ABSTRACT
This study was aimed to investigate the biological characteristics of B hematological tumor cells such as proliferation, immunological phenotype and apoptosis by silencing pax5. The specific pax5 small hairpin RNA (shRNA) was synthesized by in vitro transcription. For evaluating the inhibition efficiency, the expression change at mRNA and protein levels were assessed by real-time RT-PCR and Western blot respectively. To detect the biological characteristics of pax5-silenced hematological tumor cells, the immunological phenotype, apoptosis and cell proliferation were measured by using real-time RT-PCR, MTT assay and flow cytometry respectively. The results showed that two shRNA were synthesized, both of which were effective to block pax5 expression. After being blocked by RNAi the immunological phenotype of pax5-silenced lymphoma cells was changed, the expressions of CD19 mRNA and protein were reduced, but the expression of IgM was not changed. As compared with control group, the effect on proliferation and apoptosis of lymphoma cells not could be detected after pax5 silencing. It is concluded that the pax5 plays important role in late differentiation of B cells, and may participate in signal transduction of lymphoma cells, but the effect on proliferation and apoptosis of lymphoma cells were not detected after RNAi, which need to be elucidated further.
Subject(s)
Humans , Apoptosis , Genetics , Gene Silencing , Lymphoma, B-Cell , Genetics , Metabolism , Pathology , PAX5 Transcription Factor , Genetics , Metabolism , RNA Interference , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Tumor Cells, CulturedABSTRACT
To investigate transcription factor PAX5 expression characteristics in childhood acute leukemic cells, expression levels of PAX5 and CD19 mRNA in 6 hematological tumor cell lines and bone marrow cells of 6 normal children, 58 de novo patients and 4 relapse acute leukemic children, including 39 cases of B-ALL, 10 cases of T-ALL and 13 cases of AML, were detected by a real-time RT-PCR. The results showed that PAX5 and CD19 mRNA expression levels were 2.35% and 2.52% in Namalwa (B-cell lines) respectively, but almost not detectable in other T- and myeloid cell lines. Among clinical samples, expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML (P = 0.029 and P = 0.013 respectively). PAX5 expression was significantly lower in T-ALL and AML than that in normal controls. The difference of PAX5 mRNA expression levels between T-ALL and AML was not significant. Individual difference of PAX5 mRNA expression levels in children with B-ALL was great. Moreover, PAX5 mRNA expressions in de novo and relapse patients with B-ALL were significantly higher than those in remission (P = 0.011 and P = 0.006 respectively). As binding sites for B-cell specific activator protein have been identified in the promoter regions of CD19, the study found that in B-ALL, there was clear correlation between the expression levels of PAX5 and CD19, which was also studied by real-time RT-PCR. It is concluded that PAX5 transcripts are readily detectable and quantifiable in clinical materials with B-ALL by real-time RT-PCR. The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.
Subject(s)
Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male , Antigens, CD19 , Genetics , Cell Line, Tumor , PAX5 Transcription Factor , Genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Metabolism , Pathology , RNA, Messenger , Genetics , Transcription Factors , GeneticsABSTRACT
The research was aimed to detect the expression levels of retinoblastoma protein (pRb) in child acute leukemia cells, and to explore its possible association with leukemia cells cycle, the risk of disease, minimal residual disease (MRD) monitoring and prognosis of B-ALL. Flow cytometry (FCM) was used to detect the expression of pRb in 89 cases of acute leukemia (including 25 AML, 10 T-ALL and 54 B-ALL) and bone marrows from 7 normal children (control group). Meanwhile the cell cycle in some cases was analyzed. The results showed that (1) the FCM could accurately detect the expression of pRb in acute leukemia cells; (2) the high level of pRb expression was frequent in all types of child acute leukemias. In the same case, the expression of pRb was significantly increased in leukemia cells when compared with non-leukemia cells. And no detectable pRb protein was found in partial cases of acute leukemia; (3) there was a close relation between expression of pRb and the cell cycle of leukemia cells, the number of G(1) phase cells in pRb positive case of B-ALL was more than that in pRb negative case (92% vs 77%); (4) in B-ALL, the level of pRb expression in MRD positive group was significantly lower than that in MRD negative group (P < 0.05), but pRb expression was stable in non-leukemia cells during therapy; (5) pRb expression was related to the early response to therapy in B-ALL, the expression of pRb was significantly increased in sensitive group when compared with insensitive group (P < 0.05). It is concluded that high level or absence of pRb expression can be found in child acute leukemia cells. The expression of pRb is positively related to cell cycle of leukemia cells, MRD monitoring and the early response to therapy. In short, the detection of pRb expression level can guide the therapy and the evaluation of prognosis in B-ALL.