A mutation in the p53 tumor suppressor gene of AHH-1 tk+/- human lymphoblastoid cells.
Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G1 checkpoint and delayed apoptotic cell death, characteristics we have consistently observed in the AHH-1 tk+/- cell line following exposure to DNA-damaging agents. In order to determine the functional status of p53 in the AHH-1 tk+/- cell line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 gene. In addition, the status of the p53 gene in the closely related lymphoblast line, MCL-5, which, in our hands, has a much higher spontaneous rate of apoptosis than AHH-1 tk+/-, was also determined by molecular analysis. Initial SSCP analysis of AHH-1 tk+/- revealed an abnormal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution (CGG-->TGG) mutation had occurred at codon 282, a reported "hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 indicated any differences from wild-type DNA. These results suggest that the lack of a G1 arrest and the delayed entrance into apoptosis observed in chemically-exposed AHH-1 tk+/- cells are, at least partially, accounted for by a loss-of-function mutation in the p53 gene.
Morris
S M
SM
Division of Genetic Toxicology, Food and Drug Administration, Department of Health and Human Services, Jefferson, AR 72079, USA. smorris@nctr.fda.gov
Manjanatha
M G
MG
Shelton
S D
SD
Domon
O E
OE
McGarrity
L J
LJ
Casciano
D A
DA
D017209:Apoptosis; D002478:Cells, Cultured; D005091:Exons; D016158:Genes, p53 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA