noglobulin secreting cells would result in loss of LEF-1 expression and decreased leukemic cell survival. Agents that induce terminal differentiation of leukemic cells have been utilized therapeutically with the greatest successes realized using all-trans retinoic acid for acute promyelocytic leukemia and interferon-a for hairy cell leukemia. This strategy relies on the fact that leukemic cells are blocked at a particular stage in their development and are not truly terminally differentiated. Several agents are able to induce leukemic cells to overcome their block of differentiation resulting in inhibition of proliferation and an increase in apoptotic cell death. This alternative differentiation strategy has been combined with traditional cytotoxic therapies to increase the efficacy of treatment. Of relevance to this study, there has been an interest in utilizing toll like receptor agonists in the treatment of hematologic malignancies. TLRs are a family of pattern recognition receptors involved in the detection of pathogen associated molecular patterns and other “danger”signals. This large class of receptors plays a crucial role in both innate and adaptive immunity. The rationale for their use in hematologic malignancies has centered on the induction of and sensitization to the antileukemic immune response, as well ” as potential direct antileukemic properties of these agents. October 2011 | Volume 6 | Issue 10 | e26056 CLL Differentiation Induces LEF-1 Loss Previous studies have shown that CLL cells respond to the TLR9 agonist, CpG, with proliferation, upregulation of costimulatory molecules, and induction of apoptosis. However, to our knowledge there has been no study to date that has evaluated the ability of these agents to induce CLL B cell differentiation into ISC. Previous studies suggest that CLL 
cells can secrete antibody in response to certain stimuli such as pokeweed mitogen, or other stimuli with cytokines. TLR agonists, along with cytokines IL-2 and IL-15, are well characterized in their ability to induce terminal differentiation of normal mature B cells. We hypothesized that CpG along with cytokines IL-2 and IL-15 could be used as a tool to induce CLL cell differentiation and alter prosurvival signaling by LEF-1 and the Wnt pathway. In the present study, we addressed two critical questions; do CLL cells lose LEF-1 expression upon differentiation into ISC; and do CLL cells that differentiate into ISC lose LEF-1 prosurvival signaling and exhibit decreased survival 1 st Strand cDNA Synthesis kit. One ml of cDNA was amplified using the SYBR Green/Rox PCR reagent in a total volume of 20 ml, which included 5 pM of each primer and 1 x SYBR Green Mastermix. Amplification was carried out using a 7900 HT Applied Biosystems fast real-time PCR system as follows: Debio-1347 custom synthesis denaturation at 95uC for 15 min; 40 cycles of 15 s at 95uC; 60 s at 60uC and a melting curve cycle. Melting temperature and quantitative analysis was performed using SDS RQ software. Relative fold change was normalized against 18 s rRNA after a 1:2000 cDNA dilution and calculated using the 22DDCT method. Western blot analysis Cells were lysed for 30 min on ice in lysis buffer containing 10 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, complete mini protease inhibitor cocktail, and phosSTOP phosphatase inhibitor cocktail. An equal volume of Laemmli sample buffer was added before boiling for 5 min. Lysates were subjected to electrophoresis through a
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