Enhanced Mitochondrial DNA Repair Resuscitates Transplantable Lungs Donated After Circulatory Death.

BACKGROUND: Transplantation of lungs procured after donation after circulatory death (DCD) is challenging because postmortem metabolic degradation may engender susceptibility to ischemia-reperfusion (IR) injury. Because oxidative mitochondrial DNA (mtDNA) damage has been linked to endothelial barrier disruption in other models of IR injury, here we used a fusion protein construct targeting the DNA repair 8-oxoguanine DNA glycosylase-1 (OGG1) to mitochondria (mtOGG1) to determine if enhanced repair of mtDNA damage attenuates endothelial barrier dysfunction after IR injury in a rat model of lung procurement after DCD. MATERIALS AND METHODS: Lungs excised from donor rats 1 h after cardiac death were cold stored for 2 h after which they were perfused ex vivo in the absence and presence of mt-OGG1 or an inactive mt-OGG1 mutant. Lung endothelial barrier function and mtDNA integrity were determined during and at the end of perfusion, respectively. RESULTS AND CONCLUSIONS: Mitochondria-targeted OGG1 attenuated indices of lung endothelial dysfunction incurred after a 1h post-mortem period. Oxidative lung tissue mtDNA damage as well as accumulation of proinflammatory mtDNA fragments in lung perfusate, but not nuclear DNA fragments, also were reduced by mitochondria-targeted OGG1. A repair-deficient mt-OGG1 mutant failed to protect lungs from the adverse effects of DCD procurement. CONCLUSIONS: These findings suggest that endothelial barrier dysfunction in lungs procured after DCD is driven by mtDNA damage and point to strategies to enhance mtDNA repair in concert with EVLP as a means of alleviating DCD-related lung IR injury.

Prospects for skin cancer treatment and prevention: the potential contribution of an engineered virus.

Nonmelanoma skin cancers are among the most common human malignancies. Although typically not lethal, they are responsible for tissue deformity and substantial morbidity, particularly in high-risk populations. Solar UVB radiation-a major etiologic factor for this kind of malignancy-produces DNA lesions such as cyclobutane pyrimidine dimers and 6-4 photoproducts in skin. These lesions are removed through nucleotide excision repair because humans lack a DNA glycosylase required to initiate base excision repair of pyrimidine-pyrimidine photoproducts but produce all the other proteins required for this process. In this issue, Johnson et al. show that a DNA glycosylase derived from Chlorella virus and engineered to enhance tissue penetration and nuclear localization can remove UVB-induced DNA lesions in a human skin equivalent model and that the protein can be incorporated into a topical formulation for the prevention and treatment of UVB-induced DNA damage. These results suggest that such an enzyme may be incorporated into regimens for the chemoprevention of skin cancers.

TAT-mediated delivery of a DNA repair enzyme to skin cells rapidly initiates repair of UV-induced DNA damage.

UV light causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the United States. Although human cells possess a mechanism (nucleotide excision repair) to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated before repair of these photoproducts. Although human cells have all the enzymes necessary to complete an alternate repair pathway, base excision repair (BER), they lack a DNA glycosylase that can initiate BER of dipyrimidine photoproducts. Certain prokaryotes and viruses produce pyrimidine dimer-specific DNA glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (transcriptional transactivator, TAT). Here, we demonstrate that the Cv-pdg-NLS-TAT protein was delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. These data suggest a potential strategy for prevention of human skin cancer.

Topical treatment with OGG1 enzyme affects UVB-induced skin carcinogenesis.

Nonmelanoma skin cancer resulting from UVB exposure is a large and growing problem in the United States. Production of reactive oxygen species (ROS) during the UVB-induced inflammatory response results in the formation of oxidative DNA adducts such as 8-hydroxy-2-deoxyguanine (8-oxo-dG), which have been shown to contribute to the development of this cancer. The 8-oxoguanine DNA glycosylase (OGG1) enzyme repairs 8-oxo-dG adducts, suggesting that enhancing its activity in the skin might increase 8-oxo-dG repair thus preventing skin cancer development. We therefore used the SKH-1 murine model to examine the effect of topically applied OGG1 on UVB-induced skin cancer development. Mice were exposed three times weekly to UVB followed immediately by topical treatment with a formulation of liposome-encapsulated OGG1 enzyme for 25 weeks. While this treatment did not affect UVB-induced tumor multiplicity, it did reduce tumor size and dramatically reduced tumor progression, as indicated by tumor grade. These results suggest that oxidative DNA damage contributes to the progression of UVB-induced skin tumors and that a topical formulation containing OGG1, perhaps in conjunction with other DNA repair enzymes such as T4 endonuclease V, could be used in populations at high risk for skin cancer development.