Project description:Mutations in genes involved in dNTP metabolism can lead to tissue-specific mitochondrial depletion syndromes (MDS), likely because the expression of key enzymes is reduced to critical levels in post mitotic cells. Our goal was to establish an in vitro skeletal muscle cell model to study the muscle specificity of MDS associated with mitochondrial dNTP pool imbalance. We performed a comprehensive analysis at the mRNA level of enzymes and transporters responsible for dNTP pool imbalance in muscle cells in vitro and in vivo. Agilent Mouse Oligo Arrays 4x44K were utilized to examine expression levels in proliferating and differentiated C2C12 cells as well as in the mouse EDL (fast glycolytic) and soleus (slow oxidative) muscles. The comparison of mRNA expression profiles supports the reliability of our in vitro cell system.
Project description:Mutations in genes involved in dNTP metabolism can lead to tissue-specific mitochondrial depletion syndromes (MDS), likely because the expression of key enzymes is reduced to critical levels in post mitotic cells. Our goal was to establish an in vitro skeletal muscle cell model to study the muscle specificity of MDS associated with mitochondrial dNTP pool imbalance. We performed a comprehensive analysis at the mRNA level of enzymes and transporters responsible for dNTP pool imbalance in muscle cells in vitro and in vivo. Agilent Mouse Oligo Arrays 4x44K were utilized to examine expression levels in proliferating and differentiated C2C12 cells as well as in the mouse EDL (fast glycolytic) and soleus (slow oxidative) muscles. The comparison of mRNA expression profiles supports the reliability of our in vitro cell system. Proliferating mouse C2C12 myoblasts were collected at about 50 percent confluence. Myoblasts were then induced to differentiate in vitro into myotubes that were harvested after 96 hours and further purified to reduce the contribution of mononucleated cells present in the culture. Gene expression in C2C12 myoblasts and myotubes was compared with fully differentiated muscle fibers in vivo. To this aim, the extensor digitorum longus (EDL) and soleus hind limb muscles were isolated from adult CD1 mice. These muscles were selected because they have different metabolic (glycolytic vs. oxidative) and twitching (fast vs. slow) properties. Triplicate total RNA samples were submitted to gene expression profiling.
Project description:SILAC based protein correlation profiling using size exclusion of protein complexes derived from Mus musculus tissues (Heart, Liver, Lung, Kidney, Skeletal Muscle, Thymus)
Project description:SILAC based protein correlation profiling using size exclusion of protein complexes derived from seven Mus musculus tissues (Heart, Brain, Liver, Lung, Kidney, Skeletal Muscle, Thymus)
Project description:Perturbation of the deoxyribonucleotide triphosphate (dNTP) pool is recognized for contributing to the mutagenic processes involved in oncogenesis. The RAS gene family encodes well characterized oncoproteins whose structure and function are among the most frequently altered in several cancers. In this work, we show that fluctuation of the dNTP pool induces CG->TA mutations across the whole genome, including RAS gene at codons for glycine 12 and 13, known hotspots in cancers. Cell culture addition of the ribonucleotide reductase inhibitor thymidine increases the mutation frequency in nuclear DNA and leads to disruption of mitochondrial metabolism. Interestingly, this effect is counteracted by the addition of deoxycytidine. Finally, screening for the loss of hydrogen bonds detecting CG->TA transition in RAS gene of 135 patients with colorectal cancer confirmed the clinical relevance of this process. All together, these data demonstrate that fluctuation of intracellular dNTP pool alters the nuclear DNA and mitochondrial metabolism.
Project description:Perturbation of the deoxyribonucleotide triphosphate (dNTP) pool is recognized for contributing to the mutagenic processes involved in oncogenesis. The RAS gene family encodes well characterized oncoproteins whose structure and function are among the most frequently altered in several cancers. In this work, we show that fluctuation of the dNTP pool induces CG->TA mutations across the whole genome, including RAS gene at codons for glycine 12 and 13, known hotspots in cancers. Cell culture addition of the ribonucleotide reductase inhibitor thymidine increases the mutation frequency in nuclear DNA and leads to disruption of mitochondrial metabolism. Interestingly, this effect is counteracted by the addition of deoxycytidine. Finally, screening for the loss of hydrogen bonds detecting CG->TA transition in RAS gene of 135 patients with colorectal cancer confirmed the clinical relevance of this process. All together, these data demonstrate that fluctuation of intracellular dNTP pool alters the nuclear DNA and mitochondrial metabolism.
