The Alteration of SEPT5 Gene Expression in BCR-ABL Positive Cells and Its Possible Correlation with the Development and / or Progression of Chronic Myeloid Leukemia (CML)

Abstract 4415

The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p <0.05). The spontaneous and stimulated with IL-8 chemotaxis assays used granulocytes and were assessed using chamber containing 96 wells. However, although the results suggest an increased chemotactic activity in patients, there were no significant differences (p<0.05) between controls and patients – regardless of whether the chemotaxis was spontaneous or stimulated with IL-8. In mammals the SEPT5 gene is associated with cellular processes such as exocytosis, apoptosis, leukemogenesis, carcinogenesis and neurodegeneration. Therefore, molecules capable of interacting with the septins, either at biochemical or molecular level, can bring information about their functions in cytokinesis. Studies indicate that the human septins can interact among themselves and with other components of the cytoskeleton – this may be a relevant observation regarding the function of this gene in cancer. The SEPT5 can be activated by different pathways – this may increase expression in translocated cells. Despite major advances in the treatment of CML, the treatments available are not capable of inactivating all the signaling pathways activated by BCR/ABL. Our results demonstrate that SEPT5 may be involved in the pathophysiology of CML. Also, it is clear the importance of the study of pathways that could culminate in its high expression or the triggering of other unknown pathways involved in the development of CML. The increased expression of this gene may be related to disease progression, and finally, the identification of several important genes may lead to a better understanding of CML and helping to identify new therapeutic targets. FAPESP/INCT.