B-cell chronic lymphocytic leukemia (B-CLL IntroductionB-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal, slow-dividing CD5 ϩ B lymphocytes in the peripheral blood. [1][2][3] In most cases, these cells progressively infiltrate the bone marrow and secondary lymphoid tissue, resulting in poor prognosis. 1-3 Extravasation of B-CLL cells and migration through endothelium are mainly directed by 3 chemokines: CCL21, which is expressed in high endothelial venules (HEVs), and CCL19 and CXCL12, which are produced by stromal cells of lymph nodes and bone marrow, respectively. 4,5 The corresponding receptors for these chemokines, CCR7 (for CCL21 and CCL19) and CXCR4 (for CXCL12), are highly expressed in B-CLL with widespread involvement of lymph nodes. 5,6 Other molecules such as vascular endothelial growth factor (VEGF) and ␣L␤2/␣4␤1 integrins were also recently shown to be involved in B-CLL transendothelial migration in response to chemokines. 7 Moreover, high expression of ␣4␤1 (but not ␣L␤2) correlated with the presence of lymphadenopathy, 7 suggesting an important role for this integrin in B-CLL progression.Transendothelial migration and organ invasion of malignant cells also require proteolytic degradation of the vascular basement membrane and the extracellular matrix of lymphoid tissues. This can be accomplished by matrix metalloproteinases (MMPs), [8][9][10] in particular the gelatinases MMP-2 and MMP-9. MMPs also release matrix-bound growth factors that stimulate malignant cell expansion and angiogenesis. 11 Indeed angiogenesis is increased in the bone marrow of B-CLL patients, [12][13][14] and high levels of the angiogenic factors VEGF and basic fibroblast growth factor (bFGF) have been detected in the urine and serum of these patients. 12,15,16 Previous studies have shown that early-stage B-CLL cells produce and secrete MMP-9, which can be detected in the serum of these patients and in B-CLL cell culture supernatants. 17 It was later demonstrated that B-CLL cells constitutively produce MMP-9 in various molecular forms and that elevated levels of intracellular MMP-9 correlate with advanced stage and poor patient survival. 18 Moreover, MMP-9 was highly expressed by B-CLL cells present in the bone marrow (with a diffused pattern) and in lymph nodes, and contributed to B-CLL migration through artificial basement membranes or endothelial cells. 18 The presence of other MMPs in B-CLL cells has not been reported. performed research; R.S. performed the confocal microscopy analyses; M.J.T. contributed with patient samples and data; J.A.G.-M. contributed patient samples and data; and A.G.-P. designed and supervised research and wrote the paper.The online version of this article contains a data supplement.Reprints: Á ngeles García-Pardo, Departamento de Inmunología, Centro de Investigaciones Bioló gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; e-mail: agarciapardo@cib.csic.es.The publication costs of this article were defrayed in part by page charge payment. Therefore, an...

Purpose: Arsenic trioxide (ATO) induces B-cell chronic lymphocytic leukemia (B-CLL) cell apoptosis in vitro. We sought to study the mechanism involved in this effect and whether ATO is suitable for combination therapies with protein kinase inhibitors.Experimental Design: B-CLL cells were isolated from the peripheral blood of 28 patients. Cell viability studies with ATO alone or in combination with kinase inhibitors were done by flow cytometry, Western blotting, and immunofluorescence analyses.Results: After 48 hours, 3 μmol/L ATO induced apoptosis (average 75%) in all B-CLL samples studied and with minimal effect on normal peripheral blood lymphocytes. Apoptosis entailed Akt and NF-κB inactivation, XIAP downregulation, and PTEN upregulation, thus implying inhibition of the phosphoinositide 3-kinase (PI3K) survival pathway. Indeed, the combination of ATO and PI3K inhibitors increased the apoptotic effect of either agent alone. ATO also induced c-jun-NH 2 terminal kinase (JNK) activation, and this was crucial and required for subsequent apoptotic events, as inhibiting JNK activity by either gene silencing or specific inhibitors prevented Akt and NF-κB inactivation, caspase activation, and mitochondrial damage. Moreover, JNK activation was the earliest response to ATO, preceding and determining reactive oxygen species production.Conclusions: We identified the mechanism involved in ATO action on B-CLL cells and show that the combination of low doses of ATO and PI3K inhibitors efficiently induces B-CLL cell death. ATO may therefore constitute an efficient treatment for B-CLL, particularly in combined therapies. Clin Cancer Res; 16(17); 4382-91. ©2010 AACR.