GENES, CHROMOSOMES & CANCER 4:1-15 (1991)

REVIEW ARTICLE

TP53 Tumor Suppressor Gene: A Model for Investigating Human Mutagenesis Claude Caron de Fromentel and Thierry Soussi Unite d'Oncologie Moleculaire, UPR 275. Villejuif Cedex (C C.d.F.), and Centre de Genetique Moleculaire. Univenite P et M Curie, U30 I INSERM, Paris (T.S), France

More than 350 independent point mutations of the TP53 gene, found in a wide variety of human cancers, were compiled and analysed. From this study, we confirm the presence of four hot-spot regions which colocalize with some highly conserved domains of the protein. W e also define a new hot-spot region which is observed predominantly in lung tumors. Analysis of the mutational events suggests the direct involvement of environmental carcinogens in lung tumors and hepatocarcinomas, and spontaneous mutagenesis generating essentially CpG transitions in most of the remaining ones. Furthermore, we demonstrate in this work that the TP53 gene is an informative model with which to study the molecular mechanisms of mutagenesis in the human genome. Genes Chrorn Cancer 4 : / - / 5 (1992).

INTRODUCTION

It is generally assumed that there are two types of mutagenic events affecting DNA: external (exogenous) events, implicating environmental factors, and internal (endogenous)events, resulting from errors of the mechanisms involved in the nucleic acid metabolism. In the first case, the mutagenic agent determines the nature of the lesion: thymine dimer formation follows W irradiation, =/AT conversion occurs in the presence of nitrous acid, etc. In the second, mutations (depurination, replication errors, etc.) appear to be spontaneous. CpG dinucleotides are frequently subject to this type of mutation, explaining their underrepresentation in vertebrates (Perutz, 1990; Sved and Bird, 1990). Because cytosine in the dinucleotide is frequently methylated, this high mutability could be due to the deamination of the cytosine, leading to a thymidine (Coulondre et al., 1978). The accumulation of mutations, endogenous and/or exogenous, in the genome of a single cell can lead to the deregulation of its developmental and mitogenic programs and to cancer. Despite the diversity and multiple etiologies of the resulting human tumors, the genes targeted by the mutations fall into two general categories: proto-oncogenes,whose modification generates abnormal or overexpression of an active and dominant product; and tumor suppressor genes (antioncogenes),both copies of which, generally, must be inactivated by a mutational event. TP53, along with RBI,DCC, and several other loci, is in the second category, although the consequences of inactivation appear to be different for each gene. TP53 characteristics will not be discussed here as several recent reviews deal with this subject (Lane and Benchimol, 1990; Levine, 1990; Soussi et al., 0 1992 WILEY-LISS, INC.

199Oa; Levine et al., 1991; Michalovitz et al., 1991). According to published data, TP53 inactivation is often a two-step mechanism which involves point mutation in one allele and loss of the remaining one (Baker et al., 1990). The wide variety of these point mutations makes TP53 an extremely attractive candidate in which to allow the characterization of the mutational events responsible for the transformed phenotype (Sommer, 1990).A comparison of TP53 to members of the proto-oncogenefamily RAS is indicative: mutagenic analysis of an activated RAS gene has uncovered transforming mutations in only three out of 188 amino acids. On the contrary, independent point mutations in 95 out of 393 amino acids lead to an oncogenic TP53 variant. Therefore, alterations in TP53 sequences are potentially more diverse and should indicate mutational preference or bias, if it exists. TP53, like RAS, is altered in a wide variety of human tumors and then can indicate and distinguish tumors with different mutational origins. Two considerations must be kept in mind it is not clear yet if mutated TP53 only represents an inactivated form of wild-type TP53 or corresponds to a new product carrying distinct activities (Michalovitz et al., 1991, for discussion). Furthermore, all TP53 variants do not share the same properties (Hinds et al., 1990; Levine et al., 1991).Nevertheless, we will consider all the mutational events along the TP53 molecule generating a selected TP53 mutant involved in cellular transformation. Phylogenetic studies demonstrated that TP53 contains five domains highly conserved during the course Received July 9, 1991; accepted September 27, 1991. Address reprint requests to Thierry Soussi. Centre de Genetique Moleculaire, U30l INSERM, 27 rue J. Dodu, 75010 Paris, France.

2

CARON DE FROMENTEL AND SOUSSI

of evolution (highly conserved domains, HCD, I to V) (Soussi et al., 1987,1988a,b, 199Oa;Caron de Fromentel et al., 1992; Legros et al., 1992).In 1989, Nigro et al. identified a variety of TP53 mutations and defined four regions most frequently modified in human tumors (hot-spot regions, HSR, A to D). These authors observed a tight correlation between the HSR A-D and the HCD II-V. Extensive observations by several other groups have confirmed the localization of mutations in the HSR A-D defined by Nigro et al. (See Table 1 for references.) Thus, mutations in the conserved regions, especially HCD II-V, seem to be specifically involved in the transformation process. This review describes more than 350 independent mutations of the human TP53 gene characterized in 35 different tumor types and one familial syndrome. The analysis of these mutations has allowed us to correlate the sequence of the codon affected with the mutational event (transition or transversion) and the tumor type. First, we confirm that the overwhelming majority of missense point mutations map to four HSR and also confirm the presence of a fifth hot-spot region, which is observed predominantly in primary lung tumor cells and cell lines. These point mutations have been characterized in order to establish their probable spontaneous or carcinogenic origin and consequence for the amino acid sequence. Second, we discuss the predominance of mutations in the CpG dinucleotide. Finally, splice site mutations, insertions, and deletions are presented. STUDY O F MUTATIONS IN THE CODING SEQUENCE

Figure 1shows all the mutations that we have been able to collect to date (September 1991). (See Table 1 for references.) It is clear that most of the mutations affect the amino acids conserved either in vertebrates (red) or, at least, in mammals (green). They are clustered in five HSR. Twenty mutations were characterized in HSR A (amino acids 132-la), 40 mutations in HSR B (amino acids 172-179), 91 mutations in HSR C (amino acids 237-249), and 62 mutations in HSR D (amino acids 272-286), as shown in Table 2. A fifth HSR (designated HSR A’) is identified by our analysis; it comprises 24 mutations in amino acids 151-159 and was not detected by Nigro et al. (1989)because it is not associated with the tumor types they studied. Chiba et al. (1990) found that eight out of 24 mutations from non-small-cell lung carcinoma (NSCLC) occurred in this HSR. The five HSR are confined to exons 5-8 and account for 73% of the mutations detected. We should keep in mind that most of the PCR amplifications and sequencing were performed between these exons, thus possibly missing some mutations outside. Of the five,

only HSR A’ is not in a classical HCD, but, as shown in Figure 1, it is located in a region conserved during mammalian evolution. Among the five HCD, only HCD I1 is interrupted by an intron. Interestingly, HCD IIa, coded by exon 4, is never mutated, while HCD IIb, coded by the beginning of exon 5, corresponds to HSR A. It is surprising that HCD IIa and HCD I are spared from mutation. This amino-terminal region contains elements which activate transcription (Fields and Jang, 1990; ORourke et al., 1990; Raycroft et al., 1990),and mutations within may be incompatible with any modification of TP53 necessary for the tumorigenic process. Considering the distribution of mutations in respect of the cancer type, only Li-Fraumeni syndrome is associated with a specific region. Most of the mutations (13 out of 17)are localized in exon 7, partially overlapping HSR C. In this familial syndrome, TP53 is found in the heterozygous state in normal cells (Malkin et al., 1990; Srivastava et al., 1990).Biochemical analysis of such TP53 mutants indicates that they do not exert a dominant effect over wild-type TP53, contrary to some others mutants (Milner and Medcalf, 1991).This behaviour could be necessary for the simultaneous presence of wild-type and mutated TP53 in the cell. CpG MUTATIONS

CpG dinucleotides are preferentially involved in spontaneous mutations. Since Arg is frequently coded by CGN, it is not surprising that this amino acid is a preferential target of the spontaneous mutations. Indeed, within the five HSR defined above, the three amino acids which are the principal targets are Arg175,Argm, and Arg273.They are hit 24, 35, and 24 times, respectively (25% of total missense mutations). It should be noted that the codon for ArgB2 is hit 13 times; thus it can be considered as a hot-spot codon. Deamination of the 5-methylcytosinecan generate a C to T or G to A transition. It is exactly this type of change which is observed in most of these three cases. This finding is in agreement with the results of Rideout et al. (1990),which demonstrate that codons 175 and 273 are methylated in vivo. However, there is an unusual observation: CpG transition for Arg175involved the G nucleotide 22 times, and never the C residue. If the C to T transition was to occur, Arg would be replaced by Cys. Therefore, it may be that (1)this mutation does not alter the function of TP53 and, therefore, offers no selective advantage for growth, or (2) this mutation may be lethal for the cell. There is clearly something specific to position 175, because the two other arginines (24.8 and 273) are mutated at equal frequencies at C (to T) and G (to A) (Tables 1, 2).

HUMAN TP53 GENE MUTATIONS

3

Figure I . Localizationof TP53 mutations. The amino acids are colored t o indicate their evolutionary conservation: black for human specific residues, green and red for mammalian and vertebrate conserved residues, respectively. The five boxes (I-v) represent the five highly conserved domains. A = deletion. & = insertion. 0 = exon delineation in the gene. v = amino acid number that is a multiple of ten.

Other CpG transitions are found between exons 5 and 8 (Table 2). However, there are five adjacent CpG dinucleotides in exon 10 as well as 13 such dinucleotide pairs distributed over exons 2 and 4, which are never found altered in human cancers. These observations reinforce the idea that mutations in the central part of the TP53 are specifically selected for their behavior. Out of the 324 point mutations, 22 are nonsense, all but one affecting the first nucleotide of the wild-type codon. Eleven of these are C to T transitions converting CGA (Arg) to TGA (Stop);55% of nonsense point mutations (12)are clustered between amino acids 196 and 213. Apart from lung tumors, hepatocarcinomas (discussed below), and esophageal carcinomas, the CpG mutations are recovered equally in all types of cancer. They represent the most common mutational event,

especially in colorectal carcinomas (20 out of 34) and B-cell lymphomas (19 out of 37) (Figs. 2A, D). Given the chemical mechanism of the CpG transition, these results suggest that numerous CpG dinucleotides are methylated in the TP53 locus. Nevertheless, CpG dinucleotides are probably not equally susceptible to mutation, because they are not methylated or because flanking sequences may influence the mutation process. We should also keep in mind that not all amino acid substitutions lead to an oncogenic TP53. Most of the amino acids targeted by the CpG transition are conserved through evolution, even those localized outside the HCD. It seems paradoxical that conserved amino acids are encoded by codons which are the preferential target of spontaneous mutation. This indicates that these codons lead to amino acids essential for the function of TP53.

CARON DE FROMENTEL AND SOUSSl

4

TABLE I . Compilation and References of Data Presented in Figure la

POS

wr

MUT

Event

Tumor type

Origin

36 49 53 60 68 I10 I I3 I29 I30 I32 I32 I32 I32 I32 I33 I33 I34 I35 I35 I35 I35 I36 I38 I38 I40 141 141 I43 I43 I44 I44 151 151 151 151 I52 I52 I54 I54 I54 I54 I56 I56 I56 I56 I57 I57 I57 I57 I57 I57 I58 I58 I59 I59 I63 I63 I64 171

CCG GAT TGG CCA GAG CGT TTC GCC CTC AAG

CCA CAT TGT TCA TAG TGT TGT GAC CTG AAC CAG AAT CAG AGG TTG AAG TTA TAC TCC TAC TGG GAG GTC GGC TAC TAC TAC GCG TTG TAG CCG CAT CAC TCC TCC CTG TCG GTC GTC GTC GTC

GC -+ AT GC -+ CG GC -+ TA GC -+ AT GC -+ TA GC -+ AT Double M GC -+ TA GC -+ CG GC + CG AT -+ CG GC -+ TA A T -+ CG AT -+ GC AT -+ TA AT + TA AT -+ TA GC -+ AT GC -+ CG GC -+ AT GC -+ CG Double M GC -+ AT GC -+ CG AT -+ TA GC -+ AT GC -+ AT AT -+ GC GC -+ TA GC -+ AT AT -+ CG Double M GC -+ TA GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ TA GC -+ TA GC -+ TA GC -+ TA GC -+ CG GC -+ CG GC -+ AT GC -+ CG GC -+ T A GC -+ TA GC -+ TA GC -+ TA GC -+ TA GC -+ TA GC -+ AT GC -+AT GC -+ AT GC -+ CG AT -+ GC AT -+ GC AT -+ CG GC + TA

Lung (NSCLC) ca CML CML CML Lung (SCLC) ca Hepatoca Lung (NSCLC) ca Neurofibrosa MDS Colorectal ca Breast ca Lung (NSCLC) ca Pancreatic ca CML Colorectal ca Burkitt lymphoma Lung (SCLC) ca Colorectal ca AML Lung (NSCLC) ca MDS Breast ca Rhabdomyosa Lung (SCLC) ca CML Colorectal ca Bladder ca Colorectal ca Lung (NSCLC) ca Esophageal ca Burkitt lymphoma Leiomyosa Lung (SCLC) ca Glioblastoma Lung (NSCLC) ca Leiomyosa Breast ca Esophageal ca Lung (NSCLC) ca Lung (NSCLC) ca Lung (NSCLC) ca Rhabdomyosa Osteosa Lung (NSCLC) ca Lung (NSCLC) ca Hepatoca Lung (SCLC) ca Lung (NSCLC) ca Breast ca Lung (SCLC) ca Bladder ca Neurofibrosa Burkitt lymphoma Lung (NSCLC) ca Lung (NSCLC) ca Breast ca Burkitt lymphoma Breast ca Lung (SCLC) ca

Tumor Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Xenograft Cell line Tumor Cell line Tumor Xenograft Tumor Cell line Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Xenograft Tumor Xenograft Tumor Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Cell line Cell line Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Tumor Cell line Tumor Tumor Tumor Cell line Tumor Cell line

ATG TTT TGC

CAA GCC ACC TGC GTG CAG

ccc

CCG GGC

CGC

ccc ccc CGT

ccc GTC

CGC GCC TAC AAG GAG

TTC TTC TTC TTC TTC TTC CGT CAC GTC

ccc TGC CAC CAG TAG

References Chiba et al., I990 Ahuja e t al., I99 I Ahuja et al., 1991 Ahuja e t al., 1991 d‘Amico et al., in prep. Murakami et al., in prep. Miller e t al., in prep. Menon et al., 1990 Jonveaux e t al., I99 I Nigro et al.. 1989 Bartek et al., 1990 Chiba et al., I990 Murakami et al., I991 Feinstein et al., I99 I Nigro et al., 1989 Gaidano et al., I99 I Nigro et al.. 1989 Baker e t al., 1990 Slingerland et al., I99 I Chiba et al., I990 Jonveaux et al., I99 I Prosser et al., I990 Mulligan et al., I990 Miller et al., in prep. Ahuja e t al., I99 I Nigro et al., 1989 Sidransky et al., I99 I Baker e t al., 1989 Miller et al., in prep. Hollstein e t al., I99 I a Gaidano et al., I99 I Stratton et al., 1990 d’Amico et al., in prep. Cheng et al., in prep. Chiba et al., I990 Stratton et al., 1990 Prosser et al., I990 Hollstein et al., I990 lggo et al., 1990 Chiba et al., 1990 Chiba et al.. I990 Stratton et al., 1990 Romano e t al., 1989 Chiba et al., 1990 Chiba et al., 1990 Bressac et al., I99 I d’Amico et al., in prep. Chiba e t al., 1990 Prosser et al.. I990 Miller et al., in prep. Sidransky et al., I99 I Menon et al., 1990 Gaidano et al., 199 I Chiba e t al., 1990 Chiba et al., 1990 Davidoff e t al., I99 Ia Gaidano et al., I99 I Prosser et al., 1990 Hensel et al., 1991 (Continued on next page)

HUMAN TP53 GENE MUTATIONS

TABLE I. (continued)

POS

WT

MUT

Event

Tumor type

Origin

References

I72 I73 I73 I73 I73 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I75 I76 I76 I 76 I76 I77 I79 I79 I79 I79 I79 I 79 I81 I87 I92 I93 I93 I93 I 94 I94 I94 I94 I94 I96 I96 I96 I96 I96 I 98 I98 202 204

GTT GTG

TTT TTG TTG GGG GTA CAC CAC CAC CAC CAC CAC CAC CAC CAC CAC CAC CAC CAC CAC CAC CTC AGC CAC CAC CAC CAC CAC CAC CAC TTC TTC TTC TAC CGC TAT CAG CTT GAT CTT CTT CAC TGT TAG CGT TAT CGT TTT CGT CGT CGT CGT TGA TGA TGA TGA TGA TAA TAA CTT GGG

GC -+ TA GC -+ TA GC -+ TA AT -+ CG GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC + AT GC -+ AT GC -+ AT GC -+ TA GC -+ TA GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ TA GC + TA GC -+ TA GC -+ AT GC + CG GC -+ AT AT -+ CG AT + TA GC -+ CG AT + TA AT -+ TA GC -+ AT GC -+ TA GC -+ AT AT -+ GC GC -+ AT AT -+ GC GC -+ AT AT -+ CG AT -+ CG AT -+ CG AT -+ CG GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ TA GC -+ TA GC -+ TA AT -+ GC

Burkitt lymphoma Lung (NSCLC) ca Lung (NSCLC) ca Burkitt lymphoma Gastric ca Colorectal ad Colorectal ad Colorectal ad Colorectal ca Colorectal ca T-ALL Brain tumor Colorectal ca Colorectal ca Leiomyosa Esophageal ca Glioblastoma Colorectal ca T-ALL Breast ca Breast ca Hepatoca B-ALL B-ALL Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma Gastric ca Lung (NSCLC) ca Esophageal ca Lung (NSCLC) ca Burkitt lymphoma PTLC Neurofibrosa Lung (SCLC) ca Esophageal ca Breast ca Cholangiosa Cholangiosa Li-Fraumeni sdm Breast ca Esophageal ca Lung (SCLC) ca Esophageal ca AML Breast ca Lung (SCLC) ca Esophageal ca Esophageal ca B-CLL Colorectal ca T-ALL T-cell lymphoma Lung (SCLC) ca Bladder ca Lung (SCLC) ca Lung (SCLC) ca CML CML

Cell line Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Cell line Tumor Tumor Xenograft Cell line Tumor Tumor Xenograft Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Cell line Cell line Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Tumor Tumor Tumor Germ line Tumor Tumor Cell line Tumor Tumor Cell line Cell line Tumor Tumor Tumor Tumor Cell line Cell line Cell line Tumor Tumor Tumor Tumor Tumor

Caron de Fromentel et al., in prep. lggo et al., 1990 Chiba et al., I990 Gaidano e t al., 1991 Tamura et al., 1991 Baker et al., 1990 Baker et al., 1990 Baker et al., 1990 Baker e t al., 1990 Baker et al., I990 Cheng and Haas, 1990 Nigro et al., 1989 Nigro et al., 1989 Nigro et al., 1989 Stratton et al., 1990 Hollstein et al., I99 l a Cheng et al., in prep. Baker et al., 1989 Haas et al., in prep. Varley et al., I99 I Prosser et al., I990 Murakami e t al., in prep. Gaidano e t al., I99 I Gaidano et a[., I99 I Gaidano et al., I99 I Gaidano et al., I99 I Gaidano et al., I99 I Gaidano et al., I99 I Kim et al., 1991 lggo et al., 1990 Hollstein et al., I99 Ia Chiba e t al., 1990 Gaidano et al., I99 I Gaidano et al., I99 I Nigro et al., 1989 Takahashi et al., I989 Hollstein et al., 1991a Prosser et al., I990 Murakami e t al., in prep. Murakami et al., in prep. Friend et al., in prep. Prosser et al.. I990 Hollstein et al., 1990 Lehman et al., I99 I Hollstein et al., I99 Ia Jonveaux and Fenaux, in prep. Nigro et al.. 1989 Takahashi et al., I989 Hollstein et al., I99 Ia Hollstein et al., I99 l a Gaidano et al., 1991 Baker et al., 1990 Cheng and Haas, 1990 Cheng and Haas, 1990 Lehman et al., I99 I Sidransky et al., 1991 Miller et al., in prep. Miller et al., in prep. Ahuja et al., 1991 Ahuja et al., 1991

CGC

TGC

ccc CAT

CGC GGT CAG CAT

CTT

CGA

GAA CGT GAG

(Continued on next page)

6

CARON DE FROMENTEL AND SOUSSI

TABLE I .

(continued)

POS

WT

MUT

Event

Tumor type

Origin

205 205 205 21 I 213 213 213 213 213 213 213 213 213 213 215 216 216 216 216 220 229 232 234 234 234 236 237 237 237 237 237 237 238 238 238 239 239 239 239 239 239 24 I 24 I 24 I 242 242 242 242 244 144 244 244 245 245 245 245 245 245 245 245

TAT

TGT TGT TTT GCT TGA CAA CAA CGG CGG TGA CGG TGA TGA TGA GGT ATG GAG TTG ATG TGT TGA AGC CAC CAC TGC TGC AGG ATA ATA ATA ATA ATA TTT TAT TAT AGC AGC AGC AGC AGC AGC TTC TGC TGC TCC TTC TCC TAC TGC TGC TGC AGC GTC TGC AGC GAC AGC GCC GAC GAC

AT -+ GC AT -+ GC AT + TA AT -+ GC GC + AT GC -+ AT GC + AT AT -+ GC AT -+ GC GC + AT AT -+ GC GC -+ AT GC -+ AT GC -+ AT AT + GC GC + AT AT -+ TA GC -+ TA GC + AT AT + GC AT 4 TA AT 4 CG AT + GC AT + GC AT + GC AT + GC AT 4 CG GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ TA GC + AT GC + AT AT + GC AT -+ GC AT + GC AT -+ GC AT -+ GC AT + GC GC + AT GC -+ CG GC -+ CG GC -+ CG GC -+ TA GC -+ CG GC -+ AT GC -+ TA GC -+ TA GC + TA GC + AT GC + TA GC -+ TA GC -+ AT GC -+ AT GC + A T GC + CG GC + AT GC -+ AT

B-ALL B-CLL Gastric ca Colorectal ca Colorectal ca B-cell lymphoma Burkitt lymphoma Lung (SCLC) ca Esophageal ca Lung (NSCLC) ca Lung (NSCLC) ca Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma Colorectal ca Brain tumor Burkitt lymphoma Gastric ca Ovarian ca Colorectal ca Lung (SCLC) ca B-CLL B-cell lymphoma Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma T-ALL Lung (SCLC) ca AML Breast ca Burkitt lymphoma Richter's sdm Larynx ca Burkitt lymphoma CML Colorectal ca Colorectal ca Burkitt lymphoma CML CML B-CLL Colorectal ca Colorectal ad Bladder ca Lung (SCLC) ca Breast ca MDS Ependymoma T-ALL Esophageal ca Lung (SCLC) ca Hepatoca Esophageal ca Li-Fraumeni sdm Leyomyosa Li-Fraumeni sdm Esophageal ca Bladder ca Breast ca Li-Fraumeni sdm

Tumor Tumor Cell line Tumor Xenograft Cell line Cell line Cell line Tumor Cell line Tumor Tumor Cell line Cell line Xenograft Tumor Tumor Tumor Tumor Tumor Cell line Tumor Cell line Cell line Cell line Tumor Cell line Cell line Tumor Tumor Cell line Tumor Tumor Cell line Tumor Xenograft Tumor Tumor Tumor Tumor Tumor Cell line Tumor Tumor Cell line Tumor Tumor Germ line Cell line Tumor Tumor Tumor Cell line Germ line Cell line Cell line Tumor Tumor Tumor Germ line

ACT CGA

AGT GTG

TAT TGT ATC TAC

TAC ATG

TGT

AAC

TCC

TGC

GGC

GGC

References Gaidano et al., 1991 Gaidano et al., I99 I Kim e t al., I99 I Baker e t al., 1990 Baker et al., 1990 Cheng and Haas, 1990 May et al., in prep. Lehman et al., I99 I Hollstein e t al., 1991a d'Amico et al., in prep. Chiba et al., 1990 Gaidano et al., I99 I Gaidano et al., I99 I Gaidano et al., I99 I Shirasawa et al., I99 I Nigro et al., 1989 Gaidano et al.. I99 I Kim et al., 1991 Marks e t al., I99 I Baker e t al., 1990 Lehman et al., I99 I Gaidano et al., I99 I Cheng and Haas, 1990 May et al.. in prep. Gaidano et al.. 1991 Gaidano et al., I991 Cheng and Haas, 1990 Lehman et al., 1991 Jonveaux and Fenaux, in prep. Davidoff et al., 1991a Gaidano et al., I99 I Gaidano e t al., I99 I Maestro e t al., in prep. Gaidano e t al., 1991 Ahuja et al., I99 I Baker et al., 1990 Nigro et al., 1989 Gaidano et al., I99 I Foti et al.. 1991 Ahuja et al.. 1991 Jonveaux and Fenaux, in prep. Rodrigues e t al., I990 Shirasawa et al., I99 I Sidransky et al., I99 I Takahashi e t al., 1989 Prosser et al., in prep. Jonveaux et al., I99 I Meager e t al., I99 I Cheng and Haas, 1990 Hollstein et al., I99 Ia Miller et al., in prep. Murakami et al., in prep. Hollstein et al., 1990 Malkin et al., 1990 Stratton et al., 1990 Srivastava e t al., I990 Hollstein et al., I99 Ia Sidransky et al., I99 I Davidoff et al.. I99 Ia Friend et al., in prep. (Continued on next page)

7

HUMAN TP53 GENE MUTATIONS

TABLE I. (continued)

POS

WT

MUT

Event

Tumor type

Origin

References

245 245 246 246 246 246 247 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 248 249 249 249 249 249 249 249 249 249 249 249 249 249 249 249 250 25 I 252

GGC

TGC GTC GTG ATC GTG GTG ATC TGG TGG CAG CAG CAG CAG TGG TGG TGG TGG TGG CTG TGG CAG CTG CAG TGG CTG TGG CAG CAG TGG CAG CAG CAG TGG CAG TGG CAG TGG CAG CAG TGG CAG TGG AGT AGT AGT AGC AGT AGT AGT AGT AGT AGT AGT AGT AGC AGT AGT CTC AGC

GC -+ TA GC -+ TA AT -+ GC GC + CG AT -+ GC AT + GC AT + TA GC + AT GC -+ AT GC + AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC + AT GC + AT GC -+ AT GC -+ AT GC + TA GC -+ AT GC + AT GC + TA GC -+ AT GC -+ AT GC -+ TA GC + AT GC -+ AT GC + A T GC -+ AT GC + AT GC -+ AT GC -+ AT GC + AT GC + AT GC + AT GC + AT GC + AT GC 4 AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC + TA GC -+ TA GC -+ TA GC -+ CG GC -+ TA GC + TA GC -+ TA GC -+ TA GC -+ TA GC -+ TA GC -+ TA GC + TA GC -+ CG GC -+ TA GC + TA GC -+ AT AT -+ CG AT -+ GC

Li-Fraumeni sdm Cervical ca AML Lung (NSCLC) ca Hepatoca Bladder ca Lung (NSCLC) ca Colorectal ad Colorectal ca Colorectal ca Colorectal ca T-ALL Esophageal ca Li-Fraumeni sdm Li-Fraumeni sdm Colorectal ca Colorectal ca Rhabdomyosa Esophageal ca Lung (NSCLC) ca Lung (SCLC) ca Lung (SCLC) ca T-ALL Lung (NSCLC) ca Lung (SCLC) ca Colorectal ca Bladder ca MDS Burkitt lymphoma Breast ca B-CLL Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma Gastric ca Lung (SCLC) ca Breast ca CML Li-Fraumeni sdm Li-Fraumeni sdm Colorectal ca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Hepatoca Esophageal ca Breast ca Lung (NSCLC) ca Hepatoca Burkitt lymphoma Gastric ca Li-Fraumeni sdm

Germ line Cell line Tumor Cell line Tumor Tumor Tumor Cell line Xenograft Cell line Tumor Cell line Tumor Germ line Germ line Xenograft Cell line Cell line Tumor Cell line Cell line Cell line Cell line Tumor Tumor Cell line Tumor Tumor Cell line Tumor Tumor Cell line Cell line Cell line Cell line Cell line Tumor Tumor Tumor Germ line Germ line Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Cell line Tumor Cell line Cell line Tumor Germ line

Friend et al., in prep. Scheffner et al., 1991 Slingerland e t al., I99 I Takahashi et al., 1989 Murakami et al., in prep. Sidransky et al., I99 I Chiba et al., 1990 Baker et al., 1990 Baker et al., 1990 Baker et al., 1990 Baker et al., 1990 Cheng and Haas, 1990 Hollstein et al., I990 Malkin et al., 1990 Malkin et al., 1990 Nigro et al., 1989 Nigro e t al., 1989 Stratton et al., 1990 Hollstein et al., I99 Ia dAmico e t al., in prep. d'Amico e t al., in prep. d'Amico et al., in prep. Rodrigues et al., 1990 Chiba et al., 1990 Miller et al., in prep. Murakami et al., I99 I Sidransky et al., I99 I Jonveaux et al., I99 I Caron de Fromentel et al., in prep. Davidoff et al., I99 Ia Gaidano e t al., I99 I Gaidano et al., 1991 Gaidano et al., I99 I Gaidano et al., 1991 Gaidano et al., I99 I Kim et al., I99 I Hensel et al., 1991 Davidoff e t al., I99 Ib Feinstein et al., I99 I Friend et al., in prep. Friend et al., in prep. Baker e t al., 1990 Bressac et al.. I99 I Bressac et al., I99 I Bressac et al., I99 I Hsu et al., 1991 Hsu et al., I99 I Hsu e t al., 1991 Hsu et al., I99 I Hsu et al., 1991 Hsu et al., I99 I Hsu et at., I99 I Hsu et al., I99 I Hollstein et al., 1991a Bartek et al., 1990 Chiba et al., I990 Murakami et al., 1991 Caron de Fromentel et al., in prep. Tamura et al., I99 I Malkin et al., 1990

ATG

AAC CGG

AGG

ccc ATC CTC

ccc

(Continued on next page)

8

CARON DE FROMENTEL AND SOUSSI

TABLE I . POS

WT

252 254 254 256 258 258 258 259 260 266 266 266 267 270 270 272 272 272 272 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 273 274 276 276 277 278 278 278 278 278 280 280 28I 28 I 28 I 28 I 282 282

CTC ATC ACA GAA

GAC TCC GGA

CGG TTT GTG

CGT

GTT GCC TGT CCT

AGA GAC GAC CGG

(continued)

MUT

Event

Tumor type

Origin

References

ccc

AT -+ GC Double M AT -+ TA AT -+ GC GC -+ AT GC -+ AT GC -+ AT AT -+ GC A T -+ CG GC -+ TA GC -+ TA GC -+ TA GC + CG AT -+ CG AT -+ CG GC -+ AT GC -+ CG GC -+ AT GC -+ AT GC -+ AT GC + AT GC -+ AT GC -+ AT GC -+AT GC + TA GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ CG GC -+ TA GC -+ TA GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ AT GC -+ TA AT -+ TA GC -+ CG GC -+ TA GC -+ TA GC -+ AT GC -+ AT GC -+ CG GC -+ AT GC -+ CG GC -+ AT GC + AT AT -+ GC AT -+ GC GC -+ CG GC -+ TA GC -+ AT GC + AT

Li-Fraumeni sdm Burkitt lymphoma Breast ca T-ALL Li-Fraumeni sdm Burkitt lymphoma Li-Fraumeni sdm T-ALL T-ALL Lung (NSCLC) ca Lung (NSCLC) ca Breast ca Lung (SCLC) ca Esophageal ca T-ALL Brain tumor Lung (SCLC) ca Hepatoca AM L Colorectal ad Brain tumor Breast ca Colorectal ca Lung (NSCLC) ca Lung (SCLC) ca Colorectal ca Colorectal ca Colorectal ca Lung (NSCLC) ca Lung (NSCLC) ca Lung (NSCLC) ca Lung (NSCLC) ca Thyroid ca Lung (SCLC) ca B-cell lymphoma B-ALL Burkitt lymphoma Burkitt lymphoma Li-Fraumeni sdm Cervical ca AML B-CLL 6-CLL Erythroleukemia B-ALL Hepatoca Lung (SCLC) ca Esophageal ca Esophageal ca Breast ca Lung (SCLC) ca Ovarian ca Esophageal ca Breast ca Colorectal ca Breast ca Richter's sdm B-CLL Colorectal ad Colorectal ca

Germ line Cell line Tumor Cell line Germ line Tumor Germ line Cell line Cell line Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Tumor Tumor Tumor Xenograft Cell line Cell line Cell line Cell line Cell line Cell line Cell line Tumor Tumor Tumor Tumor Cell line Tumor Cell line Tumor Cell line Cell line Germ line Cell line Tumor Tumor Tumor Cell line Tumor Tumor Cell line Cell line Tumor Tumor Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Cell line Xenograft

Friend e t al., in prep. Gaidano et al., 1991 Davidoff et al.. 1991b Cheng and Haas, 1990 Malkin e t al., 1990 Gaidano et al., I99 I Friend e t al., in prep. Cheng and Haas, 1990 Cheng and Haas, I990 Chiba e t al., 1990 Chiba e t al., 1990 Davidoff et al., I99 I b d'Amico et al., in prep. Hollstein et al., I99 Ia Felix et al., in prep. Nigro e t al., 1989 Miller et al., in prep. Murakami et al., in prep. Jonveaux and Fenaux, in prep. Baker e t al., 1990 Nigro et al., 1989 Nigro e t al., 1989 Nigro et al., 1989 d'Amico et al., in prep. d'Amico et al., in prep. Rodrigues et al., I990 Rodrigues et al., I990 Rodrigues e t al., 1990 Chiba e t al., 1990 Chiba et al., 1990 Chiba et al., I990 Chiba et al., 1990 Wright et al., in prep. Miller et al., in prep. Caron de Fromentel e t al., in prep. Gaidano et al., I99 I Gaidano et al., I99 I Gaidano et al., I99 I Friend e t al., in prep. Scheffner et al., I99 I Jonveaux and Fenaux, in prep. Jonveaux and Fenaux, in prep. Jonveaux and Fenaux, in prep. Slingerland et al., I99 I Sugimoto et al., I99 I Murakami et al., in prep. Lehman et al., I99 I Hollstein e t al., 1990 Hollstein et al., 1990 Davidoff et al., I99 I a Hensel e t al., I99 I Marks e t al., I99 I Hollstein et al., I99 Ia Bartek et al., I990 Nigro et al.. 1989 Prosser et al.. in prep. Gaidano e t al., 1991 Jonveaux and Fenaux, in prep. Baker e t al., 1990 Baker et al., 1990

GAC AAC GCA AAA AAA AAA GGC GCC GTA GTA GTA CCG TGT TGT ATG CTG ATG ATG TGT TGT CAT CAT TGT CTT CAT CAT CAT CAT CCT CTT CTT CAT CAT TGT TGT TGT TGT CAT TGT TGT CAT CTT GAT

ccc GAC TTT TCT CTT GCT TCT CGT AAA AAA GGC GGC GAG TAC TGG TGG

(Continued on next page)

9

HUMAN TP53 GENE MUTATIONS

TABLE I. (continued) POS

WT

282 282 282 282 282 282 282 282 282 282 282 283 283 285 286 286 286 287 293 298 302 305 305 307 309 334 342

CGG

CGC

MUT TGG GGG CCG TGG TGG CTG TGG TGG TGG TGG TGG TGC

ccc GAG GAA

GAG GGG GAG GGG AAG GCA

ccc GGG CGA

AAG AAA GGA GCA TAG TGG TAG GGT TAG TAG ACA TCC GTG TGA

Event GC -+ AT GC + CG GC + CG GC + AT GC -+ AT GC -+ TA GC + AT GC -+ AT GC + AT GC + AT GC + AT GC + AT GC -+ CG GC + AT GC + AT AT + GC AT -+ CG GC -+ TA GC + TA GC -+ TA GC -+ TA AT + TA AT -+ TA GC + AT GC -+ AT GC -+ TA GC -+ AT

Tumor type Rhabdomyosa Lung (NSCLC) ca Breast ca Bladder ca AML Breast ca B-ALL Burkitt lymphoma Richter’s sdm Ovarian ca Li-Fraumeni sdm Colorectal ca Lung (NSCLC) ca Breast ca Colorectal ca Lung (SCLC) ca Li-Fraumeni sdm Burkitt lymphoma Glioblastoma Bladder ca Lung (SCLC) ca Esophageal ca Esophageal ca Breast ca Colorectal ca Lung (SCLC) ca Lung (SCLC) ca

Origin Tumor Cell line Tumor Tumor Tumor Tumor Tumor Cell line Tumor Tumor Germ line Xenograft Tumor Cell line Tumor Tumor Germ line Cell line Tumor Tumor Tumor Tumor Tumor Tumor Cell line Cell line Cell line

References Mulligan et al., I990 d‘Amico e t al., in prep. Varley et al., I99 I Sidransky et a!., I99 I Jonveaux and Fenaux, in prep. Davidoff e t al., 1991a Gaidano et al., 1991 Gaidano e t al., I99 I Gaidano et al., 1991 Marks et al., 1991 Friend et al., in prep. Baker et al., 1990 Chiba et al., I990 Bartek et al., I990 Baker et al., 1990 Miller et al., in prep. Friend et al., in prep. Gaidano e t al., 1991 Cheng et al., in prep. Sidransky et al., I99 I Miller et al., in prep. Hollstein et al.. I99 Ia Hollstein et al., I99 Ia Prosser et al., in prep. Nigro e t al., 1989 d’Amico e t al., in prep. d’Amico et al., in prep.

Deletions/insertions Codon

Event

Tumor type

Origin

References

I37 I43 I52 I67 I68 I75 I90 20 I 206 206 214 236 239 262 262 262 263 264 286 293 307 38 I Exon 5 I52 239 252

del 7 del I del 13 del I del 31 del 18 del 3 del I del I del I del I del 27 del I del I del 24 del 24 del I del I del 8 del I del I del I del 15 ins I ins I ins 4

Gastric ca Gastric ca Colorectal ad Breast ca Hepatoca Breast ca nu1 ALL Breast ca Burkitt lymphoma Burkitt lymphoma B-ALL Bladder ca Lung (NSCLC) ca Astrocytoma Gastric ca Lung (NSCLC) ca Esophageal ca AML Hepatoca Lung (NSCLC) ca Li-Fraumeni sdm Hepatoca B-ALL B-CLL Waldenstrom sdm Gastric ca

Tumor Cell line Tumor Tumor Tumor Tumor Tumor Tumor Cell line Cell line Tumor Tumor Tumor Tumor Cell line Tumor Tumor Tumor Tumor Tumor Germ line Tumor Tumor Tumor Tumor Tumor

Tamura et al., I99 I Kim et al., 1991 Shirasawa et al., I99 I Varley et al., 1991 Murakami e t al., in prep. Davidoff et al., 1991a Sugimoto et al., I99 I Prosser et al., 1990 Gaidano e t al., I99 I Gaidano et al., 1991 Felix et al., in prep. Sidransky et al., I99 I Takahashi et al., 1989 Chung et al., in prep. Kim et al., 1991 Chiba et al., I990 Hollstein et al., 1990 Jonveaux and Fenaux, in prep. Bressac et al., I99 I Nigro et al., 1989 Tsunematsu et al., in prep. Murakami e t al., in prep. Gaidano e t al., 1991 Gaidano et al., 1991 Sugimoto et al., I99 I Tamura et al., I99 I (Continued on next page)

10

CARON DE FROMENTEL AND SOUSSl

TABLE I. (continued) Codon

Event

256 275 30 I 307 Exon 8

ins I ins I ins I ins I ins 25

Tumor type

Origin

AML B-CLL MD5 Glioblastoma HCL

References

Tumor Tumor Tumor Tumor Tumor

Jonveaux and Fenaux, in prep. Gaidano et al., 1991 Jonveaux and Fenaux, in prep. Chung et al., in prep. Gaidano et al., 1991

Splice mutations lntron

Site

Event

Tumor type

Origin

References

lntron 3 lntron 4 lntron 4 lntron 5 lntron 6 lntron 6 lntron 6 lntron 7 lntron 7 lntron 7 lntron 7 lntron 9

Accept. Donor Donor Donor Donor Accept. Accept. Donor Accept. Accept. Donor Donor

GC-CG G C -TA GC+AT GC-AT AT-CG AT + TA TA AT GC+TA GC-CG C G +AT GC-TA GC-TA

Lung (SCLC) ca Lung (SCLC) ca T-ALL CML Lung (SCLC) ca Lung (SCLC) ca Lung (NSCLC) ca Lung (NSCLC) ca Lung (SCLC) ca AML Lung (SCLC) ca Lung (SCLC) ca

Cell line Cell line Cell line Tumor Cell line Cell line Tumor Cell line Cell line Tumor Cell line Cell line

Takahashi et al., 1990 dAmico et al., in prep. Soudon et al., in prep. Foti et al., 1990 d‘Amico et al., in prep. Hensel et al., I99 I Chiba e t al., I990 Takahashi et al., I990 d’Amico et al., in prep. Jonveaux and Fenaux, in prep. Sameshima et al.. I990 d’Amico et al., in prep.

-

‘POS corresponds to the position of amino acid in the human TP53 protein, WT and MUT, to wild type and mutated codon, respectively. “Event” represents the type of mutational event. Double M is mutations on two nucleotides in the same codon. Abbreviations: ca, carcinoma; sa, sarcoma; sdm, syndrome; ALL, acute lymphoblasticleukemia; AML. acute myelocytic leukemia; CLL, chronic lymphocytic leukemia; CML. chronic myelocytic leukemia; HCL, hairy cell leukemia; MDS, myelodysplastic syndrome; PTCL, peripheral T-cell lymphoma; ad, adenoma.

TABLE 2. Distribution of the Mutational Events in the Different JP53 Regionsa Region No. of CpG No. of mutated CpG

A

0 -

A‘

B

C

D

Other

6 3

I I

2 2

3 3

23 5

I 3

22 0

18 16

10 20

4

3

0 0 0

0

13 9 I I 8 25 91

CpG transition type G+A C +T Other mutations GCJAT AT/GC ATJCG ATFA GCJCG GCFA

4 If

2 3 2 8

Total mutations

23

40

Total

%

4 12

55 51

17 16

10 3 I I 9 8

13 21 II 6 3 17

48 35 17 15 30 71

15 II 5 4.5 9.5 22

62

87

324

‘A to D refer to the five HSR and Other to the remaining regions. “Other mutations” includes all the mutations except for CpG transitions.

I00

I/

HUMAN TP53 GENE MUTATIONS

40

8

30

3

I-

u. 0

20

8 10

0 GCzAT ATwGC GCzCG GCzTA ATzCG ATZTA D.mut.

QCzAT A T S C QCzCQ G G T A A T S G ATzTA

MUTATIONAL EVENT

I

D.mut.

MUTATIONAL EVENT

€1

40 v)

a

g

30

F

u. 0

20

8 10

GCzAT ATwGC

GCzCG GCsTA A T S G

ATzTA

D.mut.

MUTATIONAL EVENT

GCzAT AT>=

Q C A X QCwTA A T K G ATzTA

D.mut.

MUTATIONAL EVENT

Figure 2. Distribution of the mutational events among the different types of cancer. A All cancer types. 8:Colorectal carcinoma.C: Lung carcinoma (SCLC and NSCLC). D: All except colorectal and lung carcinomas. Hatched box in G C to AT transitions corresponds t o CpG mutation.

OTHER TYPES OF MUTATION AND THE POSSIBLE INVOLVEMENT OF CARCINOGENS

Analysis of the distribution of other types of mutation reveals a high frequency of transversions, despite the fact that transitions are normally more common (Fig. 2A). The GC to TA transversion is most frequent (70 cases), though this varies with the type of tumor. Twenty-eight (40%) were isolated from lung tumors (SCLC and NSCLC) while 14 (20%) were found in hepatmrcinomas. On the other hand, no GC to TA transversions were observed in 34 cases of colorectal carcinoma (Figs. 2B, 3). Lung carcinomas are usually associated with environmental carcinogens like benzo(a)pyrene,present in cigarette smoke, leading to G to T transversion (Eisenstadt et al., 1982). In fact, 44% of SCLC (14

cases) and 41% of NSCLC (14 cases) are affected by this transversion (Fig. 3). Furthermore, in region HSR A', described above and seen in Table 2,12 out of the 24 mutations are observed in pulmonary tumors, seven of which are GC to TA transversions. (See also Chiba et al., 1990.)No mutations were observed in this region in colorectal tumors. Twenty-five of the 27 nucleotides in this region are G or C, supporting the notion that this region is highly susceptible to chemical carcinogens. Six and seven mutations from lung carcinoma are located in codons 248 and 273, respectively, whereas none is found in codon 175. The significance of this observation is unclear, but it suggests the tissue-specific nature of some mutations. Hepatocarcinoma transversions in the TP53 gene were found to be associated predominantly with codon 249 (AGG to AGT) (11 out of 19 cases, Fig. 3)

12

CARON DE FROMENTEL AND SOUSSl

--

I

60

cn K

B3 8U

40

w

m

I 20

0

LUNGCA

HCCA

BR.CA

ES.CA.

LFS.

CML

B.Lym.

BL.CA. LA.CA.

NFSA

MDS

COLCA.

TUMOR TYPE Figure 3. Frequency of the GC to T A transversions. Black boxes correspond t o GC to T A transversions and hatched boxes to the remaining mutations. Among tumors lacking such a transversion, only data from colorectal carcinoma are shown. Abbreviations: CA. carcinoma; SA, sarcoma; BL, bladder; BR. breasc COL. colorectal; ES. esophagus; HCCA, hepatocarcinoma; LA, larynx; LFS. Li-Fraumeni syndrome; NFSA, neurofibrosarcoma.

(Bressac et al., 1991;Hsu et al., 1991).The nucleotide G in a GC-rich region is known to be a preferential target for aflatoxin B1. Patients analysed in these studies were from China and South Africa, where aflatoxin B1 is a common food contaminant.The specificity of the 249 mutation could be directed either by the high preference of aflatoxin B1 for this codon or by a high selection for this transversion as a necessary step in hepatocarcinogenesis. The possible involvement of such a carcinogen in hepatocarcinoma and the prevalence of the 249 mutation could be assessed by the study of patients from different geographic regions and ethnic origins. (See Harris, 1991, for discussion.) When we consider the 70 GC to TA transversions, we observe a disequilibrium in the frequency of mutations in one strand of the DNA as compared to the other (66 G to T substitutions in the strand corresponding to the coding sequence).It is known that the nontranscribedDNA strand is less efficiently repaired than the transcribed one (Mellon et al., 1987). Moreover, nucleotide G is the preferential target for chemical-carcinogen-inducedmutations (Kriek et al., 1984). Considering first that the coding sequence corresponds to that of the nontranscribed strand and second that the nucleotide affected is a G, it is tempting to interpret the observed disequilibrium in terms of preferential repair of one DNA strand.

MUTATIONS IN SPLICE SITES, INSERTIONS, AND DELETIONS

Other missense point mutations were found in splice donor and acceptor sites (Table 1). These less frequent mutations generate a shorter mRNA or an mRNA retaining some intronic sequence, which could lead to a truncated TP53 protein lacking part of the carboxy terminus. Nine of the 12 mutants of this type observed were found in lung tumors, though the significance of this fact is not obvious. The last category of mutations is deletions (23) or insertions (eight) (Table 1).These alterations change the reading frame, also leading to a prematurely truncated TP53. Although no particular codon is affected by this type of alteration, it should be noted that they also clustered in the central region of the molecule. Due to the small number of these types of mutation described and the difficulty of the molecular study of these mutational events, we have not analysed them further. CONCLUSIONS

In this review, it is shown that mutational alterations in TP53 sequences on the one hand provide an index of the metabolic and environmental events leading to particular cancers, and on the other hand, define the portions of the molecule which are crucial to its tumor suppressor function.

13

HUMAN TP53 GENE MUTATIONS

This study has allowed us to emphasize the following points: (1)We define a new HSR in a region of coding sequence which is conserved in mammals but is not an HCD. This HSR A' is predominantly hit in pulmonary (both SCLC and NSCLC) tumors. Furthermore, 50% of mutational events in this region are GC to TA transversions (Table a), and the tumor types affected are usually associated with exogenous factors (bladder, esophageal, lung, and hepatocarcinomas). This observation can be related to the high GC content of HSR A'. (2) We point out Arg175as being of structural significance. Unlike codons 248 and 273, where both nucleotides of CpG are equally hit, only one is hit in codon 175 (Table 2). Furthermore, codon 175 is never associated with lung carcinoma, suggesting a tissue specificity for some TP53 mutations. (3) We confirm the predominance of CpG dinucleotides in putatively endogenous mutational events. This is especially interesting as it strongly suggests that many CpGs are methylated, even in the ubiquitous TP53 expression. (4) We conclude that CpG modification is not responsible for the majority of tumors suspected to be due to chemical carcinogens as an initiating event like pulmonary and hepatic neoplasms. In these tumors, the frequent Occurrence of GC to TA transversion (otherwise a rare event) suggests the implication of external mutagens. Concerning the TP53 protein itself, this study distinguishes three regions which can be deduced from the protein primary structure. The amino terminal part which consists of an acidic region and possesses a transactivating activity (Fields and Jang, 1990; 0 Rourke et al., 1990; Raycroft et al., 1990) is never the target of mutation despite the conserved domains HCD I and IIa. The carboxy terminus, which possesses a DNA binding activity, as well as the oligomerization domain and the site of nuclear transport (see Soussi et al., 1990a, for review), are equally spared from mutations. Only the central region (exons 5-8)is compromised by mutational events. This region, which includes the HSRs A-D, is the location of HCD IIb-V. The consequences of these mutations (activation or inactivation or both) are not clearly determined. Furthermore, the functional role of this region remains unclear. Nevertheless, (1) the fact that this region of murine tp53 binds the large T antigen of SV40 uenkins et al., 1988)and (2) the fact that tp53 of Xenopus laevis and rainbow trout are also capable of specifically associating with SV40 T antigen (Soussi et al., 1989, 1990b; Caron de Fromentel et al., 1992) suggest that this is the site of fixation of a cellular T-antigen equivalent, which modulates the normal function of tp53. The identification of such a factor(s) and the study of its interaction with wild-type or

mutated TP53 should allow a better understanding of activation or inactivation of TP53 in human cancer. The data included in this review represent a compilation (September 1991) of numerous independent mutations found in a human gene. A similar analysis has been recently published by Hollstein et al. (1991b). TP53 gene mutations are a very attractive model in which, first, to assess the genetic alterations that lead to the expression of an oncogenic TP53, and second, in which to study the molecular mechanisms of mutagenesis in the human genome. ACKNOWLEDGMENTS

We are especially grateful to D. Grausz for the preparation of the manuscript, to F. Bourre, C.J. Larsen, P. May, and A. Sarasin for critical reading, and to M. Kress and Y. Legros for help in the database management. This work was made possible by the generous help of the following people who provided us with their data: S. Benchimol, B. Bressac, W. Cavenee, P.H. Cogen, C. Cooper, P. Fenaux, S. Friend, M. Haas, C.C. Harris, P. Jonveaux,P. Koeffler, D. Lane, R. Maestro, C. Miller, J. Minna, M. Ozturk, J. Prosser, T. Sasazuki, B. Seizinger, T. Sekiya, Dr Tsunematsu, J. Varley, B. Vogelstein, and D. Wyndford-Thomas. N O T E ADDED IN PROOF

The work of Murakami et al. concerning mutations in hepatocarcinoma and Wright et al. about human thyroid carcinoma are now published wurakami et al. (1991) Cancer Res 51:552&5525, and Wright et al. (1991)Oncogene 6169S16971. REFERENCES Ahuja H, Bareli M, Arlin 2, Advani S, Allen SL, Goldman J, Snyder D, Foti A, Cline M (1991) The spectrum of molecular alterations in the evolution of chronic myelocytic leukemia. J Clin Invest 8720422047. Baker SJ, Fearon ER, Nigro J, Hamilton S, Preisinger AC, Jessup JM, vanTuinen P, Ledbetter DH, Barker DF, Nakamura Y, Whyte R, Vogelstein B (1989) Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 244:217-221. Baker SJ, Preisinger AC, JessupJM, Paraskeva C, Markowitz S. Willson JKV, Hamilton S, Vogelstein B (1990) p53 gene mutations occur in combination with 17p alleiic deletions as late events in colorectal tumorigenesis. Cancer Res 97717-7722. Bartek J, Iggo R, Gannon J, Lane DP (1990) Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines. Once gene 5895-899. Bressac B, Kew M, Wands J, O m k M (1591) Selective G-mutation to T-mutation of p53 gene in hepatocellular carcinoma from Southern Africa. Nature 35042M31. Caron de Fromentel C, Padkel C, Chapus A, Baney C, May P, Soussi T (1992) Rainbow trout p53 cDNA cloning and biochemical characterization. Gene (in press). ChengJ, Haas M (1990) Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines. Mol Cell Biol 105502-5509. Chiba I, Takahashi T, Nau MM, d'Amico D, Curie1 DT, Mitsudomi T, Buchhagen DL, Carbone D, Piantadosi S, Koga H, Reissman PT, Slamon DJ, Holmes EC, Minna JD (1990) Mutations in the p53 gene are frequent in primary, resected non-small-celllung cancer. Oncogene 5:1603-1610. Coulondre C, Miller JH, Farabaugh PJ, Gilbert W (1978) Molecular basis

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CARON DE FROMENTEL AND SOUSSI

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TP53 tumor suppressor gene: a model for investigating human mutagenesis.

More than 350 independent point mutations of the TP53 gene, found in a wide variety of human cancers, were compiled and analysed. From this study, we ...
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