BCR-ABL Decreases the Expression of CCN3 Leading to Increased Clonogenic Potential and Cell Growth.

Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we have identified downregulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity and detected reduced CCN3 expression in human CML cell lines and primary human CML cells. We now report a reciprocal relationship of BCR-ABL and CCN3 expression and the functional consequence of expressing CCN3 in BCR-ABL+ cells. Real-time PCR was used to examine the relationship between BCR-ABL and CCN3 expression in human K562 cells. Parental K562 cells showed high expression of BCR-ABL (4.68 x10⁴ transcripts in 5 μL of cDNA) whilst CCN3 expression was not detected. Treatment with siRNA directed against BCR-ABL (0.5 μg per10⁶ cells) for 72 hours resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (mean Ct change 1.9 ± 0.2 and 2.6 ± 0.5 for BCR-ABL and CCN3 respectively, n=3, p=0.001). Similarly, K562 cells treated with imatinib (1 micromolar) for 96 hours showed a 5.9 fold decrease in BCR-ABL expression and a 4.2 fold increase in CCN3 expression (mean Ct change 2.5 ± 0.1 and 2.1± 0.2 for BCR-ABL and CCN3 respectively, n=3, p=0.001). To investigate CCN3 function, we expressed CCN3 in BCR-ABL+ cells using Nucleofector technology (Amaxa, GmbH). K562 cells were transfected with either the pCb6+ vector (Invitrogen,UK) or pCb6+ vector containing the CCN3 construct. Cells were analysed 24 hours post-transfection by flow cytometry and also after 7 days in methyl cellulose culture to determine clonogenicity. Cell cycle analysis was performed on 20,000 events using the winMDI software. CCN3 expression in BCR-ABL+ cells resulted in an accumulation of cells in the subG₀ phase of the cell cycle (mean for subG₀ 9.9% ± 4.6 and 21.8% ± 0.7 for the pCb6+ vector alone and pCb6+ vector containing CCN3 construct respectively). CCN3 expression significantly increased the number of cells within the subG₀ area of the cell cycle (n=3, p=0.027). In addition, CCN3 expression reduced the clonogenic capacity of BCR-ABL+ cells. K562 cells transfected with the pCb6+ vector containing CCN3 construct formed significantly fewer colonies on methyl cellulose in comparison to cells that had been transfected with the pCb6+ vector alone (n=3, p=0.027).

This study demonstrates a reciprocal relationship between CCN3 and BCR-ABL expression. CCN3 is known to be a negative growth regulator and increased expression of CCN3 in BCR-ABL+ cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.