Functional rescue of the glomerulosclerosis phenotype in Mpv17 mice by transgenesis with the human Mpv17 homologue

The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease

The human homologue of the murine glomerulosclerosis gene MPV17.

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Constitutively Activated Nuclear Factor-κB, but not Induced NF-κB, Leads to TRAIL Resistance by Up-Regulation of X-Linked Inhibitor of Apoptosis Protein in Human Cancer Cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-κB (NF-κB) has been implicated, but its exact role is controversial. We studied different cell lines displaying varying responses to TRAIL and found that TRAIL can activate NF-κB in all our cancer cell lines regardless of their TRAIL sensitivity. Inhibition of NF-κB via adenoviral expression of the IκB-α super-repressor only sensitized the TRAIL-resistant pancreatic cancer cell line Panc-1. Panc-1 cells harbor constitutively activated NF-κB, pointing to a possible role of preactivated NF-κB in protection from TRAIL. Furthermore, we could reduce X-linked inhibitor of apoptosis protein (XIAP) levels in Panc-1 cells by inhibition of constitutively activated NF-κB and sensitize Panc-1 cells to TRAIL by RNA interference against XIAP. These results implicate elevated XIAP levels caused by high basal NF-κB activity in TRAIL resistance and suggest that therapeutic strategies involving TRAIL can be abetted by inhibition of NF-κB and/or XIAP only in tumor cells with constitutively activated NF-κB. Copyright © 2006 American Association for Cancer Research

IFN-γ combined with targeting of XIAP leads to increased apoptosis-sensitisation of TRAIL resistant pancreatic carcinoma cells

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a specific and potent inducer of apoptosis in cancer cells, but the resistance of many tumour cells to TRAIL still represents a major hurdle for the clinical treatment of tumours with TRAIL. As apoptosis is regulated by the balance of activities of several anti-apoptotic factors and pro-apoptotic factors, we analysed the relative contribution of the two sides and found that down-regulation of Bcl-x L and in particular XIAP, but not c-Flip, sensitised the TRAIL resistant pancreatic cancer cell line Panc-1. A combination of both XIAP and Bcl-x L knock-downs showed no substantial added benefit indicating that both act in the same pathway. Notably, the degree of sensitisation by silencing of anti-apoptotic genes was further elevated by concomitantly increasing the pro-apoptotic potential in Panc-1 cells through over-expression of TRAIL-R1 or IFN-γ-mediated increases in caspase-8 levels. Similar sensitisation effects were obtained for another TRAIL-resistant pancreatic tumour cell line, AsPC-1. Our findings demonstrate that modulation of the balance between anti- and pro-apoptotic pathways from both sides by inhibition of apoptosis-antagonists and stimulation of pro-apoptotic factors provides the best way to enhance the anti-tumourigenic effect of TRAIL. © 2011 Elsevier Ireland Ltd

Redox Gene Therapy Protects Human IB-3 Lung Epithelial Cells Against Ionizing Radiation-Induced Apoptosis

Toxicity to nontumor-derived tissue has proven to be a significant obstacle in achieving therapeutic levels of gamma irradiation in the treatment of cancer. The formation of reactive oxygen species (ROS) such as superoxide radicals (O2-) following irradiation is thought to be a major determinant of cellular damage. To this end, we describe the generation of two recombinant adenoviral vectors expressing the radical-scavenging enzymes MnSOD and CuZnSOD to test therapeutic strategies of radioprotection. Using a human lung epithelial cell line (IB-3), we have demonstrated that infections with both Ad.CMVMnSOD or Ad.CMVCuZnSOD significantly increase both the levels of SOD protein and enzymatic activity as compared to control cells. This increase in SOD expression reduced the level of apoptosis at 72 hr post-irradiation by 50% as compared to mock- or Ad.CMVLacZ-infected cells. Such studies provide the foundation for radioprotective gene therapies in the treatment of cancer

Betulinic acid as new activator of NF-κB: molecular mechanisms and implications for cancer therapy

Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor-kappaB (NF-κB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-κB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-κB in a variety of tumor cell lines. NF-κB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-κB activation involved increased IKK activity and phosphorylation of IκB-α at serine 32/36 followed by degradation of IκB-α. Reporter assays revealed that NF-κB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-κB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-κB inhibition by transient or stable expression of IκB-α super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IκB-α super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-κB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-κB inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols. © 2005 Nature Publishing Group. All rights reserved

Age-dependent hypertension in Mpv17-deficient mice, a transgenic model of glomerulosclerosis and inner ear disease

The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/ - mice might be hypertensive. Indeed, our study revealed that Mpv17-/ - mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concommittantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension. Copyright (C) 1999 Elsevier Science Inc