THE MOUSE LYMPHOMA L5178Y TK(+ -) CELL-LINE IS HETEROZYGOUS FOR A CODON-170 MUTATION IN THE P53 TUMOR-SUPPRESSOR GENE/

The p53 tumor suppressor protein plays an important role in regulating the cellular response to DNA damage, including cell cycle arrest and apoptosis induction. Normal p53 function is critical for the maintenan ce of genomic stability. The mouse lymphoma L5178Y/TK+/--3.7.2C cell l ine is widely used in genetic toxicology for mutagenesis and clastogen esis testing. A related line L5178Y-R, has previously been shown to re act with antibodies specific for mutant as well as wild-type p53 prote in and to exhibit delayed cell death after radiation. For this reason, as well as the mouse lymphoma assay's reputation for high sensitivity of detection for genotoxic agents but low specificity, we examined se veral clones of L5178Y cells for mutations in the conserved core domai n (exons 5-8) of the p53 gene. Using single-strand conformational poly morphism analysis, we found evidence for the same mutation in exon 5 o f p53 in L5178Y-R, L5178Y-S and L5178Y/TK+/+-3.7.2C cells. The mutatio n was identified by sequencing of exon 5 as a TGC (Cys) to CGC (Arg) t ransition in codon 170 (= codon 176 in humans). Sequencing showed appr oximately equivalent signals for the mutant and normal alleles for all 3 lines. The mutation in codon 170 is adjacent to a mutation hotspot of the human p53 gene (codon 175) and eliminates a critical zinc-coord inating cysteine residue such that the mutant protein is likely to be denatured and have a dominant negative effect on normal p53 function. Western blots showed approximately 100-fold higher levels of p53 prote in in uniradiated L5178Y cells as compared to induced levels of p53 in normal mouse splenocytes 4 h after 5 Gy of gamma radiation. The high levels of p53 protein in L5178Y cells were not further inducible by ra diation, whereas an 11-fold induction was seen in the irradiated splen ocytes. These results indicate that p53 protein in L5178Y cells is dys functional and suggest that this line may therefore be abnormally susc eptible to the induction of genetic alterations.