Studies of leukotriene C4 synthase expression and regulation in chronic myeloid leukaemia

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.