SHIP-1 protein level and phosphorylation status differs between CLL cells segregated by ZAP-70 expression

GABELLONI MARÍA LAURA; MERCEDES BORGE; GALLETTI JEREMÍAS; CAÑONES CRISTIAN; NANNINI PAULA; MORANDE PABLO; BEZARES FERNANDO; GIORDANO MIRTA; GAMBERALE ROMINA

Londres

Workshop; International Workshop on Chronic Lymphocytic Leukemia; 2007

Leukemic cells from aggressive B cell chronic lymphocytic leukemia (CLL) patientstypically display B cell receptors (BCRs) encoded by unmutated immunoglobulinvariable heavy-chain genes (IgVH) and express the protein tyrosine kinase ZAP-70 (1).In contrast, mutated IgVH genes and the absence of ZAP-70 are mostly found inpatients with indolent disease (1). It has been proposed that survival differencesbetween these two subgroups are related to the differential ability of the BCR torespond to stimulation (2, 3).Thus, ZAP-70+ patients display a more effective BCRsignal transduction that could contribute to their relatively aggressive clinical behavior(3).Inhibitory phosphatases SHP-1 (SH2 domain-containing tyrosine phosphatase-1), SHIP-1 (SH2 domain-containing phosphatidylinositol 5-phosphatase-1) and SHIP-2are involved in the complex and organized machinery aimed at counterbalancing B cellactivation upon BCR engagement by restricting the duration and/or intensity of thesignaling (4, 5). Since ZAP-70- patients present an impaired BCR signaling capacity,we have explored the possibility that these phosphatases were preferentially expressedin CLL cells from this subgroup.The study was carried out with fourty nine typical CLL patients, whose medianage was 70 years old. ZAP-70 expression was evaluated by flow cytometry as it waspreviously described (1) and was found to be negative in 49% of the patients evaluated.Western blotting analysis showed that SHP-1 and SHIP-1 proteins are expressed in Bcells from ZAP-70- and ZAP-70+ CLL patients, while SHIP-2 could not be detected inany of the samples analyzed, even when the amount of protein loaded in each lanewas duplicated. Normal tonsillar B lymphocytes, used as controls, showed specificbands at 68kDa, 145kDa and 150kDa corresponding to SHP-1, SHIP-1 and SHIP-2respectively. There were no significant differences in SHP-1 expression between CLLsubgroups. By contrast, ZAP-70- CLL cells not only displayed a higher SHIP-1 proteinexpression compared to ZAP-70+ subgroup (p<0.01, Mann-Whitney nonparametrictest), but also it was constitutively tyrosine phosphorylated to a greater extent (p<0.01).Finally, we found that, in ZAP-70- patients but not in ZAP-70+ samples, BCR crosslinkingled to a time-dependent increase in reactivity of the anti-P-SHIP-1 antibodywhich was maximal at 15 minutes and then decreased back towards baseline by 30minutes (# p<0.05 stimulated vs unstimulated cells, Student’s paired t test).In conclusion, we found that the inhibitory phosphatase SHIP-1 exclusivelyparticipates in BCR signal transduction in ZAP-70- CLL cells, wherein it is expressedand constitutively tyrosine- phosphorylated to a greater extent compared to ZAP-70+samples. Taken together our data suggest that SHIP-1 might be involved in theimpaired BCR signaling commonly found in the former subgroup of patients. Moreover,given that SHIP-1 can negatively modulate not only BCR but also cytokine andchemokine receptor signaling (5), the possibility exists that ZAP-70- CLL cells, byexpressing higher SHIP-1 levels, hold higher signaling thresholds to differentmicroenvironment stimuli.