Internalisation of uncross-linked rituximab is not essential for the induction of caspase-independent killing in Burkitt lymphoma cell lines

Abstract

Characterising the mechanisms underpinning caspase-independent programmed cell death (CI-PCD) induction by uncross-linked rituximab in B-cells may positively impact upon the treatment of disease states in which the classical apoptotic pathway is disabled. The necessity of rituximab internalisation for CI-PCD induction was investigated by flow cytometry and confocal microscopy in human BL cell lines with (e.g. Mutu I) and without (Mutu III) susceptibility to rituximab-induced killing. Flow cytometry demonstrated small, significant and similar amounts of rituximab internalisation by Mutu I cells after 1, 2, 4 and 24 h (p < 0.03, n = 5) and Mutu III cells after 0.5, 2, 4 and 24 h (p < 0.05, n = 4). Confocal microscopy confirmed this. Cytochalasin B and latrunculin A significantly inhibited rituximab-induced CI-PCD (p ≤ 0.04, n = 6 and p = 0.01, n = 6, respectively) and internalisation (p = 0.02, n = 5 and p = 0.0002, n = 6, respectively) in Mutu I cells, but confocal microscopy showed no correlation between internalised rituximab and phosphatidylserine exposure. We conclude that rituximab internalisation is not essential for CI-PCD induction in BL cell lines.

Keywords:

• Rituximab
• CD20
• internalisation
• caspase-independent cell death
• Burkitt lymphoma
• endocytosis