Apoptosis of lymphoma cells is abolished due to blockade of cytochrome <Emphasis Type="Italic">c</Emphasis> release despite Nur77 mitochondrial targeting

Nur77 is reported to undergo translocation to mitochondria in response to apoptotic signaling in a variety of cancer cell lines. It was shown that on the mitochondrial membrane, Nur77 interacts with Bcl-2, leading to the conversion of this protein from a protector to a killer with subsequent release of cytochrome c to the cytosol. Here it is shown that in thymic lymphoma cells resistant to calcium-mediated apoptosis, cytochrome c release is abolished despite of Nur77 mitochondrial targeting. However, cytochrome c release and apoptosis can be restored by treatment with FK506. Hence, the molecular target regulation of the sensitivity of lymphoma cells to calcium signaling is associated with cytochrome c release and is FK506 sensitive. These results provide new insight into the role of FK506-sensitive factors as a critical link between calcium signaling and resistance of lymphoma cells to death.     

Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis

The mitochondrial localization of the membrane proteins Bcl-2 and Bcl-x(L) is essential for their anti-apoptotic function. Here we show that mitochondrial FK506-binding protein 38 (FKBP38), unlike FKBP12, binds to and inhibits calcineurin in the absence of the immunosuppressant FK506, suggesting that FKBP38 is an inherent inhibitor of this phosphatase. FKBP38 is associated with Bcl-2 and Bcl-x(L) in immunoprecipitation assays and colocalizes with these proteins in mitochondria; in addition, the expression of FKBP38 mutant proteins induces a marked redistribution of Bcl-2 and Bcl-x(L). Overexpression of FKBP38 blocks apoptosis, whereas functional inhibition of this protein by a dominant-negative mutant or by RNA interference promotes apoptosis. Thus, FKBP38 might function to inhibit apoptosis by anchoring Bcl-2 and Bcl-x(L) to mitochondria.     

FK506 affects mitochondrial protein synthesis and oxygen consumption in human cells

FK506 is an important immunosuppressive medication. However, it can provoke neurotoxicity, nephrotoxicity, and diabetes as adverse side effects. The decrease in oxygen consumption of rat cells treated with pharmacologically relevant concentrations of FK506, along with other evidences, has insinuated that some of the toxic effects are probably caused by drug-induced mitochondrial dysfunction at the level of gene expression. To confirm this suggestion, we have analyzed cell respiration and mitochondrial protein synthesis in human cell lines treated with FK506. This drug provokes an important decrease in oxygen consumption, accompanied by a slight reduction in the synthesis of mitochondria DNA-encoded proteins. These results are similar to those triggered by rapamycin, another macrolide with immunosuppressive properties, therefore insinuating a common toxic pathway.     

Ionomycin-induced apoptosis of thymocytes is independent of Nur77 NBRE or NurRE binding, but is accompanied by Nur77 mitochondrial targeting

The induction of thymocyte apoptosis through the Nur77-mediated intrinsic pathway can be of physiological importance in the clonal deletion of autoreactive thymocytes during negative selection in the thymus and/or in thymocytes undergoing oncogenic transformation. Ionomycin treatment induces endogenous Nur77 expression as well as apoptosis and cytochrome c release in thymocytes. Here it is shown for the first time that in normal thymocytes undergoing apoptosis, ionomycin induces translocation of endogenous Nur77 not only to the nucleus, but also to mitochondria. Immunosuppressant FK506 inhibits Nur77 NBRE and NurRE binding activity but has no effect on thymocytes apoptosis, the subcellular localization of Nur77, or cytochrome c release. This indicates that thymocytes can undergo apoptosis through the intrinsic Nur77-mediated mitochondrial pathway and that the transactivation activity of Nur77 monomers or dimers is not necessary for thymocyte apoptosis.     

Immunophilin deficiency augments Ca2+-dependent glutamate release from mouse cortical astrocytes

Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred to as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca(2+) dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca(2+) (Ca(cyt)(2+)) dynamics and consequential Ca(2+)-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif(-/-) displayed reduced mechanically induced Ca(cyt)(2+) increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca(2+) buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Ca(cyt)(2+) dynamics, but also to an enhanced Ca(2+)-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.