Abstract 12613: Apurinic/apyrimidinic Endonuclease/redox Factor-1 Gene Enhances Anti-apoptotic Function of Cardiac Progenitor Cells via TAK1-Activation and Promotes Cardiac Regeneration in Myocardial Infarction

Introduction: Overcoming the poor survival of cell grafts is an indispensable mission in cell therapy. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1) is known as a multifunctional enzyme to encourage cell survival, whereas the role of APE1 in cardiac cell therapy is still unknown. Hypothesis: APE1 overexpression in cardiac progenitor cells (CPC) ameliorates the effect of cardiac cell therapy. Methods: CPCs from 8-10 week-old C57BL/6 mice hearts were transfected with APE1-DsRed gene (APE1-CPC) or DsRed gene (Control [Ct]-CPC). The apoptosis induced by oxidative stress was assessed in APE1-/Ct-CPCs, and in neonatal rat cardiomyocytes (NRCM) within the co-culture system of APE1-/Ct-CPCs. Western blot analysis indicated the cellular signal to protect CPC via APE1 enzyme. To evaluate the effect of APE1 overexpression in cell therapy, we transplanted APE1-CPCs and Ct-CPCs into the mice myocardial infarction (MI) model and assessed the pathophysiological role of APE1 with functional and histological analysis. Results: Under the oxidative stress condition, APE1 overexpression inhibited the apoptosis of CPCs and accelerated TAK1 activation (Ct-CPC : APE1-CPC = 1.5±0.4 : 3.3±1.6 fold, p<0.05), and consequently NfKB phosphorylation in CPCs. In the co-culture system, the apoptosis of NRCMs was inhibited with APE1-CPCs compared to that with Ct-CPCs. In vivo, in the mice MI model, the number of total CPC grafts and cardiac α-actinin-positive graft CPCs were significantly larger in APE1-CPC injected mice (APE1 mice) compared to Ct-CPC injected mice (Ct mice) at 7 days after implantation. Eventually, the left ventricular ejection fraction of APE1 mice was significantly improved compared to Ct mice (Ct mice : APE1 mice = +3.1±6.7 : +11.3±4.0%, p<0.05) and was accompanied with the attenuation of fibrosis at 28 days after implantation. Conclusions: APE1 gene inhibited the apoptosis of CPCs and host cells against oxidative stress via the activation of TAK1-NFkB pathway, which is a novel insight into the stress response of APE1 enzyme. Furthermore, APE1-CPC grafts that effectively survived in the ischemic heart restored cardiac dysfunction and attenuated myocardial infarct size, and may be an innovative strategy to reinforce cardiac cell therapy.

Developing Kernel and Rachis Node Induce the Trichothecene Pathway of Fusarium graminearum During Wheat Head Infection

The fungal pathogen Fusarium graminearum is the most common agent of Fusarium head blight (FHB) in small grain cereals and cob rot of maize. The threat posed by this fungus is due to a decrease in yield and, additionally, mycotoxin contamination of the harvested cereals. Among the mycotoxins, trichothecenes influence virulence of F. graminearum in a highly complex manner that is strongly host- as well as chemotype-specific. The factors inducing mycotoxin production during plant infection are still unknown. To evaluate the induction of the trichothecene pathway, the green fluorescence protein (GFP) gene was fused to the promoter of the TRI5 gene coding for the trichodiene synthase and integrated into the genome by homologous integration. The resulting mutant contains a fully functional TRI5 gene ensuring virulence on wheat and exhibits GFP driven by the endogenous TRI5 promoter. We are now able to monitor the induction of trichothecenes under real-time conditions. To localize the fungus in the plant tissue, the dsRed gene was integrated under constitutive control of the glycerol-3-phosphate dehydrogenase (gpdA) promoter. We are now able to show that, first, induction of GFP as well as trichothecene production in the reporter strain reflects TRI5 induction and trichothecene production in the wild type; second, expression of TRI5 is inducible during growth in culture; and, third, trichothecene production is not uniformly induced during the onset of infection but is tissue specific during fungal infection of wheat.

Expression of Transgenes from Newcastle Disease Virus with a Segmented Genome

ABSTRACT Paramyxoviruses belong to the Paramyxoviridae family of the order Mononegavirales. They have a nonsegmented negative-stranded RNA genome and can cause a number of diseases in humans and animals. We generated a recombinant Newcastle disease virus (NDV) possessing a two-segmented genome. Each genomic segment is flanked by authentic NDV 3′ and 5′ noncoding termini allowing for efficient replication and transcription. A reporter gene encoding green fluorescent protein (GFP) was inserted into one segment, and a red fluorescent protein dsRed gene was inserted into the other segment in order to easily detect the replication and transcription of segments in infected cells. The rescued viruses grew well and were stable in embryonated chicken eggs over multiple passages. We were able to detect the expression of both reporter genes in the same cell infected with the virus possessing a segmented genome, and viral particles can contain either one or two types of RNA segments. We also rescued a two-segmented virus expressing GFP and the severe acute respiratory syndrome-associated coronavirus spike S protein, which is about 200 kDa. The chimeric virus extends the coding capacity of NDV by 30%, suggesting that the two-segmented NDV can be used for development of vaccines or gene therapy vectors carrying long and multiple transgenes.

Effect of Inhibition of Grb2 Expression on BCR-ABL Mediated Transformation of Primary Human Hematopoietic Cells.

The critical mechanisms underlying human hematopoietic stem cell (HSC) transformation by the BCR-ABL gene in chronic myeloid leukemia (CML) are still not well understood. Since treatment with imatinib fails to eliminate primitive CML hematopoietic cells, there is a pressing need to identify additional mechanisms that can be targeted to enhance elimination of CML stem cells. A tyrosine residue at position 177 (Y177) in the BCR-ABL protein binds the adapter protein Grb2 and appears to play an important role in BCR-ABL induced myeloid leukemogenesis in murine CML models. We have recently shown that a tyrosine to phenylalanine mutation of Y177 (Y177F) also results in significant reduction of abnormalities in proliferation and differentiation in BCR-ABL expressing human CD34+ cells and in BCR-ABL induced activation of Ras, Akt and STAT5 signaling (Cancer Res200767:7045). Since Grb2 signaling is implicated in several other human malignancies and likely plays an important role in signaling downstream of BCR-ABL, we were interested in investigating the role of Grb2 in BCR-ABL-mediated transformation of primary human hematopoietic cells. Cord blood (CB) CD34+ cells were transduced with bicistronic retrovirus vectors coexpressing wild type or Y177F mutated BCR-ABL genes with the GFP gene. Coimmunoprecipitation studies confirmed the association of Grb2 with BCR-ABL and was abrogated by the Y177F mutation. We next investigated the effect of inhibition of Grb2 expression in BCR-ABL transduced human CD34+ cells. CB CD34+ cells were co-transduced with retroviral vectors coexpressing the BCR-ABL and GFP genes or control vectors expressing the GFP gene alone together with lentivirus vectors coexpressing Grb2 shRNA constructs and the dsRed gene or the dsRed gene alone. Cells expressing CD34+, GFP and dsRed were selected using flow cytometry. Western blot analysis indicated that Grb2 levels were reduced by 80.8±14.5% (n=3, P < 0.014); in CD34+ cells transduced with BCR-ABL and Grb2 shRNA compared with cells expressing BCR-ABL alone. Expression of Grb2 shRNA resulted in significant reduction in expansion of BCR-ABL expressing CD34+ cells compared with cells expressing BCR-ABL alone after 7 days of culture in serum free medium (SFM) with low concentrations of growth factor similar to those present in bone marrow stroma conditioned medium (BA alone 48.5±7.7; BA+Grb2 shRNA, 16.7±4.7, n=3). Co-expression of Grb2 shRNA also resulted in significant reduction in the total number of colonies generated by BCR-ABL expressing CD34+ cells in methylcellulose progenitor culture (BA, 123±31; BA+Grb2, 40±9; n=3, p<0.03), with reduction being seen mainly for erythroid colonies. Expression of Grb2 shRNA also reduced cell expansion from control CD34+ expressing GFP alone but the difference was not statistically significant. Similarly Grb2 inhibition did not result in a significant difference in the number of colonies generated from control CD34+ cells. Grb2 inhibition was associated with reduced levels of P-MAPK, but not P-AKT in BCR-ABL expressing CD34+ cells. Interestingly Grb2 inhibition also results in decreased levels of P-STAT5 consistent with the observed reduction in erythroid colonies. In conclusion RNAi mediated inhibition of Grb2 expression results in significant inhibition of BCR-ABL induced proliferation of human CD34+ cells proliferation, indicating an important role for Grb2 in BCR-ABL mediated transformation of CML cells. These observations support further evaluation of inhibition of Grb2 signaling in targeting of CML stem cells.