Hematopoietic Stem Cell Repopulation Modulated by ROS-Detoxifying Enzymes,

Abstract 4172 A tightly regulated level of reactive oxygen species (ROS) is critical for proper functioning of hematopoietic stem cells (HSCs) and high levels of ROS were shown to be able to exhaust the HSC compartment under stress conditions. Intracellular excessive ROS are normally scavenged by antioxidant enzymes. Superoxide anion radical (O) converts into hydrogen peroxide (H2O2) by superoxide dismutase (SOD), and then detoxified by catalase and glutathione peroxidase (GPx) into water. We hypothesize that maintaining a low redox status by ectopically expressing these ROS-detoxifying enzymes, namely manganese-containing SOD (MnSOD) and catalse is able to augment HSC regeneration. In this study, we explored the potential usefulness of MnSOD-PL, a drugable MnSOD plasmid and lipfectin complex, in enhancing the efficiency of HSC transplant. We have also investigated the effects of overexprressed MnSOD or catalase via retroviral delivery on the engraftment efficiency of transduced HSCsSCs. Our results showed that the basal mRNA levels of MnSOD and catalase in long-term and short-term HSCs were less than 1–3% of that of the cellular beta-actin. The mRNA levels were 2 to 4 fold higher in the short-term repopulating HSCs than those in the long-term repopulating HSCs. Irradiation did not induce enzymes expression except catalase in megakaryocytic-erythroid progenitors (MEP) and GPx1 in granulocyte-monocytic progenitors (GMP), which were significantly increased after exposure to 800 cGy (p=0.05) and 400 cGy (p=0.046) radiations, respectively. Administration of MnSOD-PL before TBI conferred significant radiation protection for irradiated recipient mice. On the one hand, the recovery of endogenous hematopoietic could be boosted. On the other hand, the function of the donor HSCs was more preserved from the ROS damage and proliferative stress, and those preserved HSCs were able to replenish the BM when a secondary stress occurred. However, the beneficial effects of MnSOD-PL seemed to be largely via the host environment, since our subsequent experiment showed that overexpression of MnSOD in HSCs only provided minimal benefits to the hematopoietic reconstitution. This potential utility of MnSOD-PL suggests an alternative therapeutic strategy to enhance the HSCs engraftment efficiency in bone marrow transplant. In order to further explore the effects of catalase in HSC protection, human catalase was over-expressed in mouse LKS cells via retroviral delivery in comparison with MnSOD. In the colony-forming cell assay, CFU-M colonies were significantly higher in catalase or MnSOD over-expressed group than those of vector control group in the 200 cGy irradiated plates (p=0.01). Within three months after competitive transplantation, the engraftment levels were increased to 2.7–3.4 fold in catalase group than those of vector group. After 200 cGy re-irradiation engraftment levels were significantly increased to 6.4–7.9 fold in the catalase group (p<0.05). The results showed that overexpression of catalase alone could significantly improve the HSC repopulation. Despite no significant difference in the primary transplantation experiments, MnSOD over-expression also showed a positive effect on the HSC repopulation capacity in the serial transplantation. Taken together, these results demonstrate that maintaining low ROS by ectopically expressing the ROS-detoxifying enzymes is a viable approach to improve HSC functions under stress and damaging conditions. Disclosures: No relevant conflicts of interest to declare.

Retroviral Delivery of Platelet-Derived Growth Factor to Spinal Cord Progenitor Cells Drives the Formation of Intramedullary Gliomas

BACKGROUND High-grade gliomas of the spinal cord are poorly understood tumors that are very commonly associated with bad outcomes. The transforming effects of platelet-derived growth factor (PDGF) on spinal cord glial progenitor cells may play an important role in the development of these tumors. OBJECTIVE To investigate the possible tumor-initiating effects of PDGF overexpression in the spinal cord, we delivered a PDGF retrovirus directly into the substance of the spinal cord. METHODS The spinal cords of wild-type adult rats were surgically exposed and injected with 106 colony-forming units of a green fluorescent protein-tagged, PDGF-expressing retrovirus. A control virus was injected to assess the cell types that become infected during retroviral delivery to the spinal cord. RESULTS It was observed that PDGF overexpression in the spinal cord causes morbidity from high-grade intramedullary glioma formation between 27 and 49 days after PDGF retrovirus injection. Retroviral transduction was highly efficient with 100% of injected animals displaying the tumor phenotype. The tumors produced were highly proliferative, were locally invasive, and displayed the immunophenotype of virus-targeted glial progenitor cells (Olig2+PDGFR+NG2+GFAP−). CONCLUSION PDGF is capable of driving glial progenitor cells within the adult spinal cord to form high-grade gliomas. Further investigation of PDGF signaling in the spinal cord is needed to better understand and treat these devastating tumors.

Dependence of antibody gene diversification on uracil excision

Activation-induced deaminase (AID) catalyses deamination of deoxycytidine to deoxyuridine within immunoglobulin loci, triggering pathways of antibody diversification that are largely dependent on uracil-DNA glycosylase (uracil-N-glycolase [UNG]). Surprisingly efficient class switch recombination is restored to ung−/− B cells through retroviral delivery of active-site mutants of UNG, stimulating discussion about the need for UNG's uracil-excision activity. In this study, however, we find that even with the overexpression achieved through retroviral delivery, switching is only mediated by UNG mutants that retain detectable excision activity, with this switching being especially dependent on MSH2. In contrast to their potentiation of switching, low-activity UNGs are relatively ineffective in restoring transversion mutations at C:G pairs during hypermutation, or in restoring gene conversion in stably transfected DT40 cells. The results indicate that UNG does, indeed, act through uracil excision, but suggest that, in the presence of MSH2, efficient switch recombination requires base excision at only a small proportion of the AID-generated uracils in the S region. Interestingly, enforced expression of thymine-DNA glycosylase (which can excise U from U:G mispairs) does not (unlike enforced UNG or SMUG1 expression) potentiate efficient switching, which is consistent with a need either for specific recruitment of the uracil-excision enzyme or for it to be active on single-stranded DNA.