Baseline Nucleophosmin Status in Mutated(M) Versus Unmutated (U) Immunoglobulin B-CLL Is a Nuclear Reflection of Different Signal Transduction Physiology.

Aim: The difference in prognosis between B-CLL patients with (M) and wihout (U) somatic hypermutations of the immunoglobulin genes has led to a search for surrogate markers in routine practice. cDNA-Microarray differentiation between M and U led to further analysis of Zap-70, fibromodulin and LPLase, all of which succesfully discriminate the majority of M vs U.. In 1 of 2 proteomic studies searching the difference between M and U B-CLL, nucleophosmin came out as uniformly absent in 6 U patients and present in all 6 with M B-CLL. Nucleophosmin is a nucleolar non-ribosomal protein, associated with cellular proliferation. It is implicated in carcinogenesis as a differentiation blocker in K562 myeloid leukemias where its degradation from a 35 to a 21 kD form accompanies phorbol ester induced differentiation. In K562 this degradation is dependent on P-Erk-translocation to the nucleus that by itself depends on prolonged Erk-activation. We have recently demonstrated that SDF-1, an important microenvironment factor in B-CLL, is capable of inducing prolonged Erk-activation in Zap-70+ but not in Zap-70- B-CLL cells in vitro. This prompted us to study the unstimulated expression of nucleophosmin and other nuclear proteins by Westen blot in 16 patients with B-CLL.

Methods: Histones and other alkalic proteins were extracted from the nuclei (pellet fraction) upon treatment with 0.4 M HCl. Following precipitation in acetone, histones and proteins were resuspended in sample buffer and separated by SDS PAGE or acid urea gel electrophoresis. Subsequently, the proteins were electrotransferred to a nitrocellulose membrane. Western blot analysis was performed against nucleophosmin, histone H3 or H4, and final signal detection was revealed by enhanced chemiluminescence reaction.

Results: The 35 kD nucleophosmin signal was uniformly expressed in all samples and related to total histone content, irrespective of mutation status, cytogenetics and stage of disease. However, a minor band at 21 kD reflecting degradation was present in 6/6 patients with U B-CLL and absent in 8/10 with M B-CLL. Pan-histone H3 and H4 antibodies revealed similar banding patterns for histone H3 in all patients, but the presence of a heavier (probably ubiquinated) double band for histone H4 in some. This ubiquinated fraction was important in 7/16, faint in 5/16 and absent in 4/16. Morphology was atypical in 4/7 with an important fraction, and typical in all others. 5/7 with important bands and 4/5 with faint bands were M B-CLL against only 1 out of 4 in the group with absence of ubiquitination bands.

Discussion: In contrast to previous proteomic studies, we found the 35 kD form of nucleophosmin present in both M and U B-CLL. However, nucleophosmin metabolisation to the 21 kD form is observed preferentially in the U B-CLL. The increased baseline degradation of nucleophosmin in 6/6 U B-CLL all of whom are Zap-70+ can be explained as a downstream nuclear signal from constitutive increased Erk-phosphorylation upon environmental signals in Zap-70+ B-CLL.