Role of PKC during B cell development and transformation

Abstract

The objective of this thesis is to determine the role of specific PKC isotypes during B cell development and transformation. B cell generation systems were validated both in vitro and in vivo, by coculturing haematopoietic progenitor cells (HPCs) on the calvanial cell line, 0P9, or by adoptively transferring HPCs into recombinase-activating gene 1-deficient (RAG-1-/-) mice, respectively. In both cases, mature B cells were generated as determined by analysing surface B cell marker expression. Coupling of these in vitro and in vivo B cell generation systems with a retroviral gene transfer technique, plasmids-encoding PKC mutants in the retroviral vector MIEV were stably expressed in foetal liver (FL)-derived HPCs from wild type mice and cultured to assess the ability of individual PKC isoforms to modulate the development or transformation of B cells. Of note, expression of a plasmid-encoding dominant negative PKCalpha (PKCalpha-KR) in HPCs and placement in B cell generation system in vitro or in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity. Analysis of PKCalpha-KR-expressing cells in vitro revealed that these cells incorporated BrdU significantly more than the MIEV control, and unexpectedly upregulated cell cycle regulators, p21waf-1 and p27kip-1. Of surprise, PKCalpha-KR-expressing cells phenotypically resemble human B cell chronic lymphocytic leukaemia (CLL) cells. Expression of constitutively active PKCalpha, PKCalpha-CAT, or dominant negative PKCalpha, PKCalpha-KR in HPCs caused significant decrease in cell number. CLL is characterised by the accumulation of long-lived phenotypically mature B cells with the distinctive phenotype: CD19hi, CDS+, CD23+, IgMdim, which are deficient in apoptosis and have undergone cell cycle arrest in the G0/G1 phase. Closer analysis of PKCalpha-KR-expressing cells uncovered that these cells undergo cell cycle arrest in the absence of growth factors and stroma and consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 and Mcl-1 expression. Upon stimulation with IL-7, PKCalpha-KR-expressing cells showed explosive proliferation, suggesting that IL-7 is a proliferation factor for these cells. In accordance with this, IL-7R expression was upregulated in these cells, which may contribute to the increased sensitivity to IL-7. Mice injected with wildtype PKCalpha-KR-HPCs bore solid intraperitonial tumours at the injection site and the cells from both tumour and spleen showed CLL-like phenotype. Interestingly, splenocytes from these mice were cycling whereas the tumour cells were arrested at the G0/G1 stage, probably reflecting the two phases of this disease, a quiescent stage and an extensive proliferative stage, respectively. The expansion of the leukaemic cells was halted when they were cultured on 0P9-DL1, 0P9 cells with ectopic expression of Notch ligand, DL1, suggesting that Notch signalling mediates tumour suppression in CLL cells