Delineating the apoptotic mechanisms of an anti-CD19 immunotoxin used in combination with the anti-CD20 antibody rituximab against human B-lymphoma cells

Bryson, Christine Jane (2004) Delineating the apoptotic mechanisms of an anti-CD19 immunotoxin used in combination with the anti-CD20 antibody rituximab against human B-lymphoma cells. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The aim was to determine the effect of rituximab and BU12-saporin individually and in combination on B lymphoma cell lines and determine the contribution of complement mediated mechanisms. In an in vitro protein synthesis inhibition assay, using Burkitt’s lymphoma cells, the presence of rituximab caused a complement independent 5-fold additive decrease to the IC₅₀ value of BU12-saporin. Two corroborative apoptosis assays confirmed that the functional effect of BU12-saporin and rituximab in combination was the induction of apoptosis. Statistical analysis showed exposure to rituximab and BU12-saporin in combination caused a significant additive, complement-independent increase to the sub G₀/G₁ population. Annexin V/PI staining of Ramos cells treated with rituximab and BU12-saporin showed significant additive complement independent increases to the early apoptotic and secondary necrotic cells, and significantly decreased the viable cell population.

This project also aimed to examine these event at the molecular level, including PARP cleavage, caspase activation the effect of caspase inhibitors, the role of selected Bcl-2 family proteins and to determine the effectiveness of the combination against cells over expressing Bcl-2. Cleavage of the caspase substrate PARP was detected by western blot of Ramos cells exposed to rituximab and BU12-saporin. Caspase 3 and caspase 9 cleavage was detected but no caspase 8 cleavage was detected, except in late apoptotic cells. Addition of the peptide caspase inhibitor _ZVAD significantly reduced the number of apoptotic cells, and PARP and caspase 3 cleavage. Blocking TRAIL or FAS receptors did not reduce the level of PARP cleavage or caspase 3 cleavage. This suggested the mitochondrial rather than the death receptor pathway was involved in the induction of apoptosis by rituximab and BU12-saporin.

Text

945884.pdf - Version of Record

Available under License University of Southampton Thesis Licence.

Download (9MB)