Defensins are 20 to 30 amino acids long, cystine and arginine rich peptides that constitute more than 5% of the total cellular proteins in mature granulocytes and at least 30% of proteins in primary granules. Human defensins have been reported to have antimicrobial, antifungal, antiviral and tumour lysis activities. We have isolated defensin mRNA, using the differential display technique, from the well-characterised ATRA responsive acute promyelocytic leukaemia cell line, NB4. The differential display analysis showed an up-regulation of defensin mRNA in NB4 cells after treatment with 10-7M ATRA for 24 hours. This up-regulation was not seen in the NB4:R2 cell line, an ATRA resistant subclone of NB4 cells. In order to further investigate the effects of this gene, we virally infected NB4 and NB4:R2 cells with full-length defensin cDNA in either the sense and anti-sense directions. The sense defensin induced cell growth arrest and cell death in both cell lines. Whilst NB4 cells died within 48-73 hours, the NB4:R2 cells survived for at least 96 hours before dying in culture. Phenotypic analysis, (24, 36 and 48 hours), showed a constant high expression of Annexin V in sense infected cells compared to antisense and uninfected cells in both cell lines. There was no significant increase in CD11b expression in any of the two cell lines used. No obvious cellular response was observed in the anti-sense infected cells even when exposed to different concentrations of ATRA (10-7-10-9M). Molecular analysis, by Atlas cDNA array, of gene expression changes showed an overall down-regulation of the genes, on the array, in anti-sense NB4 cells compared to uninfected cells. No such effect was seen in the antisense infected NB4:R2 cells. Our data suggest that defensin is not only a reliable marker for granulocytic differentiation but can also be considered a candidate target gene for molecular therapy in acute promyelocytic leukaemia.
Full conference title:
44th Amercian Society of Hematology Annual Meeting
- ASH 44th (2002)