Abstract
Chronic Myelogenous Leukemia (CML) is associated with a chromosomal translocation, t(9;22)(q34;q11.2), that produces the Philadelphia chromosome (Ph).
The molecular consequence of this translocation is the generation of the BCR/ABL oncogene, which encodes a chimeric protein of 210 kDa (p210Bcr/Abl) with elevated tyrosine kinase activity. BCR/ABL exerts its oncogenic effect in CML cells essentially by stimulating cell proliferation, inhibiting apoptosis and altering cell adhesion to bone marrow stroma. Despite of this consistent molecular abnormality, a marked heterogeneity in prognosis and response to treatment has been reported.
Different molecular markers have been studied, such as: BMI1, ELA2, PR3, E2F1 and apoptotic genes (BCL-2, BCL-XL, BAX, BAD, BAK) in order to predict progression and overall survival in myeloid leukemia.
The polycomb group gene BMI1 plays an essential role in regulating the proliferative activity in leukemic stem cell. The expression of this gene is related to a higher degree of malignancy. On the other hand, BCL-2 family genes involved in the mitochondrial-apoptotic pathway are related with clinical response and treatment failure. Enhanced expression of the apoptotic inhibitor BCL-2 or its homolog BCL-XL lead to tumor cells having a decreased susceptibility to cell death. Other BCL-2 family members such as BAX are able to induced apoptosis, so that the ratio of expression of proapoptotic and anti-apoptotic members might determine the apoptotic potencial of cancer cells.
In this study we evaluated the expression of BMI1 and BAX/BCL-XL ratio (apoptotic index) to determine whether these genes could behave as biomarkers to predict disease aggressiveness and progression from chronic phase to more advanced phases.
Total RNA was extracted from leucocytes of peripheral blood. using Trizol method. cDNA was synthesized with random hexamer primers and reverse transcriptase. The expression was assessed by quantitative real time (QRT-PCR) using the LightCycler 2.0 instrument (Roche), based on the Syber-Green method.
All QRT-PCR reactions were performed in 20ul volume. The β-actin expression was used as the endogenous cDNA quality control. Groups of patients were compared using the Mann-Whitney test.
The study was performed in 31 patients: 16 in chronic phase (CP), 15 in advanced phases (accelerated and blast crisis) and 10 healthy donors (control group).
BMI1 expression levels were significantly lower in CP (mean ± SEM: 0.54±0.15) than in more advanced stages of CML (mean ± SEM: 4.54±1.4) (P<0.0005). In peripherical blood of healthy donors, the expression of this gene was similar to CML-CP patients (0.4±0.13). The relationship of BAX/BCL-XL values were higher in CP (mean ± SEM: 13.81± 1.85) and lower in advanced phase (mean ± SEM: 0.88±0.17) than in the control group (mean ± SEM: 4.82 ± 0.49) (P<0.0044 and P< 0.0002, respectively).
The CP patients showed a low BMI1 expression level and a high apoptotic index, this inverse correlation is associated with a benign stage of the disease and good treatment response. On the contrary, cases in more advance stage displayed overexpression of BMI1 gene and low BAX/BCL-XL ratio suggesting an aggressive stage and poor response. The identification of a genetic hostile profile in CP phase could predict an impending disease progression. Our results show that the simultaneous use of two biomarkers: BMI1 and the ratio BAX/BCL-XL represent sensitive indicators of clinical outcome in CML-CP. Therefore, the prospective screening of these biomarkers would help to refine CML disease staging and would be useful prognostic indicators for optimizing therapeutic strategies.
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