Naslov
Effect of UVC irradiation on p53 in FADD+/+ and FADD-/- cells

Autori
Matić, Igor ; Radnić, Maja ; Marijanović Inga ; Furčić, Ivana ; Nagy, Biserka

Vrsta, podvrsta i kategorija rada
Radovi u zbornicima skupova, cjeloviti rad (in extenso), znanstveni

Izvornik
IRPA12 International Congress of the International Radiation Protection Association, Proceedings / - Buenos Aires, 2008

Skup
IRPA12 International Congress of the International Radiation Protection Association

Mjesto i datum
Buenos Aires, Argentina, 19.10.2008. - 24.10.2008

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
UVC radiation; mutations in p53; Fas-associating protein with death domain (FADD); AS-PCR

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, and is frequently due to mutations in the P53 tumor suppressor gene. Fas-associated death domain (FADD) is an adapter protein that is required for apoptosis induced by all known death receptors. FADD is implicated in death receptor-independent apoptotic response, such as DNA damage. We used embryonic fibroblasts derived from FADD knockout mice and their genetic counterparts. We predicted that UV exposure induces a loss of FADD function and leads to mutations in P53. Loss of FADD expression causes deregulation of apoptosis and expansion of mutated cells and initiation of cancer. We predicted that FADD dysfunction may be potentially advantageous for tumor growth and that FADD can act as a tumor suppressor. Cells were irradiated with UVC light (254 nm) using a germicidal lamp (Upland, CA). The culture media was drained before the irradiation and fresh media was added after. In the first experiment we irradiated cells with a dose of 25 J/m2 and after 5 days we isolated genomic DNA but part of the cells were irradiated again with the same dose. After 5 days DNA were isolated so the cumulative irradiation dose was 50 J/m2. In the second experiment cells were irradiated ones with the dose of 40 J/m2 and DNA was isolated after 18 days. Lethal dosage for each cell line is 50 J/m2. Genomic DNA was analyzed by allele-specific polymerase chain reaction (AS-PCR) for CC to TT mutation at codons 154-155 and 175-176 in exon 5 and for C to T mutations at codons 270 and 275 in exon 8 of the P53 gene. The mutant-specific forward primer was used for each mutation. The reverse primers for amplification of mutations were not mutant-specific. Allele-specific PCR detection of p53 in genomic DNA from UV-irradiated knockout and wild type cells were analyzed by gel electrophoresis. The results indicated that the mutant-specific primers (154-155, 175-176 and 275) didn’ t amplified 119-, 55- and 134-base pair (bp) products, unexpectedly, from irradiated FADD+/+ and FADD-/- cell DNAs as well as expected from non-radiated FADD+/+ and FADD-/- cell DNAs. In contrast, the mutant-specific primer for codon 270 amplified 134-base pair product from irradiated from FADD+/+ and FADD-/- cell DNAs. Because FADD is required for elimination of UV-irradiated cells, its loss could inhibit apoptosis and lead to expansion of apoptosis defective cells that contain p53 mutations. The data presented here suggest that loss of FADD expression and loss of wild type p53 function due to mutations can inhibit apoptosis and lead to the expansion of UV carcinogenesis.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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