Carcinogen-induced impairment of enzymes for replicative fidelity of DNA and the initiation of tumours

Leon P. Bignold

Institute of Medical and Veterinary Science, Adelaide, South Australia and Department of Pathology, University of Adelaide, SA 5005, Australia Email: leon.bignold{at}adelaide.edu.au


    Abstract
 Top
 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
Not all carcinogens are mutagens, and many mutagens are not carcinogens. Among related chemicals, small changes of structure can markedly influence carcinogenic potency. Many tumours are genetically unstable, but some, especially ‘benign’ types, rarely exhibit ‘progression’ or show other evidence of genetic instability. Cells of particular tumour types exhibit identifiable particular ‘sets’ of phenotypic abnormalities (e.g. rapid growth, uniform nuclei, little cytoplasm and occasionally production of adrenocorticotrophic hormone by anaplastic small-celled carcinoma of the bronchus). Tumour cells pass their abnormalities on to their daughter cells, indicating that a genomic alteration probably underlies tumour formation. A possible mechanism, which might explain these phenomena is carcinogen-induced reduction of fidelity of replication of DNA polymerase complexes during S phase of normal tissue stem cells. A single ‘hit’ by a reactive agent (chemical or physical) on one of the major enzymic sites (synthesis, proofreading, mismatch repair—MMR) could cause multiple sequence abnormalities in the length of DNA synthesized by one DNA polymerase complex. Because this length of DNA (half a replication ‘bubble’) averages 15 000–150 000 nucleotides, the affected DNA could include two or more significant genomic elements (genes, especially for tumour suppression, regulatory loci and other elements). The particular mutant elements in the affected DNA could then determine the ‘set’ of phenotypic abnormalities exhibited by a resulting tumour. Non-genotoxic carcinogenicity, non-carcinogenic mutagenicity, structure-dependent chemical carcinogenicity and the phenomenon of ‘sets’ of phenotypic abnormalities could thus be accommodated. In experimental studies, the ‘hallmark pattern’ of mutation caused by this mechanism would be multiple mainly point mutations clustered within the length of half a replication ‘bubble’. Such a ‘hallmark pattern’ of mutation might be detectable in carcinogen-treated cell cultures by the use of cycle-synchronized cultures, single cell subculturing, restriction (endonuclease) fragment length analysis of the clones and nucleotide sequencing of abnormal bands for localization in the human genome. If the mechanism is important to carcinogenesis generally, then non-carcinogenic mutagens should not cause the ‘hallmark pattern’ of mutations in either in vitro or in vivo systems. In human tumour cells, the ‘hallmark pattern’ of mutations may be demonstrable in genetically stable human tumours, but might well be lost or obscured by secondary mutations in genetically unstable tumours. Among different cases of the same type of human tumour, the clustered point mutations might be tumour-type specific in their location in the genome, but vary case-to-case in the precise ‘points’ mutated in the cluster region. New assays for assessing the carcinogenic potential of environmental and synthetic substances for human and animal populations may result. The hypothesis is not put forward to the exclusion of some established mechanisms of carcinogenesis for particular human tumours: for example, the ‘two-hit’ mutational hypothesis for retinoblastoma, the ‘multiple sequential mutational’ hypothesis for UV-induced lesions of the epidermis, and the possibility of adduct-induced frameshift mutations by some chemical carcinogens for experimental tumours.

Abbreviations: RFL, restriction fragment length


    Introduction
 Top
 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
From the 1930s, when pure chemical carcinogens became available (1), the investigation of the possibility of carcinogen-induced somatic mutation as the basis of tumours has followed several lines.

First, it was found that some chemical carcinogens are mutagens (2,3). The methods of detection of mutations in early studies were largely limited to chromosomal morphology and/or limited phenotypic changes and the results were thought unconvincing by some authors of the period (2,4,5).

Secondly, numerous attempts were made to establish particular ‘carcinogenic structural characteristics’ among categories of chemical carcinogens, especially polycyclic hydrocarbons, and aromatic amines (69). This search illuminated exquisite sensitivity of the carcinogenic potency of some molecular species to structural changes. For example, 3,4-benzpyrene is a potent carcinogen while 1,2-benzpyrene has only a weak carcinogenic effect (the difference between the molecules being the position of one benzene ring) (7). Similarly, 3'-methyl-N,N-dimethyl-4-aminoazobenzene is a potent carcinogen, while 4'-methyl-dimethylaminoazobenzene is a weak carcinogen (the difference between the two compounds being the position of one methyl group) (7). Specific interactions of carcinogens with nucleotides have since been established, for example between ultraviolet light and covalent binding of pyrimidines (10) and between chemical carcinogens and purines and pyrimidines (11), but no general relationships between structure and carcinogenicity have been established for any chemical group of carcinogens.

Thirdly, the actions of carcinogens were investigated in relation to putative intracellular ‘targets’. Studies of protein ‘targets’ (7,9,1214) showed that carcinogens or their active metabolites (15) are reactive species and can bind to most intracellular macromolecules, including cytosolic and nuclear proteins. Ketterer (14) observed that transcription of chromatin seemed particularly vulnerable to carcinogens, but no general mechanism based on interactions of carcinogens with proteins emerged.

Attention turned to DNA as the ‘target’ of carcinogens, especially after the discoveries that carcinogens can cause strand breaks in DNA in cells (13,16,17) and that the inherited mutation of xeroderma pigmentosum is of the gene for an enzyme associated with repair of damaged DNA (18). Farber (17) found that most carcinogens (ethionine being one exception) induced strand breaks in the DNA of hepatocytes. However, this occurred whether the carcinogen caused tumour in the liver or not (17). Farber (17) suggested that the basis of this cell type-specificity of action might be related to the observation that strand breaks caused by agents that are carcinogenic in the liver are only slowly repaired in that organ, while breaks caused by non-hepatic carcinogens are rapidly repaired in this organ.

However, in the same period, it was noted in other studies using the experimental hepatocarcinogenesis model (mice and rats), that prior partial hepatectomy enables a single dose of carcinogen to cause tumours in adult animals (19). Otherwise, the carcinogen could only cause tumours when given as a single dose to non-hepatectomized newborn animals, or when given chronically in the diet to adult animals (19). Craddock (19) commented that an event during cell replication might be the critical point of action of the carcinogen.

In the 1970s, concerted study began of particular nucleotide-carcinogen binding products (‘adducts’) in the DNA of organs of animals exposed to carcinogens, including alkylating agents, polycyclic hydrocarbons, aromatic amines and mycotoxins (2026). However, the biologic significance of adduct formation has proved difficult to establish. Adducts are found both in organs in which tumours form and in organs in which tumours do not form, so that the detection of adducts in a tissue does not necessarily indicate a specific tumourigenic risk for that tissue (27). Single carcinogens, especially ‘bulky’ ones often cause a variety of adduct types (28), and the potencies of particular types of adducts vary unpredictably by several orders of magnitude (27). The possibility that particular types of adducts might cause particular types of mutation (29) has been investigated by adduct site-specific techniques, but without conclusive outcome. Loechler (28) indicates that the types of mutation caused by adducts are influenced by experimental parameters, including ratio of adducts to genome and the nucleotide sequence context of the adduct. He also noted that the types of mutation caused by adducts appear to vary with cell type. No general relationship between carcinogenic potency and tendency to cause particular types of mutations has been established.


    Genetic instability: not a feature of all tumours
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 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
Until the 1960s, tumours were thought to be composed of genetically fairly uniform masses of cells which grow excessively possibly because of loss of gene(s) for regulation of cell growth (30), and possibly having a disorder of ‘differentiation’ as the basis of at least some of their complex morphologic and behavioural variability (8).

The concept that tumours are not genetically homogenous came from work showing variability of metastatic properties of cells cultured from single tumours in vivo (31,32), and this phenomenon came to be termed ‘tumour cell heterogeneity’ (3234). Busch in 1990 (34) noted that heterogeneity exists with respect to virtually all features of cancers: growth, invasiveness, metastasis, pigment production (in melanomas), surface markers, chromosomal variants, markers of differentiation, secretion (of hormones and other products) and drug resistance.

Meanwhile the work on strand breaks of DNA and Cleaver's discovery of the basis of xeroderma pigmentosum (see above) together with the renewed attention to chromosomal instability in tumours (35,36) led to genetic instability being recognized as the probable basis of both tumour cell heterogeneity and tumour progression (32,33,37).

Subsequently, numerous mechanisms of induction of genetic instability have been identified (3844).

  1. Template DNA: exogenous genotoxic ‘adducts’ (see above); depurination, deamination and oxidation; alkylation.
  2. Nucleotides: imbalanced concentrations; inappropriate analogues and enzymes normally degrading these.
  3. Error-prone DNA polymerases (including proofreading and MMR) and accessory proteins.
  4. Presence of DNA polymerases which synthesize across abnormal templates (‘translesional synthesis’) so that the usual arrest of synthesis does not occur (44).
  5. Strand misalignments.
  6. Abnormalities of DNA repair enzymes.
  7. Abnormalities of cell cycle checkpoints (45).
  8. Mechanisms affecting chromosomal integrity and ploidy (46,47).
  9. Epigenetic mechanisms, including DNA methylation (48).
  10. Telomerase dysfunction (49).
  11. Other mechanisms.

The importance of this list is that it indicates that a large number of genes are required for preservation of the genome, and correspondingly, that random mutational events are likely to affect one of these genes (and hence cause genetic instability) more often than they will affect a single gene for a specific phenotypic feature.

Loeb (5053) was among the first to suggest that genetic instability might be a necessary aspect of tumour formation, and has proposed that normal cells acquire a ‘mutator phenotype’ early in the neoplastic process. This is supported by recent discoveries that tumour cells can contain up to 105 genomic events (54) (such numbers can only arise by enhanced mutation rates among cells) and that genetic instability may occur in tissue cells adjacent to tumours (55,56). Genetic instability may also provide possible explanations of general features of tumours, especially their cell-to-cell and focus-to-focus variability of cytologic and architectural morphology, and the occasionally poor correlations of degrees of abnormal morphologies with aggressive clinical behaviour (57).

Notwithstanding the foregoing, genetic instability may not be an essential aspect of all tumours and not an essential initiating step. Although genetic instability has been documented in many malignant tumours (5153,55,56), many human tumours (mainly ‘benign’ ones, for example, lipomas, neurilemomas, seborrhoeic keratoses) show few abnormalities other than excess growth, and do not show ‘progression’ to suggest an unstable genome. Some ‘low grade’ malignant tumours, such as basal cell carcinoma of epidermis and intestinal carcinoid tumour do not undergo ‘progression’. Specific studies of large numbers of ‘benign’ and ‘low grade malignant’ tumours for genetic instability have not been reported, and little evidence of the phenomenon has been found in the limited studies of basal cell carcinoma (58) and intestinal carcinoid (59) so far published.

Furthermore, experimental evidence has been published, which weighs against genetic instability as the basis of all tumour types. Many ‘immortal’ or transformed cell lines remain stable in culture for long periods of time and several studies have shown that tumourigenic human cell lines are no more susceptible to mutation than their non-tumourigenic counterparts (6063). In one report, extracts of transformed cells showed no difference in replicative fidelity of DNA compared with non-transformed cells (64).


    Carcinogen-impaired DNA polymerase complexes as a mechanism of the initiation of tumour formation
 Top
 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
One mechanism of carcinogenesis, which can potentially explain a variety of tumour phenomena is that the carcinogen binds to enzymic sites of the DNA polymerase complex during S phase of the cell cycle, and causes multiple mutations in DNA. This idea was mentioned by Speyer (65) in 1965 and discussed in some detail by Nelson and Mason (66) in 1972. Loeb and co-workers in 1974 (49) discussed the hypothesis in relation to early evidence of error-prone DNA polymerases in tumour cells. The present author (67) drew attention to possible explanations provided by the hypothesis of phenomena such as time delays in tumour development after exposure to carcinogens, non-chemical promoters of carcinogenesis and hormonal initiators and promoters of carcinogenesis.

The attraction of this notion is that if a single ‘hit’ of carcinogen on an enzymic site for DNA genomic fidelity (base selection, proofreading or MMR) during S phase, then multiple mutations could be inflicted on a length of nucleotide sequence equal to a half replication ‘bubble’ (15 000–150 000 nt) (68) (Figure 1). There would be a significant chance of ‘effective’ (rather than ‘silent’) mutations of the genomic elements in the affected length of DNA without any damage to the remainder of the genome. The resulting phenotypic abnormalities of the cell would depend on the genomic composition of the affected half replication ‘bubble’, and two or more such abnormalities would appear in a cell at the same time if two genomic elements (genes and gene regulators) were present in the affected length of DNA.



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Fig. 1. A non-genotoxic mechanism by which a carcinogen could cause one or more mutations in one cell at the same time. If the half replication ‘bubble’ includes only a tumour-suppressor gene, only excessive growth might result. If the half replication ‘bubble’ includes a gene supporting replication fidelity, the daughter cell could be genetically unstable. More complex tumours could arise if other appropriate genes are present in the half replication ‘bubble’. N.b. The carcinogen in the diagram is placed in the site of synthesis by the DNA polymerase, implying base selection is impaired. However, sites of proofreading or mismatch repair could serve as alternative sites (see text).

 
All of the relevant enzymic sites of the DNA polymerase complex (for base selection, proofreading and mismatch repair) are extremely dependent on precise structural determinants, perhaps corresponding to the precise structural characteristics required of carcinogens (see above). In particular, to be a carcinogen, a molecule must derange the fidelity mechanism without arresting synthesis of DNA.

Furthermore, the affected DNA need not contain a gene necessary for fidelity of replication of DNA for tumour to form. Thus, if the affected half replication ‘bubble’ included only a tumour suppressor gene, the daughter cell would be only hyperproliferative, and remain genetically stable. This then could account for genetically stable tumours and cell lines deriving from carcinogen-exposed cells in vivo and in vitro.

It can also be noted that a gene cluster containing genetic stability genes, but not a growth suppressor could lead to genetically unstable cells with little accumulation of cell numbers. This combination is that of ‘in-situ’ tumours, which are recognized in many tissues, including epidermis, bladder epithelium and other tissues (69).


    Experimental evidence for direct actions of carcinogens on DNA polymerases
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 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
Much of the experimental basis for this notion derives from studies of the loss of fidelity of replication of DNA induced by carcinogenic metal ions such as those of cadmium, manganese and cobalt in cell-free systems. For general references see (7073) and for specific reviews see (7481).

Sirover and Loeb in 1977 (82) showed that infidelity of replication of DNA by DNA polymerase from avian myeloblastosis virus in the presence of Mn2+ or Co2+ is not likely to be due to interactions of the ions either with the nucleotides being added to the new strand of DNA during DNA synthesis or with the template strand of DNA. Loeb, Sirover and co-workers (83) in the same year suggested that induction of infidelity of replication might be a useful test of carcinogenicity. Snow and co-workers (84,85) considered that chromium ions and nickel ions might cause mutations and hence carcinogenesis by increasing the DNA polymerase processivity and the rate of polymerase bypass of DNA lesions.

Pelletier and co-workers (86) used X-ray crystallography to study the effects of Mg2+, Ca2+, Mn3+, Co2+, Cr3+ and Ni2+ on the active sites of human DNA polymerase beta. These authors found that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. In addition, all metal ions tested, with the exception of Mg2+, promoted a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues.

Further evidence of non-genotoxic carcinogenesis has come from a recent study of the mutagenicity of cadmium by Jin and co-workers (87) who found that the mutagenic effects of cadmium ions in yeast cultures derive from the ability of the cation to interfere with the fidelity of replication of DNA in target cells. Jin et al. (87) found that large numbers of illegitimate nucleotide additions go uncorrected but the synthesis of DNA is not blocked. They suggest that the precise site of the impairment of the fidelity of replication is the MMR mechanism, because the basis that the pattern of uncorrected mismatches which they observed in Cd2+-exposed cells is similar to the pattern in MMR-deficient strains of yeast. Extracts of human cell lines showed direct inhibition of correction of heteroduplexes containing one base-loop (a function of MMR enzymes) by Cd2+.

McMurray and Tainer in a commentary (88) mention many mechanisms by which Cd2+ can interfere with the functions of proteins. These authors note that Cd2+ may replace Zn2+ in tissues, and that perhaps the Cd2+ ion disrupts a currently unknown ‘zinc finger’ structure of MMR sites.

In whole animal studies, Chan and Becker (89) reported that DNA polymerase alpha isolated from rat livers after feeding with N-2-fluorenylacetamide is significantly error-prone in replication of synthetic polynucleotide templates compared with the same enzyme from untreated controls. These authors apparently did not obtain a similar result when a different carcinogen (N-2-aminoacetamide) was used (90).

In contrast to the above, essentially negative results were obtained in studies of the effects of carcinogens on cells in culture described by Brucker, Loeb and Thielmann (91). These authors applied carcinogens [Me(NO)(NO2)Gdn and MeNOUr] to living cells, and then extracted the DNA polymerase alpha–primase complexes from the cells and tested the fidelity of replication of bacteriophage DNA (as template) in cell-free systems. They found that the fidelity of DNA polymerase alpha–primase complexes was similar in carcinogen-treated and -untreated cells. However, increased synthesis of DNA occurred in carcinogen-treated cells compared with untreated cells, which the authors thought might be due to a carcinogen-induced alteration of an accessory protein of the complex. The fidelity of replication by the DNA polymerase was assessed in a cell-free system, however, so that a functional defect might have been present but undetected owing to the assay conditions.


    Discussion: comparison with other models
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 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
‘Tumours’ comprise a large number of lesions of the body, which vary markedly in the degrees and natures of their morphological and behavioural abnormalities. For some particular tumour types, there may be particular mechanisms. For example, for retinoblastoma, for which no role of any environmental carcinogen is suspected, there is little reason to doubt Knudson's ‘two (mutational) hit’ hypothesis (92–94, and see below). The ‘first hit’ is inherited in most affected individuals, and spontaneous mutations, being relatively common in rapidly dividing tissues, can provide the ‘second hit’. As another example, some chemical carcinogens may act principally by adducts (see Introduction), which induce frameshift mutations. These mutations, by affecting lengths of DNA between the site of an adduct and the site of termination of DNA synthesis, could have a mutational impact similar to that of impairment of DNA fidelity-of-replication sites. Yet again, time-sequentially accumulated ‘point’ mutations (‘sequential multi-hit’ model, see below) may well be the mechanism by which ultraviolet light (which occurs in humans as large numbers of exposures over decades) induces solar keratoses and squamous celled carcinomas (both of which are often preceded by an in situ phase—i.e. Bowen's disease).

However, in the consideration of claims that any particular theory may have universal relevance, the following comments may be relevant.

  1. Theories involving the direct mutagenic activity (genotoxicity) of carcinogens do not easily explain non-mutagenic carcinogens, non-carcinogenic mutagens or the exquisite structure-dependency of potency of chemical carcinogens. Furthermore:
    1. Simple one-mutation hypotheses do not explain the variability (heterogeneity) of tumours, because all daughter cells after a mutation should be alike (5).
    2. The ‘two-hit’ hypothesis of Knudson (93–95), and the ‘sequential multi-hit’ models [put forward on epidemiologic grounds by Nordling in 1953 (96), modified by Armitage (97) and more recently espoused by Vogelstein and Kinzler (98,99)] do not easily explain tumours which are inducible experimentally by single doses of chemical carcinogen [see Craddock (19) and above], or the multiplicity of phenotypic abnormalities in ‘sets’ exhibited by many human tumours.

  2. Genetic instability, whether induced by direct mutation of a genome-preserving gene, or by a non-genotoxic mechanism, appears to provide a basis for morphologic and behavioural heterogeneity of tumours (3234,57). However, the phenomenon may not serve as an initiation event associated with all tumours, because many tumours (especially benign ones) do not exhibit the features of genetic instability. Furthermore, without a concurrent mutation resulting in increased growth, genetic instability is likely to result only in ‘in situ’ cytologically abnormal cells, rather than a hyperproliferative mass of cells.
  3. Non-genotoxic mechanisms of carcinogenesis other than direct action of carcinogen on DNA polymerases do not readily offer explanation ‘sets’ of phenotypic abnormalities (including occasional additional features) of tumours. Specifically:
    1. Notions of abnormal ‘differentiation’ (8,100–102) (meaning either abnormal local specialization, or abnormal local development), do not offer any explanation of phenotypic features which are unrelated to differentiation (such as adrenocorticotrophic hormone production by anaplastic small-celled carcinoma or bronchus). Further, the hypotheses are vague, because no particular biochemical target of carcinogens is suggested.
    2. Recent suggestions concerning the roles of particular proteins, including ligand-activated transcription factors (103) and peroxisome proliferators (104) and ‘gap junctions’ (105) as well as abnormalities of transcription (106) or translation (107) do not provide for the immortal genetic abnormalities of tumours, and frequent genetic instability. Hypotheses that carcinogens affect methylation of DNA and hence gene expression (108112) do not easily account for the permanently inheritable nature of the change in the affected cell without some early mutation occurring. A hypothesis, which suggests that a mutation of gene(s) for an epigenetic mechanism as the first event of tumour formation, is not fundamentally an ‘epigenetic hypothesis’ but a mutational hypothesis, and subject to the difficulties mentioned above.
    3. The notion that carcinogens may act by generation of endogenous genotoxic substances (113116) is difficult to evaluate because the nature of all of the possible ultimate endogenous genotoxins is unclear. No endogenous genotoxin or its precursor suggested so far is characterized by exquisite structure-dependence of their carcinogenic activation or actions.


    Further investigation of carcinogen-induced impairment of DNA fidelity of replication
 Top
 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
For the purposes of investigation, the hypothesis that carcinogens may act on the fidelity of replication mechanisms of DNA polymerase complexes suggests that a ‘hallmark pattern’ of mutation might result in the genome of daughter cells. In experimental studies, the features of this pattern would be: multiple mainly point mutations clustered within the length of half a replication ‘bubble’. In human tumour cells (if the pattern could be identified), the clusters might be tumour type-specific in their location in the human genome, but varying case-to-case in the precise ‘points’ mutated in the cluster region. This pattern is distinct both from frameshift mutations, in which the nucleotide sequence is preserved, but displaced 1 or 2 nt places in the DNA chain, and from random point mutations throughout the genome.

At the present time, there is very little strong direct, published experimental evidence to support the notion of carcinogen-binding to DNA polymerases of local cells (see above).

While various types of experiments have been reported, without conclusive results, it is possible that the methodology used has been insufficiently sensitive to detect the genomic changes, which might have occurred. Especially since the 1970s, there have been numerous improvements of biochemical and molecular biologic techniques, which could now be employed in re-visiting these basic experiments. These are now mentioned according to their experimental type: cell-free, cell culture, whole animal and human lesional studies.

Cell-free studies
Some details of technique in the published studies involving cell-free systems might possibly be improved. First the purification of the DNA polymerases may have involved use of denaturing precipitants and dissociating agents [see Kornberg and Tate (117)] (details of these preparative methods are not available in many papers). Methods for producing functionally superior (less denatured) DNA polymerase preparations are now available (118).

Secondly, the conditions of incubation may have been suboptimal. For example, the addition of a protein such as serum albumin can limit the non-specific damage of toxins to sensitive chemical structures.

Thirdly, the method of detecting illicit nucleotide additions by these methods involved measuring the inclusion of illicit labelled nucleotide in new chains synthesized on artificial polynucleotide templates [for example C or G additions to a chain synthesized on poly A-T (82,83)]. Currrently, short ‘natural’ template DNA could be used, and the product could be nucleotide-sequenced by current standard automated methods and compared with the original template DNA.

It should be noted that cell-free systems lack the indirect factors, which may affect replicative fidelity of DNA (see section on genetic instability), so that their results cannot be taken as conclusive evidence that the same effects of the substances occur in vivo.

Cell cultures (Figure 2)
The major technical requirements of studies of the effects of carcinogens on fidelity of replication of DNA in cell cultures are seen to be:

  1. To accommodate possible cell type-specificity of effects of some carcinogens and provide a stable genome with which abnormal restriciton fragment length (RLF) bands can be compared (see below).
  2. To limit the cell toxicity side effect of the carcinogen.
  3. To maximize the number of DNA polymerase complexes relative to other proteins in the culture.
  4. To detect the putative cluster of point mutations over a range of 15 000–150 000 nt in the whole genome of the daughter cells.
  5. If genotoxic substances are also to be tested, to distinguish genomic changes produced by DNA-adducts from mutations caused by impaired DNA polymerase complexes.



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Fig. 2. Suggested scheme of investigation of hypothesis using cell cultures. Carcinogen is added to a synchronized cell culture during S phase. Single cell subculturing and restriction (endonuclease) fragment length RFL analysis provides some subcultures with abnormal bands. Sequencing of the regions of the bands from the various digests would show that they are paired. For each sub-culture the bands of each pair would be found to come from a region of high incidence of mainly point mutations (the ‘hallmark pattern’ of mutations) induced by a single impaired DNA polymerase complex—see Figure 1 and text. Different subcultures would have different sites of the abnormal region, because there is no specificity of action of carcinogens on DNA polymerases according to which part of the genome the polymerase synthesizes. Excess bands in the DNA of a clone would indicate a genetically unstable clone [see text and (67)]. In corresponding studies of genetically stable human tumour tissue, sequencing of abnormal bands in comparison with the individual patient's normal cells may show tumour type-specific location in the human genome, but varying case-to-case in the precise ‘points’ mutated in the cluster region.

 
First, the genetically stable cell cultures used could be as closely related as possible to the desired ‘target’ cell type. Primary explants of human and animal cells may be useful.

Secondly, efforts to reduce cell toxicity should be made by investigating the effects of cell-protective additives to the culture system. Even cultures in connective tissue matrices, such as of collagen could be considered.

Thirdly, in the published reports (see above), randomly multiplying cultures were used. However, methods for synchronizing cell cultures have been available for many years (119), and have been used occasionally in studies of experimental carcinogenesis [e.g. Meuller and co-workers (120)]. Synchronizing the target cell culture would have two methodological benefits. Because the carcinogen could be added at the beginning and removed at the end of S phase, the exposure of the cells to carcinogen for non-specific damage and cell toxicity, would be limited. Also, because the proportion of the target molecule (the DNA polymerase) relative to irrelevant molecules to which the carcinogen might bind would be increased, lower concentrations of carcinogen could be used, and hence non-specfic cell toxicity reduced.

Fourthly, to detect patterns of mutations, after the completion of the S phase, the culture could be single-cell subcultured, and these subcultures could be analysed for mutations (when sufficiently multiplied) by restriction fragment length (RFL) analysis in comparison with the genome of the original cells. It is assumed the clustered multiple mutations would cause change of a site of action of at least one restriction enzyme. Batteries of such enzymes may be needed to demonstrate the mutant length of DNA. Once detected, abnormal bands could be sequenced and then compared with the human genome data for localization. Different clones should have clusters of mutations at different loci in the genome. The results of nucleotide sequencing should allow distinction both from the ‘hallmark pattern’ of mutation both from frameshift mutations, and from genome-wide random mutations. The dosage of carcinogen would be adjusted so that excessive number of ‘hits’ per cell do not make the results of subsequent sequencing difficult to interpret.

The bacteria-based assay methods of Kunkel and co-workers (118,121) might not be easily applicable to this type of experiment.

Some subcultures are expected to show genetic instability [due to the original impaired fidelity of replication by DNA polymerase affecting genes for genetic instability in the daughter cell (67)] and such clones could be expected to have large numbers of mutations throughout their genome, and correspondingly large numbers of abnormal bands of their DNA after RFL.

Fifth, to limit numbers of adduct-derived mutations, non-genotoxic carcinogens (such as ethionine) could be tested, because these would not cause possibly confusing concurrent adduct-based mutations. For genotoxic carcinogens, the dose should be kept as low as possible, to minimize these adduct-based mutations.

Controls would consist of cell cultures treated with non-carcinogenic mutagens. If the direct impairment of DNA mechanism is important, these agents will be found not to cause the ‘hallmark pattern’ of mutations.

Whole animal studies
For these studies, the general plan would be similar to that described for cell culture experiments. In animal tumours, it may be important to use an early carcinogen-induced neoplastic lesion, before genetic instability has created spurious additional mutations. An obvious animal experimental lesion is the pre-malignant hepatic nodule of carcinogen-treated rats (17). These lesions could be harvested, preferably at the earliest possible time after administration of carcinogen, grown in single cell sub-culture if insufficient in amount, and then assessed by RFL analysis, sequencing and comparison with the animal's genome as described above.

Human lesions
The same general plan may be usefully applied to human tumours. Investigation of genomic abnormalities of ‘benign’ and other tumours, which do not show behavioural evidence of genetic instability may be more appropriate than such studies of genetically unstable tumours, because the former may more clearly demonstrate the mutational ‘hall mark pattern’ (multiple mainly point mutations clustered within the length of half a replication ‘bubble’, being tumour type specific in their location in the human genome but varying case-to-case in the precise ‘points’ mutated in the cluster region). It is possible that once a tumour-type locus is identified, genomic changes in various cases of tumours of this type could be analysed by PCR using genome site-specific primers upstream of the affected locus. The resultant product could be compared to the product from the normal cells from the individual patient in whom the tumour arose.


    Conclusions
 Top
 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 
The somatic mutation theory of carcinogenesis has had a chequered history even since pure carcinogens have become available. The major difficulties have been the lack of explanations of non-genotoxic carcinogenicity, non-carcinogenic mutagenicity and structure-dependence of carcinogenic potency. Furthermore, there has been little suggested of mutational mechanisms to explain the occurrence of ‘sets’ of phenotypic features (e.g. hyperproliferative and/or cytologically abnormalities), which characterize the various tumour types.

The present genetic hypothesis offers a possible explanatory basis for many of these observations, in particular, non-genotoxic induction of tumours (which adduct-based mechanisms do not easily provide) and the phenomenon of ‘sets’ of phenotypic abnormalities occurring in one cell at one time, which appears to be the manner in which most tumour types arise.

Nevertheless, the hypothesis is not put forward to the exclusion of other established mechanisms of carcinogenesis, which are believed to have particular roles in particular tumour types. These mechanisms may also contribute (as does genetic instability) to the general case-to-case and other variabilities within tumour types.

The studies outlined above may lead to the development of additional methods for the testing of possible carcinogenic effects of non-genotoxic as well as possibly genotoxic environmental and synthetic substances for their carcinogenic risk to human and animal populations.

Finally, more detailed study of the human genome for proximities of genes for tumorous phenomena, which occur in common combination may well be valuable.


    References
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 Abstract
 Introduction
 Genetic instability: not a...
 Carcinogen-impaired DNA...
 Experimental evidence for direct...
 Discussion: comparison with...
 Further investigation of...
 Conclusions
 References
 

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Received August 20, 2003; revised October 19, 2003; accepted October 28, 2003.





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