Upregulation of matrix metalloproteinase 1 and disruption of mitochondrial network in skin fibroblasts of patients with MERRF syndrome.

By using cDNA microarray and RT-PCR techniques, we investigated the genome-wide alteration of gene expression in skin fibroblasts from patients with myoclonic epilepsy and ragged-red fibers (MERRF) syndrome. By screening for the genes with altered levels of expression, we first discovered that matrix metalloproteinase 1 (MMP1) was highly induced in the primary culture of skin fibroblasts of a female patient in a four-generation family with MERRF syndrome. This phenomenon was confirmed in skin fibroblasts from three other MERRF patients harboring about 85% of mtDNA with A8344G mutation. A further study revealed that the expression of MMP1 could be further induced by treatment of the skin fibroblasts with 200 microM hydrogen peroxide (H2O2) and inhibited by 1 mM N-acetylcysteine. Moreover, the intracellular level of H2O2 in skin fibroblasts of the female MERRF patient was higher than those of the asymptomatic family members and age-matched healthy controls. These findings imply that the increase in the expression of MMP1 may represent one of the responses to the increased oxidative stress in the skin fibroblasts of MERRF patients. We suggest that in affected tissues the oxidative stress-elicited overexpression of MMP1, and probably other matrix metalloproteinases involved in cytoskeleton remodeling, may play an important role in the pathogenesis and progression of mitochondrial encephalomyopathies such as MERRF syndrome.

Chromosome positional effects of gene expressions after cellular senescence.

Normal human fibroblasts stop dividing after a limited number of cell divisions termed cellular senescence. Telomere shortening has been shown to be the main factor that causes cellular senescence, however, the molecular mechanism of how telomere shortening causes cellular senescence is unclear. Here we analyze the relationship between gene expressions and their chromosomal locations during cellular senescence. It appears that the expression of genes located in chromosome 4 is preferentially altered after senescence. Moreover, we identify four chromosomal loci in which gene expressions are affected by senescence. Finally, we show that there is no preferential alteration of telomere-proximal genes during cellular senescence, implying that cellular senescence is not caused by derepression of telomere-proximal genes.

Increased expression of manganese-superoxide dismutase in fibroblasts of patients with CPEO syndrome.

Alterations in the expression of free radical scavenging enzymes and production of reactive oxygen species (ROS) in tissue cells may contribute to the pathogenesis of mitochondrial diseases such as chronic progressive external ophthalmoplegia (CPEO) syndrome. Since the mitochondria with impaired respiratory function in affected tissues generate more ROS via electron leakage, we examined the expression levels of free radical scavenging enzymes in primary culture of muscle fibroblasts of eight patients with CPEO syndrome. The results showed that the enzyme activity and protein levels of Mn-SOD of the fibroblasts from CPEO patients were significantly increased but those of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) were not increased compared with controls. A similar pattern was observed in the mRNA levels of Mn-SOD and GPx in muscle fibroblasts of all CPEO patients. The activity ratios of Mn-SOD/catalase and Mn-SOD/GPx in muscle fibroblasts of the CPEO patients were increased 1.7-3.4 and 1.8- to 5.3-fold, respectively, compared to those of the controls. Moreover, by using flow cytometry we found that the production of O2(*-) and H2O2 in the fibroblasts was about 2 times higher than those of controls. The 8-OHdG/dG ratios in total DNA of muscle biopsies from three CPEO patients were much higher than those of age-matched controls as determined by high performance liquid chromatography (HPLC). In the light of these findings, we suggest that the increase in expression of Mn-SOD, ROS production and oxidative damage in affected tissues may play an important role in the pathogenesis and progression of the CPEO syndrome.

Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer.

BACKGROUND: Molecular analysis of mitochondrial DNA (mtDNA) has provided a final diagnosis for many of the mitochondrial diseases. We evaluated the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) to determine whether the system could replace the conventional restriction fragment length polymorphism (RFLP) analysis by the agarose gel electrophoresis for the detection of the mtDNA mutation. METHODS: Three members of a family with MELAS syndrome and four members of a family with MERRF syndrome were recruited for this study. After PCR and restriction enzyme digestion, DNA fragments were separated on the Agilent 2100 bioanalyzer in conjunction with the DNA 500 and DNA 1000 Labchip kits and by electrophoresis on precast 3% agarose gels. RESULTS: The data generated by the DNA 500 and DNA 1000 assays using the Agilent 2100 bioanalyzer showed a lower percentage error and a better reproducibility as compared to those obtained by the conventional method. CONCLUSION: Based on the performance of the bioanalyzer, we suggest that this novel Labchip is adequate to replace the current RFLP analysis by the agarose gel electrophoresis for mtDNA mutation detection.