American Society of Hematology

Figure 2

$Antiapoptotic Bcl-xL protects B-NHL cells to rituximab-induced apoptosis in vitro and in vivo. (A) Rituximab-sensitive SU-DHL-4 cells were retrovirally transduced to express the indicated genetic inhibitors of apoptosis and EGFP via a bicistronic cassette. EGFP-expressing cells were isolated by fluorescence-activated cell sorting followed by treatment with cross-linked rituximab for 48 hours. The fraction of cells with apoptotic DNA fragmentation was quantified flow cytometrically after hypotonic lysis and staining with PI; mean values plus SD of 3 independent experiments are given. (B) Three rituximab-sensitive B-NHL cell lines were retrovirally transduced to express antiapoptotic Bcl-xL and EGFP or control vector, and were sorted as in panel A. The fraction of cells with apoptotic DNA fragmentation was quantified flow cytometrically after treatment with cross-linked rituximab for 48 hours; mean values plus SD of 3 independent experiments are shown. (C) Histopathologic analysis of NOD/SCID mice that were intravenously inoculated with human Ramos B-NHL cells. Representative photomicrographs from organ sections stained with hematoxylin/eosin and antibodies against the human CD20 antigen or the mitotic marker Ki-67. Note the presence of mitotically active human CD20-positive B-NHL cells demonstrating engraftment in various murine organs. Slides were viewed with a Zeiss Axioplan upright research-grade microscope for brightfield and fluorescence applications (Carl Zeiss, Jena, Germany) using a Plan-NEOFLUAR lens at 10 ×/0.30, 20 ×/0.50, and 40 ×/0.75. Images were acquired using the Axio Cam HRc camera (Zeiss), and were processed with Axio-Vision Rel. 4.6 software (Zeiss). (D) Kaplan-Meier plots of overall survival of NOD/SCID mice after intravenous inoculation of 107 Ramos (top panel) or Bcl-xL–expressing Ramos cells (bottom panel). Starting on day 5 after tumor inoculation, the mice received intraperitoneal injections of rituximab (+ R, red line) or vehicle (black line); 10 mice per group. Note that rituximab treatment significantly (P = .001) prolonged survival of Ramos lymphoma-bearing, but not of Ramos-Bcl-xL lymphoma-bearing mice.$

Antiapoptotic Bcl-xL protects B-NHL cells to rituximab-induced apoptosis in vitro and in vivo. (A) Rituximab-sensitive SU-DHL-4 cells were retrovirally transduced to express the indicated genetic inhibitors of apoptosis and EGFP via a bicistronic cassette. EGFP-expressing cells were isolated by fluorescence-activated cell sorting followed by treatment with cross-linked rituximab for 48 hours. The fraction of cells with apoptotic DNA fragmentation was quantified flow cytometrically after hypotonic lysis and staining with PI; mean values plus SD of 3 independent experiments are given. (B) Three rituximab-sensitive B-NHL cell lines were retrovirally transduced to express antiapoptotic Bcl-xL and EGFP or control vector, and were sorted as in panel A. The fraction of cells with apoptotic DNA fragmentation was quantified flow cytometrically after treatment with cross-linked rituximab for 48 hours; mean values plus SD of 3 independent experiments are shown. (C) Histopathologic analysis of NOD/SCID mice that were intravenously inoculated with human Ramos B-NHL cells. Representative photomicrographs from organ sections stained with hematoxylin/eosin and antibodies against the human CD20 antigen or the mitotic marker Ki-67. Note the presence of mitotically active human CD20-positive B-NHL cells demonstrating engraftment in various murine organs. Slides were viewed with a Zeiss Axioplan upright research-grade microscope for brightfield and fluorescence applications (Carl Zeiss, Jena, Germany) using a Plan-NEOFLUAR lens at 10 ×/0.30, 20 ×/0.50, and 40 ×/0.75. Images were acquired using the Axio Cam HRc camera (Zeiss), and were processed with Axio-Vision Rel. 4.6 software (Zeiss). (D) Kaplan-Meier plots of overall survival of NOD/SCID mice after intravenous inoculation of 10⁷ Ramos (top panel) or Bcl-xL–expressing Ramos cells (bottom panel). Starting on day 5 after tumor inoculation, the mice received intraperitoneal injections of rituximab (+ R, red line) or vehicle (black line); 10 mice per group. Note that rituximab treatment significantly (P = .001) prolonged survival of Ramos lymphoma-bearing, but not of Ramos-Bcl-xL lymphoma-bearing mice.

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