Novel siRNA Expressing Lymphoma Model Identify Distinct Molecular Mechanisms Regulating NFkB1 and NFkB2 Induced Cell Proliferation.

The NFkB pathway has been implicated in tumorigenesis of several lymphoid malignancies. Most aggressive lymphomas have a constitutively active NFkB. Activation of NFkB results in processing of p105 and p100 to produce p50 and p52, respectively, and degradation of IkB. As a result, heterodimers of p65 and p50 or p52 and REL-B are formed and translocated to the nucleus where they induce transcription. Previously, it has been suggested that NFkB regulation of cyclin D and Myc expression is important for cell proliferation. However, these studies fail to explain all the NFkB regulatory effects, as well as the role of each individual pathway. To address these questions we tested the effect of NFkB on proliferation in two virally mediated murine lymphoma cell lines over-expressing Tax/HTLV-1 oncoprotein (SC and BLA) after knocking down (KD) p105 or p100, using a lentivirus expressing siRNAs. After synchronization, cells were released for 24 hrs and the rate of proliferation and cell cycle analysis were measured by thymidine incorporation and PI staining (4,8,16,24 hrs), respectively. As it is seen below, KD p100 or p105 cell lines had a reduced rate of thymidine incorporation compare to controls (Luciferase-siRNA). However, minimal changes were observed between cell lines in cell cycle analysis. To confirm these findings, xenograft experiments with siRNA expressing cell lines were developed. These experiments demonstrated that KD p100 or p105 in xenografts, prevented (in some) or delayed tumor formation, reduced tumor size and prolonged disease free survival. We then investigated NFkB regulation of cell proliferation by assessing gene expression in each cell line. Our results revealed a distinct proliferation-related gene profile between pathways: KD p105 dependent genes include:

• Cell cycle: Cyclin D2, B2 and G, CDK-8, P27, RB6, Transcription factor Dp2 and AKT-substrate-1,

• Growth factors: Platelet derived growth factor and RhoG,

• RNA synthesis: RNA Pol III.

On the other hand KD p100 dependent genes include:

1. Cell cycle: Cyclin D1,B2, retinoblastoma-like 1 (p107), p16, ring box-1 and histone deacetylase 2,

2. Growth factors: HRAS and RhoG,

3. RNA/DNA synthesis: RNA pol III and DNA pol (p17), histone 1 and 2, and thymidine kinases.

Interestingly, both KD cell lines share many over-expressed genes such as TGFB, p21, p53, RB7 and BRCA1. In conclusion, our novel experimental model demonstrates:

1. the main effect of both NFkB pathways involves DNA replication and early S phase.

2. KD of either p100 or p105 reduces tumor development.

3. The mechanism of cell proliferation regulated by each NFkB pathway is more complex than what was previously suspected; we suggest that RNA, DNA synthesis and regulation of p21, BRCA-1 and other cell cycle regulatory proteins also play an important role.