Display Settings:

Format

Send to:

Choose Destination
Haematologica. 2006 Jun;91(6):765-71.

The effects of siRNA-mediated inhibition of E2A-PBX1 on EB-1 and Wnt16b expression in the 697 pre-B leukemia cell line.

Author information

  • 1Laboratory of Hematology and Oncology, Department of Pediatrics, University of Padova, Italy.

Abstract

BACKGROUND AND OBJECTIVES:

A common non-random translocation in childhood pre-B acute lymphoblastic leukemia (ALL) is t(1;19)(q23;p13), usually resulting in the expression of the chimeric gene E2A-PBX1. The role of this fusion gene during leukemogenesis is not yet fully understood; one approach to investigate its function is to selectively deplete the E2A-PBX1 protein and examine the consequences.

DESIGN AND METHODS:

We tested the efficacy of anti-E2A-PBX1 siRNA in the 697 pre-B leukemia cell line. Transfection was monitored by fluorescence microscopy and FACS, while E2A-PBX1 mRNA expression was measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The reduction of the level of the corresponding fusion protein was assessed by western blot analysis and the expression of putative downstream target genes was detected by SYBR Green PCR.

RESULTS:

We demonstrated efficient downregulation induced by anti-E2A-PBX1 siRNA in 697 t(1;19)-positive leukemia cells. In particular, E2A-PBX1 silencing affected the EB-1 gene, which encodes for a protein that could contribute to the transformed phenotype of pre-B ALL. The detected EB-1 expression was reduced to 25% of the normal expression level in non-transfected 697 cells. Furthermore, the significant decrease in Wnt16b mRNA levels (but not of the Wnt16a isoform of the Wnt16 gene), observed following depletion of the fusion gene, confirms the hypothesis that Wnt16b is a target of E2A-PBX1. The siRNA inhibition was followed by an increase in apoptosis and similar results were obtained in two other ALL cell lines, one with and one without the t(1;19) translocation.

INTERPRETATION AND CONCLUSIONS:

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

Comment in

PMID:
16769578
[PubMed - indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for HighWire
    Loading ...
    Write to the Help Desk