Naslov
Absence or low expression of Fas-associated protein with death domain in acute leukemia and lymphoma cell lines

Autori
Matić, Igor ; Radnić, Maja ; Marijanović, Inga ; Caput-Mihalić, Katarina ; Furčić, Ivana ; Nagy, Biserka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
European Journal of Cancer Supplements, Vol. 8, Issue 5 / - : Elsevier, 2010

Skup
20th European Association for Cancer Research conference

Mjesto i datum
Oslo, Norveška, 26.07.2010. - 29.07.2010

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
FADD; lymphoma; leukemia

Sažetak
The dominant paradigm of tumor biology is that evasion from apoptosis is one of the crucial features of malignant diseases and that the efficiency of cancer therapy depends on p53-dependent apoptosis. Because of the importance of apoptotic pathways in protecting cells against malignant transformation, disruption of apoptosis is extremely common in cancer cells. In acute leukemia and lymphoma apoptotic death receptor signalling pathway is disrupted. We predicted that absence or low expression of Fas-associated death domain (FADD) protein could be found in leukemic and lymphoma cell lines. FADD is an adapter protein that is required for apoptosis induced by all known death receptors, expression of FADD was analyzed by Western blot in two types of leukemic and two types of lymphoma cell lines. In our experiment we used MOLT-4 (human acute lymphoblastic leukemia), Jurkat (human T cell leukemia), RAJI (Burkitt’s lymphoma) and U-937 (histiocytic lymphoma) cell lines. Cells were maintained by the addition of fresh medium or replacement of medium and were cultured at 37 °C in a 5% CO2 atmosphere. Cell density was between 4 X 105 and 1.5 X 106 viable cells/ml in complete RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cultured cells were collected together with medium. After centrifugation, the pellet was washed with cold PBS and then lysed in a lysis buffer. Samples were agitated on ice for 1 hour. After centrifugation, supernatants were collected, and the protein extracts were quantified using the BCA protein assay kit (Pierce BCA Protein Assay Kit). Equal amounts of protein (30 µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes using XCell Blot Module. Nonspecific binding was blocked by TBST with 5% nonfat milk overnight at 4°C. Incubation with a rabbit polyclonal antibody FADD (H-181 , sc-5559, Santa Cruz Biotechnologz Inc.), diluted 1:200 lasted for 2 hours at room temperature with agitation. As a secondary antibody was used anti-rabbit, HRP-linked whole antibody from donkey (Amersham Biosciences) diluted 1:5000. Visualization was done by Lumi-light Western Blotting Substrate (Roche). The results indicated that in two cell lines we found absence, in one low and in second normal expression of FADD protein. The data presented here suggest that apoptotic death receptor signalling pathway in leukemic and lymphoma cells is disrupted due absence or low expression of FADD protein.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti

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