Modulation of NFkB activity and its relationship with apoptosis in a murine T cell lymphoma

LAURA ALANIZ; MARIANA GARCÍA; VICTORIA CAVALIERE; ELIDA ALVAREZ; SILVIA HAJOS

Montreal, Quebec

Congreso; 12th International Congress of Immunology (ICI) and 4th Annual Conference of FOCIS; 2004

LB is a T cell spontaneous murine lymphoma characterized by a high degree of invasiveness. Previous phenotypic characterization showed expression of CD8+, CD4-, CD3-, CD25+, gp70-, J22d.2+ markers as well as class I but not class II MHC. Expression of CD18 and CD25 make this tumor model comparable to Adult T-cell leukemia. Tumor cells produce IL-2, IL-15, IL-10, TNF-a and TGF-b, but no immune response against the tumor was evidenced.  Several cell lines have been established: LBLc (cluster) and LBLa (adherent) with differences in their invasive behaviour and migratory capacity toward HA; LBR-V160 and LBR-D160, resistant to Vincristine (VCR) and Doxorubicin (DOX). The aim of this work was to investigate NFkB activity and its modulation by specific inhibitors BAY 11-7082 (BAY) and CAPE, PI3K inhibitor wortmannin, extracellular matrix component hyaluronic acid (HA) and antineoplastic agents VCR and DOX. Since NFkB activation can suppress cell death pathways and protect tumor cells from the apoptosis, we studied the relationship between the activity of this transcription factor and cell survival. Constitutive NFkB activity was determined by EMSA demonstrating high activity in LB, LBLa, LBR-V160 and LBR-D160, while LBLc and LBR- showed lower activity. Composition of the NFkB complex was assayed using supershift assay with anti-p65 or anti-p50; both subunits were detected. NFkB activity was completely inhibited after treatment with BAY, partially with CAPE but not modified by wortmannin. Treatment with VCR, DOX or with HA enhanced NFkB activity. To establish the correlation between NFkB activity and cell survival, apoptosis was analyzed by morphological features and Anexin V. Results indicated that BAY, CAPE and wortmannin induced apoptosis (50-80%, p< 0.05) on the cell lines studied. Treatment with HA or with VCR or DOX in combination with BAY had not significant effect. However HA fragments induced 20 % ( p<0.05) apoptosis. Conclusion: In the tumor model studied: a) inhibition of NFkB activity by BAY was related with cell mortality while induction of apoptosis by CAPE and wortmannin was independent of NFkB signaling pathway, b) HA fragments behaved in a similar way, c) induction of NFkB activity by VCR, DOX or HA did not affect cell survival.