Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromsomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.

We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina. Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan. This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase. It resembles the retrograde response in Saccharomyces cerevisiae. In P. anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase. It is not induced as the result of a general decline of mitochondrial functions during senescence. We cloned and characterized PaAox. Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised. Copper also controls transcript levels of PaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2). PaSod2 is a target of transcription factor GRISEA. During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases. Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress. Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria. The involved mechanism is unknown. However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released.

The completion of the human genome sequence and the development of new techniques, which allow the visualisation of comprehensive gene expression patterns, has led to the identification of a large number of gene products differentially expressed in tumours and corresponding normal tissues. The task at hand is the sorting of these genes into correlative and causative ones. Correlative genes are merely changed as a consequence of transformation and have no decisive effects upon transformation. In contrast, causative genes play a direct role in the process of cellular transformation and the maintenance of the transformed state, which can be exploited for therapeutic purposes. Oncogenes and tumour suppressor genes are prime targets for the development of new inhibitors and gene therapeutic strategies. However, many target oncogene products do not exhibit enzymatic activity that can be inhibited by conventional small molecular weight compounds. They exert their functions through regulated protein-protein or protein-DNA interactions and might require other compounds for efficient interference with such functions. Peptides are emerging as a novel class of drugs for cancer therapy, which could fulfil these tasks. Peptide therapy aims at the specific inhibition of inappropriately activated oncogenes. This review will focus on the selection procedures, which can be employed to identify useful peptides for the treatment of cancer. Before peptide-based therapeutics can become useful, it will be necessary to increase their stability by modifications or the use of scaffolds. Additionally, various delivery methods including liposomes and particularly the use of protein transduction domains (PTDs) have to be explored. These strategies will yield highly specific and more effective peptides and improve the potential of peptide-based anti-cancer therapeutics.

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Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5 upstream region of the grisea ORF. One mutation is a G 3 A transition at the 5 splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.Aging of biological systems, as the progressive functional impairment of living beings leading to an increase in agerelated mortality, is a complex process that is dependent on both environmental and genetic factors. With regard to the genetic basis, instabilities of the mitochondrial DNA (mtDNA) were reported in various organisms to be involved in aging leading to mitochondrial dysfunction and degeneration (for reviews, see refs. 1 and 2). Moreover, increased instabilities of the mtDNA were found to lead to premature degeneration processes. Examples of this type are premature death syndromes in fungi and various neuromuscular diseases in humans (for reviews, see refs. 3-5). Interestingly, in some cases, these syndromes are due to the mutation of a nuclear gene that demonstrate that the integrity of the mtDNA is under the control of nuclear factors (6-10). Unfortunately, until now, the molecular mechanisms involved in this maintenance function have not been elucidated in any case.In the filamentous fungus Podospora anserina, all wildstrains are characterized by a limited life span. After prolonged vegetative growth, they display various symptoms of senescence. Since the first description of this syndrome (11), it has become clear that nuclear-mitochondrial interactions are crucially involved in its control (for reviews, see refs. 12-15). Detailed molecular investigations revealed that gross reorganizations of the mtDNA occur during senescence. These reorganizations are almost quantitative and lead to impairment of the energy-generating system located in mitochondria. Similar processes were subsequently reported to occur in senescing strains of Neurospora (16)(17)(18). In all cases, the activity of mobile genetic elements of circular or linear structure were found to be relevant. More specifically, in P. anserina, it appears that the mobility of a circular plasmid, termed plasmid-like DNA (plDNA) or ␣senDNA (19,20), which is a derivative of the first intron of the cytochrome oxidase gene subunit I (COI), is responsible f...

Signaling components, which confer an "addiction" phenotype on cancer cells, represent promising drug targets. The transcription factor signal transducers and activators of transcription 3 (STAT3) is constitutively activated in many different types of tumor cells and its activity is indispensible in a large fraction. We found that the expression of the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3 (PIAS3), positively correlates with STAT3 activation in normal cells. This suggests that PIAS3 controls the extent and the duration of STAT3 activity in normal cells and thus prevents its oncogenic function. In cancer cells, however, the expression of PIAS3 is posttranscriptionally suppressed, possibly enhancing the oncogenic effects of activated STAT3. We delimited the interacting domains of STAT3 and PIAS3 and identified a short fragment of the COOH-terminal acidic region of PIAS3, which binds strongly to the coiled-coil domain of STAT3. This PIAS3 fragment was used to derive the recombinant STAT3-specific inhibitor rPP-C8. The addition of a protein transduction domain allowed the efficient internalization of rPP-C8 into cancer cells. This resulted in the suppression of STAT3 target gene expression, in the inhibition of migration and proliferation, and in the induction of apoptosis at low concentrations [half maximal effective concentration (EC 50 ), <3 μmol/L]. rPP-C8 did not affect normal fibroblasts and represents an interesting lead for the development of novel cancer drugs targeting the coiled-coil domain of STAT3. Mol Cancer Res; 8(4); 539-53. ©2010 AACR.

The initial characterization of Grisea suggested that this gene codes for a transcription factor involved in the genetic control of cellular copper homeostasis in Podospora anserina. Here we demonstrate that GRISEA activates in vivo gene expression in Saccharomyces cerevisiae and is characterized by a modular organization. The DNA-binding domain was mapped to the first 168 N-terminal amino acids and the transactivation domain to the C-terminal half of the protein. Increased levels of copper in the growth medium lead to repression of the transactivation function possibly via intramolecular interactions between parts of the DNA-binding domain and the transactivation domain. The wild-type copy of Grisea was found to complement the phenotype of the mac1-1 mutant of S. cerevisiae. GRISEA is able to bind to the promoter of CTR1, a MAC1 target gene that encodes a high-affinity copper transporter. Taken together, the data reported here and in earlier investigations indicate that GRISEA is an ortholog of the yeast transcription factor MAC1 and suggest at least a partial conservation of the molecular machinery involved in the control of cellular copper homeostasis in eukaryotes. Remarkably, in P. anserina, the spectrum of phenotypes affected by this regulatory protein is much broader than that known in yeast and includes morphogenetic traits as well as lifespan and senescence.