| Key regulators in inflammation and apoptosis | |
Abstract/OtherAbstract
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Mitochondrial localization of p53 in the absence of apoptosis The tumor suppressor p53 is known to trigger apoptosis in response to various death stimuli by transactivation of target genes. Additionally, a transcription-independent mechanism was postulated recently to contribute to p53-induced apoptosis. With regard to this, it was for instance demonstrated that stress-activated p53 translocates to the mitochondria only under conditions that lead to apoptosis, but not when cell cycle arrest is induced. At the mitochondria, p53 was shown to directly interact with the anti-apoptotic Bcl-2 and Bcl-xL proteins leading to activation of the pro-apoptotic Bax protein and subsequent activation of the mitochondrial death pathway. As MCF-7 breast carcinoma cells that harbour a fully active transcriptional p53 protein are sensitive to apoptosis induction by chemotherapeutic drugs, but remarkably resistant to gamma-irradiation (IR)-induced apoptosis, it was feasible to speculate that the radio-resistant phenotype of these cells might be caused by an improper or non-functional translocation of p53 to the mitochondria. In order to elucidate such a scenario, the subcellular localisation of p53 in MCF-7 cells that were either exposed to IR or to different chemotherapeutic drugs was compared. Intriguingly, similar amounts of p53 were found to be associated with mitochondria in cells treated with apoptosis-inducing chemotherapeutic drugs and in cells that were arrested in the G2/M phase of the cell cycle following exposure to IR. Similar results were obtained using other cell systems that were either resistant or sensitive to these stimuli. Furthermore, in contrast to expression of nuclear p53, targeted expression of p53 at the mitochondria was not able to induce apoptosis. Together, these data demonstrate that a stress-induced translocation of p53 to mitochondria is not necessarily associated with apoptosis induction and that other, yet undefined, mechanisms must contribute to the radio-resistant phenotype of these cells. IκBζ and its function as a key regulator in inflammation and apoptosis In the second part of this PhD thesis, a possible role of the novel IκB protein IκBζ in the induction of cytokines that are crucial mediators of inflammatory processes was investigated. IκBζ was first identified in our laboratory by a subtractive hybridization approach using two HeLa cell lines with different apoptosis susceptibilities. Similiar to all IκB proteins, IκBζ contains six C-terminal ankyrin repeats that mediate binding to the p65 and p50 subunits of the transcription factor NF-κB, thereby inhibiting its transcriptional activity. In contrast to other IκB proteins, however, IκBζ is localized in the nucleus where it associates with matrix-associated deacetylase nuclear bodies. Interestingly, and in contrast to other IκB proteins that merely act as transcriptional repressors of NF-κB, it was found that IκBζ augmented expression of various cytokines such as IL-6, IL-8, GM-CSF and MCP-1. This effect could be attributed to the N-terminal domain of IκBζ that was shown in parallel studies to contain a transactivation domain. However, studies using p50- or p65-deficient mouse embryonic fibroblasts demonstrated that the increased cytokine production was not mediated by IκBζ alone, but critically depends on its interaction with a functional NF-κB. These findings were also verified in endothelial cells using micro-array analyses that in addition revealed activation and repression of multiple apoptosis- and mitosis- relevant genes. Indeed it could be demonstrated that IκBζ activates caspases and finally induces apoptosis. In summary, unlike classical NF-κB inhibitors such as IκBα, IκBζ plays a central role in the induction and repression of a selective pool of genes critical for diverse processes by differentially controlling NF-κB activity with either its N- or C-terminus. The role of the tyrosine kinase Lck in apoptosis Over the past 15 years, the function of the tyrosine kinase Lck in vital cellular processes such as T-cell growth and differentiation has been intensively investigated. Recent studies indicated that Lck is also involved in apoptosis induction of T-lymphocytes which was attributed to its stimulatory effect on the expression of the pro-apoptotic Bcl-2 protein Bak. However, this conclusion was solely reached by using a single Lck-deficient cell line (JCaM) that did not express Bak and, in addition, no experiments were provided in which re-introduction of the Lck gene resulted in Bak expression. To re-evaluate the role of Lck in the regulation of Bak expression and instigation of the mitochondrial death pathway, three individual Lck-deficient JCaM cell clones (JCaM, JCaM/Abr, JCaM/ATCC) obtained from different sources were investigated. Surprisingly, only the JCaM cells were resistant to apoptosis induced by either anticancer drugs or tumor necrosis factor (TNF) that engage the mitochondrial and the death receptor pathway, respectively. JCaM/Abr and JCaM/ATCC cells, in contrast, were as sensitive as wild-type Jurkat cells. This was confirmed by immunoblot analyses as well as by substrate cleavage assays showing that caspase-3 activation was only induced by these treatments in the latter two cell lines, but not in JCaM cells. Consistent with these findings, Bak expression was found in apoptosis-sensitive Lck-deficient cells, but was undetectable in the JCaM cell line. Together, these data clearly demonstrate the absence of any correlation between Lck and Bak expression, and therefore suggest that the apoptosis resistance of JCaM cells is solely dependent on the loss of Bak that must have occurred independently of the loss of Lck. |
Authors
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Pohlmann, Stephan |
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Contributors
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Schulze-Osthoff, Klaus, Mehlhorn, Hans-Peter Heinz |
Publication Detail
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Publisher : - Type : Dissertation Format : Text |
Date Detail
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2007-06-22 |
Subject
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57, Apoptose, NFkappaB, Tyrosinkinase, p53, IkappaBzeta |
Coverage
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Relation
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Source
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Copyright Information
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free |
Other Details
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Languages : eng |
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