Function and regulation of anionic phospholipids in mitochondria of Saccharomyces cerevisiae

As the major anionic phospholipids predominantly found in the mitochondrial inner membrane of eukaryotic cells, cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are of great importance in many critical mitochondrial processes. Pgs1Δ cells of Saccharomyces cerevisiae lacking both PG and CL display severe mitochondrial defects. Translation of several proteins including products of four mitochondrial DNA (mtDNA) encoded genes (COX1, COX2, COX3, and COB ) and one nuclear-encoded gene (COX4) is inhibited. The molecular basis of this phenotype was analyzed using a combined biochemical, molecular and genetic approach. ^ Using a mitochondrial targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5′ and 3′ untranslated regions (UTRs), lack of mtGFP expression independent of carbon source and strain background was confirmed to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but rather caused directly by the lack of PG/CL in the mitochondrial membrane. Re-introduction of a functional PGS1 gene restored PG synthesis and expression of the above mtGFP. Deletional analysis of the 5′ UTR of COX4 mRNA revealed the presence of a 50 nt sequence as a cis-acting element inhibiting COX4 translation. Using similar constructs with HIS3 and lacZ as reporter genes, extragenic spontaneous mutations that allowed expression of His3p and β-galactosidase were isolated, which appeared to be recessive and derived from loss-of-function mutations as determined by mating analysis. Using a tetracycline repressible plasmid-borne PGS1 expression system and an in vivo mitochondrial protein translation method, the translation of mtDNA encoded COX1 and COX3 mRNAs was shown to be significantly inhibited in parallel with reduced levels of PG/CL content. Therefore, the cytoplasmic translation machinery appears to be able to sense the level of PG/CL in mitochondria and regulate COX4 translation coordinately with the mtDNA encoded subunits. ^ The essential requirement of PG and CL in mitochondrial function was further demonstrated in the study of CL synthesis by factors affecting mitochondrial biogenesis such as carbon source, growth phase or mitochondrial mutations at the level of transcription. We have also demonstrated that CL synthesis is dependent on the level of PG and INO2/INO4 regulatory genes. ^

Mitochondrial defects in primary leukemia cells: Biological consequences and therapeutic implications

Mitochondria are actively engaged in the production of cellular energy sources, generation of reactive oxygen species (ROS), and regulation of apoptosis. Mitochondrial DNA (mtDNA) mutations/deletions and other mitochondrial abnormalities have been implicated in many diseases, especially cancer. Despite this, the roles that these defects play in cancer development, drug sensitivity, and disease progression still remain to be elucidated. The major objective of this investigation was to evaluate the mechanistic relationship between mitochondrial defects and alterations in free radical generation and chemosensitivity in primary chronic lymphocytic leukemia (CLL) cells. This study revealed that the mtDNA mutation frequency and basal superoxide generation are both significantly higher in primary cells from CLL patients with a history of chemotherapy as compared to cells from their untreated counterparts. CLL cells from refractory patients tended to have high mutation frequencies. The data suggest that chemotherapy with DNA-damaging agents may cause mtDNA mutations, which are associated with increased ROS generation and reduced drug sensitivity. Subsequent analyses demonstrated that CLL cells contain significantly more mitochondria than normal lymphocytes. This abnormal accumulation of mitochondria was linked to increased expression of nuclear respiratory factor-1 and mitochondrial transcription factor A, two key free radical-regulated mitochondrial biogenesis factors. Further analysis showed that mitochondrial content may have therapeutic implications since patient cells with high mitochondrial mass display significantly reduced in vitro sensitivity to fludarabine, a frontline agent in CLL therapy. The reduced in vitro and in vivo sensitivity to fludarabine observed in CLL cells with mitochondrial defects highlights the need for novel therapeutic strategies for the treatment of refractory disease. Brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport that is being developed as an anticancer agent, effectively induces apoptosis in fludarabine-refractory CLL cells through a secretory stress-mediated mechanism involving intracellular sequestration of pro-survival secretory factors. Taken together, these data indicate that mitochondrial defects in CLL cells are associated with alterations in free radical generation, mitochondrial biogenesis activity, and chemosensitivity. Abrogation of survival signaling by blocking ER to Golgi protein transport may be a promising therapeutic strategy for the treatment of CLL patients that respond poorly to conventional chemotherapy. ^

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^