Expression Of BAG-1 In Patients With Acute Leukemia And Its Relationship With BCL-2 And BAX

Background and ObjectiveAcute leukemia is a clonal malignant disease of hematopoietic cells characterized by the excessive proliferation and inhibition of apoptosis. The deregulation of many oncogenes, tumor suppressor genes 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 initiataed. Apoptosis plays a crucial role in carcinogenesis in various tumors and is regulated by several genes. Anti-apoptotic gene BAG-1(Bcl-2-associated athanogene-1) which has been identified at chromosome 9, was first cloned from mouse cells in 1995. Overexpression of anti-apoptotic protein BAG-1 has been found in many human cancers, particularly leukemia, breast, prostate and colon cancers. Four isoforms of BAG-1 protein have been described: p50(BAG-1L), p46(BAG-1M), p33(BAG-1S) and p29. These isoforms are produced from a common mRNA by use of alternative translation initiation codon. BAG-1 protein contains BAG domain, ubiquitin-like domain, nuclear localization sequences and TXSEEX repeat sequences. All four BAG-1 isoforms share a common C-terminus which contains the BAG domain that interacts with Bcl-2, Hsp70 and Raf-1. They also possess a common ubiquitin-like domain that binds to proteasome and target the molecular chaperones Hsp70 and Hsc70 to the protein degradation machinery. The N-terminus seems to be important for BAG-1 to bind to nuclear hormone receptors. Therefore, BAG-1 may inhibit apoptosis through its interaction with other protein such as Bcl-2 protein, heat shock proteins, protein kinase and nuclear hormone receptor, et al. BAG-1 may play a key role in oncogenesis and progression of human cancers through Hsp70/Hsc70 pathway, signal transduction pathway, transcriptional pathway, protein degradation pathway, et al. Bcl-2 gene which has been identified at chromosome 18, was first described in follicular lymphomas with t(14;18) translocation. Bcl-2 gene family play an important role in apoptosis. Anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax are belong to Bcl-2 gene family. Anti-apoptotic protein BAG-1 not only blocks apoptosis alone, but also enhances the ability of Bcl-2 to prevent apoptosis. To explore the expression of BAG-1 and its relationships with Bcl-2 and Bax in acute leukemia, the expression of BAG-1,Bcl-2 and Bax in bone marrow mononuclear cells from untreated acute leukemia patients, AL-CR patients and control group were detected by immunocytochemical staining(S-P three-step method). Their function in the leukemogenesis was further discussed.Materials and method1. Patients: (1) De novo leukemia group: Samples from 54 cases of de novo acute leukemia were collected. Patients were 10-75 years (mean 35 years old) with 32 males and 22 females. The whole patients were divided into two groups:â‘ Acute Myelogenous Leukemia (n=33), including M₀ 2 cases, M₁ 2 cases, M₂ 16 cases, M₃ 5 cases, M₄ 3 cases, M₅ 3 cases, M₆ 1 cases,M₇ 1 case;â‘¡Acute lymphocytic Leukemia (n=21); The acute leukemias were diagnosed by French-American-British classification. (2) AL-CR group: bone marrow samples from patients (n=30) who reached CR after chemotherapy. It was further divided into AML-CR and ALL-CR. (3) The control group: Bone marrow samples from non-malignant hematologic diseases (n=19). All the samples were obtained from the patients treated in the Hemotologic Department of the First Affiliated Hospital of Zhengzhou University through May to December of 2006.2. Sample processing: bone marrow mononuclear cells were separated from 1ml bone marrow of untreated Acute Leukemia patients and control patients. The BMNC were loaded on slides and detected by immunocytochemical staining(S-P three-step method) for the expressions of BAG-1, Bcl-2 and Bax.3. Statistical analysis: The data were analyzed with Chi-square test and Spearman correlation by SPSS10.0 and the standard of statistic significance was established asα=0.05.Results1. The expression of BAG-1 in control group was dim or negative. The positive rate of BAG-1 in initialed novo AL was 66.7% as positively or strong positively expressed in nuclear or cytoplasm with 63.6% in AML, 71.4% in ALL. There was no significance between the positive rate of AML and ALL (P>0.05). The positive rate of BAG-1 in AL-CR was 13.3% ,5.3% in AML-CR and 27.3% in ALL-CR. There was no significance between the positive rate of AML-CR and ALL-CR (P>0.05). The expression of BAG-1 in de novo AL was significantly higher than that in AL-CR and control (P<0.05). And the expression of BAF-1 in AML and ALL was significantly higher than that in AML-CR and ALL-CR, respectively (P<0.05).2. The expression of Bcl-2 in control group was weak or negative. The positive rate of Bcl-2 in initialed novo AL was 55.6% as positively or strong positively expressed in membrane or cytoplasm with 54.5% in AML, 57.1% in ALL. There was no significant difference between the positive rate in AML and in ALL (P>0.05). The positive rate of Bcl-2 in AL-CR was 10.0%,5.3% in AML-CR and 18.2% in ALL-CR. There was no significant difference between the positive rate in AML-CR and in ALL-CR (P>0.05). The expression of Bcl-2 in de novo AL was significantly higher than that in AL-CR and control (P<0.05). The expression of BCl-2 in AML and ALL was higher than that in AML-CR and ALL-CR, respectively (P<0.05).3. The expression of Bax in control group was weak . The positive rate of Bax in initialed novo AL was 29.6% as positively or strong positively expressed in cytoplasm with 36.4% in AML, 19.0% in ALL. There was no significant difference between the positive rate in AML and in ALL (P>0.05). The positive rate of Bax in AL-CR was 40.0%, 42.1% in AML-CR and 36.4 in ALL-CR. There was no significant difference between the positive rate in AML-CR and in ALL-CR (P>0.05). Also, there was no significant difference between de novo A1 and control, de novo AL and AL-CR, AL-CR and control, AML and AML-CR, ALL and ALL-CR (P>0.05).4. Devide BAG-1, Bcl-2, Bax into high group (score≧4) and low group (score<4) according to the positive scores. There was no significant correlation between the level of BAG-1, Bcl-2, Bax and metastasis out of bone marrow, age, sex, WBC(P>0.05). The complete remission is lower in high BAG-1 expression compared with the low BAG-1 expression(P<0.05), and Bcl-2 is also, but the complete remission is higher in high Bax expression compared with the low Bax expression(P<0.05).5. BAG-1 level correlated with Bcl-2 positively(r=0.515,P<0.05),Bax negtively(r=-0.313,P<0.05) respectively.Conclusion1. Both BAG-1 and Bcl-2 expressions were significantly higher in AML and ALLthan that in control and AL-CR, and had an inverse relationship with complete remission, which suggested that BAG-1,Bcl-2 participate in leukemogenesis through promoting malignant proliferation of leukemia cells, and could be a potential prognostic biomarker for acute leukemia.2. Furthermore, this study also showed that BAG-1 level correlated with Bcl-2, whichsuggests that up-regulation of BAG-1 enhances the expression of Bcl-2 and the ability of Bcl-2 to prevent apoptosis.3. Third,this study showed that BAG-1 level correlated with Bax negatively, whichsuggests that they have an inverse role in regulation of apoptosis...