Biomedical subjects

Michael V Miceli

Publications and source records attributed to Michael V Miceli.

4 recordsLinked to original sources

PubMed2005

Common and cell type-specific responses of human cells to mitochondrial dysfunction.

In yeast, mitochondrial dysfunction activates a specific pathway, termed retrograde regulation, which alters the expression of specific nuclear genes and results in increased replicative life span. In mammalian cells, the specific nuclear genes induced in response to loss of mitochondrial function are less well defined. This study characterizes responses in nuclear gene expression to loss of mitochondrial DNA sequences in three different human cell types: T143B, an osteosarcoma-derived cell line; ARPE19, a retinal pigment epithelium cell line; and GMO6225, a fibroblast cell population from an individual with Kearns-Sayre syndrome (KSS). Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure gene expression of a selection of glycolysis, TCA cycle, mitochondrial, peroxisomal, extracellular matrix, stress response, and regulatory genes. Gene expression changes that were common to all three cell types included up-regulation of GCK (glucokinase), CS (citrate synthase), HOX1 (heme oxygenase 1), CKMT2 (mitochondrial creatine kinase 2), MYC (v-myc myelocytomatosis viral oncogene homolog), and WRN (Werner syndrome helicase), and down-regulation of FBP1 (fructose-1, 6-bisphosphatase 1) and COL4A1 (collagen, type IV, alpha 1). RNA interference experiments show that induction of MYC is important in rho0 cells for the up-regulation of glycolysis. In addition, a variety of cell type-specific gene changes was detected and most likely depended upon the differentiated functions of the individual cell types. These expression changes may help explain the response of different tissues to the loss of mitochondrial function due to aging or disease.

Cell Line↗

PubMed2005

Nuclear gene expression changes due to mitochondrial dysfunction in ARPE-19 cells: implications for age-related macular degeneration.

PURPOSE: To measure changes in nuclear gene expression resulting from mitochondrial dysfunction in retinal pigment epithelial cells. METHODS: ARPE-19 retinal pigment epithelial cells were depleted of their mitochondrial (mt)DNA by passaging in a low concentration of ethidium bromide. Loss of mitochondrial DNA was determined by uridine auxotrophy and quantitative real-time polymerase chain reaction of isolated DNA. Loss of mitochondrial membrane potential was estimated by uptake of JC-1. Changes in nuclear gene expression were determined by quantitative real-time reverse transcription-polymerase chain reaction of isolated total RNA from ethidium-bromide-treated and untreated cells. Morphologic and phenotypic changes were determined by phase-contrast microscopy, sensitivity to the oxidant tert-butyl hydroperoxide (tBH), and invasion assay. RESULTS: ARPE-19 cells became auxotrophic for growth on uridine after eight passages in 50 ng/mL ethidium bromide. Quantitative PCR revealed almost complete loss of mitochondrial DNA (rho(0) cells). Uptake of JC-1 was reduced in the rho(0) cells, indicating reduction of mitochondrial membrane potential. Quantitative RT-PCR measured increased expression of genes coding for drusen components, lipid transport, extracellular matrix components, and responses to inflammation in the rho(0) cells. The rho(0) cells also exhibited an increased sensitivity to killing by tBH and increased migration and invasion through solubulized basement membrane-coated tissue culture inserts. CONCLUSIONS: ARPE-19 cells respond to loss of mitochondrial function by changes in nuclear gene expression that resemble changes observed in age-related macular degeneration. The results lead to the hypothesis that loss of mitochondrial function with age and resultant changes in nuclear gene expression may explain some of the changes in the macula that are associated with the known clinical manifestations of age-related macular degeneration.

Benzimidazoles↗

PubMed2003

Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging.

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

Fungal Proteins↗

PubMed2002

Expression of metallothionein isoforms in human chorioretinal complex.

PURPOSE: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex. METHODS: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression. RESULTS: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina. CONCLUSIONS: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.

Adult↗