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Unlimited self-renewal is an absolute prerequisite for any malignancy, and is the ultimate arbiter of the continuous growth and metastasis of tumors. It has been suggested that the self-renewal properties of a tumor are exclusively contained within a small population, i.e., the so-called cancer stem cells. Enhanced self-renewal potential plays a pivotal role in the development of leukemia. My data have shown that APL associated translocation products PML/RARalpha and PLZF/RARalpha increased the replating efficiency of mouse lin-/Sca1+ hematopoietic stem cells (HSCs). This effect is partly mediated by induction of gamma–catenin which is an important mediator of the Wnt signaling pathway and has been shown to be up regulated by the AML associated translocation products(AATPs). Suppression of gamma–catenin by siRNA can abrogate the increased replating efficiency induced by AATPs. Transduction of gamma–catenin in lin-/Sca1+ HSCs led to increased replating efficiency and the expression of stem cell markers Sca1 and c-kit. Additionally it induced accelerated cell cycle progression of mouse bone marrow HSCs. Transduction/transplantation mouse models have shown that ectopic expression of gamma–catenin in HSCs led to acute myeloid leukemia without maturation. These data suggest important roles of Wnt signaling pathway in the leukemogenesis induced by PML/RARalpha, PLZF/RARalpha and AML1/ETO. In contrast to AATPs, CML and Ph+-ALL associated translocation products p185(BCR-ABL) and p210(BCR-ABL) did not affect the self-renewal potential of hematopoietic stem/progenitor cells. However my studies indicated that their reciprocal translocation products p40(ABL/BCR) and p96(ABL/BCR) actually increased the replating efficiency of hematopoietic stem/progenitor cells. The effect is stronger when induced by p96(ABL/BCR) than by p40(ABL/BCR). It is very intriguing that p96(ABL/BCR) can activate Wnt signaling and up regulate the expression of HoxB4. Transduction/transplantation mouse model has shown that p40(ABL/BCR) and p96(ABL/BCR) both have their own leukemogenic potential. Given the fact that leukemic stem cells maintain the growth of tumor and are the origin of relapse, the cure of leukemia is dependent on the eradication of the leukemic stem cell and abrogation of aberrantly regulated self-renewal capability. Both t-RA and As2O3 have been shown to induce complete remission in APL patients with PML/RARalpha translocation product. However, t-RA as a single agent achieves completeremission (CR) but not complete molecular remissions (CMR). Therefore, virtually all patients will experience a relapse within a few months. In contrast to t-RA, As2O3 as a single agent is able to induce CR as well as CMR followed by long-term relapse-free survival in about 50% of APL patients even if relapsed after treatment with t-RA-containing chemotherapy regimens. Nothing is known about the mechanisms leading to the complete different clinical outcomes by the two compounds although both have been shown to induce differentiation of blast cells, proliferation arrest, induction of apoptosis and degradation of PML/RARalpha. We investigated the effect of t-RA and arsenic on PML/RARalpha-expressing cell population with stem cell capacity derived from the APL cell line NB4 as well as Sca1+/lin- murine bone marrow cells. We found that t-RA did not reduce the replating efficiency in PML/RARalpha- and PLZF/RARalpha-infected Sca1+/lincells whereas it selected small compact colonies representing very early progenitor cells. T-RA was unable to reduce the capacity to form colony forming units-spleen (CFU-S) of Sca1+/lin-cells expressing PML/RARalpha, additionally t-RA did not impair the capability of engraftment of NB4 cells in NOD/SCID mouse. On the contrary to t-RA, As2O3 abolished the aberrant self-renewal potential of Sca1+/lin- cells expressing PML/RARalpha. As2O3 not only abolished the replating efficiency of PML/RARalpha positive cells but also completely abrogated the ability of PML/RARalpha-positive HSC to produce CFU-S in vivo. On the contrary to As2O3, t-RA increased the absolute cell number and the percentage of cells in the side population with respect to the whole cell population in NB4 cells. Taken together these data suggest that arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha positive leukemic stem cells. My data prove for the first time that there is a direct relationship between the capacity of compounds to effectively target the LSC and their capacity to eradicate the leukemia, and, thereby, to induce complete molecular remission and long-term relapse-free survival. Thus, in order to increase the curative potential of leukemia therapies, future studies need to include the effect of given compounds on the stem cell compartment to determine their ability to eradicate the LSC.
B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
As one of the most widespread infectious diseases in the world, it is currently estimated that approximately 296 million people globally are chronically infected with Hepatitis B virus (HBV), the consequences of HBV infection cause more than 620,000 deaths each year. Although safe and effective HBV vaccines have reduced the incidence of new HBV infections in most countries, there are still around 1.5 million new infections each year. HBV remains a major health problem because there is no large-scale effective vaccination strategy in many countries with a high burden of disease, many people with chronic HBV infection are not receiving effective and timely treatment, and a complete cure for chronic infection is still far from being achieved.
Since its discovery, HBV has been identified as an enveloped DNA virus with a diameter of 42 nm. For efficient egress from host cells, HBV is thought to acquire the viral envelope by budding into multivesicular bodies (MVBs) and escape from infected cells via the exosome release pathway. It is clear that HBV hijacks the host vesicle system to complete self-assembly and propagation by interacting with factors that mediate exosome formation. Consequently, the overlap with exosome biogenesis, using MVBs as the release platform, raises the possibility for the release of exosomal HBV particles. Currently, virus containing exosomal vesicles have been described for several viruses. In light of this, this study explored whether intact HBV-virions wrapped in exosomes are released by HBV-producing cells.
First, this study established a robust method for efficient separation of exosomes from HBV virions by a combination of differential ultracentrifugation and iodixanol density gradient centrifugation. Fractionation of the density gradient revealed that two populations of infectious viral particles can be separated from the culture fluids of HBV-producing cells. The population present in the low-density peak co-migrates with the exosome markers. Whereas the population that appeared in the high-density fractions was the classical HBV virions, which are rcDNA-containing nucleocapsids encapsulated by the HBV envelope.
Subsequently, the characterization of this low-density population was performed, namely the highly purified exosome fraction was systematically investigated. Relying on the detergent sensitivity of the exosome membrane and the outer envelope of the HBV virus, disruption of the exosome structure by treatment with limited detergent revealed the presence of HBsAg in the exosomes. At the same time, mild and limited NP-40 treatment of highly purified exosomes and a further combination of density gradient centrifugation resulted in the stepwise release of intact HBV virions and naked capsids from the exosomes generated by HBV-producing cells. This implies the presence of intact HBV particles encapsulated by the host membrane.
The presence of exosome-encapsulated HBV particles was consequently also verified by suppressing the morphogenesis of MVBs or exosomes. Impairment of MVB- or exosome-generation with small molecule inhibitors has significantly inhibited the release of host membrane-encapsulated HBV particles as well. Likewise, silencing of exosome-related proteins caused a diminution of exosome output, which compromised the budding efficiency of wrapped HBV.
Moreover, electron microscopy images of ultra-thin sections combined with immunogold staining visualized the hidden virus in the exosomal structure. Additionally, the presence of LHBs on the surface of exosomes derived from HBV-expressing cells was also observed.
As expected, these exosomal membrane-wrapped HBV particles can spread productive infection in differentiated HepaRG cells. In HBV-susceptible cells, as LHBs on the membrane surface, this type of exosomal HBV appeared to be uptaken in an NTCP receptor-dependent manner.
Taken together these data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV. Exosomes hijacked by HBV act as a transporter impacting the dissemination of the virus.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
The Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. The majority of symptoms disappear after about one week. However, arthritis can last for months or even years (in about 30% of cases), which makes people unable to work during this period. The virus is endemic in Sub-Saharan Africa, the Indian Ocean islands, India, and Southeast Asia. It has additionally caused several large outbreaks in the last few years, affecting millions of people. The mortality rate is very low (0.1%), but the infection rates are high (sometimes 30%) and the number of asymptomatic cases is rare (about 15%). The first CHIKV outbreak in a country with a moderate climate was detected in Italy in 2007. Furthermore, the virus has spread to the Caribbean in late 2013. Due to climate change, globalization, and vector switching, the virus will most likely continue to cause new worldwide outbreaks. Additionally, more temperate regions of the world like Europe or the USA, which have recently reported their first cases, will likely become targets. Alarmingly, there is no specific treatment or vaccination against CHIKV available so far.
The cell entry process of CHIKV is also not understood in detail, and was thusly the focus of study for this project. The E2 envelope protein is responsible for cell attachment and entry. It consists of the domain C, located close to the viral membrane, domain A, in the center of the protein, and domain B, at the distal end, prominently exposed on the viral surface.
In this work, the important role of cell surface glycosaminoglycans (GAGs) for CHIKV cell attachment was uncovered. GAGs consist of long linear chains of heavily sulfated disaccharide units and can be covalently linked to membrane associated proteins. They play an important role in different cell signaling pathways. So far, solely cell culture passage has revealed an increased GAG-dependency of CHIKV due to mutations in E2 domain A, which was associated with virus attenuation in vivo. However, in this work it could be shown that cell surface GAGs promote CHIKV entry using non-cell culture adapted CHIKV envelope (Env) proteins. Transduction and infection of cell surface GAG-deficient pgsA-745 cells with CHIKV Env pseudotyped vector particles (VPs) and with wild-type CHIKV revealed decreased transduction and replication rates. Furthermore, cell entry and transduction rates of GAG-containing cells were also dose-dependently decreased in the presence of soluble GAGs. In contrast, transduction of pgsA-745 cells with CHIKV Env pseudotyped VPs was enhanced by the addition of soluble GAGs. This data suggests a mechanism by which GAGs activate CHIKV particles for subsequent binding to a cellular receptor. However, at least one GAG-independent entry pathway might exist, as CHIKV entry could not be totally inhibited by soluble GAGs and entry into pgsA-745 was, albeit at a lower rate, still possible. Further binding experiments using recombinant CHIKV E2 domains A, B, and C suggest that domain B is responsible for the GAG binding, domain A possibly for receptor binding, and domain C is not involved in cell binding. These results are in line with the geometry of CHIKV Env on the viral surface. They altogether reveal that GAG binding promotes viral cell entry and that the E2 domain B plays a central role for this mechanism.
As no vaccine against CHIKV has been approved so far, another goal of this project was to test new vaccination approaches. It has been published that a single linear epitope of E2 is the target of the majority of early neutralizing antibodies against CHIKV in patients. Artificial E2-derived proteins were created, expressed in E.coli, and successfully purified. They consisted of 5 repeats of the mentioned linear epitope (L), the surface exposed regions of domain A linked by glycine-serine linkers (sA), the whole domain B plus a part of the β-ribbon connector (B+), or a combination of these 3 modules. Vaccination experiments revealed that B+ was necessary and sufficient to induce a neutralizing immune response in mice, with the protein sAB+ yielding the best results. sAB+, as a protein vaccine, efficiently and significantly reduced viral titers in mice upon CHIKV challenge, which was not the case for recombinant Modified Vaccinia virus Ankara (MVA; MVA-CHIKV-sAB+), as a vaccine platform expressing the same protein. These experiments show that a small rationally designed CHIKV Env derived protein might, after optimization of some vaccination parameters, be sufficient as a safe, easy-to-produce, and cheap CHIKV vaccine.
Epigallocatechin-3-gallate (EGCG) is a catechin found in green tea and was, in this work, found to inhibit the CHIKV life cycle at the entry state in in vitro experiments using CHIKV Env VPs and wild-type virus. EGCG was recently published to inhibit attachment of several viruses to cell surface GAGs, which is in line with the role for GAGs in CHIKV entry revealed in this work. EGCG might serve as a lead compound for the development of a small molecule treatment against CHIKV.
Hematopoietic stem cells (HSCs) have the unique abilities of life-long self-renewal and multi-lineage differentiation. They are routinely used in BM or stem cell transplantations to reconstitute the blood system of patients suffering from malignant or monogenic blood disorders. For an adequate production of each blood cell lineage in homeostasis and under stress conditions, the fate choice of HSCs to either self-renew or to differentiate must be strictly controlled. The incomplete understanding of the molecular mechanisms that control this balance makes it still impossible to maintain or expand undifferentiated HSCs in culture for advanced regenerative medical purposes.
The aim of this thesis was the identification and molecular characterisation of mechanisms that control the decision of HSCs to self-renew or to differentiate, and how they are connected to extrinsic cytokine signaling control. Prior to this thesis, a screening for genes upregulated under self-renewal promoting thrombopoietin (TPO) signaling via the transcription factors STAT5A/B in HSCs was conducted, and Growth arrest and DNA damage inducible 45 gamma (Gadd45g) was one of the regulated genes. GADD45G was described as stress sensor, DNA-damage response and tumor suppressor gene, that is epigenetically silenced in many solid tumors and leukemia. Furthermore, Gadd45g is upregulated in aged HSCs with impaired multi-lineage reconstitution abilities, and it is induced by differentiation promoting cytokines in GM-committed cells. However, the function of GADD45G in LT-HSCs was unknown. All these points warrant further investigation to unravel the function of GADD45G on early cell fate decisions of HSCs in hematopoiesis.
The expression of Gadd45g was stimulated by hematopoietic cytokines TPO, IL3 and IL6 both in HSCs and MPPs, making GADD45G an interesting target to focus on. To simulate the cytokine-induced expression GADD45G was lentivirally transduced in HSCs. Surprisingly, GADD45G did not induce cell cycle arrest or cell death in hematopoietic cells neither in vitro nor in vivo, as reported in many cell lines. Instead GADD45G revealed an enhanced and markedly accelerated differentiation of HSCs into mainly myelomonocytic cells, similar as observed for IL3 and IL6 containing cultures. Also in vivo, GADD45G rapidly initiates the differentiation program in HSCs at the expense of self-renewal and long-term engraftment, as shown by serial HSC transplantation experiments. Along the same line, HSCs from Gadd45g-knock out mice exhibited an increased self-renewal. In vitro, Gadd45g-/- progenitors showed higher and prolonged colony formation potential and slower expansion after cytokine stimulation. The loss of Gadd45g increased HSC self-renewal and improved repopulation in secondary recipients, determined by serial competitive transplantations. Taken together, GADD45G could be identified as molecular link between differentiation-promoting cytokine signaling and rapid differentiation induction in murine LT-HSCs.
As presented in this thesis the differentiation induction of GADD45G was mediated by the activation of the cascade of MAP3K4 – MKK6 –p38 MAPK. Small molecule inhibition of p38, but not JNK, blocked the GADD45G-induced differentiation. GADD45G binds to MAP3K4 and releases its auto-inhibitory loop by a change in confirmation, initiating this cascade. Phosphoflow cytometry demonstrated the activation of p38 and a downstream kinase MK2 by GADD45G expression in MPPs. Furthermore, the expression of constitutive active MAP3K4 and MKK6 were able to phenocopy GADD45G-induced differentiation, which could be blocked by p38 inhibition.
The other two family members GADD45A and B also induced accelerated differentiation in LT-HSCs. Interestingly, only GADD45G suppressed the differentiation into megakaryocyte and erythrocyte (Mek/E) lineage cells suggesting a role of GADD45G in lineage choice. Long-term time-lapse microscopy-based cell tracking of single LT-HSCs and their progeny revealed that, once GADD45G is expressed, the development of LT-HSCs into granulocyte-macrophage-committed progeny occurred within 36 hours, and uncovered a selective lineage choice with a severe reduction in Mek/E cells. Furthermore, no megakaryocytic-erythroid progenitors (MEPs) could develop from HSPCs in BM 2 weeks after transplantation suggesting a very early selection against Mek/E cell fates. In line with these findings, GADD45G-transduced MEPs could not expand or form colonies in vitro, demonstrating that the differentiation program induced by GADD45G is not compatible with Mek/E lineage fate. Gene expression profiling of HSCs indicated that GADD45G promotes myelomonocytic differentiation programs over programs for self-renewal or megakaryo-/ erythropoiesis. The here identified differentiation induction potential of GADD45G is so strong that the expression of GADD45G in primary acute myeloid leukemia (AML) cells inhibited their expansion accompanied by enhanced differentiation and increased apoptosis.
The here presented work shows that IL3 and IL6 induce a differentiation program in HSCs via GADD45G and p38 closing the link of extrinsic cytokine signaling and differentiation induction. Since the loss of Gadd45g increased the self-renewal and slowed HSC differentiation, this may be utilized, i.e. by p38 inhibition, to ex vivo maintain and expand HSCs by preventing cytokine-induced differentiation. Furthermore, Re-expression of GADD45G may overcome the differentiation block in leukemia to eliminate these cells by driving them into terminal differentiation and apoptosis.
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all "cancer genes" were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: proximity of genes able to produce prematurely terminated transcripts, which leads to the production of transspliced fusion RNAs, and finally, the induction of DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced.
Chronic inflammation is considered to be a cause of the autoimmune diseases such as rheumatoid arthritis, Alzheimer’s disease, multiple sclerosis, etc. The search for effective compounds with anti-inflammatory properties to combat these diseases is still ongoing. Natural compound narciclasine, derived from plants of Narcissus species, demonstrated its anti-inflammatory activity in in vivo arthritis models. Further investigation of narciclasine’s anti-inflammatory activity together with its impact on the interaction between leukocytes and endothelial cells was the main focus of this PhD thesis.
Narciclasine reduced the infiltration of monocytes and neutrophils to the abdomen and the concentration of the pro-inflammatory cytokines TNF, IL-6 and IL-1β. Together with this, it reduced acute visceral pain caused by zymosan injection. Narciclasine interfered with leukocyte-endothelial cell interaction in both in vivo and in vitro models. In vivo microscopy revealed that the compound reduced rolling, adhesion and transmigration of leukocytes in the vessels of an injured murine cremaster muscle. This observation was confirmed in the in vitro models for adhesion and transmigration where narciclasine reduced the level of leukocyte’s interaction with HUVECs. Narciclasine demonstrated profound anti-inflammatory properties based on its interference with leukocyte-endothelium interaction by downregulation of endothelial cell adhesion molecules expression (ICAM-1, VCAM-1, E-selectin, CX3CL1) and shutdown of NF-κB pathway. All these effects were a result of the TNF receptor 1 protein translation blocking by narciclasine.
In this work the ability of the compound to reduce visceral pain, downregulate the expression of the endothelial cell adhesion molecules and to interfere with the interaction between leukocytes and endothelial cells was demonstrated for narciclasine for the first time. Obtained results open a promising insight into the understanding of narciclasine’s anti-inflammatory properties and justify further investigation of its potential for treatment of inflammatory diseases.
Orthopoxviruses are large DNA viruses that replicate within the cytoplasm of infected cells encoding over a hundred different proteins. The orthopoxviral 68k ankyrin‐like protein (68k‐ank) is highly conserved among orthopoxviruses, and this study aimed at elucidating the function of 68k‐ank. The 68k‐ank protein is composed of four ankyrin repeats (ANK) and an F‐box‐like domain; both motifs are known proteinprotein interaction domains. The F‐box is found in cellular F‐box proteins (FBP), crucial components of cellular E3 ubiquitin (Ub) ligases. With yeast‐two‐hybrid screens and subsequent co‐immunoprecipitation analyses, it was possible to identify S‐phase kinase‐associated protein 1a (Skp1a) as a cellular counterpart of 68k‐ank via binding to the F‐box‐like domain. Additionally, Cullin‐1 was co‐precipitated, suggesting the formation of a viral‐cellular SCF E3 Ub ligase complex. Modified Vaccinia virus Ankara (MVA) ‐ being attenuated and unable to replicate in most mammalian cell lines due to a block in morphogenesis – nevertheless, expresses its complete genetic information attributing to its properties as promising vector vaccine. Conservation of 68k‐ank as the only ANK protein encoded by MVA implied a substantial role of this viral factor. Hence, its function in the viral life cycle was assessed by studying a 68k‐ank knock‐out MVA. A mutant phenotype manifested in nonpermissive mammalian cells characterized by a block succeeding viral early gene expression and by a reduced ability of the virus to shutoff host protein synthesis. Studies with MVA encoding a 68k‐ank F‐box‐like domain truncated protein revealed that viral‐cellular SCF complex formation and maintenance of viral gene expression are two distinct, unrelated functions fulfilled by 68k‐ank. Moreover, K1, a well‐described VACV host range factor of the ANK protein family, is able to complement 68k‐ank function. This suggests that gene expression of MVA putatively depends on the ANKs encoded in 68k‐ank. In addition to the important findings in vitro, first virulence studies with the mouse pox agent, ectromelia virus (ECTV) deleted of the 68k‐ank ortholog (C11) suggested that this factor contributes to ECTV virulence in vivo.
Pretubulysin (PT), a biosynthetic precursor of the myxobacterial compound tubulysin D, was recently identified as a novel microtubule-targeting agent (MTA) causing microtubule destabilization. MTAs are the most frequently used chemotherapeutic drugs. They are well studied regarding their direct cytotoxic effects against various tumors as well as for their anti-angiogenic and vascular-disrupting action addressing endothelial cells of the tumor vasculature. However, the impact of MTAs on endothelial cells of the non-tumor vasculature has been largely neglected, although tumor cell interactions with the healthy endothelium play a crucial role in the process of cancer metastasis. Besides their use as potent anti-cancer drugs, some MTAs such as colchicine are traditionally used or recommended for the therapy of inflammatory diseases. Here, too, the role of endothelial cells has been largely neglected, although the endothelium is crucially involved in regulating the process of inflammation.
In the present study, the impact of PT on tumor-endothelial cell interactions was therefore analyzed in vitro to gain insights into the mechanism underlying its anti-metastatic effect that was recently confirmed in vivo. In the second part of this work, the influence of PT and other MTAs, namely the microtubule-destabilizing compounds vincristine (VIN) and colchicine (COL) and the microtubule-stabilizing drug paclitaxel (PAC), on leukocyte-endothelial cell interactions was investigated in vitro and in vivo (only PT). It is important to mention that in all in vitro experiments solely endothelial cells and not tumor cells or leukocytes were treated with the MTAs to strictly focus on the role of the endothelium in the action of these compounds.
The impact of PT on tumor-endothelial cell interactions was analyzed in vitro by cell adhesion and transendothelial migration assays as well as immunocytochemistry using the breast cancer cell line MDA-MB-231 and primary human umbilical vein endothelial cells (HUVECs). The treatment of HUVECs with PT increased the adhesion of MDA cells onto the endothelial monolayer, whereas their transendothelial migration was reduced by the compound. Thereafter, the influence of PT on the endothelial cell adhesion molecules (CAMs) E-selectin, N-cadherin, ICAM-1, VCAM-1 and galectin-3 and on the CXCL12/CXCR4 chemokine system was examined, since they might be involved in the PT-triggered tumor cell adhesion. Interestingly, although PT induced the upregulation of ICAM-1, VCAM-1, N-cadherin and CXCL12, cell adhesion assays using neutralizing antibodies or the CXCL12 inhibitor AMD3100 revealed that all these molecules were dispensable for the PT-evoked tumor cell adhesion. As PT induces the formation of interendothelial gaps and MDA cells might adhere onto components of the underlying extracellular matrix (ECM), the precise location of MDA cells attached to the PT-treated endothelial monolayer was investigated. Instead of a direct interaction between tumor and endothelial cells, this work showed that MDA cells preferred to adhere to the ECM component collagen that was exposed within PT-triggered endothelial gaps. Both the PT-evoked increase in tumor cell adhesion onto and the decrease in trans-endothelial migration were completely abolished when β1-integrins were blocked on MDA cells. Similar results were obtained when endothelial cells were treated with VIN and COL but not PAC, indicating that the observed effects of PT depend on its microtubule-destabilizing activity.
The impact of PT, VIN, COL and PAC on leukocyte-endothelial cell interactions was analyzed in vivo (only PT) by intravital microscopy of the mouse cremaster muscle and in vitro by cell adhesion assays using the monocyte-like cell line THP-1 and TNFα-activated human dermal microvascular endothelial cells (HMEC-1). While PT did not affect the rolling of leukocytes on the endothelium, their firm adhesion onto and transmigration through the activated endothelium was reduced by PT in vivo. In accordance, the treatment of HMEC-1 with PT, VIN and COL decreased the TNFα-induced adhesion of THP-1 cells onto the endothelial monolayer, whereas PAC had no influence on this process. Thereafter, the influence of PT, VIN, COL and PAC on endothelial ICAM-1 and VCAM-1 was examined, since these molecules are substantially involved in the firm adhesion of leukocytes onto the endothelium. The cell surface protein expression of ICAM-1 and VCAM-1 was reduced by PT, VIN and COL in activated endothelial cells, whereas PAC did only slightly affect the TNFα-induced upregulation of VCAM-1. As the pro-inflammatory transcription factor NFκB plays a crucial role in the TNFα-induced expression of these CAMs, the impact of the MTAs on the NFκB promotor activity was investigated. While PT, VIN and COL decreased the activation of NFκB in activated endothelial cells, PAC did not affect this process. However, in contrast to the strong effects regarding the cell surface protein expression of ICAM-1 and VCAM-1, the effects of PT, VIN and COL on the NFκB activity was rather low. Thus, the used MTAs might also affect other relevant signaling pathways and/or the intracellular transport of CAMs might be influenced by the impact of the MTAs on the microtubule network.
Taken together, the current study provides – at least in part – an explanation for the anti-metastatic potential of PT and gives first insights into the use of PT and VIN as anti-inflammatory drugs. Moreover, this work highlights the endothelium as an attractive target for the development of new anti-cancer and anti-inflammatory drugs.