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

GABELLONI MARÍA LAURA; BORGE MERCEDES; MORANDE PABLO; BEZARES FERNANDO; GIORDANO MIRTA; GAMBERALE ROMINA

London

Workshop; XII International Workshop on Chronic Lymphocytic Leukemia; 2007

Leukemic cells from aggressive B cell chronic lymphocytic leukemia (CLL) patients typically display B cell receptors (BCRs) encoded by unmutated immunoglobulin variable 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 in patients with indolent disease (1). It has been proposed that survival differences between these two subgroups are related to the differential ability of the BCR to respond to stimulation (2, 3).Thus, ZAP-70+ patients display a more effective BCR signal 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-2 are involved in the complex and organized machinery aimed at counterbalancing B cell activation upon BCR engagement by restricting the duration and/or intensity of the signaling (4, 5). Since ZAP-70- patients present an impaired BCR signaling capacity, we have explored the possibility that these phosphatases were preferentially expressed in CLL cells from this subgroup. The study was carried out with fourty nine typical CLL patients, whose median age was 70 years old. ZAP-70 expression was evaluated by flow cytometry as it was previously 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 B cells from ZAP-70- and ZAP-70+ CLL patients, while SHIP-2 could not be detected in any of the samples analyzed, even when the amount of protein loaded in each lane was duplicated. Normal tonsillar B lymphocytes, used as controls, showed specific bands at 68kDa, 145kDa and 150kDa corresponding to SHP-1, SHIP-1 and SHIP-2 respectively. There were no significant differences in SHP-1 expression between CLL subgroups. By contrast, ZAP-70- CLL cells not only displayed a higher SHIP-1 protein expression compared to ZAP-70+ subgroup (p<0.01, Mann-Whitney nonparametric test), 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 cross-linking led to a time-dependent increase in reactivity of the anti-P-SHIP-1 antibody which was maximal at 15 minutes and then decreased back towards baseline by 30 minutes (# p<0.05 stimulated vs unstimulated cells, Student’s paired t test). In conclusion, we found that the inhibitory phosphatase SHIP-1 exclusively participates in BCR signal transduction in ZAP-70- CLL cells, wherein it is expressed and constitutively tyrosine- phosphorylated to a greater extent compared to ZAP-70+ samples. Taken together our data suggest that SHIP-1 might be involved in the impaired BCR signaling commonly found in the former subgroup of patients. Moreover, given that SHIP-1 can negatively modulate not only BCR but also cytokine and chemokine receptor signaling (5), the possibility exists that ZAP-70- CLL cells, by expressing higher SHIP-1 levels, hold higher signaling thresholds to different microenvironment stimuli. REFERENCES 1.         Crespo M et al. N Engl J Med. 2003 May 1;348(18):1764-75. 2.            Stevenson FK, Caligaris-Cappio F. Blood. 2004 Jun 15;103(12):4389-95. 3.         Chen L et al. Blood. 2002 Dec 15;100(13):4609-14. 4.         Zhang J et al. 2000 Aug;12(4):361-78. 5.         March ME, Ravichandran K. Semin Immunol. 2002 Feb;14(1):37-47.