ABSTRACT
BACKGROUND: Apoptosis is known to be a crucial process involved in embryogenesis, development and homeostasis of the immune system. Impaired apoptosis causes dysfunction of lymphocyte homeostasis, growth advantage of tumor cells as well as resistance to current treatment protocols. To investigate the role of the apoptosis adaptor molecules TRADD and FADD in the development of hematological diseases, patient samples were screened for mutations in these genes. PROCEDURE: Genomic DNA from 51 children suffering from B-lineage-ALL (n = 17), T-lineage-ALL (n = 24), ALPS Type Ia (n = 3) and ALPS Type III (n = 7) were analyzed. Genomic DNA from 50 unrelated donors without hematological diseases served as controls. Identified mutations were cloned and their influence on cell viability and NFkappaB activation was analyzed by flow cytometry and luciferase assay, respectively. RESULTS: In the FADD gene no genetic alteration could be detected. However, three novel missense mutations in the TRADD gene could be detected. They are located within a region of TRADD known to exert mainly anti-apoptotic effects for example through the activation of the NFkappaB pathway. Functional analysis of cells overexpressing mutant TRADD cDNA demonstrated a reduced NFkappaB activity and consequently increased cell death compared to wild-type TRADD. CONCLUSION: Mutations in the TRADD gene may contribute to the development of different hematological diseases. The identified mutations demonstrate a putative impact on TRADD signaling and cell survival but may not mainly explain the pathology of the diseases investigated.
Subject(s)
Autoimmune Diseases/genetics , Lymphoproliferative Disorders/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , TNF Receptor-Associated Death Domain Protein/genetics , Animals , Autoimmune Diseases/metabolism , Base Sequence , Cell Death/physiology , Cell Survival , Child , Humans , Lymphoproliferative Disorders/metabolism , Mice , Mutation, Missense , NF-kappa B/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , TransfectionABSTRACT
Phytohemagglutinin-activated peripheral CD95+ T cells (day 1 T cells) are resistant to CD95-mediated apoptosis. After prolonged interleukin-2 treatment, these T cells become CD95-mediated apoptosis-sensitive (day 6 T cells). To elucidate the molecular mechanism of apoptosis resistance, day 1 and day 6 T cells were tested for formation of the CD95 death-inducing signaling complex (DISC). DISC-associated active Fas-associated DD protein (FADD)-like interleukin-1 beta-converting enzyme-like protease (FLICE) also referred to as MACH/caspase 8 was only found in apoptosis-sensitive day 6 T cells. Further-analysis of mRNA and protein expression levels of apoptosis-signaling molecules FADD, receptor interacting protein, hematopoietic cell protein tyrosine phosphatase, Fas-associated phosphatase-1, FLICE, bel-2, bcl-xL, and, bax-alpha showed that only the expression level of bcl-xL correlated with T cell resistance to CD95-mediated apoptosis (day 1 T cells: bcl-xhiL; day 6 T cells: bcl-XloL). In T cells activated in vitro, up-regulation of bcl-xL, has previously been correlated with general apoptosis resistance. However, the experiments presented suggest that resistance to CD95-mediated apoptosis in T cells can also be regulated at the level of recruitment of FLICE to the DISC.
