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.