Bnnsh Mtdicnl BtdUtin (1991) Vol. 47, No. 1, pp 3-20 (D The Brituh Council 1991

Carcinogenesis: Molecular defences against carcinogens P J Smith MRC Clinical Oncology and Radiothcrapeutics Unit, MRC Centre, Cambridge, UK.

It has long been recognized that exposure to specific chemical and physical agents can result in the appearance of tumours in both man and animals. There is substantial evidence that DNA is the ultimate cellular target of carcinogens and that DNA repair is an important cellular defence against the effects of carcinogen exposure. Some insight into the molecular processes involved in cellular responses to carcinogens is provided by the occurrence of rare human disorders that display complex abnormalities in processing DNA damage or maintaining genomic integrity. These disorders include xeroderma pigmentosum, Cockayne syndrome, ataxia telangiectasia, dysplastic nevus syndrome and Bloom syndrome. Such disorders have provided clues to some of the basic mechanisms involved in carcinogenesis.

Many types of cancer in humans may be preventable since epidemiological studies reveal that diet, customs and environmental factors are predominant influences on tumour incidence. In some cases, such as cancer of the skin or lung, the causal relationship between exposure to a known agent, namely sunlight or cigarette smoke respectively, and tumour incidence is clear. In other cases the nature of the initiation event is not known but is presumed to involve changes in DNA—a concept reflected in the somatic mutation theory of carcinogenesis. A causal relationship between exposure to a carcinogen and the appearance of cancer in an individual is only clear when the exposure is at a high enough level to overwhelm host defence mechanisms or if the individual is unusually susceptible. In surveying a wide range of synthetic and

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natural chemicals Ames has reported1 that about half of all chemicals tested could act as carcinogens and has suggested that exposure to chemicals at high levels (close to the maximum tolerated doses) may be carcinogenic due to the generation of additional risk factors for cancer such as inflammation and cell proliferation. The study implies that we are well buffered against toxicity at low doses from both man-made and natural chemicals and that low doses of carcinogens appear to be both much more common but less hazardous than is generally thought. A vital buffer to carcinogen attack is provided by the operation of active cellular processes for the repair of DNA damage. -•-• -Much of our understanding about the action of carcinogens at the target level arises from animal studies. However, the many anatomical, physiological, and biochemical differences among various mammalian species make it difficult to extrapolate findings in animals to humans. Most malignant tumours in animals and humans demonstrate a multistep origin due to the interaction of target cells with both endogenous and exogenous factors and the relative importance of such factors will vary according to the species studied.2 Understanding the molecular basis for genetic predisposition to cancer in man offers the opportunity ofJ "determining the fundamental mechanisms of carcinogenesis pertinent to human populations. A group of naturally occurring genetic disorders in man, characterized by biochemical defects in DNA repair or DNA processing have provided clues to the cellular factors that limit the carcinogenic consequences of DNA damage.3 This chapter considers the general events involved in neoplasia, the cellular mechanisms responsible for maintaining the integrity of damaged DNA and how DNA processing abnormalities increase the risk of cancer in man. SEQUENCE OF EVENTS IN NEOPLASIA - • The whole neoplastic process can be divided into' three major stages: initiation, promotion, and progression. The initiation event involves some genetic alteration which through promotion allows the clonal expansion of cells and progression to malignancy. In attempting to view the temporal relationship between initiation and subsequent events in man it is important to appreciate that the time course may be stretched considerably since prenatal factors (e.g. embryonic or prezygotic exposure to carcinogens) may contribute to the occurrence of cancer.4 In experimental systems

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5

the process of oncogenic transformation can be followed using a variety of assays5 that detect a sequence of cellular changes, including: altered morphology, growth in soft agar, 'unlimited' growth, transplantability, invasion and metastasis. One of the most studied carcinogens is ionizing radiation and Table 1 shows the approximate time course of molecular and cellular events involved in radiogenic transformation. Here pivotal roles are played by DNA damage and DNA repair pathways.

EXOGENOUS AND ENDOGENOUS INDUCERS OF DNA DAMAGE The ubiquitous nature of sunlight and the carcinogenic potential of its UV component have prompted many studies on the types of UV-induced DNA lesions. The majority of work has concentrated on the effects of far-UV (200-290 run wavelengths), in particular 254 nm UV radiation emitted by germicidal lamps was highly efficient at inducing intrastrand cyclobutane pyrimidine dimers in cellular DNA. For many years the view has existed that pyrimidine dimers are the major cytotoxic and mutagenic photoproducts. However, the pyrimidine-pyrimidone (6-4) photoproduct is now considered to be an important determinant of the lethal and mutagenic effects of UV radiation.7 A further complication is that solar Table 1 Temporal sequence of carcinogenic and anticarcinogenic events in radiation-induced neoplastic transformation1 Approximate Process duration (s) 10~ 18 -10~ 3

io-33-io° io- -io° 10" 2 -10" 10°-103 104-10* 103-105 104-107 5

10 -10

7

Ionizing radiation energy deposition and generation of free radicals Radical scavenging

Transformation stage

INITIATION

Induction of DNA damage: (e.g. base modifications and strand breaks) Error free or error prone DNA repair

Chromosomal aberrations: (e.g. deletions and translocations)

FIXATION

Inhibition of early stages in transformation: (e^;. action of protease inhibitors)

Oncogene(s) activation Inhibition of effects of promoters (e.g. action of retinoids)

Abnormal growth control

"Table adapted from ref 6

PROGRESSION

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radiation, incident at the Earth's surface is essentially devoid of the far UV component since stratospheric ozone filters out UV radiation of wavelengths shorter than 295 nm. Thus the majority of studies have been carried out using environmentally irrelevant UV wavelengths. Furthermore, there is evidence that the near-UV (320-400 nm) component of sunlight can both induce biologically important non-dimer damage and inhibit DNA repair processes in human cells.8 Ionizing radiation induces a wide range of DNA lesions including single and double strand breaks, and a plethora of base and sugar modifications. Cytogenetic studies have underlined a predominant role for chromosome aberrations in the cytotoxic, mutagenic and tumourigenic effects of ionizing radiation leading to the conclusion that mis-repaired or persistent DNA strand breaks are the progenitor lesions.5 Many factors determine whether a particular electrophilic chemical will exert a carcinogenic effect. For example, the physical form of the carcinogen and the route of exposure will influence the target tissue affected.9 Cellular capacity to metabolize a procarcinogen to its ultimate carcinogenic form and the action of enzymatic and non-enzymatic detoxifying pathways can influence the impact of a chemical on a given target tissue. Many chemical carcinogens appear to act as mutagens by the generation of nucleotide adducts. The form of adduct ranges from discrete covalent linking of atkyl groups with specific nitrogen and oxygen positions on DNA bases (e.g. MNNG; Table 2) to the linking of relatively large molecules such as the aromatic amine N-acetoxy-2-acetylaminofluorine. The existence of enzyme systems for the repair of oxidative damage to DNA in both prokaryotic and eukaryotic organisms may be taken as evidence that cellular DNA is continuously subject to endogenous oxidative stress. Indeed the endogenous background level of oxidant-induced DNA damage such as 8-hydroxydeoxyguanosine is significant, representing 1/130,000 bases in nuclear DNA of normal rat liver.10 Apurinic/apyrimidinic sites in DNA can result from the spontaneous cleavage of the N-glycosylic bond connecting the base to the sugar, the latter moiety being unstable and undergoing rapid hydrolysis. The process leaves the phosphodiester backbone intact until the site undergoes endonucleolytic incision during the course of repair (Tables 2 and 3). Depurination is likely the most frequent spontaneous alteration that occurs in DNA under physiological con-

MOLECULAR DEFENCES AGAINST CARCINOGENS Table 2 Routes for the generation of DNA damage Agent or Mechanism 1.

UV radiation

2.

Ionizing radiation

Examples

Types of DNA damage

254-320 nm wavelengths X-rays, y-rays

Cyclobutane pyrimidine dimcrs; (6—4) pyrimidine-pyrimidones Single & double strand breaks; base damage Nucleotide adducts

Electrophilic agents MNNG1 aflatoxin B, 4. Spontaneous 5. Oxi dative metabolism 6. Enzyme dysfunction 3.

7.

Cytotoxic drugs

Cisplatin & DNA intercalators

AP & APy sites'; strand breaks modified bases DNA topoisomerase II generated breaks or recombination; DNA polymerase replication errors DNA-DNA crosslinks; DNAprotein crosslinks; strand breaks

•MNNG* N-methyl-AT-nitro-N-nitroiogianidine. 11 AP ind APy lite*, apurinic tmd apymwdmic lira respectively.

ditions (104 depurinations per day occurring in the DNA of a mammalian cell), providing a potential source of spontaneous mutations.11 A recent study12 has raised the possibility that action of a DNA processing enzyme, DNA topoisomerase II, at unusual sites on DNA may play a role in the generation of endogenous 'DNA damage' and genetic instability. Topoisomerase enzymes, found in prokaryotes to human cells, control conformational changes in DNA and aid the orderly progression of DNA replication, gene transcription and the separation of daughter chromosomes at cell division.13 Sperry et al. have noted12 that a family of A + T-rich sequences termed 'matrix association regions' mediate chromosomal loop attachment and that several regions both specifically bind and contain multiple sites of cleavage by topoisomerase II. A dysfunction of such regions is illegitimate recombination providing the breakpoint of a chromosomal translocation which could lead to the genetic instability required for neoplastic progression. Interestingly, topoisomerase II may also be a target for some carcinogens since several classes of cytotoxic drugs are now recognized as topoisomerase poisons because of their ability to trap enzyme molecules on DNA. Thus, this enzyme is an important factor both in maintaining the functional integrity of DNA and in determining sensitivity to antitumour agents with mutagenic potential.14

Distorted helix or AP/APy site

Strand break

2. Strand cleavage

3. Repair replication

Gap in daughter strand

2. Post-replication repair

T i b l c adapted from Ref. 3

Persistent damage

1. Translesion synthesis

Tolerance

Modified base

Pyrimidine dimer Adduct

Substrate

1. Base removal

Excision Repair:

1. Reversal 2. Removal

Direct Repair:

Process

Recombination & repair replication enzymes

Polymerase complex

Spontaneous; glycosylase Specific endonuclease or spontaneous Exonuclease, polymerase & ligase

Photolyase Alkyltransferase

Repair enzymes

Table 3 Cellular pathways for the repair and tolerance of DNA lesions*

Error-prone bypass of lesion and replicative dilution Intact daughter strand

Lesion excision or generation of substrate for repair replication Lesion removal and insertion of repair patch

AP/APy site

Light-dependent splitting Methyl group removal

DNA modification/product

jo

n m r

n m

m

00

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9

CELLULAR STRATEGIES FOR COPING WITH DNA DAMAGE It is not surprising, given the hostile environments experienced by primordial organisms, that there is an evolutionary tendency to elaborate cellular pathways for coping with DNA damage. Lesions in DNA can lead to mutations or undesirable genetic instability—both processes providing opportunities for a cell to undergo neoplastic progression. However a cell must be capable of dealing with a wide diversity of chemical and structural modifications to DNA with a limited number of repair enzymes. To solve this problem prokaryotic and eukaryotic cells have evolved strategies for repairing or tolerating the most frequently occurring types of damage.15 Parts of these pathways employ lesion-recognition enzymes that tend to detect structural distortions in the DNA helix rather than the precise molecular form of a damaged site. Table 3 shows the various pathways for the processing of DNA damaged by UV-radiation, ionizing radiation, electrophilic agents and by spontaneous alterations that occur under physiological conditions. Human cells deal with UV-induced pyrimidine dimers through an excision repair pathway or tolerate their persistence by postreplication repair (Table 3). Non-placental mammals appear to have vestiges of the prokaryotic capacity to split the linkage between pyrimidine dimers upon activation with visible light. The excision repair process for base damage in man has a general similarity to the mechanism of the bacterial uvr ABC exonuclease, but it is becoming increasingly clear that many genes are involved in the regulation of the process in human cells. Mammalian cells also appear to be capable of selective repair of active genes16, this factor affecting the potential for DNA damage to disrupt the integrity or coding fidelity of an essential gene.17 The removal of (6-4) photoproducts appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of blocks to replication. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined, making it difficult to identify the relative importance of each type of photoproduct. Brash et al. have investigated18 the role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells. They reported considerable variation in the frequencies of cyclobutane dimers and (6-4) photoproducts at different dipyrimidine sites within a specific gene.18

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All transition mutations occurred at dipyrimidine sites, predominantly at cytosine, however, at individual sites neither the frequency of dimers nor the frequency of the (6-4) photoproducts correlated with the mutation frequency. The authors18 conclude that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts. In other words the context of lesion within a gene is of vital importance and measuring the genomic load of damage may not provide, even in the relatively well characterized UV radiation system, a true insight into the mutagenic consequences. Base damage and DNA strand breaks induced by ionizing radiation are essentially handled via an excision repair process and strand ligation. Unlike the 'long patch' excision repair of pyrimidine dimers, the exonuclease and polymerase steps in the processing of radiogenic lesions result in the incorporation of only a few nucleotides. Many of the lesions introduced by ionizing are identical to those introduced into DNA by reactive oxygen species generated by activated white cells, and are substrates for repair enzymes including DNA exo- and endonucleases and DNA glycosylases which remove oxidatively damaged portions of the DNA molecule, thereby initiating excision-repair. DNA damage resulting from attack by electrophilic methylating agents can be repaired by the discrete removal, by an alkyl transferase, of a methyl group from the O6position on guanine or attached to a phosphate in the backbone of DNA. Other types of damage induced by electrophilic agents are generally directed through the excision repair pathway.15 DNA PROCESSING ABNORMALITIES AND CARCINOGENESIS The identification of human genetic defects in the processing of DNA damage has helped greatly in forging links between the induction of DNA damage, cellular repair capacity, hypermutation and predisposition to cancer.3>15'19 This section reviews several of these disorders, the characteristics of which are shown in Table 4. Xeroderma pigmentosum (XP) XP is a rare autosomal recessive human disease, clinically characterized by the early onset of severe photosensitivity of exposed

Acute sun sensitivity Neurological defects Immune deficiencies

Acute sun sensitivity No neurological defects

Cerebellar ataxia Telangiectasia Hypersensitivity to radiotherapy Partial immunodeficiency

Dwarfism Mental retardation Sun sensitivity

Growth retardation Sunlight sensitivity

Abnormal nevi

Xeroderma pigmentosum (classical XP)

Xeroderma pigmentosum (XP variant)

Ataxiatelangiectasia

Cockayne syndrome

Bloom syndrome

Dysplastic nevus syndrome

'Adapted from Reference 3

Major clinical features

Disorder

Hypomutable by X & f-rays Possible defects in processing base damage and double strand breaks Reduced radiogenic DNA synthesis inhibition

Ionizing radiation

UV and UV mimetic chemicals

Lymphomas

Normal

No characteristic sensitivity Variable UV and and carcinogen sensitivity

Acute leukaemia Melanoma

Hypermutable by UV

High spontaneous rate of chromosome aberrations DNA ligase defect

Depressed recovery of RNA synthesis following UV-irradiation Hypermutable by UV

Hypermutable buy UV Defective replication of damaged DNA

Cutaneous

Cutaneous

Slight sensitivity to UV and UV mimetic chemicals

DNA processing defect Hypermutable by UV Defective incision step

In vitro carcinogen sensitivity UV and UV-mimeticchemicals

Cancer risk

Table 4 Human autosomal recessive disorders displaying in vitro or in vivo carcinogen sensitivity1

R

§ LAR DEFENCES AGAINSTCARC tNOGENS

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THE CANCER CELL

skin to sunlight, a very high incidence of skin cancers and frequent neurological abnormalities. XP patients have more than a 1000fold increased risk of cutaneous melanoma. In recent years the use of effective sun-screen preparations, frequent clinical examinations to detect early signs of neoplasia and changes in life-style to avoid sunlight exposure have helped to extend the life expectancy of XP patients. In contrast to the risk of cutaneous neoplasia in XP there is a relatively low risk of developing internal malignancies.20"22 Cells from classical XP patients are hypersensitive to 'killing by UV-radiation and UV-mimetic chemicals, because they have a defect in an early step in the excision repair of pyrimidine. dimers.23 Complementation studies carried out with cell free extracts have demonstrated that XP cell lines fail to act on UVdamaged DNA, but are proficient in repair synthesis of UV irradiated DNA once die incision step has been provided by an exogenously supplied prokaryotic enzyme.24 All studies to date indicate that all tissues from a given XP patient, including specific target tissues for neoplastdc transformation such as melanocytes and nevus cells, express the characteristic XP DNA repair defect.25 Cells derived from a subgroup of XP patients, showing the variant form of the disorder (Table 4), show normal excision repair capacity, efficient removal of (6-4) photoproducts but impaired post-replication repair.26'27 The complexity of die repair process in man is underlined by the observation that phenotypic reversion can occur in classical XP cell lines and two UV-resistant XP revertants that do not remove pyrimidine dimers have been shown to repair non-dimer photoproducts.28 Evidently, excision of pyrimidine dimers from the whole genome is not necessary for the survival of human cells after UV irradiation rather repair of non-dimer photoproducts in the genome as a whole or pyrimidine dimers in a small number of genes may be more biologically important.28 Genetic complementation analysis by cell fusion has led to the identification of at least eight complementation groups, designated as groups A through H and a variant.29'30 Complementation of die repair defect in XP group A cells has been achieved by die transfer of human chromosome 9.31 Likewise, human chromosome 15 has been found to confer partial complementation of phenotypes to XP group F cells.32 However die observation diat phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion diat the XP group F locus is carried on chromosome 15. Another approach in

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the search for the genetic defect in XP involves identifying rodent genes that confer resistance on XP cells of specific complementation groups. For example, a mouse gene that complements the defect of an XP Group A cell line has been cloned and was found to confer UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G.33 These results suggest that this cloned DNA repair gene is specific for group A XP and may be the mouse homologue of the group A XP human gene: Similarly, a hamster DNA repair gene has been isolated by cosmid rescue after transfection of an immortalized XP complementation group D cell line with normal hamster DNA. 34 A parallel approach to gene cloning involves the identification of proteins, defective or deficient in XP cells, that are involved in cellular responses to UV-radiation. Chu and Chang have recently reported35 that group E XP cells lack a nuclear factor capable of binding to UV damaged DNA and probably involved in the molecular recognition process for DNA damage. XP skin tumors demonstrate mutations in the N-ras gene, c-myc amplification and over transcription, and Ha-ras gene rearrangement and amplification.36 However normal skin fibroblasts from XP patients show normal patterns and levels of N-ras, c-myc and Ha-ras sequences. A model has been proposed36 for proto-oncogene activation by point mutation in XP cells in which UVinduced pyrimidine dimers or (6—4) photoproducts remain unrepaired and replication synthesis results in a point mutation opposite the lesion. When a point mutation occurs in a crucial codon, activation of the gene ensues, reducing the activity of the ras protein in the modulation of signal transduction through transmembrane signalling systems.37 Ataxia telangiectasia (A-T) A-T is a multifaceted genetic syndrome, the major clinical features including: progressive cerebellar ataxia, oculocutaneous telangiectasis beginning in infancy, repeated sinopulmonary infections, immune defects associated with thymus underdevelopment, and an abnormal endocrine system. The disorder is autosomal recessive and based on US incidence data, the minimum frequency of a single hypothetical A-T gene in the white population has been estimated to be 0.0017.38 This neurological syndrome is of considerable interest because homozygotes are highly predisposed to cancer and show untoward response to standard radiotherapy.39

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THE CANCER CELL

The cancer excess in A-T is most pronounced for lymphoma, with 252- and 750-fold excesses observed for US whites and blacks, respectively.40 Heterozygous carriers of the gene also have an excess risk of cancer, particularly breast cancer in women (rate ratio, 3.0; heterozygote relative risk, 6.8).41 On the basis of this relative risk of 6.8 and an estimated heterozygote frequency in the general population of 1.4%, it has been calculated41 that 8.8% of patients with breast cancer in the US white population would be heterozygous for A—T. Genetic linkage analysis of 31 families with A-T-affected members has allowed a gene for A-T to be localized to chromosomal region llq22-23. 42 Although it is thought that the immune defects contribute significantly to cancer proneness, A-T homozygotes have been shown43 to have an elevated frequency of somatic cell mutation in erythroid precursor cells in vivo, supporting a causal link between susceptibility to somatic mutation and the predisposition to cancer. Consistent with the clinical findings, cultured cells from A-T patients are sensitive to ionizing radiation and this phenotype has been associated39>4i4 with a putative defect in DNA repair although there has been no definitive evidence of the biochemical nature of such a defect. Interestingly, following radiation exposure, the rate of DNA replication is inhibited to a lesser extent than in normal cells, whereas the frequency of chromosomal aberrations is enhanced.45 Genetic complementation studies based on the radiation resistance phenotype and comparisons with the related disorder of Nijmegen breakage syndrome have suggested that a defect in one of at least six different genes may underlie inherited radiosensitivity in humans.46 Discovery47 that the heterozygous state of the A-T gene can confer partial sensitivity to ionizing radiation has suggested that some abnormality in the processing of DNA damage may be expressed in the tissues of apparently normal but cancer prone A-T heterozygotes. Bloom syndrome (BS) and genetic instability BS is a rare disease associated with increased frequency of leukaemia. Although some BS patients present with sunlight sensitivity, the characteristic cytological abnormalities (increased numbers of homologous chromatid interchange figures and sisterchromatid exchanges) have led to the theory that cancer predisposition relates to some underlying genetic instability. Kuhn and Therman have suggested48 that the high (25%) incidence of cancer

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in BS may depend on: (a) the high rate of homozygosity resulting from mitotic crossing-over, which would allow the expression of recessive cancer genes; (b) unequal crossing-over would amplify these genes; (c) chromosome structural changes that might transfer oncogenes to new locations and, thus, activate them; and (d) immunodeficiency, which would promote malignant growth. Support for the above view has been provided by the results of direct measurements of the frequencies of variant erythrocytes in BS patients in attempts to assess the frequency of putative mutational or recombinational events in erythroid precursor cells.49 Samples of blood from persons with BS show dramatic increases in the frequency of homozygous variant erythrocytes providing evidence for increased somatic crossing-over in vivo. In vitro studies on BS cells have revealed abnormally slow repliconfork progression and retarded rates of DNA-chain maturation, suggesting that the primary defect affects DNA replication. Prime candidates for the defective protein(s) in BS are DNA ligases and DNA polymerases, but various studies of DNA polymerases in BS cells have disclosed no abnormalities. Two reports 50 ' 51 indicate that a defective DNA ligase may be responsible for the abnormalities in semi-conservative DNA replication. The BS defect may also be heterogeneous since not all transformed or virus-transformed human cell lines from BS patients show a ligase defect.52 It will be important in future studies to attempt to correlate the expression of this new molecular marker with the cytological and clinical findings. Dysplastic nevus syndrome (DNS), sunlight and melanoma Cumulative total lifetime exposure to sunlight appears to be a major risk factor with the non-melanoma forms of skin cancer whereas melanoma causation is multifactoral. Important risk factors for melanoma include intense and episodic exposures to sunlight early in life and the prevalence of precursor nevi.53 Melanoma and non-melanoma skin cancer is increasing in several industrialized nations and the United States is currently experiencing an average increase in rate of about 4% per year. It is probable that changes in life-style habits resulting in an increasing exposure to mid-UV radiation present in sunlight is partly responsible for this dramatic increase in incidence. Increasing levels of mid-UV radiation would be one effect of depletion of stratospheric ozone

16

THE CANCER CELL

and it has been estimated that a 1% depletion could result in increases of 1-2% in melanoma incidence and 0.8-1.5% in melanoma mortality.5* DNS is an autosomal dominant disorder in which affected individuals have increased numbers of dysplastic (premalignant) nevi and a greater than 100-fold increased risk of developing cutaneous melanoma. Cells from patients with hereditary DNS have been shown53"57 to be hypermutable to UV radiation and in some cases sensitive to UV radiation and a UVmimetic carcinogen, 4-nitroquinoline-l-oxide. Identification of the primary defect in DNS promises to uncover a non-XP-like, tissue specific pathway for cancer predisposition in man. Cockayne syndrome (CS) and hypermutation This is a rare autosomal recessive human disorder characterized by progressive neurological disease, dwarfism, skeletal abnormalities and frequent hypersensitivity of the skin to sunlight.58 In some cases the photosensitive dermatitis is manifest only as blistering and excessive redness but no increased incidence of skin cancer.59 Cultured cells from CS patients are hypersensitive to UV radiation. This phenotype has been associated60 with some abnormality in the post-incision processing of photolesions in DNA resulting in a pronounced and sustained suppression of RNA synthesis after UV irradiation. Three CS complementation gToups have been identified.61 The difference between XP and CS in the feature of cancer predisposition contrasts with the observations that both CS and XP cells are hypermutable by UV radiation. Thus the in vitro data obtained for CS and XP challenges the simple interpretation of predisposition to skin cancer in XP relating simply to the increased mutational load. An important difference between CS and XP is that only the latter present deficiencies in the immune system. The current supposition62 is that further suppression of the immune response by sunlight63 may be responsible for the actual development of cancers in XP patients. CONCLUDING REMARKS In viewing the incidence of cancer in man the 'DNA repair deficiency disorders' provide instances where the underlying influence of a class of carcinogens can be glimpsed. Although these human disorders provide vital clues to the nature and prevalence

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of oncogenic initiation events in man, it is also clear that they reveal the importance of post-initiation events in neoplastic progression. The studies on Bloom's syndrome in particular indicate the importance of genetic stability in limiting progression to malignancy. The current concept of the neoplastic process being a sequence of discrete events offers the possibility of interrupting the chain at certain points. The spectrum and mutagenic potential of DNA lesions occurring in DNA, especially within proto-oncogene sequences, are critical early factors in carcinogenesis. An important step would be to identify the molecular nature and consequences of the initiating lesions so that strategies could be devised to avoid or protect against the inducing agent or to abrogate the early events leading to cancer development. REFERENCES 1 Ames BN. Endogenous DNA damage as related to cancer and aging. Environ Mol Mutagen 1989; 14: 66 2 Slaga TJ. Interspecies comparisons of tissue DNA damage, repair, fixation, and replication. Environ Health Perspect 1988; 77: 73 3 Hanawalt PC, Sarasin A. Cancer prone hereditary diseases with DNA processing abnormalities. Trends Genet 1986; 2: 124 4 Tomatis L. Overview of perinatal and multigeneration carcinogenesis. In: Napalkov NP, Rice JM, Tomatis L, Yamasaki H, eds. Perinatal and multigeneration carcinogenesis. Lyon: IARC, 1989: p 1 5 Chadwick KH, Seymour C, Bamhart B, eds. Cell transformation and radiationinduced cancer. Bristol: Adam Hilger, 1989 6 Yang TC, Craise, LM, Tobias CA. Molecular lesions important for neoplastic transformation of mouse (C3H10T1/2) and human epithelial cells by ionising radiation. In: Chadwick KH, Seymour C, Barnhart B, eds. Cell transformation and radiation-induced cancer. Bristol: Adam Hilger, 1989: p 301 7 Mitchell DL, Nairn RS. The biology of the (6-4) photoproduct. Photochem Photobiol 1989; 49: 805 8 Smith PJ, Paterson MC. Lethality and the induction and repair of DNA damage in far, mid or near UV-irradiated human fibroblasts: comparison of effects in normal, xeroderma pigmentosum and Bloom's syndrome cells. Photochem Photobiol 1982; 36: 33 9 Jeffrey AM. DNA modification by chemical carcinogens. Pharmacol Ther 1985; 28: 237 10 Ames BN. Endogenous DNA damage as related to cancer and aging. Mutat Res 1989; 214: 416 11 Loeb LA, Preston BD. Mutagenesis by apurinic/apyrimidinic sites. Ann Rev Genet 1986; 20: 201 12 Sperry AO, Blasquez VC, Garrard WT. Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerasc II. Proc Nad Acad Sci USA 1989; 86: 5497 13 Wang JC. DNA topoisomerases. Annu Rev Biochem 1985; 54: 665 14 Liu LF. DNA topoisomerase poisons as antitumour drugs. Annu Rev Biochem 1989; 58: 351 15 Hanawalt PC, Cooper PK, Ganesan A, Smith CA. DNA repair in bacteria and mammalian cells. Ann Rev Biochem 1979; 48: 783

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16 Bohr VA, Smith CA, Okumot DS, Hanawalt PC. DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 1985; 40: 359 17 Bohr VA, Evans MK, Fornace AJ Jr. DNA repair and its pathogenetic implications. Lab Invest 1989; 61: 143 18 Brash DE, Scetharam S, Kraemer KH, Seidman MM, Bredberg A. Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc Natl Acad Sri USA 1987; 84: 3782 19 Cleaver JE. DNA repair in man. Birth Defects 1989; 25: 61 20 Kraemer K, Myung M, Scotto J. DNA repair protects against cutaneous and internal neoplasia: evidence from xeroderma pigmentosum. Carcinogenesis 1984; 5: 511 21 English JS, Swerdlow AJ. The risk of malignant melanoma, internal malignancy and mortality in xeroderma pigmentosum patients. Br J Dermatol 1987; 117: 457 22 Pippard EC, Hall AJ, Barker DJ, Bridges BA. Cancer in homozygotes and heterozygotes of ataxia-telangiectasia and xeroderma pigmentosum in Britain. Cancer Res 1988; 48: 2929 23 Cleaver JE. Defective repair replication of DNA in xeroderma pigmentosum. Nature 1968; 218: 652 24 Wood RD, Robins P, Lindahl T. Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell 1988; 53: 97 25 Kraemer KH, Herlyn M, Yuspa SH et al. Reduced DNA repair in cultured melanocytes and nevus cells from a patient with xeroderma pigmentosum. Arch Dermatol 1989; 125: 263 26 Lehmann AR, Kirk-Bell S, Arlett CF, Paterson MC, Lohman PHM, Bootsma D. Xeroderma pigmentosum cells with normal levels of excision have a defect in DNA synthesis after UV-irradiation. Proc Natl Acad Sci USA 1975; 72: 219 27 Mitchell DL, Haipek CA, Clarkson JM. Xeroderma pigmentosum variant cells are not defective in the repair of (6—4) photoproducts. Int J Radiat Biol 1987; 52: 201 28 Cleaver JE. DNA damage and repair in normal, xeroderma pigmentosum and XP revertant cells analyzed by gel electrophoresis: excision of cyclobutane dimers from the whole genome is not necessary for cell survival. Carcinogenesis 1989; 10: 1691 29 Bootsma D, Keijzer W, Jung EG, Bohnert E. Xeroderma pigmentosum complementation group XP-I withdrawn. Mutat Res 1989; 218: 149 30 Bootsma D, Westerveld A, Hoeijmakers JH. DNA repair in human cells: from genetic complementation to isolation of genes. Cancer Surv 1988; 7: 303 31 Kaur GP, Athwal RS. Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9. Proc Natl Acad Sci USA 1989; 86: 8872 32 Saxon PJ, Schultz RA, Stanbridge EJ, Fricdberg EC. Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells. Am J Hum Genet 1989; 44: 474 33 Tanaka K, Satokata I, Ogita Z, Uchida T, Okada Y. Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum. Proc Natl Acad Sri USA 1989; 86: 5512 34 Arrand JE, Bone NM, Johnson RT. Molecular cloning and characterization of a mammalian excision repair gene that partially restores UV resistance to xeroderma pigmentosum complementation group D cells. Proc Natl Acad Sri USA 1989; 86: 6997 35 Chu G, Chang E. Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA. Science 1988; 242: 564 36 Suarez HG, Daya Grosjean L, Schlaifer D, Nardeux P, Renault G, Bos JL,

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