Daniela B. Zanatta

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-β-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

BackgroundReactivation 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.MethodsB16 (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.ResultsHere 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.ConclusionsTo 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.

Bone marrow stem cells in facial nerve regeneration from isolated stumps

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. Muscle Nerve 48: 423–429, 2013

Reestablishment of p53/Arf and interferon- β pathways mediated by a novel adenoviral vector potentiates antiviral response and immunogenic cell death

Late stage melanoma continues to be quite difficult to treat and new therapeutic approaches are needed. Since these tumors often retain wild-type p53 and have a strong immunogenic potential, we developed a gene transfer approach which targets these characteristics. Previously, we have shown that combined gene transfer of p19Arf and interferon-beta (IFNβ) results in higher levels of cell death and superior immune-mediated antitumor protection. However, these experiments were performed using B16 cells (p53wt) with forced expression of the adenovirus receptor and also the mechanism of death was largely unexplored. Here we take advantage of a novel adenoviral vector (AdRGD-PG), presenting an RGD-modified fiber as well as a p53-responsive promoter, in order to investigate further potential benefits and cell death mechanisms involved with the combined transfer of the p19Arf and IFNβ genes to the parental B16 cell line. Simultaneous p19Arf and IFNβ gene transfer is more effective for the induction of cell death than single gene treatment and we revealed that p19Arf can sensitize cells to the bystander effect mediated by secreted IFNβ. Strikingly, the levels of cell death induced upon activating the p53/p19Arf and interferon pathways were higher in the presence of the AdRGD-PG vectors as compared to approaches using pharmacological mimetics and this was accompanied by the upregulation of antiviral response genes. Only combined gene transfer conferred immunogenic cell death revealed by the detection of key markers both in vitro and in vivo. Finally, whole-genome transcriptome analysis revealed unique expression profiles depending on gene function, including immune activation, response to virus and p53 signaling. In this way, cooperation of p19Arf and IFNβ activates the p53 pathway in the presence of an antiviral response elicited by IFNβ, culminating in immunogenic cell death.

Intratumoral Immunization by p19Arf and Interferon-β Gene Transfer in a Heterotopic Mouse Model of Lung Carcinoma

Therapeutic strategies that act by eliciting and enhancing antitumor immunity have been clinically validated as an effective treatment modality but may benefit from the induction of both cell death and immune activation as primary stimuli. Using our AdRGD-PG adenovector platform, we show here for the first time that in situ gene transfer of p19Arf and interferon-β (IFNβ) in the LLC1 mouse model of lung carcinoma acts as an immunotherapy. Although p19Arf is sufficient to induce cell death, only its pairing with IFNβ significantly induced markers of immunogenic cell death. In situ gene therapy with IFNβ, either alone or in combination with p19Arf, could retard tumor progression, but only the combined treatment was associated with a protective immune response. Specifically in the case of combined intratumoral gene transfer, we identified 167 differentially expressed genes when using microarray to evaluate tumors that were treated in vivo and confirmed the activation of CCL3, CXCL3, IL1α, IL1β, CD274, and OSM, involved in immune response and chemotaxis. Histologic evaluation revealed significant tumor infiltration by neutrophils, whereas functional depletion of granulocytes ablated the antitumor effect of our approach. The association of in situ gene therapy with cisplatin resulted in synergistic elimination of tumor progression. In all, in situ gene transfer with p19Arf and IFNβ acts as an immunotherapy involving recruitment of neutrophils, a desirable but previously untested outcome, and this approach may be allied with chemotherapy, thus providing significant antitumor activity and warranting further development for the treatment of lung carcinoma.

Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.

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.

Abstract 1236: Combined transfer of p19Arf and interferon-beta genes to mouse melanoma cells causes LC3B- and caspase-3-independent cell death and alters the expression of critical genes

Tumors cells are able to resist cell death and evade the immune system. This resistance can be caused by alterations in Trp53 itself or p19Arf loss and the reduced anti-tumor immune response may be caused by loss of interferon-β (IFN), an important immune-stimulatory cytokine. Here, we investigate the impact of p19Arf and IFN gene transfer in melanoma cells with regards to the expression profile of genes responsible for the induction of cell death. Recombinant adenoviral vectors with a p53-responsive promoter (PG) driving expression of p19Arf or IFN were applied alone or in combination in B16F10 cells in vitro (mouse melanoma, p53 wt). Cell death was evaluated by annexin/PI staining and MTT assays. LC3B expression was verified by western blot analysis of cells extracts. Caspase-3 activity was evaluated using a lentiviral vector which constitutively expressing luciferase protein linked to an ubiquitin site that can be cleaved by caspase-3. RNA expression profile was evaluated by microarray analysis of treated samples and validation of gene expression was performed by real-time PCR. We verified that combined gene transfer of p19Arf and IFN resulted in 74% hypodiploid cells whereas single gene transfer yielded only half of this number, as shown by cell cycle and MTT analysis. Although there was a high rate of cell death, only 25% of cells were stained for annexin/PI and activation of caspase-3 was reduced in cells treated with p19Arf and IFN combination compared to treatment of p19Arf alone. Also, there was no increase in LC3B expression when cells were treated with the p19Arf and IFN combination. These observations suggest that B16F10 cells are dying by other mechanisms rather than apoptosis or autophagy. Transduced cells were analyzed for gene expression profile and critical genes were selected for validation. Combination of p19Arf and IFN transduction in B16F10 cells revealed a synergistic effect modifying exclusively the expression of 1054 genes involved in cell death, immune system activation and cell cycle arrest. There was an increase of 4-20 fold in expression of MDM2, p53, p21Waf1, PUMA, MDM4, FOXO1, NR3C1, PHLDA3, RANBP9 and WDR46, and also an increase of 400-fold in p73 expression in cells treated with p19Arf and IFN combination. Conclusions: The use of p53-responsive vectors to express p19Arf and IFN promotes complementation of the p53/Arf and IFN pathways, resulting in a synergistic gene regulation of many essential functions in cancer cells, destabilizing cell cycle and cell organization in general, while promoting expression of genes related to cell death. As p73 is a transcription factor of caspase-1 expression, we hypothesize that this could be the major participant involved in death of these cells. Financial support: 2011/10656-5, 2011/50911-4 and 2013/25167-5 (FAPESP). Citation Format: Aline H. Ribeiro, Paulo R. Del Valle, Ruan F.V. Medrano, Daniel G. Ferrari, Daniela B. Zanatta, Bryan E. Strauss. Combined transfer of p19Arf and interferon-beta genes to mouse melanoma cells causes LC3B- and caspase-3-independent cell death and alters the expression of critical genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2015-1236

Abstract 3539: Modifications of adenoviral structure and genome improves transduction efficiency and transgene expression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Innovative strategies are required for cancer treatment, especially evident given that many patients relapse or do not respond to current therapeutics. Adenoviral vectors are powerful tools for gene transfer and our laboratory employs a vector where the fiber was modified to present the RGD peptide, considerably improving cell transduction. We developed a system that controls transgene expression by the PG promoter, which is responsive to p53 transcriptional activity. We utilize this system to deliver the human p53 and interferon beta cDNAs. The present study evaluated the transduction efficiency, the dependence of transgene expression on p53 status in human colorectal cancer cell lines HCT116 (p53 wt), HCT116p53-/- and HT29 (p53 mut) and the influence of the transgenes on cell viability. The vectors efficiently transduced all of the tested cell lines. Even at MOIs as low as 10, it was possible to see 42% of cells HCT116 positive for GFP using the AdRGDPGeGFP vector. Higher MOIs (50 and 100) did not present major differences in transduction efficiency (approximately 85%), but the GFP expression intensity increased approximately 14 fold. Cells were treated with doxorubicin (1μM) in order to stimulate p53 activity and when a MOI of 10 was used, 54% of cells were GFP positive with a 16 fold increase in the expression level, yet expression intensity rose 16 fold (MOI 50) and two fold (MOI 100), when compared with non-stimulated with doxorubicin. Since p53 expression is absent in the HCT116p53-/- cell line, we would not expect to observe GFP expression when using the PG promoter. However, very low level GFP activity was detected and was slightly induced upon treatment with doxorubicin, suggesting a p53-independent mechanism for transgene expression. However, restoring p53 in this cell line led to higher expression levels of GFP. We observed an important benefit mediated by our vectors (MOI of 25) for the reduction of cell viability mediated by doxorubicin, cisplatin and 5-fluorouracil (5-FU). The effect of doxorubicin was especially interesting, even at a low dose (0.625μM) the co-transduction with both the p53 and IFN-β vectors conferred a benefit higher than that seen with isolated transductions. The dose of 10μM cisplatin and 6.25μM 5-FU also provided a major benefit in reducing cell viability upon co-transduction. Both HCT116 and HCT116p53-/-responded in a similar way to gene transfer combined with chemotherapeutics, but HT29 was more sensitive to IFN-β when associated with doxorubicin (0.625μM) and cisplatin (doses range 10 - 1.25 μM). Thus, our system reveals an opportunity to improve transduction efficiency and transgene expression and, as a consequence, increase sensitivity to genotoxic stress. Moreover, the co-transfection of p53 and IFN-β may improve the efficacy of chemotherapeutics in colorectal cancer. Financial support: Sao Paulo Research Foundation (FAPESP) 2013/16074-3; 2013/25167-5. Citation Format: Paulo Roberto Del Valle, Daniela B. Zanatta, Bryan E. Strauss. Modifications of adenoviral structure and genome improves transduction efficiency and transgene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3539. doi:10.1158/1538-7445.AM2015-3539

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.