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- Frauenklinik und Poliklinik (2)
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- 668036 (1)
Background:
Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects.
Results:
We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors.
Conclusion:
Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.
In contrast to c-Myc, a deregulated expression of the MYCN gene is restricted to human neuroendocrine tumours. In most cases, the excessive activity of N-Myc results from a MYCN amplification. In neuroblastoma, amplification of MYCN is a predictor of poor prognosis and resistance to therapy. The inability to target the N-Myc protein directly necessitates the search for alternative targets. This project aimed at identifying genes specifically required for growth and survival of cells that express high levels of N-Myc using high-throughput shRNA screening combined with next generation sequencing. The identification and analysis of these genes will shed light on functional interaction partners of N-Myc.
We screened a shRNA library containing 18,327 shRNAs and identified 148 shRNAs, which were selectively depleted in the presence of active N-Myc. In addition, shRNAs targeting genes that are involved in p53 and ARF turnover and apoptosis were depleted in the cell population during the screen. These processes are known to affect N-Myc-mediated apoptosis. Consequently, these results biologically validated the screen. The 148 shRNAs that showed a significant synthetic lethal interaction with high levels of N-Myc expression were further analysed using the bioinformatics program DAVID. We found an enrichment of shRNAs that target genes involved in specific biological processes. For example, we validated synthetic lethal interactions for genes such as, THOC1, NUP153 and LARP7, which play an important role in the process of RNA polymerase II-mediated transcription elongation. We also validated genes that are involved in the neddylation pathway.
In the screen we identified Cullin 3, which is a component of the BTB-CUL3-Rbx1 ubiquitin ligase that is involved in the turnover of Cyclin E. Depletion of cullin 3 and activation of N-Myc was found to synergistically increase Cyclin E expression to supraphysiological levels, inducing S-phase arrest and a strong DNA damage response.
Together with results from a proteomics analysis of N-Myc associated proteins, our results lead us to the following hypothesis: In a neuroblastoma cell, the high levels of N-Myc result in a conflict between RNA polymerase II and the replication machinery during S-phase. The newly identified interaction partners of N- Myc are required to solve this conflict. Consequently, loss of the interaction leads to a massive DNA damage and the induction of apoptosis. In addition, inhibition or depletion of the essential components of the neddylation pathway also results in an unresolvable problem during S-phase.
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Glucocorticoids (GCs) are small lipophilic compounds that mediate a plethora of biological effects by binding to the intracellular glucocorticoid receptor (GR) which, in turn, translocates to the nucleus and directly or indirectly regulates gene transcription. GCs remain the cornerstone in the treatment for a number of hematological malignancies, including leukemia, lymphoma and myeloma. Extensive literature suggests that the efficacy of GCs stems from their ability to mediate apoptosis. Despite the enormous strides made in our understanding of regulated cell death, the exact mechanism by which GCs cause apoptosis is still unknown. The data obtained so far provide strong evidence that gene transactivation by the GR underlies the initiation phase of GC-induced thymocyte apoptosis. Furthermore, the multicatalytic proteasome, several members of the Bcl-2 family, changes in calcium flux as well as caspases have been identified as important players in the execution phase of GC-mediated cell death. However, the exact sequence of events in this process still remains elusive. A major problem of the current discussion arises from the fact that different cell types, such as thymocytes, peripheral T cells and lymphoma cells are compared without acknowledging their different characteristics and gene expression profiles. Although it is generally assumed that GCs induce apoptosis via a conserved mechanism, this is not supported by any data. In other words, it is possible that thymocytes, peripheral T cells and lymphoma cells may undergo cell death along different pathways. We therefore wondered whether a unique signal transduction pathway is engaged by GCs to initiate and execute cell death in all types of T lymphocytes or whether distinct pathways exist. Therefore, we compared the role of the proteasome, various caspases, the lysosomal compartment and other factors in GC-induced apoptosis of murine thymocytes and peripheral T cells as well as T-ALL lymphoma cells. Our findings show that the initiation phase of GC-induced apoptosis is similar irrespective of the differentiation state of the cell. Apoptosis in both thymocytes and peripheral T cells is mediated by the GR and depends on gene transcription. In contrast, the execution phase significantly differs between thymocyte and peripheral T cells in its requirement for a number of signal transduction components. Whilst in thymocytes, the proteasome, caspases 3, 8 and 9 as well as cathepsin B play an important role in GC-induced apoptosis, these factors are dispensable for the induction of cell death in peripheral T cells. In contrast, changes in the expression and intracellular location of Bcl-2 family members do not appear to contribute to GC-induced apoptosis in either cell type. Importantly, our observation that GC treatment of thymocytes leads to an activation of the lysosomal protease cathepsin B and that this is an essential step in the induction of cell death by GCs, is the first indication that a lysosomal amplification loop is involved in this process. Analysis of GC-induced apoptosis in several T-ALL cell lines further indicates that the signaling pathway induced by GCs in thymocytes but not in peripheral T cells is shared by all lymphoma cell-types analyzed. Given the therapeutic importance of high-dose GC-therapy for the treatment of hematological malignancies, this finding could potentially form a basis for new anti-cancer strategies in the future, which specifically target tumor cells whilst leaving peripheral T cells of patients untouched.
Macrophages stand in the first line of defense against a variety of pathogens but are also involved in the maintenance of tissue homeostasis. To fulfill their functions macrophages sense a broad range of pathogen- and damage-associated molecular patterns (PAMPs/DAMPs) by plasma membrane and intracellular pattern recognition receptors (PRRs). Intriguingly, the overwhelming majority of PPRs trigger the production of the pleiotropic cytokine tumor necrosis factor-alpha (TNF). TNF affects almost any type of cell including macrophages themselves. TNF promotes the inflammatory activity of macrophages but also controls macrophage survival and death. TNF exerts its activities by stimulation of two different types of receptors, TNF receptor-1 (TNFR1) and TNFR2, which are both expressed by macrophages. The two TNF receptor types trigger distinct and common signaling pathways that can work in an interconnected manner. Based on a brief general description of major TNF receptor-associated signaling pathways, we focus in this review on research of recent years that revealed insights into the molecular mechanisms how the TNFR1-TNFR2 signaling network controls the life and death balance of macrophages. In particular, we discuss how the TNFR1-TNFR2 signaling network is integrated into PRR signaling.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application.
The pro-inflammatory phase of bone healing, initiated by platelet activation and eventually hematoma formation, impacts bone marrow mesenchymal stromal cells (MSCs) in unknown ways. Here, we created platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of encapsulated MSCs in comparison to a non-inflammatory fibrin (FBR) hydrogel environment. MSCs were isolated from human bone marrow, while PRP was collected from pooled apheresis thrombocyte concentrates and used for hydrogel preparation. After their encapsulation in hydrogels for 72 h, retrieved MSCs were analyzed for immunomodulatory activities, apoptosis, stem cell properties, senescence, CD9\(^+\), CD63\(^+\) and CD81\(^+\) extracellular vesicle (EV) release, and metabolism-related changes. PRP-hydrogels stimulated immunosuppressive functions of MSCs, along with their upregulated susceptibility to cell death in communication with PBMCs and augmented caspase 3/7 activity. We found impaired clonal growth and cell cycle progression, and more pronounced β-galactosidase activity as well as accumulation of LC3-II-positive vacuoles in PRP-MSCs. Stimuli derived from PRP-hydrogels upregulated AKT and reduced mTOR phosphorylation in MSCs, which suggests an initiation of survival-related processes. Our results showed that PRP-hydrogels might represent a metabolically stressful environment, inducing acidification of MSCs, reducing polarization of the mitochondrial membrane and increasing lipid accumulation. These features were not detected in FBR-MSCs, which showed reduced CD63\(^+\) and CD81\(^+\) EV production and maintained clonogenicity. Our data revealed that PRP-derived hematoma components cause metabolic adaptation of MSCs followed by increased immune regulatory functions. For the first time, we showed that PRP stimuli represent a survival challenge and “apoptotic priming” that are detrimental for stem cell-like growth of MSCs and important for their therapeutic consideration.
To combine the CD27 stimulation inhibitory effect of blocking CD70 antibodies with an antibody-dependent cellular cytotoxicity (ADCC)-independent, cell death-inducing activity for targeting of CD70-expressing tumors, we evaluated here fusion proteins of the apoptosis-inducing TNF family member TRAIL and a single-chain variable fragment (scFv) derived from a high-affinity llama-derived anti-human CD70 antibody (lαhCD70). A fusion protein of scFv:lαhCD70 with TNC-TRAIL, a stabilized form of TRAIL, showed strongly enhanced apoptosis induction upon CD70 binding and furthermore efficiently interfered with CD70-CD27 interaction. Noteworthy, introduction of recently identified mutations that discriminate between TRAILR1 and TRAILR2 binding into the TRAIL part of scFv:lαhCD70-TNC-TRAIL resulted in TRAIL death receptor-specific fusion proteins with CD70-restricted activity.
The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.
In this study, we have analysed the apoptotic effects of the ubiquitous environmental toxin benzo[ a] pyrene (BP) in HaCaT cells and human keratinocytes. Although prolonged exposure to BP was not cytotoxic on its own, a strong enhancement of CD95 (Fas)-mediated apoptosis was observed with BP at concentrations activating the aryl hydrocarbon receptor (AhR). Importantly, the ultimately mutagenic BP-metabolite, that is, (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE), failed to enhance CD95-mediated cell death, suggesting that the observed pro-apoptotic effect of BP is neither associated with DNA adducts nor DNA-damage related signalling. CD95-induced apoptosis was also enhanced by beta-naphtoflavone, a well-known agonist of the AhR that does not induce DNA damage, thus suggesting a crucial role for AhR activation. Consistently, BP failed to sensitise for CD95L-induced apoptosis in AhR knockdown HaCaT cells. Furthermore, inhibition of CYP1A1 and/or 1B1 expression did not affect the pro-apoptotic crosstalk. Exposure to BP did not increase expression of CD95, but led to augmented activation of caspase-8. Enhancement of apoptosis was also observed with the TRAIL death receptors that activate caspase-8 and apoptosis by similar mechanisms as CD95. Together, these observations indicate an interference of AhR signalling with the activity of receptor-associated signalling intermediates that are shared by CD95 and TRAIL receptors. Our data thus suggest that AhR agonists can enhance cytokine-mediated adversity upon dermal exposure.
Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.
Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.
Tumor necrosis factor (TNF) receptor 1 (TNFR1), TNFR2 and fibroblast growth factor-inducible 14 (Fn14) belong to the TNF receptor superfamily (TNFRSF). From a structural point of view, TNFR1 is a prototypic death domain (DD)-containing receptor. In contrast to other prominent death receptors, such as CD95/Fas and the two TRAIL death receptors DR4 and DR5, however, liganded TNFR1 does not instruct the formation of a plasma membrane-associated death inducing signaling complex converting procaspase-8 into highly active mature heterotetrameric caspase-8 molecules. Instead, liganded TNFR1 recruits the DD-containing cytoplasmic signaling proteins TRADD and RIPK1 and empowers these proteins to trigger cell death signaling by cytosolic complexes after their release from the TNFR1 signaling complex. The activity and quality (apoptosis versus necroptosis) of TNF-induced cell death signaling is controlled by caspase-8, the caspase-8 regulatory FLIP proteins, TRAF2, RIPK1 and the RIPK1-ubiquitinating E3 ligases cIAP1 and cIAP2. TNFR2 and Fn14 efficiently recruit TRAF2 along with the TRAF2 binding partners cIAP1 and cIAP2 and can thereby limit the availability of these molecules for other TRAF2/cIAP1/2-utilizing proteins including TNFR1. Accordingly, at the cellular level engagement of TNFR2 or Fn14 inhibits TNFR1-induced RIPK1-mediated effects reaching from activation of the classical NFκB pathway to induction of apoptosis and necroptosis. In this review, we summarize the effects of TNFR2- and Fn14-mediated depletion of TRAF2 and the cIAP1/2 on TNFR1 signaling at the molecular level and discuss the consequences this has in vivo.
Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) has been originally identified as a protein interacting with TNF receptor 2 (TNFR2) but also binds to several other receptors of the TNF receptor superfamily (TNFRSF). TRAF2, often in concert with other members of the TRAF protein family, is involved in the activation of the classical NFκB pathway and the stimulation of various mitogen-activated protein (MAP) kinase cascades by TNFRSF receptors (TNFRs), but is also required to inhibit the alternative NFκB pathway. TRAF2 has also been implicated in endoplasmic reticulum (ER) stress signaling, the regulation of autophagy, and the control of cell death programs. TRAF2 fulfills its functions by acting as a scaffold, bringing together the E3 ligase cellular inhibitor of apoptosis-1 (cIAP1) and cIAP2 with their substrates and various regulatory proteins, e.g., deubiquitinases. Furthermore, TRAF2 can act as an E3 ligase by help of its N-terminal really interesting new gene (RING) domain. The finding that TRAF2 (but also several other members of the TRAF family) interacts with the latent membrane protein 1 (LMP1) oncogene of the Epstein–Barr virus (EBV) indicated early on that TRAF2 could play a role in the oncogenesis of B-cell malignancies and EBV-associated non-keratinizing nasopharyngeal carcinoma (NPC). TRAF2 can also act as an oncogene in solid tumors, e.g., in colon cancer by promoting Wnt/β-catenin signaling. Moreover, tumor cell-expressed TRAF2 has been identified as a major factor-limiting cancer cell killing by cytotoxic T-cells after immune checkpoint blockade. However, TRAF2 can also be context-dependent as a tumor suppressor, presumably by virtue of its inhibitory effect on the alternative NFκB pathway. For example, inactivating mutations of TRAF2 have been associated with tumor development, e.g., in multiple myeloma and mantle cell lymphoma. In this review, we summarize the various TRAF2-related signaling pathways and their relevance for the oncogenic and tumor suppressive activities of TRAF2. Particularly, we discuss currently emerging concepts to target TRAF2 for therapeutic purposes.
Dysregulation of the apoptotic pathway is widely recognized as a key step in lymphomagenesis. Notably, LITAF was initially identified as a p53-inducible gene, subsequently implicated as a tumor suppressor. Our previous study also showed LITAF to be methylated in 89.5% B-NHL samples. Conversely, deregulated expression of BCL6 is a pathogenic event in many lymphomas. Interestingly, our study found an oppositional expression of LITAF and BCL6 in B-NHL. In addition, LITAF was recently identified as a novel target gene of BCL6. Therefore, we sought to explore the feedback loop between LITAF and BCL6 in B-NHL. Here, our data for the first time show that LITAF can repress expression of BCL6 by binding to Region A (−87 to +65) containing a putative LITAF-binding motif (CTCCC) within the BCL6 promoter. Furthermore, the regulation of BCL6 targets (PRDM1 or c-Myc) by LITAF may be associated with B-cell differentiation. Results also demonstrate that ectopic expression of LITAF induces cell apoptosis, activated by releasing cytochrome c, cleaving PARP and caspase 3 in B-NHL cells whereas knockdown of LITAF robustly protected cells from apoptosis. Interestingly, BCL6, in turn, could reverse cell apoptosis mediated by LITAF. Collectively, our findings provide a novel apoptotic regulatory pathway in which LITAF, as a transcription factor, inhibits the expression of BCL6, which leads to activation of the intrinsic mitochondrial pathway and tumor apoptosis. Our study is expected to provide a possible biomarker as well as a target for clinical therapies to promote tumor cell apoptosis.
Mechanisms underlying therapy resistance of tumor cells include protein kinase Akt. Putative Akt targets include store-operated \(Ca^{2+}\)-entry (SOCE) accomplished by pore forming ion channel unit Orai1 and its regulator STIM1. We explored whether therapy resistant (A2780cis) differ from therapy sensitive (A2780) ovary carcinoma cells in Akt, Orai1, and STIM1 expression, \(Ca^{2+}\)-signaling and cell survival following cisplatin (100µM) treatment. Transcript levels were quantified with RT-PCR, protein abundance with Western blotting, cytosolic \(Ca^{2+}\)-activity ([\(Ca^{2+}\)]i) with Fura-2-fluorescence, SOCE from increase of [\(Ca^{2+}\)]i following \(Ca^{2+}\)-readdition after Ca2+-store depletion, and apoptosis utilizing flow cytometry. Transcript levels of Orai1 and STIM1, protein expression of Orai1, STIM1, and phosphorylated Akt, as well as SOCE were significantly higher in A2780cis than A2780 cells. SOCE was decreased by Akt inhibitor III (SH-6, 10µM) in A2780cis but not A2780 cells and decreased in both cell lines by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-ABP, 50µM). Phosphatidylserine exposure and late apoptosis following cisplatin treatment were significantly lower in A2780cis than A2780 cells, a difference virtually abolished by SH-6 or 2-ABP. In conclusion, Orai1/STIM1 expression and function are increased in therapy resistant ovary carcinoma cells, a property at least in part due to enhanced Akt activity and contributing to therapy resistance in those cells.
p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context-and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53's apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing.
Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.
Inflammation is a central aspect of tumour biology and can contribute significantly to both the origination and progression of tumours. The NFκB pathway is one of the most important signal transduction pathways in inflammation and is, therefore, an excellent target for cancer therapy. In this work, we examined the influence of four NFκB inhibitors — Cortisol, MLN4924, QNZ and TPCA1 — on proliferation, inflammation and sensitisation to apoptosis mediated by the death ligand FasL in the HNSCC cell lines PCI1, PCI9, PCI13, PCI52 and SCC25 and in the human dermal keratinocyte cell line HaCaT. We found that the selection of the inhibitor is critical to ensure that cells do not respond by inducing counteracting activities in the context of cancer therapy, e.g., the extreme IL-8 induction mediated by MLN4924 or FasL resistance mediated by Cortisol. However, TPCA1 was qualified by this in vitro study as an excellent therapeutic mediator in HNSCC by four positive qualities: (1) proliferation was inhibited at low μM-range concentrations; (2) TNFα-induced IL-8 secretion was blocked; (3) HNSCC cells were sensitized to TNFα-induced cell death; and (4) FasL-mediated apoptosis was not disrupted.
We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.