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Partially transformed T3T3 cells express high levels of mutant p53 in the 'wild-type' immunoreactive form with
J Milner1, Y S Chan, E A Medcalf
1Department of Pathology, Cambridge University, UK.
Abstract:
High levels of wild-type p53 suppress transformed growth of many cell lines and yet murine T3T3 cells shown partially transformed growth despite high endogenous levels of phenotypically 'wild-type' p53. On sequencing T3T3 p53 was found to encode missense mutations at codons 230 and 287 and, although endogenous T3T3 p53 is 'wild type', the protein adopted the mutant phenotype when expressed in vitro. Size fractionation of T3T3 cell lysate indicated monomeric p53 possibly in complex with a low molecular weight protein. When expressed in vitro T3T3 p53 formed dimers and higher order structures. Thus T3T3 cells appear (i) to drive endogenous mutant p53 to adopt conformational epitopes characteristic of the 'wild-type' protein, and (ii) to interfere with normal assembly of p53 quaternary structure. Phosphopeptide mapping of p53 from 3T3x cells, T3T3 cells and SV3T3 cells indicated reduced amino terminal phosphorylation of the mutant p53 phenotype. Alternative splicing of p53 was also detected in 3T3x cells; similar splicing occurs in wild-type p53 (Han & Kulesz-Martin, 1992; Nucl. Acids Res., 20, 1979-1981) and a possible regulatory function is discussed.
Insights
Murine T3T3 cells exhibit partial transformation despite wild-type p53. This study reveals mutations in T3T3 p53, affecting its structure and phosphorylation, leading to a mutant phenotype and altered quaternary assembly.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Wild-type p53 protein typically suppresses cell transformation.
- Murine T3T3 cells display partial transformation despite high levels of wild-type p53.
- The underlying mechanisms for this phenomenon require investigation.
Purpose of the Study:
- To investigate the molecular basis of partially transformed growth in murine T3T3 cells.
- To analyze the p53 protein in T3T3 cells for mutations and altered phenotypes.
- To understand the impact of cellular context on p53 structure and function.
Main Methods:
- DNA sequencing to identify p53 mutations.
- In vitro protein expression to assess p53 phenotype.
- Size fractionation of cell lysates to analyze p53 complex formation.
- Phosphopeptide mapping to examine p53 phosphorylation patterns.
- Analysis of alternative splicing in p53.
Main Results:
- T3T3 p53 encodes missense mutations at codons 230 and 287.
- Expressed T3T3 p53 protein adopted a mutant phenotype in vitro.
- T3T3 p53 formed dimers and higher-order structures in vitro, unlike the monomeric form in cell lysate.
- Reduced amino-terminal phosphorylation was observed in T3T3 p53.
- Alternative splicing of p53 was detected in T3T3 cells.
Conclusions:
- Murine T3T3 cells possess mutations in p53 that confer a mutant phenotype.
- The cellular environment of T3T3 cells influences p53 conformation and quaternary structure assembly.
- Altered p53 phosphorylation and alternative splicing may play roles in T3T3 cell transformation.