Role in tumorigenesis of silent mutations in the TP53 gene.

Over 10,000 mutations in the TP53 suppressor gene have been recorded in the International Agency for Research on Cancer (IARC) tumor data base. About 4% of these mutations are silent. It is a question whether these mutations play a role in tumor development. In order to approach this question, we asked whether the reported silent mutations are randomly distributed throughout the TP53 gene. The p53 data base was searched exon by exon. From the frequency of codons with no silent mutations, the average number of silent mutations per codon for each exon was calculated using the Poisson distribution. The results indicate the distribution to be non-random. About one-third of all silent mutations occur in "hot-spots" and after subtraction of these hot-spots, the remaining silent mutations are randomly distributed. In addition, the percentage of silent mutations among the total in the silent mutation hot-spots is close to that expected for random mutation. We conclude that most of the silent mutations recorded in tumors play no role in tumor development and that the percentage of silent mutation is an indication of the amount of random mutation during tumorigenesis. Silent mutations occur to a significantly different extent in different tumor types. Tumors of the esophagus and colon have a low frequency of silent mutations, tumors of the prostate have a high frequency.

Role of the dinB gene product in spontaneous mutation in Escherichia coli with an impaired replicative polymerase.

We isolated several new mutator mutations of the Escherichia coli replicative polymerase dnaE subunit alpha and used them and a previously reported dnaE mutation to study spontaneous frameshift and base substitution mutations. Two of these dnaE strains produce many more mutants when grown on rich (Luria-Bertani) than on minimal medium. A differential effect of the medium was not observed when these dnaE mutations were combined with a mismatch repair mutation. The selection scheme for the dnaE mutations required that they be able to complement a temperature-sensitive strain. However, the ability to complement is not related to the mutator effect for at least one of the mutants. Comparison of the mutation rates for frameshift and base substitution mutations in mutS and dnaE mutS strains suggests that the mismatch repair proteins respond differently to the two types of change. Deletion of dinB from both chromosome and plasmid resulted in a four- to fivefold decrease in the rate of frameshift and base substitution mutations in a dnaE mutS double mutant background. This reduction indicates that most mistakes in replication occur as a result of the action of the auxiliary rather than the replicative polymerase in this dnaE mutant. Deletion of dinB from strains carrying a wild-type dnaE had a measurable effect, suggesting that a fraction of spontaneous mutations occur as a result of dinB polymerase action even in cells with a normal replicative polymerase.

Frameshift mutation, microsatellites and mismatch repair.

The structure of eukaryotic DNA, with its repeated sequences, makes base addition and loss a major obstacle to the maintenance of genetic stability. As compared to the bacteria, much of the mismatch repair capacity of the eukaryotic cell must be devoted to the surveillance of frameshift changes. Any alteration in the activity of proteins which recognize frameshifts or which hold the DNA in place during replication is likely to result in genomic instability.

Hypermutability in carcinogenesis.

The presence of numerous chromosomal changes and point mutations in tumors is well established. At least some of these changes play a role in the development of the tumors. It has been suggested that the number of these genetic changes requires that tumorigenesis involves an increase in mutation rate. However, the presence of numerous changes can also be accounted for by efficient selection. What is required to settle the issue is some measure of nonselected mutations in tumors. In order to determine whether the tumor suppressor TP53 (coding for the protein p53) is hypermutable at some stage of carcinogenesis, the frequency of silent and multiple mutations in this gene has been examined. Silent mutations make up approximately 3% of the total recorded but constitute 9.5% of the mutations found in tumors with multiple mutations. Multiple closely linked mutations are also observed. Such multiple mutations suggest the operation of an error-prone replication process in a subclass of cells. The published data indicate that TP53 is hypermutable at some stage of tumor development. It is not yet clear whether TP53 is unique or whether other genes display a similar pattern of silent and multiple mutations.

Hypermutability and silent mutations in human carcinogenesis.

Silent or synonymous mutations are nucleotide changes which do not result in an amino acid change. Such alterations make up approximately 4% of the total TP53 mutations in a database of almost 10,000 mutations. Insofar as these changes do not confer a selective advantage, silent mutations make it possible to estimate an unbiased mutation frequency. Since this frequency is at least 100 times higher than would be expected for normal tissue, tumors must be hypermutable. In addition, a class of tumors may be supermutable with a mutation frequency comparable to that observed in the generation of immune diversity.

Silent and multiple mutations in p53 and the question of the hypermutability of tumors.

Published data on TP53 mutations can be used to examine the question of whether generalized hypermutability is a necessary condition for tumorigenesis. Although individual mutations do play an etiologic role in tumor formation, the evidence so far does not make it necessary to assume a general mutability. Silent and multiple mutations in the TP53 data set indicate that a special hypermutability process operates on this gene during the generation of tumors. The percentage of silent p53 mutations observed (3%) is at least 20 times greater than would be expected and indicates hypermutability for this gene. The greater proportion of silent mutations among multiple p53 mutations (10%) indicates that the mutations occur nonselectively. The presence of silent mutations implies that not all mutations observed in tumors have an etiologic role. Analysis of the distribution of tumors with two, three, four and more p53 mutations suggests that mutations in some tumors occur in clusters possibly as a result of 'stuttering' in DNA synthesis. It is argued that the most likely alternative explanations of the data, polymorphism and/or a selective role for silent mutations, are not correct. It remains possible that the hypermutability process is restricted to particular genes or to regions of the genome as, for example, in antibody production. There is a surprising paucity of data on human polymorphism and nucleotide diversity which makes the analysis difficult.

Role of proofreading and mismatch repair in maintaining the stability of nucleotide repeats in DNA.

The role of the proofreading exonuclease in maintaining the stability of multiply repeated units in DNA was studied in Escherichia coli. Reversion of plasmids in which the beta-galactosidase alpha complementing sequence was moved +2 out of frame by inserts containing (CA)14, (CA)5, (CA)2 or (TA)6 or +1 by creating a run of 8 C was compared in mutS and mutSdnaQ strains. Proofreading corrects at least half of the frameshift errors for all the plasmids and at least 99% of the errors in the (CA)2 plasmid. The (CA)2 plasmid reverts mostly by +1 frameshifts in the restriction sites flanking the insert. With the (CA)14, (TA)6, (CA)5 and 8C plasmids, reversion is mainly by loss of a repeat unit. The data support the hypothesis that the dnaQgene product recognizes frameshifts close to the DNA growing point. Frameshifts distal to the growing point are mainly corrected by mismatch repair. We speculate that mismatches in mononucleotide repeats are susceptible to proofreading because they can either migrate to a point where they are recognized by the exonuclease or, alternatively, because single nucleotide distortions are more readily detected than dinucleotides.

Laser ablation of prostatic hypertrophy.

Laser prostatectomy offers the urologist a new tool for the management of symptomatic benign prostatic hypertrophy. Future studies will determine the long-term benefits of this new procedure in a properly selected group of patients as a possible alternative to transurethral prostatectomy.

The 'A rule' of mutagen specificity: a consequence of DNA polymerase bypass of non-instructional lesions?

The replicative bypass of lesions in DNA and the induction of mutations by agents which react with DNA to produce damaged bases can be understood on the basis of a simple kinetic model. Bypass can be analyzed by separately considering three processes: a) addition of a base opposite a lesion, b) a proofreading excision process, and c) a rate limiting elongation step. Adenine nucleotides are preferentially added opposite many lesions making it possible to predict mutational specificity. Replicative bypass (translesion synthesis) is dependent on modulation of proofreading exonuclease activity but loss of exonuclease activity alone is not sufficient to ensure bypass. Frameshift mutation is the result of the failure of translesion synthesis accompanied by rearrangement of the template, particularly at repetitive sites.

The use of a multimodal image, the apple technique, to facilitate clinical hypnosis: a brief communication.

A 1- to 3-minute exercise involving imagination (of an apple) and ideomotor ideation (hand levitation) is a simple, benign technique that is useful for illustrating to patients the nature of imagery and hypnosis. It avoids power struggles and allows a reasonable approximation of the patient's capacity for imagery and hypnotic responsiveness, without emphasizing the use of a hypnotic procedure. When administered to 35 college students, the hand levitation component of this exercise correlated with the Stanford Hypnotic Susceptibility Scale, Form C (Weitzenhoffer & E. R. Hilgard, 1963) (r = .66, p less than .001) and with the Stanford Hypnotic Clinical Scale: Adult (Morgan & J. R. Hilgard, 1975, 1978/79) (r = .60, p less than .001).

Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA.

N-(Deoxyguanosin-8-yl)-2-(acetylamino)fluorene (AAF-G) adducts in the DNA of bacteriophage M13 can be converted to N-(deoxyguanosin-8-yl)-2-aminofluorene (AF-G) adducts in situ by treatment with 1.0 M NaOH for 45 min at room temperature. The conversion is accompanied by a dramatic increase in the transfection activity of the samples which is correlated with the measured deacetylation of the acetylaminofluorene adduct. The pair of substrates (AAF-G/AF-G) with adducts at identical places in the DNA has been used to study bypass synthesis catalyzed by T7 DNA polymerase, an altered T7 DNA polymerase from which the 3'----5' exonuclease has been genetically removed by an 84 nucleotide deletion (Sequenase 2), T4 DNA polymerase and Escherichia coli DNA polymerase I. All polymerases appear blocked at acetylaminofluorene lesions. Sequenase 2 is apparently able to add nucleotides opposite the acetylaminofluorene lesion but is unable to catalyze further elongation. T7 DNA polymerase, including thioredoxin and with an active 3'----5' exonuclease, is unable to bypass aminofluorene adducts, whereas Sequenase 2 bypasses the lesions readily. The data support the view that the elongation step is rate limiting in synthesis past lesions and that low 3'----5' exonuclease activity allows the priming nucleotide opposite the altered template site to remain in position long enough for elongation past particular adducts.