B-cell CLL is characterized by the accumulation of mononuclear B cells that are resistant to apoptosis as a result of bcl-2 oncogene overexpression. Nucleolin has recently been identified as a bcl-2 mRNA stabilizing protein that binds specifically to a 139 base AU-rich instability element (ARE) (

Sengupta et al.,
J. Biol. Chem.
). Thus, studies were done to address the question whether the increased levels of bcl-2 mRNA in CLL are related to stabilization of bcl-2 mRNA by nucleolin. B cells were isolated from the blood of 9 patients with CLL and 5 normal volunteers by density gradient centrifugation followed by positive selection with CD19 immuno-magnetic microbeads. Flow cytometric analysis indicated that greater than 90% of the CLL and normal B cells were CD19 positive and CD3 negative. Western blotting revealed that cytoplasmic nucleolin and total cellular bcl-2 protein levels were elevated 18-fold (p<0.001) and 5-fold (p<0.001) respectively, in CLL compared to normal B cells. To directly examine the ability of nucleolin to stabilize bcl-2 mRNA, in vitro RNA decay assays were carried out using capped and polyadenylated bcl-2 mRNA transcripts. The average half-life of the bcl-2 mRNA transcript containing the ARE instability element was 12.5 min in S100 extracts of normal B cells, but was increased to 39.7 min in CLL cell extracts. Purified recombinant nucleolin (280 nM) stabilized bcl-2 mRNA when added to S100 extracts of normal B cells, but had no significant effect on bcl-2 mRNA stability in CLL cell extracts. The results are consistent with the hypothesis that resistance of CLL cells to apoptosis is related to overexpression of cytoplasmic nucleolin and increased stability of bcl-2 mRNA. These findings warrant further investigation of nucleolin as a potential therapeutic target in CLL.

Author notes

Corresponding author

Sign in via your Institution