Abstract
PAX5 gene is a paired-box PAX gene family member,and encodes the transcription factor BSAP(B-cell specific activator protein) which is a key regulator of B-cell development and differentiation.Dysregulation of PAX5 gene function may contribute to tumorigenesis in lymphoid malignancies.But up to now,a detailed examination of PAX5/BSAP expression in acute leukemia(mainly acute B-lineage lymphoblastic leukemia) has not been reported.In this study,a real-time RT-PCR assay for the relative quantitation of PAX5 and CD19 mRNA expression was developed.It was applied on 6 haematological tumor cell lines and bone marrow cells of 6 normal children,58 previously untreated and 4 relapse acute leukemic children,including 39 cases of B-ALL,10 cases of T-ALL,and 13 cases of AML.PAX5 and CD19 mRNA expression were detected in B-cell lines tested,but almost not 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 normal controls.The mRNA levels of PAX5 between T-ALL and AML had not any difference.Individual difference of PAX5 mRNA expression levels in children with B-ALL was great.Because binding sites for BSAP have been identified in the promoters of CD19,the study found that in B-ALL,there was clear correlation between the level of PAX5 expression and that of CD19,which was also studied by real-time RT-PCR.BSAP expression by Western Blotting analysis was also performed in haematological tumor cells,including 6 haematological tumor cell lines and 4 clinical samples(2 cases of B-ALL,1 case of T-ALL,and 1 case of AML).The results of Western Blotting analysis showed a 52-KD BSAP band in B lineage cells,but not in T- and myeloid lineage cells.The intensity of BSAP bands was in accordance with PAX5 mRNA expression level detected by real-time RT-PCR.It was concluded that PAX5 transcripts are readily detectable and quantified 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.
Real-time live-cell analysis system for screening single tumor cell clones and analyzing their colony-forming ability