P53 integrity in the genetically engineered mammalian cell lines AHH-1 and MCL-5.
Recently, a C to T transition mutation in exon 8 of the p53 gene has been identified in a subculture of the genetically engineered human lymphoblastoid cell line AHH-1 and this mutation was proposed to cause a loss of function of the p53 suppressor protein and may limit the use of this cell culture in genotoxicology test assays. This led us to investigate early passage cultures of AHH-1 and its derivative MCL-5 to determine the distribution of the mutation. In order to characterise the presence of mutations at the p53 locus, exon 8 was analysed using restriction enzyme analysis and automated sequencing to locate possible changes of sequence. Mutations were identified at codon 282, and treatment with the Msp1 restriction enzyme led to incomplete digestion suggesting the presence of heterozygosity at the site which was confirmed by sequencing. Our results indicate that the p53 gene is heterozygous at the interface between the codons 281 and 282 in both AHH-1 and MCL-5. An Annexin V labeling study was carried out and both AHH-1 and MCL-5 cell lines were shown to undergo DNA damage induced cell death after a 1-h exposure to MNNG.
Guest
R D
RD
School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea, Wales SA2 8PP, UK.
Parry
J M
JM
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002461:Cell Line, Transformed; D004252:DNA Mutational Analysis; D005818:Genetic Engineering; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity; D015183:Restriction Mapping; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D017304:Annexin A5; D009153:Mutagens; D016159:Tumor Suppressor Protein p53; D008769:Methylnitronitrosoguanidine