Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.
SummaryAlthough skeletal muscle cells can be generated from human induced pluripotent stem cells (iPSCs), transgene-free protocols include only limited options for their purification and expansion. In this study, we found that fluorescence-activated cell sorting-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 × 1011-fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of acid α-glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/Cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies.
Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. They resemble developing skeletal muscle and are histologically divided into two main subtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with their corresponding expression profiles. Genes altered at the transcriptional level by genomic imbalances were identified and the effect on expression was proportional to the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal compared with alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors.
RAS oncogenes are among the most frequently mutated genes in human cancer, but effective strategies for therapeutic inhibition of the RAS pathway have been elusive. Sprouty1 (SPRY1) is an upstream antagonist of RAS that is activated by extracellular signal-related kinase (ERK), providing a negative feedback loop for RAS signaling, and other evidence suggests that SPRY1 may have a tumor suppressor function. Studies of RAS status in the human childhood tumor rhabdomyosarcoma (RMS) indicated mutations in approximately half of the tumors of the embryonal rhabdomyosarcoma subtype (ERMS) but not the alveolar subtype (ARMS). ERMS tumors also showed overexpression of SPRY1, which was indeed upregulated by mutant RAS. However, we found that, in the presence of mutant RAS, the function of SPRY1 was changed from an antagonist to an agonist of RAS signaling. Thus, SPRY1 supported formation of activated ERK and mitogen-activated protein/ERK kinase and was essential for ERMS cell proliferation and survival. Conversely, silencing of SPRY1 in ERMS cells (but not ARMS cells) abolished their tumorigenicity in mice. Moreover, silencing of SPRY1 caused regression of established ERMS tumors (but not ARMS tumors) formed in xenograft settings. Our findings argue that SPRY1 inhibition can offer a therapeutic strategy to treat childhood RMS and possibly other tumors carrying oncogenic RAS mutations. Cancer Res; 70(2); 762-71. ©2010 AACR.
Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Improved treatment strategies have increased overall survival, but the response of approximately one-third of the patients is still poor. To increase the knowledge of RMS pathogenesis, we performed the first full transcriptome analysis of RMS using serial analysis of gene expression (SAGE). With a G-test for the simultaneous comparison of subsets of SAGE libraries of normal skeletal muscle, embryonal (ERMS) and alveolar (ARMS) RMS, we identified 251 differentially expressed genes. A literature-mining procedure demonstrated that 158 of these genes have not previously been associated with RMS or normal muscle. Gene Ontology (GO) analysis assigned 198 of the 251 genes to muscle-specific classes, including those involved in normal myogenic development, as well as tumor-related classes. Prominent GO classes were those associated with proliferation and actin reorganization, which are processes that play roles during early muscle development, muscle function, and tumor progression. Using custom microarrays, we confirmed the (up- or down-) regulation of 80% of 98 differentially expressed genes. Another SAGE library of 19- to 22-week-old fetal skeletal muscle was compared with the RMS and normal muscle transcriptomes. Cluster analysis showed that the RMS and fetal muscle SAGE libraries formed one cluster distinct from normal muscle samples. Moreover, the expression profile of 86% of the differentially expressed genes between normal muscle and RMS was highly similar in fetal muscle and RMS. In conclusion, the G-test is a robust tool for analyzing groups of SAGE libraries and correctly identifies genes marking the difference between fully differentiated skeletal muscle and RMS. This study not only substantiates the close association between embryonic myogenesis and RMS development but also provides a rich source of candidate genes to further elucidate the etiology of RMS or to identify diagnostic and/or prognostic markers.
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