Raffaella Iannone

Tumour p53 mutations exhibit promoter selective dominance over wild type p53.

The tumour suppressor gene p53 is frequently mutated in human cancer. Tumour derived p53 mutants are usually transcriptionally inactive, but some mutants retain the ability to transactivate a subset of p53 target genes. In addition to simple loss of function, some p53 mutants may be carcinogenic through a dominant negative mechanism. Aiming at a more general classification of p53 mutants into predictive functional categories it is important to determine (i) which p53 mutants are dominant, (ii) what features characterize dominant mutants and (iii) whether dominance is target gene specific. The ability of 71 p53 mutants to inhibit wild type p53 was determined using a simple yeast transcriptional assay. Approximately 30% of the mutants were dominant. They preferentially affect highly conserved amino acids (P<0.005), which are frequently mutated in tumours (P<0.005), and usually located near the DNA binding surface of the protein (P<0.001). Different tumour-derived amino acid substitutions at the same codon usually have the same dominance phenotype. To determine whether the ability of p53 mutants to inhibit wild type p53 is target gene specific, the dominance towards p21, bax, and PIG3 binding sites was examined. Approximately 40% of the 45 mutants examined were dominant for the p21 (17/45) or PIG3 (20/45) responsive elements and 71% (32/45) were dominant for the bax responsive element. These differences are statistically significant (p21 vs bax, P<0.003; bax vs PIG3, P<0.02, Fishers exact test) and defined a hierarchy of dominance. Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours, some of which retained wild type level of transcription in yeast as well as in human cells, but show gain of function in transformation assays. Since transformation assays require transdominant inhibition of the endogenous wild type allele, one possible explanation for the behaviour of the BRCA-associated mutants is that they adopt conformations able to bind DNA alone but not in mixed tetramers with wild type p53. The yeast data do not support this explanation, because all BRCA-associated mutants that behaved as wild type in transcription assay were recessive in dominance assays.

Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology

In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants. Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GC>AT transitions represented 73% of all single base pair substitutions. In 70% of the cases the mutated G was preceded 5′ by a purine. The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P<0.0003) and CCNU induced only GC>AT transitions at those positions. The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion. One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations. This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.

Characterization of the p53 mutants ability to inhibit p73β transactivation using a yeast-based functional assay

p53 is the most frequently altered tumor suppressor gene in a wide spectrum of human tumors. The large majority of p53 mutations observed in tumors are missense mutations. The p73 gene, encoding a protein with significant sequence similarity to p53, expresses multiple transcription-competent spliced variants, or transcription-incompetent forms (i.e. ΔNp73). It was clearly shown that p73 transactivation from a p53-responsive promoter is inhibited by some tumor-derived p53 mutants in eucaryotic cells. In this study, we adapted a yeast-based p53 functional assay for the analysis of the influences of different p53 mutants on the activity of one of the p73 isoforms, namely p73β. We determined the ability of a panel of 61 p53 mutants to inhibit p73β activity following the net transcription of the ADE2 color (red/white) reporter gene driven by a p53-responsive promoter. By analysing a large number of mutants, we could conclude that interference: (a) is a quite frequent phenomenon (more than 70% of p53 mutants analysed are interfering); (b) is not confined to p53 mutations located in particular topological regions of the DNA binding domain; (c) does not appear to be dependent on the kind of side chains introduced at a specific position; (d) appears to significantly correlate with evolutionary conservation of the mutated p53 codon, frequency of occurrence of the mutation in tumors. The influence of a common R/P polymorphism at codon 72 on the ability of p53 mutants to interfere with p73β was also studied. Two sets of polymorphic variants (R and P) for 14 mutants were constructed and analysed. In all cases, the R/P 72 polymorphism was phenotypically irrelevant. In conclusion, our results suggest that the interpretation of the biological effects of p53 mutants should take into consideration the possibility that p53 mutants show loss or gain of function also through the interference with p53 family members.

Study on aneuploidy and p53 mutations in astrocytonias

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence selective N3-adenine methylating agent

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: Δmag1, Δapn1apn2, and Δapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in Δmag1, Δapn1apn2, and Δapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < Δmag1< Δapn1apn2 = Δapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in Δmag1, and 23 in Δapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in Δmag1, and 23 in Δapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in Δmag1; and 14/23, 61%, in Δapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.

Mutation spectra analysis suggests that N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli

To determine the influence of some bacterial DNA report pathways on the mutagenic and the lethal effects of N‐(2‐chloroethyl)‐N′‐cyclohexyl‐N‐nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr−ada+ogt+, and uvr−ada−ogt−). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty‐six CCNU‐induced supF mutants were isolated and sequenced. All mutants were characterized by single base‐pair substitutions, most of which (more than 96%) were GC→AT transitions (the mutated G being almost exclusively preceded 5′ by a purine). Mutation distribution was not random. Position 160 (5′‐GGT‐3′, nontranscribed (NT) strand) was a uvr+ada+ogt+‐specific hot‐spot. Position 123 (5′‐GGG‐3′, NT strand) was a common hot‐spot but significantly more mutable in repair‐proficient strains than in repair‐deficient strains. Conversely, position 168 (5′‐GGA‐3′, transcribed (T) strand) was significantly more mutable in repair‐deficient strains than in repair‐proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr− strains, whereas in the uvr− background, no difference was observed between mutation spectra in ada+ogt+ versus ada−ogt− strains. Our results are consistent with the hypothesis that O6‐alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC→AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr− strains suggests that CCNU‐induced premutational lesions are susceptible to strand‐preferential repair in E. coli. Mol. Carcinog. 19:39–45, 1997.

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene.

Forward mutations induced by 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea (CCNU) in the supF gene of Escherichio coli were recovered from bacteria deficient in nucleotide excision repair and in DNA‐alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7‐fold above the spontaneous value. A total of 73 independent supF− mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle‐vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C → A:T transitions. A substantial proportion (6/18) of CCNU‐induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage‐misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C → A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O6‐chloroethylguanine lesion with thymine. Most G:C → A:T transitions (34/39) were located at three 5′‐GG‐3′ hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C → A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF‐bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6‐chloroethylguanine formation, reducing its incidence in the transcribed DNA strand. Environ. Mal. Mutagen. 30:65–71, 1997.