores de Bicho-da-Seda de Nova Esperança e Regiões Sericícolas do Paraná - ACESP, were evaluated regarding biological and productive traits economically important. The Chinese C122-B and C121-A, and the Japanese HA-A and HA-B strains yielded the highest cocoon weight, which is related to the raw silk percentage. Our data will be useful in breeding programs for the production of superior silkworm strains and hybrids.

A classificação e caracterização de linhagens de Bombyx mori L é importante para a sericicultura, uma vez que essa atividade é sustentada pelo constante desenvolvimento de novos híbridos da espécie. Neste trabalho, 16 linhages parentais de B. mori do banco de germoplasma da Universidade Estadual de Maringá - UEM, e da Associação dos Criadores de Bicho-da-Seda de Nova Esperança e Regiões Sericícolas do Paraná - ACESP, foram avaliadas em relação às características biológicas e produtivas consideradas economicamente importantes. As linhagens C122-B e C121-A, de origem Chinesa, e as HA-A e HA-B, Japonesas, apresentaram o maior peso de casulo (CW), o qual é associado ao teor de seda (RSP). Os resultados apresentados neste trabalho podem ser utilizados em vários programas de melhoramento visando à produção de linhagens e híbridos geneticamente superiores.