An anti-CD19 antibody inhibits the interaction between P-glycoprotein (P-gp) and CD19, causes P-gp to translocate out of lipid rafts, and chemosensitizes a multidrug-resistant (MDR) lymphoma cell line by Maria-Ana Ghetie, Radu Marches, Stephanie Kufert, and Ellen S. Vitetta Blood Volume 104(1):178-183 July 1, 2004 ©2004 by American Society of Hematology

Expression of CD19 and P-gp as determined by the binding of anti-CD19 (HD37) and anti–P-gp (UIC2) MAbs to Namalwa/MDR1 cells. Expression of CD19 and P-gp as determined by the binding of anti-CD19 (HD37) and anti–P-gp (UIC2) MAbs to Namalwa/MDR1 cells. (A) Cell surface P-gp expression on Namalwa (dashed line) versus Namalwa/MDR1 (solid line), detected with PE-mouse anti–P-gp (UIC2) and analyzed on a FACScan, and (B) the corresponding total P-gp protein level, detected by Western blotting of the 1% Triton X-100 cell lysates (100 μg protein/lane). The binding affinities of HD37 (○) and UIC2 (•) for P-gp on Namalwa/MDR1 cells were calculated from the saturation curve expressed as (C) percent positive cells and (D) mean fluorescence intensity (MFI). Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

Rhodamine 123 efflux as a functional test for the P-gp pump. Rhodamine 123 efflux as a functional test for the P-gp pump. Namalwa/MDR1 cells at 5 × 105 per milliliter were treated for one hour at 37°C with 1.3 μM rhodamine 123 alone or in combination with 10 μM verapamil, 10–8 M HD37, or UIC2. Then cells were washed twice with RPMI 1640 without FBS, resuspended in FBS-free medium, and incubated for 2 more hours at 37°C to measure efflux. Namalwa cells were used as a positive control for the retention of rhodamine 123. (A) Namalwa/MDR1 cells in media (1) without rhodamine 123, (2) with rhodamine 123, and (3) with verapamil and rhodamine 123; and (4) Namalwa cells with rhodamine 123. (B) Namalwa/MDR1 cells (1) without rhodamine 123, and with rhodamine 123 in the (2) absence and (3) presence of verapamil. (4) Cells were pretreated for 10 minutes at 37°C with 10 mM MBCD prior to conducting the rhodamine 123 assay. (C) Namalwa/MDR1 cells with media (1) without rhodamine 123, (2) with rhodamine 123, (3) treated with verapamil and rhodamine 123, (4) treated with anti-CD19 (HD37) and rhodamine 123, and (5) treated with anti–P-gp (UIC2) and rhodamine 123. The histograms in each panel depict 1 representative experiment of 3 performed. Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

The chemoresistance of Namalwa/MDR1 cells can be overcome in the presence of anti-CD19. The chemoresistance of Namalwa/MDR1 cells can be overcome in the presence of anti-CD19. Cells were cultured in media ± vincristine (VC; 25 and 50 nM). Cells were counted daily and viability was determined by trypan blue exclusion. (A) Namalwa/MDR1 cells cultured with vincristine for 3 weeks survived, whereas parental Namalwa cells died after 3 days. This is 1 representative experiment of 3 performed. (B) The cytotoxic effect of doxorubicin in Namalwa/MDR1 cells in media (▾) or media supplemented with HD37 (15 μg/mL; □) versus parental Namalwa cells (•). Cells were incubated overnight with 15 μg/mL HD37, then plated in triplicates in 96-well plates and incubated for 72 hours in the presence of different concentrations (5-50 nM) of doxorubicin (triplicates were used for each concentration). The cells were then pulsed with 3H-thymidine for 18 hours, harvested, and counted. The radioactivity in treated cells versus control cells was then determined. This is a representative experiment of 5 performed. Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

Association of CD19 and P-gp in Namalwa/MDR1 cells. Association of CD19 and P-gp in Namalwa/MDR1 cells. (A) Coprecipitation of CD19 and P-gp in 1% Triton X-100 lysates of Namalwa/MDR1 cells. Equal amounts of the total cell lysate proteins were immunoprecipitated with the indicated MAbs and analyzed by Western blotting for the presence of P-gp, CD19, and CD22. (B-C) Flow cytometric analysis of FRET between PE anti–P-gp and Cy5 anti-CD19 or Cy5 anti-CD22 in Namalwa/MDR1 cells. Cells (106) were incubated for 30 minutes on ice with 1 μg PE anti–P-gp, washed, and then incubated for another 30 minutes on ice in the absence or presence of 1 μg Cy5 anti-CD22 MAb or anti-CD19. The cells were washed, fixed with 1% paraformaldehyde, and analyzed on a FACS-Calibur flow cytometer. The bold line histogram represents background PE fluorescence in the absence of Cy5 anti-CD19 or anti-CD22, whereas filled histograms represent PE fluorescence in the presence of Cy5 anti-CD19 (B) or Cy5 anti-CD22 (C). PE fluorescence quenching in the presence of Cy5 anti-CD19 corresponds to the percent of energy transfer efficiency (E%) of 49.9%. Cy5 anti-CD22 did not induce fluorescence quenching (E = 1.1%). At least 3 independent experiments were performed. IP indicates immunoprecipitation. Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

Detection of GM1 ganglioside as a marker for lipid rafts. Detection of GM1 ganglioside as a marker for lipid rafts. (A) GM1 on intact cells. Cells were treated with FITC-ChB in a direct binding assay and the cells were analyzed on FACScan. Namalwa/MDR1 cells (1) in media, (2) stained with FITC-ChB, (3) pretreated with anti-CD19, or (4) pretreated with anti–P-gp prior to staining with FITC-ChB. (B) Distribution of GM1 ganglioside in the lipid fractions isolated by sucrose gradient ultracentrifugation of the Triton X-100 lysates of Namalwa/MDR1 cells. Cells were incubated for one hour at 37° C in the absence or presence of HD37, UIC2, or RFB4 (15 μg/106 cells), and 10 μM verapamil or 10 mM MBCD, lysed, and fractionated in a sucrose gradient. Combined raft fractions and soluble fractions from 5 × 108 cells were resolved (25 μL/lane) by 12% SDS-PAGE under nonreducing conditions and immunoblotted with ChB–horseradish peroxidase. Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

Distribution of P-gp and CD19 in the lipid raft fractions of the plasma membrane. Distribution of P-gp and CD19 in the lipid raft fractions of the plasma membrane. The 12 fractions isolated by sucrose gradient ultracentrifugation of the 1% Triton X-100 cell lysates from Namalwa/MDR1 cells before and after treatment with HD37 or RFB4 were analyzed for the presence of (A) P-gp and (B) CD19. The combined lipid raft fractions from the gradient (3-6) and soluble fractions from the gradient (9-12) were analyzed for the presence of GM1 and CD45, respectively (C). The proteins were electrophoresed on a 6% (P-gp), 7.5% (CD45), 8% (CD19), and 12% SDS-PAGE (GM1), and analyzed by Western blotting using either specific antibodies or HRP-ChB, as indicated in “Materials and methods.” Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

Anti-CD19 does not induce the internalization of P-gp. Anti-CD19 does not induce the internalization of P-gp. Cells (106) were incubated for 30 minutes on ice in the absence or presence of 15 μg anti-CD19, anti-CD22 (negative control), or anti–P-gp (positive control). The excess unbound antibody was removed by 2 washes in PBS, and then the cells were incubated at 37° C for 4 hours. The level of cell surface P-gp was then detected using PE-UIC2 and cells were analyzed by FACS. WT indicates wild type Namalwa cells. Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology

Anti-CD19 inhibits cell growth. Anti-CD19 inhibits cell growth. The number of cells (top panels) and the viability detected by trypan blue exclusion (bottom panels) are shown. Namalwa/MDR1 cells (A) and parental Namalwa cells (B) were cultured either in media (control) (•) or in the presence of 15 μg/mL of HD37 (○) and RFB4 (▾), and the number of cells was determined daily. The viability of Namalwa/MDR1 cells (C) and parental Namalwa cells (D) was also determined for cells grown in the presence of 25 nM vincristine in media only (•) or in media supplemented with 15 μg/mL of either HD37 (○) or RFB4 (▾). Viabilities of both cell lines treated with HD37 alone are also shown (▿). Maria-Ana Ghetie et al. Blood 2004;104:178-183 ©2004 by American Society of Hematology