Screening, identification, and functional analysis of three novel missense mutations in the TRADD gene in children with ALL and ALPS.

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.

Induction of apoptosis by IFNgamma in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit.

The CD95 (APO-1/Fas) system can mediate apoptosis in immune cells as well as in tumour cells, where it may contribute to tumour immune-escape. On the other hand, its induction by anticancer drugs may lead to tumour reduction. Interferongamma (IFNgamma) increases the sensitivity of tumour cell lines to anti-CD95 antibody-mediated apoptosis. We describe induction of apoptosis by IFNgamma through the expression of CD95 and its ligand (CD95L) in human neuroblastoma cell lines. Neuroblastoma cells showed low constitutive expression of CD95 and CD95L. Subsequent to IFNgamma-modulated increase in CD95 and CD95L mRNA as well as protein levels, apoptosis was observed. Our results demonstrated that cytokine-mediated apoptosis was mediated through the activation of the CD95/CD95L autocrine circuit since: (i) cell death occurred following CD95/CD95L expression and correlated with CD95 and CD95L expression levels, (ii) failed to occur in a clone which weakly upregulated CD95 and lacked CD95L induction after IFNgamma stimulation, (iii) was at least partially inhibited by using blocking F(ab')2 anti-CD95 antibody fragments and the recombinant Fas-Fc protein, that prevented the interaction between CD95 and CD95L. The intracellular molecular mechanisms elicited by IFNgamma are clearly highly complex, with several signalling pathways being activated, including the CD95 system. These findings suggest that IFNgamma may have a significant potential in the therapy of neuroblastoma in vivo.

Modulation of resistance to anti-APO-1-induced apoptosis in osteosarcoma cells by cytokines.

The CD95/APO-1 Fas receptor/ligand system plays a crucial role in growth control by mediating apoptosis in lymphoid and non-lymphoid cells. To investigate the role of CD95-mediated apoptosis in osteosarcoma, we studied 3 human osteosarcoma cell lines (HOS/TE 85, MG 63 and Saos-2) and osteoblasts derived from bone biopsies. In contrast to osteoblast-like cells, all cell lines were resistant to anti-APO-1-induced apoptosis despite constitutive CD95 expression at intermediate levels. Blocking of macromolecular synthesis by cycloheximide or actinomycin D or modulation of CD95 expression by cytokines (TNF-alpha and/or gamma-interferon) restored sensitivity to anti-APO-1-induced cell death. PCR analysis of the CD95 transcripts revealed the production of a truncated splice variant that codes for a soluble form of the CD95 receptor. Synthesis and secretion of soluble CD95 protein into the culture supernatant was demonstrated by Western blot analysis. Treatment with sensitizing cytokines led to up-regulation of full-length CD95 transcripts and the encoded membrane-bound CD95 protein but not the truncated mRNA splice variant and the corresponding soluble receptor, as shown by PCR and Western blot analysis. The biological activity of soluble CD95 secreted by osteosarcoma cells was demonstrated by the ability of osteosarcoma supernatants to protect the sensitive T-cell line Jurkat from anti-APO-1-mediated apoptosis. Our results suggest that the production of soluble CD95 by osteosarcoma cell lines that may block physiological death signals and the production of membrane-bound CD95 are differently regulated by cytokines via modulation of RNA splicing.

Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex.

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.