| Backgroud and ObjectiveAcute leukemia is a clonal malignant blood diseases of hematopoietic stem cell. The deregulation of many oncogenes, and their related genes produce abnormal proteins and lead to disruption of cell cycle, abnormal cells proliferation, suppression of apoptosis and as a result, leukemogenesis is initiated. Abnormality of apoptosis is one mechanism of carcinogenesis in various tumors and is regulated by several genes. P63 gene is a member of P53 family, sharing high homogeneous in gene sequence with different structure and function. The role of P63 in tumor is unclear. Study by Frida Lantner et al shows that TAp63 upregulate the expression of Bcl-2 in mRNA and protein level in normal cells. This function is important in maintaining the survival and proliferation of matured B lymphocytes. Bcl-2 gene is oncogene discovered by Tsujimto et al in follicular lymphoma with t(14;18).The overexpression of Bcl-2, Bcl-xl and other apoptosis regulating genes in leukemic cells is associated with drug resistance. So far, the researches on P63 are focused in solid tumor. The role of P63 gene in acute leukemia is still open. We investigate the expression and significance of TAp63 and Bcl-2 gene in bone marrow mononuclear cells from acute leukemia in different stage, e.g. de novo, complete remission and relapsed. The results will be compared to that in non-malignant hematopoietic disease as control. The relationship of TAp63 and Bcl-2 expression will be analyzed and the role of them in the development of acute leukemia will be discussed.Material and methods1. A total of 81 cases of AL, including 50 cases of ALL and 31 cases of AML, were enrolled. A total of 27 cases of adults without malignance disease served as control group. These patients hospitalized in the Department of Hematology of the First Affliated Hopsital of ZhengZhou Univesrity from March to September,2010. The diagnosis of acute leukemia is made based on complete blood count, bone marrow morphology, cytochemistry, cytogenetics and immunophenotype, according to the criteria for diagnosis of acute leukemia. These patients have a mean age of 42 years old (ranged from 14 to 70 years old), with 48 males and 33 females.31 cases are AML (14 cases of M2,6 cases of M3,4 cases of M4,7 cases of M5) and 50 cases are ALL (32 cases of B-ALL,18 cases of T-ALL).27 patients with non-malignant hematological diseases were set up as control. These patients have a mean age of 38 years old (ranged from 21 to 57 years old), with 10 males and 17 females.2. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR): Bone marrow mononuclear cells were separated;total RNAs were extracted by TRIZOL method. cDNA were reverse transcripted, and PCR were performed in the presence of primers specific for TAp63 and Bcl-2 mRNA. The products of PCR were electrophoresed in agarose gel, the bands were visualized by UV light and the optic density of each band was measured. The ratio of TAp63 mRNA/β-actin mRNA and Bcl-2 mRNA/β-actin mRNA was defined as the expression level of TAp63 and Bcl-2 mRNA.3. Statistical analysis:The data were analyzed using software SPSS10.0. Numeric data were expressed as mean±standard deviative (x±s). Paired means were analyzed by Independent-Samples t test, and multiple means were analyzed by ANOVA. The standard of significant level was a=0.05.Results1. Expression of TAp63 mRNA in acute leukemia and control groupTAp63 mRNA was expressed in 49 out of 50 cases of ALL and was expressed in all of 31 cases of AML. A total of 27 cases of adults without malignant hematological disease do not express TAp63 mRNA. The expression of TAp63 mRNA in acute leukemia group was significantly higher than that in control group (P <0.05).2. Expression of TAp63 mRNA between ALL and AMLThe relative expression of TAp63 mRNA in 50 ALL and 31 AML was compared and the results showed that the expression of TAp63 mRNA in ALL (0.92±0.42) was significantly higher than that in AML (0.73±0.22) (P<0.05).3. Expression of TAp63 mRNA in subtypes of ALLThirty-one out of 32 B-ALL cases expressed TAp63 mRNA and all 12 cases of T-ALL expressed TAp63 mRNA. The expression of TAp63 mRNA in B-ALL (1.11±0.40) was significantly higher than that in T-ALL (0.60±0.18) (P<0.05).4. Statistical analysis on the expression of TAp63 mRNA in B-ALL, T-ALL and AMLThe expression of TAp63 mRNA in B-ALL, T-ALL and AML was analyzed by F test with a significant difference (F=20.148,P<0.05). Further analysis using LSD and Bonferroni showed that the expression of TAp63 mRNA in B-ALL was significantly higher than that in T-ALL group (P<0.05) and B-ALL was significantly higher than that of AML group (P<0.05).But there is no statistical significance of TAp63 mRNA expression between AML and T-ALL(P>0.05). 5. Expression of TAp63 mRNA in different stages of ALLBy sequential observation of TAp63 mRNA expression in different stages of ALL in 5 cases, TAp63 mRNA expression in patients who were in de novo and relapsed stages was significantly higher than that in complete remission stage (P=0.00,< 0.05). There is no statistical significance of TAp63 mRNA expression between ALL in complete remission stage and control group with non-malignant hematological diseases (P>0.05).6. Expression of Bcl-2 mRNA in acute leukemia and control groupBcl-2 mRNA was expressed in 30 out of 50 cases (60%) of ALL and 20 out of 31 cases (64.5%) of AML. A total of 27 cases of non-malignant hematological disease were negative of Bcl-2 mRNA. The expression of Bcl-2 mRNA in acute leukemia was significantly higher than that in control (P<0.05). There was no statistical significance between AML and ALL (P>0.05).7. Correlation of the expression TAp63 mRNA and Bcl-2 mRNAThe relationship between the expression of TAp63 mRNA and Bcl-2 mRNA is positive correlation (r=0.631,P=0.00, P<0.05)Conclusions1. The expression of TAp63 and Bcl-2 in acute leukemia is significantly higher than that in non- malignant hematological disease. The expression of TAp63 and Bcl-2 in acute leukemia is positive correlated.2. The expression of TAp63 in ALL is significantly higher than that in AML and B-ALL higher than that in T-ALL.There is no difference of Bcl-2 expression between AML and ALL.3. The expression of TAp63 is high in patients who were in de novo;low in complete remission, and upregulated in relapsed stages. |
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