Carlo Dufour, M.D, Haematology Unit, Department of Paediatric Haemato-Oncology, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy. E-mail: email@example.com
Tumour necrosis factor (TNF) contributes to the pathogenesis of bone marrow failure in Fanconi anaemia (FA) patients. The sensitivity of haematopoietic cells from FA, complementation group A (FANCA) subjects, who represent the majority of FA patients, to TNF-related apoptosis-inducing ligand (TRAIL) is unknown. The human lymphoblastoid FANCA HSC072 cell line and the genetically corrected counterpart HSC072FANCA-neo were tested for apoptoptic response to TRAIL using flow cytometry and Western blotting. FANCA cells were more sensitive to TRAIL-induced apoptosis than their corrected counterparts, indicating that TRAIL negatively regulates haematopoietic FANCA cell lines. This effect involved poly(ADP-ribose) polymerase-1 cleavage and caspase-8 activation.
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Fanconi anaemia (FA) cells display increased sensitivity to apoptosis, either spontaneously or induced by tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) (Haneline et al, 1998; Rathbun et al, 2000).
The TRAIL does not induce apoptosis of human and murine FA, complementation group C (FANCC) cell lines (Platzbecker et al, 2004). As FA patients of diverse complementation groups have different clinical and haematological phenotypes (Faivre et al, 2000), we investigated the sensitivity of FA complementation group A (FANCA) cells to TRAIL.
Materials and methods
Human HSC072 and HSC072FANCA cell lines were provided by H. Joenje (VU University Medical Centre, Amsterdam, the Netherlands). HSC536 and HSC536FANCC cell lines were from Coriell Cell Repositories (Camden, NJ, USA). Mitomycin C test was performed as reported (Clarke et al, 2004).
Recombinant (r) SuperKiller TRAIL (100 ng/ml) was from Alexis (Grünberg, Germany). Anti-TRAIL receptors (TRAIL-R)-1 to -4 monoclonal antibodies were from Apotech (Epalinges, Switzerland). Apoptosis was evaluated using the Annexin V kit (Bender, San Bruno, CA, USA).
For Western blot experiments, cells were lysed, subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters. Anti-poly(ADP-ribose) polymerase (PARP)-1 mouse monoclonal antibody was from Becton-Dickinson (San Jose, CA, USA). Peroxidase-conjugated goat–anti-mouse IgG was from Vector (Peterborough, UK) and SuperSignal® west pico chemiluminescent from Pierce (Rockford, IL, USA).
The FLICA Kit for activated Caspase-8 detection was from Immunochemistry Technologies (Bloomington, MN, USA). In some experiments, cells were pre-treated for 1 h with Z-IETD-FMK caspase-8 inhibitor (Alexis), cultured with rTRAIL, stained with Annexin V and analysed using a FACScan (Becton-Dickinson) with the Cell Quest Software (Becton-Dickinson).
Mann–Whitney test was used to perform statistical analysis. A P-value lower than 0·05 was considered statistically significant.
The HSC072 and HSC536 cells were mitomycin C sensitive, as opposed to their corrected counterparts (Fig. 1A, left).
A volume of 100 and 300 ng/ml TRAIL-induced higher apoptosis in HSC072 over corrected cells (P = 0·0016 and P = 0·0023 respectively) (Fig. 1A, right). Upon exposure to TRAIL for 4 or 16 h, HSC072 cells showed an increment in apoptosis over HSC072FANCA cells (P = 0·002 and P = 0·041 respectively). HSC536 and corrected cells did not differ in TRAIL-induced apoptosis (Fig. 1B). A volume of 100 ng/ml TRAIL and 4-h incubation were used in all subsequent experiments.
Expression of TRAIL-R1 and TRAIL-R2 death receptors and TRAIL-R3 and TRAIL-R4 decoy receptors was similar in FANCA and FANCC cells and their corrected counterparts (Fig. 1C).
The intensity of the 89-kDa PARP-1 band cleaved following TRAIL treatment was higher in HSC072 than in HSC072FANCA cells, but similar in HSC536 and HSC536FANCC cells (Fig. 2A). Accordingly, TRAIL-induced caspase-8 activation was higher in HSC072 than HSC072FANCA cells, but not in HSC536 versus HSC536FANCC cells (Fig. 2B).
Treatment of HSC072 cells with 50 μmol/l Z-IETD-FMK, a specific caspase-8 inhibitor, abrogated caspase 8 activation (Fig. 2C).
The TRAIL-induced apoptosis of FANCA cells was independent of TRAIL death or decoy receptor expression and occurred through caspase-8 activation and PARP-1 cleavage. In contrast, FANCC cells were unaffected by TRAIL, as previously reported (Platzbecker et al, 2004).
Fanconi anaemia patients belonging to different complementation groups display different clinical and haematological phenotypes. Thus, it can be speculated that FANCA and FANCC gene products may be involved in diverse signalling pathways, resulting in an apoptotic response to TRAIL in the case of FANCA protein.
Bone marrow failure in FA may be related to excessive apoptosis of haematopoietic progenitor cells in response to different stimuli, including inhibitory cytokines IFN-γ and TNF-α. The latter was shown to have a detrimental in vitro effect on the growth of erythroid marrow progenitors of FA patients, thus confirming a role for this cytokine in bone marrow damage (Dufour et al, 2003).
As TRAIL belongs to the TNF superfamily and has pro-apoptotic activity, our results indicate this effector as another negative agent on haematopoietic FANCA cells, potentially involved in marrow failure in FANCA patients. Experiments on primary cells may confirm this role in the pathogenesis of marrow failure of FA patients.
We thank Dr G. Bagby for comments and to Dr H. Joenje for providing the cell lines. The secretarial assistance of Mrs Chiara Bernardini is acknowledged. Grant support from Italian Ministry of Health grant no. 132/03; Compagnia di San Paolo; Fondazione Carige; ERG S.p.A.; SAAR Depositi Oleari Portuali Genova; AIRFA (Associazione Italiana per la Ricerca sull'Anemia di Fanconi); Fondazione Berlucchi.