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| Author(s): |
Carolina Yaeko Namasu
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2006-07-17 |
| Examining board members: |
Fernando Lopes Alberto;
Carmen Silvia Passos Lima;
Maria Ines de Moura Campos Pardini
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| Advisor: | Fernando Lopes Alberto; Fernando Ferreira Costa |
| Abstract | |
Chronic myeloid leukemia (CML) is a clonal proliferative disorder of the hematopoietic stem cell cytogenetically characterized by the Philadelphia (Ph) chromosome, a result of chromosomal translocation t(9;22)(q34;q11). At molecular level, the Ph chromosome results in a fusion gene, the chimerical BCR-ABL which has constitutive tyrosine kinase activity and is detected in virtually all cases at diagnosis. Indeed, the BCR-ABL gene expression has a pivotal role in the known pathogenetic mechanisms in CML cell proliferation and disease progression. Conversely, BCR-ABL inhibition with imatinib mesilate (Glivec®) efficiently produces disease remission, since it is capable of selectively block the protein through occupying its ATP binding site. However, resistance to imatinib mesilate do occur and, although acquired mutations in the tyrosine kinase domain of BCR-ABL have been described, it seems that the appearance of acquired mutations, which result in gain of function, does not suffice for the resistant phenotype. Active BCR Related (ABR) gene is similar to BCR and both have a GTPase-activating protein (GAP) domain. Increased ABR activity has been detected in different solid tumors and more recently we detected over-expression of ABR in CML cDNA (EST) library. The aim of this study was to investigate the expression levels of the ABR gene in peripheral blood and bone marrow samples of CML patients in chronic phase (CP) at diagnosis and in patients who had received bone marrow transplantation (BMT) and achieved remission by using real-time quantitative RT-PCR (qPCR). ABR gene was expressed in higher levels in 8 of 9 samples derived from CP patients evaluated at diagnosis (median fold change = 5.483) when compared to a pool of healthy subjects, according to Wilcoxon signed rank test (p-value = 0.01563), what correlates with our previous results (Alberto & Costa, 2003). These data could suggest a possible regulatory function of ABR gene on disease evolution. In all cases, qPCR efficiency ranged above 98%. ABR gene expression was normalized with the best performing control genes, ACTB and GAPD (geometric mean; geNorm algorithm). Reduction of ABR gene expression was apparently correlated with the remission status of the patients evaluated an observation that was further confirmed in patients in molecular remission after allogeneic bone marrow transplantation. Analytical sensitivity in GeneAmp 5700 Sequence Detector System was tested by amplification with ABR, BCR, ACTB e GAPD primers and resulted in minimal detection of de 1, 12, 10 e 9 molecules, respectively. The normalization strategy adopted in this work was helpful for accurate qPCR profiling, and is especially suitable for studying the relevance of biological signals of low intensities (AU) | |