An MTT assay on control or drug-treated cells was performed as described elsewhere (23). promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors. studies with cells derived from patients suffering from various B-cell malignancies, a positive correlation between CD20 levels and rituximab sensitivity was found (5, 6). van Meerten (7) have exhibited a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC (R-CDC) but not ADCC. In this experimental model, the level of CD20 expression was the only variable, and it was clearly shown that reduced CD20 expression leads to impaired CDC. A direct correlation between R-CDC and the number of CD20 molecules in primary NHL cells was also found by Bellosillo (6). Therefore, strategies that lead to up-regulation of CD20 expression may improve R-CDC against low CD20-expressing cells and provide a rationale for overcoming rituximab resistance. Accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Several case or retrospective studies reported that rituximab treatment may result in CD20-unfavorable relapses (8C15), although their prevalence and duration are currently unknown. A number of mechanisms that account for the modulation of CD20 levels have been proposed. Most likely their occurrence and significance vary depending on the type of malignancy. In CLL, rituximab-mediated down-modulation of CD20 is associated with reduced levels of CD20 mRNA both (16) and (17), indicating regulation at the level of transcription. For example, activated Flt3 signaling cascade has been reported to inhibit expression of PU.1, a transcription factor involved in the expression of gene (18). Down-regulation of CD20 mRNA has been also observed in CD20-unfavorable cells obtained from patients after relapse of rituximab-treated B-cell malignancies (15). Several studies revealed that CD20 can undergo shaving (19) or lysosomal internalization (20) following rituximab exposure. Anisotropine Methylbromide (CB-154) Epigenetic mechanisms also play an emerging role in the regulation of CD20 levels (15, 21, 22). We have observed previously that statins impair detection of CD20 in NHL cells and impair R-CDC and ADCC (23). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl pyrophosphates), which are necessary for posttranslational modification of 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20, we observed that neither geranylgeranyltransferase (GGTI) nor farnesyltransferase (FTI) inhibitors could mimic the effect of statins. On the contrary, prenyltransferase inhibitors improved R-CDC. FTIs were initially developed to target tumors with Ras mutation (24). However, subsequent studies revealed their activity in tumors with normal Ras that seems to result from inhibition of prosurvival signaling mediated by other prenylation-dependent pathways. Importantly, tipifarnib, a farnesyltransferase inhibitor, was recently shown to exert some therapeutic activity in patients with relapsed and refractory lymphomas (25). Therefore, we decided to investigate in more detail the influence of prenyltransferase inhibitors on antitumor activity of anti-CD20 mAbs. EXPERIMENTAL PROCEDURES Cell Culture Human Burkitt lymphoma (Raji and HYRC Ramos) and human follicular lymphoma (DoHH2) cell lines (purchased from American Tissue Culture Collection), HEK-293T cells (purchased from DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal Anisotropine Methylbromide (CB-154) bovine serum (FBS), 100 g/ml streptomycin, 100 units/ml penicillin, and 250 ng/ml amphotericin B (Invitrogen). Cells were cultured at 37 C in a humidified atmosphere of 5% CO2 and passaged approximately every other day. Leukocyte Isolation from Blood and in Vitro Culture Primary cells from patients with B-cell tumors (NHL and CLL) were isolated from full blood using Histopaque-1077 (Sigma-Aldrich) as described elsewhere (23). Anisotropine Methylbromide (CB-154) Cells were cultured with increasing concentrations of L-744,832 for 48 h in Iscove’s modified Dulbecco’s medium supplemented with 10% heat-inactivated FBS, 100 g/ml streptomycin, 100 units/ml penicillin, and 250 ng/ml amphotericin B (Invitrogen) at 37 C in a humidified atmosphere of 5% CO2. Approval for the study was obtained from the Institutional Review Board of the Medical University of Warsaw and was conducted according to the Declaration of Helsinki. Each patient gave a written informed consent for the procedures. Reagents Rituximab, a chimeric IgG1, was purchased from Roche Applied Science. Ofatumumab (2F2; HuMax-CD20) and FITC-conjugated ofatumumab were generous Anisotropine Methylbromide (CB-154) gifts from Genmab A/S (Utrecht, The Netherlands). Farnesyltransferase inhibitors (FTI-276 and FTI-277) and geranylgeranyltransferase inhibitors (GGTI-286, GGTI-298, and GGTI-2133) were from Calbiochem (Merck LGaA). L-744,832 was purchased from Enzo Life Sciences (Plymouth Getting together with, PA). FTI-277 was dissolved in water, whereas other inhibitors were dissolved in dimethyl sulfoxide. Bortezomib obtained from Millenium Pharmaceuticals was dissolved in 0.9% NaCl. Cycloheximide (Sigma-Aldrich) was freshly dissolved before each.

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