A UVB fingerprint mutation on the p53 tumor suppressor gene decreases in vitro.
The culture of tissue-derived human cells is an important aspect of tissue engineering. Prior to transplantation, the quality of the cultured cells/tissue should be routinely tested so that any enrichment of procarcinogenic cells can be excluded. On this account, a UVB-induced p53 fingerprint mutation would be a valuable quality control marker for skin cells cultured for use in tissue engineering. We developed an allele-specific real-time polymerase chain reaction assay based on SYBR Green which can quantitatively detect CC-TT transitions in the tumor suppressor gene p53. To analyze the transition 281/282, we used DNA from HaCaT cells, a keratinocyte cell line containing the investigated mutation, as a standard to determine the mutation frequency in cultured cutaneous cells. We analyzed a variety of skin cells grown in culture and found a notable decrease in the UVB fingerprint mutation in fibroblasts during proliferation. Furthermore, we quantified the total amount of mutated DNA in different cutaneous cells and detected a significantly higher level in melanocytes. These results are consistent with findings obtained in our laboratory concerning the common deletion, the most frequently reported mutation in the mitochondrial genome, which suggest a positive influence of prolonged in vitro cell proliferation on the quality of genomic DNA.
Zhang
Zhiyuan
Z
Biomedical Engineering, Albstadt-Sigmaringen University, Sigmaringen, Germany.
Burger
Katharina
K
Hellwig
Sven
S
Bergemann
Jörg
J
D000368:Aged; D015139:Blotting, Southern; D002460:Cell Line; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D008297:Male; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D008875:Middle Aged; D009154:Mutation / Q000528:radiation effects*; D016133:Polymerase Chain Reaction; D014466:Ultraviolet Rays