Metabolic Mechanisms for Programmed Cell Death

Basic Sciences

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Abstract

In previously published studies, we have shown that overexpression of POX activates the intrinsic (mitochondrial) pathway for apoptosis. Features of this effect include DNA fragmentation, increased sub-Go/G1 fraction on flow cytometry, release of cytochrome c into cytosol, and activation of caspase 9. The mechanism of this effect is due to the generation of proline-dependent superoxide which was assayed using hydroethidine. Importantly, the co-expression of, MnSOD which is localized in mitochondria, abolished the POX-dependent apoptotic effects. We asked whether the extrinsic (death receptor) pathway is also affected by overexpression of POX. We found that POX expression upregulated the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), DR5 and activated caspase-8, a caspase specifically activated through the death receptor pathway. Importantly, apoptosis measured by flow cytometry was partially inhibited by Z-IETD-FMK, a specific inhibitor of caspase-8. Furthermore, the mechanism of this effect may be modified by nuclear factor of activated T-cells (NFAT. Additionally, POX expression markedly decreased the phosphorylation of MEK and ERK and this decrease was partially reversed by expression of MnSOD. These data showed that POX may induce apoptosis through both intrinsic and extrinsic pathways and may modulate apoptosis signals induced by p53 or other anti-cancer agents. To test whether this POX-dependent apoptotic mechanism can be translated to animal models, we performed studies using DLD-tet-off-POX xenograft tumors in athymic mice differentially administered Doxycycline (100 micrograms/ml) in their drinking water. First, we injected DLD-tet-off-vector cells (106) into the lumbar region of athymic mice and found that tumors rapidly grew unaffected by Doxycycline. By the end of week 3, all the animals were euthanized because of the size of the tumors. When DLD-tet-off-POX cells were injected, tumor formation was directly related to intake of Doxycycline. In animals on Doxycycline (POX suppressed), all animals (n=22) rapidly formed tumors in a fashion not different from those injected with DLD-tet-off-vector cells and required euthanization by the end week 3. In contrast, animals off Doxycycline (POX induced), had marked retardation of tumor growth. In fact, by the end of week 3, tumors were palpable in only 14% of the animals (3/22). Even by the end of week 5, palpable tumors were found in only 32% (7/22). These studies suggested that POX expression and the resultant proline-dependent apoptosis markedly decreased the formation of tumors in athymic mice. To extend our findings into POX-dependent suppression of specific tumors, we will develop POX transgenic mice and cross them with mice with known tumor susceptibilities to determine whether POX expression affects specific tumor phenotypes.