Figure 1.
The kinetics of changes in CD20 expression on Daudi cells following exposure to 0.5 Gy γ-radiation.
(A and B) Total CD20 levels: The changes in CD20 expression were measured using anti-CD20 antibodies. Results are expressed as ratio of levels of CD20 (CD20/β-Actin) with respect to 0 hr control. (C) CD20 expression on cell surface: The levels of CD20 at cell surface levels were calculated using quantiBRITE beads and expressed as numbers of CD20 molecules/cell. The index histogram is showing levels of CD20 at 20 hr (0.5Gy) with respect to 0 hr. Statistical analysis was done using Student's t-test (***p<0.001).
Figure 2.
The changes in ROS and MMP (ΔΨm).
(A, B and C) To measure changes ROS levels, fluorescence of CM-DCF was acquired at Ex λ of 488±10 nm and Em λ 517–527 nm and the results are expressed as the mean fluorescence ±SD of three independent experiments and statistical analysis was performed using ONE way ANOVA. Significant values are represented as; ***p<0.001 for Rtx vs 0.5Gy+Rtx or 1.5Gy+Rtx, ***p<0.001 for Tst vs 0.5Gy+Tst or 1.5Gy+Tst, ##p<0.01 for sham irradiated control vs 0.5Gy, ##p<0.001 for sham irradiated control vs 1.5Gy. Corresponding Isotype controls antibodies were used to measure changes in ROS levels. (D and E) The changes in ΔΨm were expressed from the mean fluorescence ±SD of three independent experiments and statistical analysis was performed using ONE way ANOVA. Significant values represents as; #p<0.05 for control vs 1.5Gy, *p<0.05 for control vs Rtx or 0.5Gy+RTX or 0.5Gy+Tst or 1.5Gy+Tst, **p<0.01 for control vs 1.5Gy+Rtx. Corresponding Isotypic controls were used to measure changes in ΔΨm levels [Human IgG1 (for Rtx) and Mouse IGG2a (Tst)].
Figure 3.
Cross-linking or homotypic adhesions (aggregations) and cell death.
(A–B) The binding of Fab regions of Anti-CD20 mAbs (Rtx and Tst) on CD20 and Fc region binding to FcγRIIB1 thereby induction of cross-linking or homotypic adhesions (aggregations) as well as extra cross-linking induced by corresponding secondary antibodies were observed microscopically (20x). Isotype control antibodies were taken separately to measure non-specific cross-linking. (C, D and E) Anti-CD20 mAbs induced cell death was measured using PI uptake by flowcytometrically. PI is membrane impermeable, generally excluded from viable cells and therefore, commonly used for identifying alterations in biological membrane and thereby death of cells in a population. The fold induction cell death was measured from mean fluorescence ±SD and statistical analysis was performed using ONE way ANOVA. Significant values represented as; ***p<0.001 for Rtx vs 0.5Gy+Rtx and 1.5Gy+Rtx. ***p<0.001 for Tst vs 1.5Gy+Tst, **p<0.01 Tst vs 0.5Gy+Tst. Further, ###p<0.001 for sham irradiated control vs 1.5Gy, and $$$p<0.001 for sham irradiated control vs Tst. Isotype control antibodies were taken separately to measure non-specific induction of cell death. (F) Induction of cell death by extra cross-linking using secondary antibodies: Cells expressing different levels of CD20 and treated with monoclonal anti-CD20 antibodies or isotypes (separately) were further incubated with corresponding secondary antibodies. PI uptake was also measured flowcytometrically to measure extra crosslinking induced cell death. (G) Cell cycle analysis: For cell cycle analysis, binding of PI with DNA were used as a marker of DNA content which depicts phase of cells in cell cycle.
Table 1.
Effects of anti-CD20 mAbs on the cell cycle phases.
Figure 4.
(A) Samples were subjected to alkaline comet assay to monitor DNA damage stained with SYBR Green I and Exλ 488 nm, Emλ 520 nm. Images were capture using Olympus BX60 florescence microscope at 20x. (B) Results are expressed as % DNA content in head and tail region.
Figure 5.
Cell death induced by monoclonal anti-CD20 Abs: Cell death was measured in cells expressing different levels of CD20 followed by treatment with monoclonal anti CD20 Antibodies.
(A) For measurement of apoptosis cells were lysed and equal quantity of proteins loaded and resolved in a 12% Tris–HCl gel, followed by transfer onto nitrocellulose membrane, and immunoblotted for (cleavage of both PARP and caspase 3) (B) p38 at site Thr180/Tyr 182 also were immunoblotted as described in methods section,(C) Phosphorylation of p53 at site ser15 and β-actin were used as loading control.
Figure 6.
Anti-CD20 mAbs-induced programed cell death.
Schematic diagram illustrating the kinetics of changes in CD20 expression followed 0.5Gy γ-radiation and sequence of events in the proposed possible cell death pathway evoked by type I anti-CD20 mAbs (Rtx) and type I anti-CD20 mAbs (Tst). Anti-CD20 mAbs ligation results in cross-linking and homotypic adhesions followed by generation of ROS and create genotoxic stress which ultimately culminates in apoptotic and non-apoptotic cell death. *SB = Strand Break.