Die frühe Strahlenreaktion der Mundschleimhaut ist eine regelmäßige und dosislimitierende Nebenwirkung der Strahlenbehandlung fortgeschrittener Kopf-Hals-Tumoren. Trotz einer Vielzahl von experimentellen und klinischen Ansätzen gibt es bis jetzt keine allgemein gültige Prophylaxe oder ein Behandlungskonzept. Die molekularen Mechanismen, die der Entstehung der radiogenen Mucositis enoralis zugrunde liegen, sind allerdings noch nicht geklärt. Die Beteiligung apoptotischer Prozesse an der Entstehung der radiogenen Mucositis enoralis wird derzeit kontrovers diskutiert. Das Signalmolekül Ceramid, welches bei Bestrahlung durch das Enzym neutrale Sphingomyelinase (nSMase) aus dem Membranbaustein Sphingomyelin freigesetzt wird, gilt als ein Initiator der Apoptose. Ziel der vorliegenden Arbeit war es, zu prüfen, welchen Einfluss die Inhibition der Ceramidsynthese durch Glutathion auf die Ausprägung der radiogenen Mucositis enoralis hat. Ergänzt wurden die experimentellen Arbeiten durch die Auswertung histologischer Präparate, welche bei einer fraktionierten Bestrahlung ohne bzw. mit verschiedenen Inhibitoren der Ceramidsynthese (Glutathion, Fumonisin B1, Desipramin) behandelt wurden. Alle Untersuchungen wurden am etablierten Tiermodell der Zungenunterseite der Maus (Stamm C3H/Neu) durchgeführt. Die Bestrahlung erfolgte als lokale Einzeitapplikation (Tag 0) oder als perkutane fraktionierte Bestrahlung mit 5 x 3 Gy/ Woche über eine Woche (Tag 0-4) oder zwei Wochen (Tag 0-4, 7-11), gefolgt von einer lokalen Testbestrahlung an Tag 7 bzw. Tag 14. Als klinisch relevanter Endpunkt diente die Ausbildung ulzerativer Läsionen der Schleimhaut. Zur Inhibition der nSMase1 wurde Glutathion (1 µmol/Tag, subkutan) nach verschiedenen Protokollen verabreicht. In Verbindung mit einer Einzeitbestrahlung wurde Glutathion von Tag -3 bis zur ersten Diagnose der Ulzera (-3/D) bzw. deren kompletten Heilung (-3/H) appliziert. Bei einwöchiger fraktionierter Bestrahlung erfolgte die Applikation von Tag -3 bis zum Tag der Testbestrahlung (-3/7) oder bis zur Diagnose (-3/D) bzw. bis zur Heilung (-3/H) der ulzerativen Läsionen. Die Protokolle für die fraktionierte Bestrahlung über zwei Wochen umfassten die Applikation während der ersten Bestrahlungswoche (-3/7), der zweiten Bestrahlungswoche (7/14), des gesamten Bestrahlungszeitraumes (-3/14) sowie bis zur Diagnose (-3/D) und bis zur Heilung (-3/H). In begleitenden histologischen Untersuchungen wurden die Zellzahlen im Zungenepithel bestimmt, sowie verschiedene Marker der Proliferation (Ki-67), der Apoptose (cleaved Caspase-3, Bcl-2, Bax) und des Sphingolipidmetabolismus (nSMase1, Ceramid) immunhistochemisch erfasst. Es wurden dafür sowohl histologische Präparate der eigenen experimentellen Arbeit (Glutathion), als auch Präparate aus der Arbeitsgruppe (Kontrollen, Fumonisin B1, Desipramin) ausgewertet. Die Behandlung mit Glutathion führte bei Einzeitbestrahlung in keinem der Protokolle zu einer signifikanten Veränderung der ED50 im Vergleich zur alleinigen Bestrahlung (Kontrolle: 13,0 ± 0,7 Gy; -3/D: 12,8 ± 0,7 Gy; -3/H: 12,3 ± 0,6 Gy). Bei der fraktionierten Bestrahlung über eine Woche mit Glutathiongabe von Tag -3 bis zur Erstdiagnose (-3/D) bzw. Heilung (-3/H) der ulzerativen Läsionen wurde eine signifikante Verringerung der Ulkushäufigkeiten beobachtet; die Applikation bis Tag 7 (-3/7) führte dagegen zu keiner signifikanten Veränderung der Ulkushäufigkeit (Kontrolle: 11,1 ± 0,1 Gy; -3/D: 14,6 ± 1,8 Gy; -3/H: 13,1 ± 0,6 Gy; -3/7: 12,0 ± 0,0 Gy). Auch die Protokolle der zweiwöchigen fraktionierten Bestrahlungen zeigten keine signifikanten Unterschiede der Strahlentoleranz bei Behandlung mit Glutathion (Kontrolle 12,1 ± 0,1 Gy; -3/14: 12,4 ± 0,7 Gy; -3/7: 13,2 ± 1,0 Gy; -3/D: 14,1 ± 0,7 Gy; -3/H: 13,5 ± 0,8 Gy; 7/14: 13,7 ± 0,6 Gy). Die histologischen Untersuchungen zeigten, dass die Applikation von Inhibitoren der Ceramidsynthese zu keiner systematischen Veränderung der epithelialen Zelldichte im Vergleich zur alleinigen Bestrahlung führt. Das Minimum der Zellzahl der nur bestrahlten Proben betrug 68,6 % an Tag 5, vergleichbar mit dem Zellverlust der mit Glutathion, Desipramin und Fumonisin B1 behandelten Proben. Auf die Epitheldicke konnte ebenfalls kein systematischer Einfluss der getesteten Inhibitoren nachgewiesen werden. Die immunhistochemische Detektion des Proliferationsmarkers Ki-67 zeigte, dass sowohl Glutathion als auch die anderen verwendeten Inhibitoren im Vergleich zur alleinigen Bestrahlung keinen Einfluss auf die Zellproliferation haben. Der Anteil apoptotischer Zellen (Nachweis des Spaltprodukts der Caspase-3) blieb bei Inhibitor-Applikation in der ersten Woche der fraktionierten Bestrahlung relativ konstant auf dem Niveau der alleinigen Bestrahlung, in der zweiten Woche stieg er um den Faktor 10 an. In guter Übereinstimmung zu dem geringen Apoptoseindex der Mundschleimhaut nach Bestrahlung wurde das proapoptotischen Protein Bax nicht nachgewiesen. Das Enzym nSMase1 wurde in den Zellen im unteren Teil des Stratum spinosum des Schleimhautepithels nachgewiesen; eine Zunahme von Ceramid während der alleinigen Bestrahlung konnte immunhistochemisch nicht beobachtet werden. Die Behandlung mit den Inhibitoren hatte keinen nachweisbaren Einfluss auf das Vorkommen von Ceramiden im Zungenepithel. Die Ergebnisse der histologischen und immunhistochemischen Untersuchungen erbrachten keine Hinweise auf die Beteiligung von Ceramiden bzw. apoptotischer Prozesse an der Strahlenreaktion der Mundschleimhaut. Wäre dies die Ursache, so müsste auch in allen Protokollen – insbesondere der Einzeitbestrahlung – eine Erhöhung der Strahlentoleranz durch die Applikation von Glutathion beobachtbar gewesen sein. Der mukoprotektive Effekt von Glutathion in den Versuchsarmen der fraktionierten Bestrahlung über eine Woche ist deshalb möglicherweise auf eine Stimulation der Repopulierung zurückzuführen. Für eine vollständige Klärung der zugrunde liegenden Mechanismen bedarf es weiterer präklinischer Studien.

Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 cassette of the caspase 9 gene identified five possible high affinity sequences for interaction with the SR protein, SRp30a, a well-established regulator of exon inclusion/exclusion. Replacement mutagenesis identified purine-rich sequences between exons 4 and 5 and wthin exon 6 as important for binding SRp30a and required for expression of the caspase 9a splice variant. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-acting proteins and SRp30a with these sequences. Furthermore, SDS-PAGE analysis of cross-linked RNA trans-acting factors with these possible RNA cis-elements revealed the specific binding of an approximate 66, 56, 45, and 38 kDa protein/protein complex to these sequences. A previous application of RNAi technology to downregulate SRp30a in A549 lung adenocarcinoma cells induced an approximately 75% decrease in SRp30a expression and induced a dramatic change in the ratio of caspase 9a/caspase 9b. Therefore, these studies have identified SRp30a as a major regulator of the alternative splicing of caspase 9 directly linking de novo ceramide generation, PP1, and SRp30a as the signal transduction pathway regulating the expression of caspase 9.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in a number of models of cardiovascular disease. Previously we showed that angiotensin II (Ang II), endothelin-1 (ET-1), endothelial growth factor receptor (EGFR), and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and mitochondria are involved in the activation of ICl,swell by both osmotic swelling and Beta1 integrin stretch. Sphingolipid metabolism is modulated in several cardiopathologies and because sphingolipids are bioactive lipids involved in signaling cascades that overlap significantly with these modulators of ICl,swell, we investigated the role of sphingolipids in the regulation of ICl,swell in cardiac ventricular myocytes. Under isoosmotic conditions that isolate anions currents, addition of exogenous, cell permeant C2-ceramide (C2-Cer) elicited an outwardly-rectifying Cl− current that reversed near the Cl− equilibrium potential (ECl) in both physiological and symmetrical Cl− gradients. This current was inhibited by the ICl,swell-specific blockers DCPIB. Dihydro-C2-ceramide (C2-H2Cer), the inactive analogue of C2-Cer, failed to elicit current. These data strongly suggest that the identity of C2-Cer-induced Cl− current is ICl,swell and indicate that sphingolipid signaling pathways may be involved. Bacterial sphingomyelinase (SMase), which converts endogenous sphingomyelin in the outer leaflet of the sarcolemmal membrane to native chain-length ceramides, elicited a DCPIB-sensitive Cl− current. SMase-induced current is also suppressed by tamoxifen, which under conditions that isolate anion currents is a specific inhibitor of ICl,swell. SMase-induced ICl,swell was abrogated by ebselen, a membrane permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O. This suggests that ROS are required mediators of SMase-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes and both have been implicated in modulating ICl,swell. Blocking NOX with apocynin or the NOX fusion peptide inhibitor gp91ds-tat had no effect on SMase-induced current. However, pretreatment of cardiomyocytes with gp91ds-tat reduced the maximum current amplitude of SMaseinduced ICl,swell, indicating that NOX may play a time-dependent role in this mechanism. By contrast, the mitochondrial Complex I blocker rotenone, which suppresses extramitochondrial ROS release by Complex III, completely suppresses SMase-induced ICl,swell. Additionally, SMase-induced ICl,swell is partially inhibited by blockade of mitochondrial KATP (mitoKATP) channels with 5-hydroxy-decanoic acid (5-HD). MitoKATP channels have been implicated as modulators of mitochondrial ROS release. Thus these data suggest that mitochondrial ROS generation is required for SMaseinduced activation of ICl,swell. Ceramides are metabolized to form several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase, which converts ceramide to sphingosine, with Derythro-MAPP. SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine. These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P elicited an outwardly rectifying Cl− current that was fully inhibited by DCPIB. As seen with SMaseinduced ICl,swell, S1P-induced ICl,swell was fully inhibited by rotenone. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NOX with apocynin. These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, the fact that exogenous C2-ceramide also activates ICl,swell even though C2-ceramide may not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors PD98059 and U0126 on the activation of ICl,swell. Both of these agents partially inhibited SMase-induced activation of ICl,swell, indicating SMase acts through both ERK-dependent and ERK-independent signaling pathways. HL-1 cells are derived from a murine atrial cell line that retains phenotypic characteristics of adult cardiomyocytes. Recently, ICl,swell has been observed in HL-1 cells with similar regulatory mechanisms to those seen in native cells. We showed that SMase elicits an outwardly-rectifying, DCPIB-sensitive Cl− current that reverses near ECl in HL-1 cells. Finally, we confirmed the production of ROS by SMase-induced signaling by flow cytometry in HL-1 cells using the nominally H2O2-selective fluorescent probe CH2DCFDA-AM. Exposure to SMase increased ROS production, as did the positive control H2O2. SMase-induced ROS generation was suppressed by pretreatment with rotenone but was unaffected by pretreatment with gp91ds-tat. These data indicate that exogenous and endogenous sphingolipids elicit ICl,swell in cardiomyocytes by stimulating mitochondrial ROS production. NOX may contribute to the ROS generation, but is not a required step in this mechanism. Sphingolipid signaling is likely to play an important role in stimulating ROS production and activating ICl,swell in a number of cardiovascular diseases.

Visando o tratamento tópico de tumores cutâneos, desenvolvemos micro e nanoemulsões para a co-localização cutânea de paclitaxel e ceramida C6. A nanoemulsão mostrou-se mais eficaz em promover penetração de ambos os compostos nas camadas viáveis da pele, com aumentos de 11,5 (paclitaxel) e 3,5 (ceramida) vezes comparado a uma solução controle, e não reduziu significativamente a viabilidade de equivalentes epidérmicos, sugerindo sua segurança para aplicação tópica. A incorporação na nanoemulsão diminuiu as concentrações de paclitaxel e ceramida necessárias para reduzir a viabilidade de células de melanoma a 50% em 6 a 15 vezes, respectivamente, sendo que a co-encapsulação desses compostos promoveu uma nova redução (4 vezes) nessas concentrações, demonstrando aumento da eficácia decorrente da associação dos compostos. A administração tópica do nanocarreador contendo paclitaxel e ceramida co-encapsulados promoveu desorganização e morte celular em equivalentes cutâneos com melanoma, demonstrando o potencial desta formulação para o tratamento tópico de tumores cutâneos. / Nano and microemulsions were developed in this study for cutaneous co-localization of the active agents paclitaxel and C6 ceramide for topical treatment of skin cancer. The nanoemulsion was more effective in promoting the penetration of the compounds into viable skin layers, with increases of 11.5- (paclitaxel) and 3.5- fold (ceramide) being observed. The nanoemulsion did not reduce the viability of human epidermis equivalents, suggesting its safety for topical application. Incorporation in the nanoemulsion reduced the concentration of paclitaxel and ceramide required to decrease viability of human melanoma cells to 50% by 6 to 15-fold compared with their solutions; co-encapsulation of the compounds promoted a further reduction of 4-fold. Topical administration of the nanocarrier with co-encapsulated paclitaxel and ceramide to skin equivalents with melanoma promoted disorganization and intense cytotoxicity. These results demonstrate the potential of nanoemulsions for association of paclitaxel and ceramide C6 to improve their cytotoxic effect and skin localization.

Alterations to sphingolipid metabolism are associated with increased pulmonary inflammation, but the impact of inflammatory mediators, such as neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. NE is a protease associated CF lung disease progression, and can be found in up to micromolar concentrations in patient airways. While sphingolipids have been investigated in the context of CF, the focus has been on loss of cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we present a novel observation: oropharyngeal aspiration of NE increases airway ceramides in mice. Using a previously characterized mouse model of NE-induced inflammation, we demonstrate that NE increases de novo ceramide production, which is likely mediated via increased SPTLC2 levels. Inhibition of de novo sphingolipid synthesis using myriocin, an SPT inhibitor, decreases airway ceramide as well as the release of pro-inflammatory signaling molecules induced by NE. Furthermore, in a retrospective study of the sphingolipid content of CF sputum—the largest of its type in this patient cohort to date, we investigated the association between NE and sphingolipids. There were linear correlations between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA) positive culture and female gender both strengthened the association of NE and sphingolipids, but higher FEV1 % predicted weakened the association, and Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. These data suggest that NE may increase sphingolipids in CF airways as it did in our in vivo model, and that this association is stronger in patients that have worse lung function, are female, and whose lungs are colonized with MRSA. Modulating sphingolipid homeostasis could provide novel pharmacological approaches for alleviating pulmonary inflammation.

In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

Besides playing its structural function in cellular membranes, ceramide has been recognized as a bioactive signalling molecule playing roles in the regulation of cell growth, differentiation, senescence and programmed cell death. Apoptosis can be induced in cancer cells by elevation of endogenous ceramide levels in response to a variety of apoptotic stimuli such as cytokines (TNF, IL-1), death receptor ligands (Fas ligand), heat stress, oxidative stress, chemotherapeutic agents, and ionizing or ultraviolet radiation. It was shown that use of exogenous cell-permeable short-chain ceramide can also promote apoptotic pathways in cancer cells. Several studies have attempted to further define the specific role of ceramide in cell death. However, the mechanisms by which ceramide mediates antiproliferative pathways or inhibits prosurvival effects are not yet well-defined. So, we investigated the signalling pathways triggered by exogenously-supplied natural long chain ceramide, especially C16-ceramide, to better understand how this messenger induces its biological effects in cancer cells. We first showed that C16-ceramide induced a decrease in viability of adenocarcinoma cells (HCT116), partly due to apoptosis. Using two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic approach, we identified new proteins involved notably in cell proliferation, apoptosis, protein transport and transcriptional regulation in response to exogenous C16-ceramide. Among them, the death promoting factor Btf (Bcl-2-associated transcription factor) was found to be involved in the ceramide-dependent pro-apoptotic signalling pathway. Indeed, Btf-depleted colon cancer cells were found to be more resistant to death triggered by C16-ceramide. Transfection of GFP-Btf expression plasmid up-regulated p53 and BAX protein levels whereas pBcl-2 and Mdm2 expression were down-regulated. Furthermore, we identified a new signalling pathway specifically induced by C16-ceramide, depending on Btf and leading to down-regulation of the Mdm2 protein expression and MDM2 promoter activity. Thus, we provided new information on molecular mechanisms involved in the ceramide-mediated cell death. Then, we investigated the regulation of Emerin expression and its post-translational modifications induced by ceramide. We found that cAMP-dependent protein kinase A (PKA) could be involved in the C16-ceramide induced-Emerin phosphorylation. However, we did not demonstrate the interaction between Btf and phosphorylated-Emerin upon ceramide treatment. Nevertheless, we showed that one of the pathway induced by ceramide implies Emerin and leads to down-regulation of the MDM2 promoter activity. We also hypothesized that GCL (germ-cell-less) could be an intermediate in the Emerin-Mdm2 pathway triggered by C16-ceramide. Furthermore, we showed that Emerin-depleted cells were not more sensitive to apoptosis induced by C16-ceramide. These results should allow us to further explore the potential functions of Emerin in a ceramide-dependent pathway.

Human pathogens survive anti-pathogen host immune assault by either circumventing or evading the host immune response. Bartonella henselae, an intracellular pathogen previously shown to disrupt intrinsic apoptotic messengers to enhance its survival, exploits multiple facets of the cellular apoptotic mechanisms. Cellular pathways affected by apoptotic processes were assessed using real-time reverse-transcriptase-polymerase-chain-reaction (rRT-PCR) to measure the effect of B. henselae on cell regulator gene expression (TRADD, FADD, caspase-8 and caspase-3), caspase activity, DNA cell cycle analysis, cell regulator protein expression and overall cell viability and morphology. The presence of B. henselae suppresses overall gene expression for TRADD and FADD and it dramatically suppresses ceramide-induced TRADD and FADD gene expression. The presence of B. henselae has a noticeable effect on ceramide-induced caspase-8 and caspase-3 gene expression. Only caspase-3 enzymatic activity was ceramide-induced and likewise supressed by the presence of B. henselae, whereas caspase-6 and caspase-8 were unaffected and equivalent to controls. The presence of B. henselae inhibits ceramide-induced DNA fragmentation, maintains overall cell morphology and enhances host cell viability. Lastly, B. henselae inhibits the time-dependant ceramide-induction of TRADD protein and suppresses ubiquitous FADD protein expression. We demonstrated that B. henselae inhibits apoptotic induction in a systematic manner following exogenous apoptotic induction. B. henselae protection of microvascular endothelial cells from apoptosis induction begins at the modulation of cell surface receptor-dependent signaling. B. henselae minimizes, but does not completely abrogate, the cytotoxic effect of the apoptogenic shingolipid ceramide on human microvascular endothelial cells (CDC.EU.HMEC-1). Broadening our understanding of the sequence of cell regulator suppression events by intracellular pathogens will provide insight into disease manifestation. Further, understanding how infected cells initiate and conclude apoptosis will open new avenues into the study of disease treatment.

In this study, mechanisms of resistance to Imatinib-induced apoptosis in human K562 and Meg-1 chronic myeloid leukemia (CML) cells were examined. Continuous exposure of cells to step-wise increasing concentrations of Imatinib resulted in the selection of 0.2 and 1 &amp / #956 / M imatinib resistant cells. Measurement of endogenous ceramide levels showed that treatment with Imatinib increased the generation of C18-ceramide significantly, which is mainly synthesized by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Mechanistically, analysis of mRNA and enzyme activity levels of hLASS1 in the absence or presence of Imatinib did not show any significant differences in the resistant cells when compared to its sensitive counterparts, suggesting that accumulation and/or metabolism, but not the synthesis of ceramide, might be altered in resistant cells. iv Indeed, further studies demonstrated that expression levels, and enzyme activity of sphingosine kinase-1 (SK-1), increased significantly in resistant K562 or Meg-1 cells. The expression levels of glucosyl ceramide synthase (GCS) also increased in resistant cells, comparing to the sensitive counterparts, which indicates conversion of pro-apoptotic ceramide to glucosyl ceramide. Expression analyses of BCR-ABL gene demonstrated that expression levels of BCR-ABL gene increased gradually as the cells acquired the resistance. However, Nucleotide sequence analyses of ABL kinase gene revealed that there was no mutation in Imatinib binding region of the gene in resistant cells. There was also an increase in expression levels of MDR1 gene in resistant cells, which transport the toxic substances outside of cells. In conclusion, these data show, for the first time, a role for endogenous ceramide synthesis via hLASS1 in Imatinib-induced apoptosis, and those alterations of the balance between the levels of ceramide and S1P. Mainly the overexpression of SK-1 seems to result in resistance to Imatinib in K562 cells. The cellular resistance may also result from conversion of ceramide to glucosyl ceramide, from overexpression of BCR-ABL and MDR1 genes but not due to mutations in Imatinib binding site of ABL kinase.

名古屋大学博士学位論文 学位の種類:博士（医療技術学） (課程) 学位授与年月日　平成22年3月25日