Signaling is even more complicated than we have so far discovered. Augmented PTK signaling in CLL cells may also result from decreased expression of negative regulators.
For example, p66Shc blocks Syk phosphorylation, in response to BCR ligation, by competing with p52Shc, the pro-signalling isoform. But p66Shc is strikingly under-expressed in CLL cells relative to normal B cells, with lowest expression in the poor prognosis group. Reconstitution of p66Shc expression by nucleofection (Nucleofection refers to electroporation, a transfection method which enables transfer of nucleic acids such as DNA, RNA, or small interfering RNA into cells) dramatically reduces anti-apoptotic Bcl-2 and Bcl-XL and increases expression of pro-apoptotic Bax and Bak proteins, suggesting that low p66Shc expression skews Bcl-2 family protein expression towards cytoprotection. Patients with Bcl-2/ p66Shc ratios above the median have shorter treatment free intervals and overall survival durations than do patients with ratios below the median, suggesting that p66Shc influences the clinical behaviour of the malignancy. The mechanism underlying low p66Shc expression in CLL is unclear, but epigenetic mechanisms may be involved.
Another example is the leucocyte-associated immunoglobulin-like receptor-1 (Lair-1), which recruits the Shp phosphatase via ITIMs in its cytosolic domain. Deactivation of PTKs by Shp, negatively regulates BCR signalling. Expression of Lair-1 is low in cells from poor risk patients compared to those from better risk patients. Ligation of Lair-1 resulted in a reduction in activity of constitutive Akt and NF-κB in Lair-1-expressing CLL but not in non-expressing isolates, suggesting that decreased expression of Lair-1 contributes to augmented activation of pro-survival pathways in poor-risk patients.