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- Roos, Cecilia, 1977-
(författare)
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Studies of leukotriene C4 synthase expression and regulation in chronic myeloid leukaemia
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Leukotriene (LT) C4 is generated through conjugation of LTA4 with glutathione by the actions of leukotriene C4 synthase (LTC4S). This biologically potent mediator of asthma and inflammation has also been suggested to play a regulatory role in human myelopoiesis and is overproduced by blood and bone marrow cells from patients with chronic myeloid leukaemia (CML). We investigated the expression and activity of LTC4S in normal and leukemic myeloid cells of different degrees of maturity. Normal cells displayed the highest expression and activity in the most immature cells, and it decreased with increasing maturity. The expression and activity of LTC4S followed the same pattern in corresponding CML cell fractions, however with a consistently higher expression and activity than seen in the normal cells. LTC4S could not be detected at the protein level in normal mature neutrophils but LTC4S mRNA could be amplified from total RNA. However, in cytoplasmic extracts from these cells LTC4S mRNA could not be detected. To study a possible posttranscriptional regulation we synthesised a LTC4S RNA. Cytosolic extracts from CML neutrophils, in contrast to normal neutrophil extracts, degraded this RNA, suggesting that the aberrant expression of LTC4S in CML neutrophils is not caused by stabilisation of LTC4S mRNA in the cytosol. We also show the first in vivo evidence of an increased LTC4S activity. Thus, CML patients were found to have significantly higher levels of the LTC4 metabolite LTE4 in their urine compared to healthy individuals. Imatinib is an inhibitor of BCR-ABL, a tyrosine kinase believed to induce and maintain the malignant transformation in CML. In vivo treatment with imatinib normalised the aberrant LTC4S expression seen in CML neutrophils. We could detect a significant decrease in LTC4S activity already within two weeks of therapy, without any effect on the presence of BCR-ABL. To further explore the possible connection between BCR-ABL and LTC4S, a CML cell line that expresses active LTC4S was identified. Surprisingly, upon treatment of KCL22 cells with imatinib there was a dose- and time-dependent increase in the enzyme activity. Real-time RT-PCR showed an increase in LTC4S mRNA levels, which could explain the increased enzyme activity. Taken together, the expression pattern of LTC4S during myeloid differentiation further implies a role for LTC4 in normal and leukemic myelopoiesis. The upregulated expression of LTC4S in CML seems to be a down-stream step in BCR-ABL-induced signalling.
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- Tornhamre, Susanne
(författare)
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Studies on the formation of leukotriene C4 and lipoxins from leukotriene A4 in human platelets
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human platelets lack 5-lipoxygenase activity, but possess enzymatic capacity to transform the unstable epoxide leukotriene (LT)A4 to the potent inflammatory agent LTC4 and lipoxins, a group of trihydroxylated tetraene-containing eicosanoids, which recently have been suggested to have an anti-inflammatory role. A novel pathway for the formation of lipoxin (LX)A4 via platelet lipoxygenation of SS,6R-diHETE and 5S,6S-diHETE, enzymatic/non-enzymatic hydrolysis products of LTA4, was discovered in human platelets. In contrast, human platelets did not transform cysteinyl leukotrienes to the corresponding cysteinyl lipoxins. The results indicated that platelet-dependent lipoxin formation from LTA4 can proceed both via a direct lipoxygenation, yielding 15-OH-LTA4, and through initial hydrolysis to 5,6-diHETEs. The hydroxylation of LTA4 at C-15 was more efficiently catalysed by human platelet and porcine leukocyte 12-lipoxygenasesthan by rabbit reticulocyte and soybean 15-lipoxygenases. In kinetic studies, human platelet 12-lipoxygenase was found to have a high affinity for LTA4, with a Km value comparable to those earlier reported for arachidonic acid. Platelet activation resulted in elevated lipoxin synthesis. This activation was due to stimulation of 12-lipoxygenase by endogenous 12-HPETE, formed after calcium-dependent phospholipase activation and liberation of arachidonic acid. In contrast to the increased lipoxin formation, platelet activation attenuated the conversion of LTA4 to LTC4. This inhibition of platelet LTC4 synthase activity was observed both after receptor-mediated activation of human platelets as well as after direct stimulation of PKC with PMA. These effects were blocked by pretreatment with the protein kinase inhibitor stauro-sporine, supporting the possible regulation of LTC4 synthase through phosphorylation pathways. This was further suggested by kinetic studies demonstrating non-competitive inhibition of the enzyme after incubation of intact platelets with thrombin or PMA. However, receptor-mediated attenuation of LTC4 formation could not be reversed by specific PKC inhibitors, even though these drugs efficiently prevented the PMA-induced effect. These results indicate that the LTC4 synthase activity in platelets is phosphoregulated, both via PKC-dependent and via receptor-mediated, PKC-independent mechanisms. Involvement of protein tyrosine phosphorylations in the latter process was suggested by the finding that a tyrosine phosphatase inhibitor induced dose-dependent inhibition of LTC4 production in platelet sonicates. In agreement with the findings in platelets, PKC-induced down-regulation of the LTC4synthase activity was also demonstrated in human granulocytes. Aspirin treatment in vivo was found to block attenuation of platelet LTC4 synthase activity induced by arachidonic acid or collagen ex vivo. The results suggest that aspirin treatment can uncouple mechanisms that normally restrict the production of cysteinyl leukotrienes, which are important bronchoconstrictors and asthma mediators. These findings may therefore be relevant for our understanding of the pathophysiology of aspirin-induced asthma. The effects of the anti-inflammatory drug sulfasalazine on the arachidonic acid cascade inhuman platelets and leukocytes were investigated. The drug was found to inhibit the production of leukotrienes and tromboxanes but allowed formation of prostaglandin E2. This pharmacological profile may contribute to the clinical mechanism of action of this drug. In conclusion: Platelet 12-lipoxygenase is well suited to initiate the conversion of LTA4 tolipoxins, indicating a physiological role for this enzyme in lipoxin formation. Also, platelet activation may lead to elevated formation of putative anti-inflammatory lipoxins paralleled by suppressed formation of the pro-inflammatory LTC4. According to the present findings, regulation of LTC4 synthase activity can be exerted via receptor-mediated, phosphoregulatory mechanisms and may be of importance in aspirin-induced asthma.
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