siRNA-Inhibition OF E2A-PBX1 in Pre-B Leukemia Cells

Reviewer: Eric Shinohara MD, MSCI
Abramson Cancer Center of the University of Pennsylvania
Ultima Vez Modificado: 23 de marzo del 2007

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Presenter: Casagrande, G
Presenter's Affiliation: University of Padova - Lab. Pediatric Onco-Hematology, Italy
Type of Session: Scientific

Background

  • The t(1;19)(q23;p13) chromosomal translocation is detected in ~5-6% of childhood pre-B acute lymphoblastic leukemias (ALLs) and usually results in E2A-PBX1 gene expression.
  • The role of this chimeric gene during leukemogenesis is not yet fully understood.  The purpose of this study is to investigate the function of the t(1;19)(q23;p13) chromosomal translocation utilizing the selective depletion of the E2A-PBX1 protein in pre-B leukemic cells and studying the consequences of this depletion.

Materials and Methods

  • siRNA was introduced into 697 pre-B ALL cell lines.  Delivery was validated by confirming cellular uptake using fluorescent confocal microscopy and FACS analysis.
  • Reduced expression of E2A-PBX1 mRNA was measured by real-time RT-PCR to confirm siRNA inhibition.  This was further confirmed by determining E2A-PBX1 protein levels 24 hours after transfection using western blotting.
  • In order to determine how the t(1;19) translocation affects gene expression in human pre-B cells leukemogenesis, changes in gene expression related to the t(1;19) translocation, and their transcription was detected by SYBR Green PCR.

Results

  • A decrease in E2A-PBX1 expression of 85-90% corresponded with a notable reduction in various protein levels.  Specifically, E2A-PBX1 silencing affected the EB-1 gene reducing its expression to 25%. It is possible that this protein could contribute to the transformed phenotype of pre-B ALL.
  • There was also a significant decrease in Wnt-16b mRNA levels in response to siRNA depletion of the E2A-PBX1.  However, there was no effect on the Wnt16a isoform of the Wnt16 gene.  This suggests that Wnt-16b is a target of E2A-PBX1.
  • The silencing of the E2A-PBX1 also induced apoptosis (2-fold increase of apoptotic cells percent, compared with control). The effect on apoptosis was confirmed by silencing two other pre-B ALL cell lines, with and without the t(1;19) translocation.

Author's Conclusions

  • Targeted inhibition of E2A-PBX1 leads to an increase in apoptosis and to reduced expression of the EB-1 and Wnt16b genes.
  • This suggests that the aberrant expression of the EB-1 and Wnt16b genes may be a key-step in leukemogenesis in t(1;19)-positive pre-B leukemia.

Clinical/Scientific Implications

  • This study determined the effects of t(1;19)(q23;p13) chromosomal translocation using siRNA to deplete E2A-PBX1 protein expression.  It was shown that inhibition of E2A-PBX1 protein expression induced a 2 fold increase in apoptosis and decreased expression of EB-1 and Wnt16b genes.
  • Depletion of E2A-PBX1 followed by examination of the changes in gene expression that this induced may not be the ideal way to determine how the translocation affects gene expression.  Rather by examining differences in gene expression in childhood pre-B ALLs with and without overexpression of E2A-PBX1 and comparing how gene expression is altered may be more accurate.  If overexpression of EB-1 and Wnt16b are found by doing this, it would confirm the results of this study.
  • Some childhood pre-B (ALLs) arise without increased expression of E2A-PBX1.  Determining what is driving oncogenesis in these patients as well as why E2A-PBX1 overexpression is not necessary for the oncogenesis in some patients could further elucidate the mechanism by which malignancy develops.
  • This study suggests that E2A-PBX1 may be a target for a therapeutic agent.  If such agents were to be available, further studies in in vivo models would be of great interest to determine efficacy and toxicity. 



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