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

ALANIZ LAURA; GARCÍA MARIANA; GRECKZANIK SOFÍA; ALVAREZ ELIDA; HAJOS SILVIA E

Montréal, Québec. Canada

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

IUIS/FOCIS

L Resumen B lymphoma is a T cell spontaneous non immunogenic murine lymphoproliferative disorder characterized by a high degree of invasiveness. Previous phenotypic characterization showed expression on tumor cells of CD8+, CD4-, CD3-, CD25+, gp70-, J22d.2+ markers as well as class I but not class II mayor histocompatibility complex antigens (MHC). Expression of b2 integrin (CD18) and the a chain of the IL-2 receptor (CD25) make this tumor model comparable to Adult T-cell leukemia (ATL). Tumor cells produce constitutively IL-2, IL-15, IL-10, TNF-a and TGF-b, but neither humoral nor cellular immune response against the tumor was evidenced.  From the parental LB tumor 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)  respectively, and the sensitive cell line LBR- The aim of this work was to investigate NFkB activity and its modulation by specific NFkB inhibitors BAY 11-7082 (BAY) and caffeic acid phenethyl ester (CAPE), PI3K inhibitor wortmannin, the extracellular matrix component hyaluronic acid (HA) and the antineoplastic agents VCR and DOX. Since NFkB activation can suppress cell death pathways and protect tumor cells from the apoptotic cascade, 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 the parental tumor LB, LBLa and the resistant cell lines LBR-V160 and LBR-D160, while LBLc and LBR- showed low activity. The composition of the NFkB active complex was assayed using supershift assay with anti-p65 or anti-p50 antibodies. Both the 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 while HA fragments, obtained by enzymatic digestion, were unable to modulate  constitutive NFkB activity. To establish the correlation between NFkB activity and cell survival, apoptosis induction 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.