Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

TNF-receptor-1 adaptor protein FAN mediates TNF-induced B16 melanoma motility and invasion.

BACKGROUND: Locomotion of cancer cells can be induced by TNF and other motogenic factors secreted by cells of the tumour microenvironment such as macrophages. Based on our recent findings that the TNF receptor adaptor protein FAN mediates TNF-induced actin reorganisation and regulates the directed migration of immune cells responding to chemotactic cues, we addressed the role of FAN in cancer cell motility and the formation of invadopodia, a crucial feature in tumour invasion. METHODS: In B16 mouse melanoma cells, FAN was downregulated and the impact on FAN on cell motility and invasion was determined using in vitro assays and in vivo animal models. RESULTS: Like FAN(-/-) murine embryonic fibroblasts, FAN-deficient B16 melanoma cells showed defective motility responses to TNF in vitro. In vivo FAN-deficient B16 melanoma cells produced significantly less disseminated tumours after i.v. injection into mice. Danio rerio used as a second in vivo model also revealed impaired spreading of FAN-deficient B16 melanoma cells. Furthermore, FAN mediated TNF-induced paxillin phosphorylation, metalloproteinase activation and increased extracellular matrix degradation, the hallmarks of functionally active invadopodia. CONCLUSION: The results of our study suggest that FAN through promoting melanoma cellular motility and tumour invasiveness is critical for the tumour-promoting action of TNF.

Elevated XIAP expression alone does not confer chemoresistance.

BACKGROUND: In various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated. METHODS: To better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression. RESULTS: Our data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits. CONCLUSION: Our results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.

Site- and strand-specific mismatch repair of human H-ras genomic DNA in a mammalian cell line.

Defective mismatch repair has recently been implicated as the major contributor towards the mutator phenotype observed in tumour cell lines derived from patients diagnosed with hereditary non-polyposis colon cancer (HNPCC). Cell lines from other cancer-prone syndromes, such as xeroderma pigmentosum, have been found to be defective in nucleotide excision repair of damaged bases. Some genetic complementation groups are defective specifically in transcription-coupled excision repair, although this type of repair defect has not been associated with cancer proneness. Mechanisms contributing to the high incidence of activating point mutations in oncogenes (such as H-ras codon 12) are not understood. It is possible that novel mechanisms of misrepair or misreplication occur at these sites in addition to the above DNA repair mechanisms. In this study, we have compared the rate of strand-directed mismatch repair of four mispairs (G:A, A:C, T:C and G:T) at the H-ras codon 12, middle G:C position. Our results indicate that, although this location is not a 'hot spot' for bacterial mismatch repair, it is a 'hot spot' for decreased repair of specific mismatched bases within NIH 3T3 cells. NIH 3T3, unlike Escherichia coli, have an extremely low repair rate of the G:A mispair (35%), as well as the A:C mispair (58%) at this location. NIH 3T3 also have a moderately low repair rate of the T:C mispair (80%) at the codon 12 location. Conversely, NIH 3T3 repair of G:T (100%) is comparable to E. coli repair (94%) of this mismatch. These results demonstrate that a mismatch containing an incorrect adenine on either strand at the H-ras codon 12 middle base pair location is most likely to undergo a mutational event in NIH 3T3 cells. Conversely, a mismatch containing an incorrect thymine in the transcribed strand is least likely to undergo a mutational event.