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The mouse lymphoma L5178Y Tk+/- cell line is heterozygous for a codon 170 mutation in the p53 tumor suppressor gene
R D Storer1, A R Kraynak, T W McKelvey
1Department of Genetic and Cellular Toxicology, Merck Research Laboratories, West Point, PA 19486, USA. richard_storer@merck.com
Abstract:
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 maintenance of genomic stability. The mouse lymphoma L5178Y/TK(+/-)-3.7.2C cell line is widely used in genetic toxicology for mutagenesis and clastogenesis testing. A related line L5178Y-R, has previously been shown to react with antibodies specific for mutant as well as wild-type p53 protein 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 several clones of L5178Y cells for mutations in the conserved core domain (exons 5-8) of the p53 gene. Using single-strand conformational polymorphism analysis, we found evidence for the same mutation in exon 5 of p53 in L5178Y-R, L5178Y-S and L5178Y/TK(+/+)-3.7.2C cells. The mutation was identified by sequencing of exon 5 as a TGC (Cys) to CGC (Arg) transition in codon 170 (= codon 176 in humans). Sequencing showed approximately 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-coordinating 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 protein in unirradiated 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 radiation, whereas an 11-fold induction was seen in the irradiated splenocytes. These results indicate that p53 protein in L5178Y cells is dysfunctional and suggest that this line may therefore be abnormally susceptible to the induction of genetic alterations.
Insights
Dysfunctional p53 protein in L5178Y cells, identified by a specific mutation, leads to increased susceptibility to genetic alterations. This finding impacts genetic toxicology testing and understanding of genomic stability.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- The p53 tumor suppressor protein is crucial for maintaining genomic stability by regulating cellular responses to DNA damage.
- The L5178Y mouse lymphoma cell line is widely used in genetic toxicology but has shown limitations in specificity.
- Previous studies indicated abnormal p53 reactivity and delayed cell death in L5178Y-R cells after radiation.
Purpose of the Study:
- To investigate mutations in the p53 gene's conserved core domain (exons 5-8) in various L5178Y cell clones.
- To characterize the functional consequences of identified p53 mutations in L5178Y cells.
- To assess the implications of p53 dysfunction for the sensitivity and specificity of the mouse lymphoma assay.
Main Methods:
- Single-strand conformational polymorphism (SSCP) analysis was used to screen for p53 gene mutations.
- DNA sequencing was employed to identify the specific mutation in exon 5 of the p53 gene.
- Western blotting was utilized to compare p53 protein levels and inducibility in L5178Y cells versus normal mouse splenocytes.
Main Results:
- A consistent mutation (TGC to CGC transition at codon 170) was identified in exon 5 of p53 across L5178Y-R, L5178Y-S, and L5178Y/TK(+/+)-3.7.2C cells.
- This mutation eliminates a critical cysteine residue, likely denaturing the p53 protein and conferring a dominant-negative effect.
- L5178Y cells exhibited significantly higher basal levels of p53 protein, which were not inducible by radiation, unlike normal splenocytes.
Conclusions:
- The p53 protein in L5178Y cells is dysfunctional due to a specific mutation in the p53 gene.
- This p53 dysfunction may render L5178Y cells abnormally susceptible to the induction of genetic alterations.
- The findings suggest that p53 mutations contribute to the high sensitivity but low specificity observed in the mouse lymphoma assay.