Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family

Abstract

Previous investigations of a Li ± Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was con®rmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.Keywords: p53; Li ± Fraumeni syndrome; oligomerisation domain; functional assaysThe tumour suppressor gene, p53, was ®rst discovered in 1979 (Lane and Crawford, 1979;Linzer and Levine, 1979). p53 is a 393 amino acid nuclear phosphoprotein which plays a central role in the cellular processes involved in the response to DNA damage (reviewed Kastan et al., 1995). It is made up of at least four domains, a transactivation domain, a central DNA binding domain, an oligomerisation domain and a regulatory domain, the latter two residing at the carboxy end of the protein.Following initial studies by Malkin et al. (1990) it is now established that germline p53 mutations are associated with Li ± Fraumeni syndrome (LFS), a dominantly inherited syndrome ®rst proposed by Fraumeni in 1969 (Li andFraumeni, 1969). However mutations in the coding region of p53 have only been con®rmed in about 50% of families with LFS (Birch et al., 1994). Families with this syndrome exhibit multiple primary neoplasms in childhood and early adulthood and the spectrum of cancers which commonly occurs in LFS families includes brain tumours, breast tumours, sarcomas, leukaemia and adrenal cortical tumours (Li et al., 1988). Families have now been recognised in which there is a similar spectrum of malignancies to LFS but the families do not ®t exactly into the criteria of LFS. These families have been named Li ± Fraumeni like (LFL) (Birch et al., 1994) and p53 germline mutations have been found in 7 ± 20% of these families (Eeles, 1995; Varley et al. manuscript in preparation). The patient who is the subject of this study is a member of a LFL family and meets the criteria for LFL families as set out by Birch et al. (1994).In 1992 Barnes et al. described a member of a LFL ...

Characterization of the oligomerization defects of two p53 mutants found in families with Li–Fraumeni and Li–Fraumeni-like syndrome

“…Relatively few tumour-derived mutations have been reported for the tet domain (Cariello, 1994) suggesting that either it is more tolerant of mutations than the core domain (Ishioka et al, 1995;Je rey et al, 1995;Waterman et al, 1995), or that tetramerization is not necessary for tumour suppressor function (Ishioka et al, 1997;Tarunina and Jenkins, 1993). However, the fact that germline mutations in the tet domain have recently been documented for two families with Li ± Fraumeni and Li ± Fraumeni-like syndromes (inherited susceptibility to certain cancers associated with a germline mutation in p53) support a role for the tet domain in tumour suppression (Lomax et al, 1997;Varley et al, 1996). Indeed, Lomax et al (1997) suggest that the paucity of mutations in the Cterminal (and N-terminal) domains of p53 may be exaggerated by the fact that many researchers do not sequence these regions of the gene when looking for tumour-related or germline mutations.…”

“…However, the fact that germline mutations in the tet domain have recently been documented for two families with Li ± Fraumeni and Li ± Fraumeni-like syndromes (inherited susceptibility to certain cancers associated with a germline mutation in p53) support a role for the tet domain in tumour suppression (Lomax et al, 1997;Varley et al, 1996). Indeed, Lomax et al (1997) suggest that the paucity of mutations in the Cterminal (and N-terminal) domains of p53 may be exaggerated by the fact that many researchers do not sequence these regions of the gene when looking for tumour-related or germline mutations. We report here the biophysical and biochemical characterization of these two Li ± Fraumeni mutations in the tet domain.…”

“…Mutation of Leu344 to Pro (L344P; Varley et al, 1996) in the center of the ahelix of each subunit would be expected to severely disrupt the structure of the helix. Mutation of Arg337 to Cys (R337C; Lomax et al, 1997) removes a salt bridge and hydrophobic interactions between opposite subunits of each dimer. In order to better understand the e ects these mutations have on the structure and function of p53 we expressed and puri®ed recombinant WTp53, L344P and R337C as well as C-terminal constructs containing the oligomerization domain of each protein.…”

“…R337C has been reported as a sporadic mutation (Hollstein et al, 1996) and as a familial germline mutation in two families with Li ± Fraumeni-like syndrome (Lomax et al, 1997; S Verselis personal Figure 5 Assessment of hetero-oligomerization between WTp53 and R337C or WTp53 and L344P run on 15% SDSpolyacrylamide glycine or tricine gels, respectively. (a) Equimolar samples of (His) 6 -R337C(310 ± 393) (lane 1) and WTp53(310 ± 360) with no (His) 6 -tag (lane 2) were incubated at 378C overnight (lane 3) and loaded onto a 200 ml bed of QIAGEN Ni-NTA agarose beads, and subsequently washed (lanes 4 ± 8) with 3 ml of 30 mM imidazole, 500 mM NaCl, 40 mM Tris-HCl pH 7.5, and eluted (lanes 9 ± 12) with 2 ml of 500 mM imidazole, 500 mM NaCl, 40 mMTris-HCl pH 7.5.…”

“…Samples were loaded onto a pre-equilibrated 0.256TBE 5% polyacrylamide gel and electrophoresed for 2 h at room temperature communication). In a yeast functional assay for transactivation that usually yields large white colonies for active p53 and small red colonies for inactive p53, R337C produced medium-sized pink colonies (Lomax et al, 1997). Likewise in assays for the ability of p53 to trigger apoptosis (Lomax et al, 1997), activate a reporter gene or suppress growth (Lomax et al, 1998) R337C had an activity intermediate between WT and completely inactive p53.…”