Historical Perspective

Skin cancers occur in uniquely accessible sites and are caused by well-defined environmental agents; consequently, their formation illustrates numerous salient features of carcinogenesis. Although most cases are not reported, the annual incidence is estimated at about 1 million, accounting for about 45% of all malignancies.^1a Skin tumors in man account for about 30% of all new cancers reported annually.^1,2 Epidemiologic and laboratory studies provide evidence for a direct causal role of sunlight exposure in the induction of cancer,³ and the high rate of skin carcinogenesis is a direct result of the high dose rate from this causative agent. Both basal cell and squamous cell carcinomas are found on sun-exposed parts of the body (e.g., the face and trunk in men, face and legs in women); and their incidence is correlated with cumulative sunlight exposure. Tumor incidence and mortality increase with decreasing latitude, corresponding to exposure; skin cancers are less frequent in dark-skinned populations than in lighter-skinned peoples; and tumor incidence increases with occupational exposure, such as in ranchers and fishermen. Melanoma, although also associated with sunlight exposure, shows a weaker dependence on total exposure to sunlight and a distribution over the body that is correlated to intermittently rather than continually exposed areas.⁴

Exposure to direct sunlight in the mid–United States latitudes results in the accumulation of a mean lethal dose to unprotected human cells within approximately 30 minutes.⁵ The only other carcinogen to which we are exposed that even approaches these exposure levels would be cigarette smoke in very heavy smokers. Variations in individual susceptibility are also clearly observed in skin carcinogenesis. Human skin can be classified into types I to IV, ranging from individuals who always burn and never tan, to those who tan but never burn; skin cancer susceptibility varies accordingly.⁶ But the most dramatic examples of variations in human susceptibility occur in the human genetic disorders that show increased responses to sunlight exposure.⁷ These include xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), basal cell nevus syndrome (BCNS), dysplastic nevus syndrome, Rothmund-Thompson syndrome, the porphyrias, and phenylketonuria. Some other disorders are associated with an acquired sun sensitivity, including polymorphous light eruption, actinic reticuloid, solar urticaria, lupus erythematosus, and Darier’s disease. Less specific factors contributing to sun sensitivity include skin type, race, eye and hair color, and tendency for freckling; additional factors can include medication and immunologic status. Sunlight exposure also has a major immunosuppressive effect leading to loss of antigen-presenting Langerhans’ cells and the appearance of dyskeratotic keratinocytes (apoptotic sunburn cells) in the upper epidermis, together with the erythemal sunburn response associated with vasodilation caused by a release of prostaglandin.⁸

Epidemiology

Skin Cancer Frequency and Age of Onset

Sunlight (UVB) is the major environmental agent that precipitates the clinical symptoms of skin carcinogenesis. This is well established for squamous and basal cell cancers but some controversy remains regarding melanoma.⁹ Nonmelanoma skin cancers are by far the most common cancers that occur in the United States each year.^2,10 They make up about 45% of all cancers and the incidence is increasing at an alarming rate^11,12 and may be considered a quiet 20^th century epidemic. The role of the sun in inducing these cancers was suggested by a number of astute clinical observations around the turn of the century and has been confirmed subsequently by epidemiologic studies. As a consequence, there is a wealth of human epidemiologic data on skin cancer risks that are associated with geographic locations, skin type, and various photosensitizing, enhancing, and protective applications.^12–16 There is also a possibility of greater risk when the exposure is received during childhood and adolescence than later.¹⁷ Nonmelanoma skin cancer is therefore one of the few malignancies for which there is a clear evidence for the identification of the initiating agent: the UVB component of sunlight. The relationship of melanoma skin cancer to sun exposure and the possible action spectrum is less clear ⁹ but appears to be related to acute burns rather than accumulated dose.

The importance of DNA as a chromophore for the shorter wavelengths is illustrated by the autosomal recessive disease XP. In this disease, a failure in one cellular protective mechanism, DNA repair, is associated with a major increase in the rate of onset of squamous and basal cell carcinoma and melanoma.⁷ Median onset for skin cancer in the general United States population occurs at 50 to 60 years of age; in XP patients carcinogenesis is accelerated and median onset is within the first decadeFig. 15.1. This early onset is a direct consequence of sunlight-induced changes in the DNA of skin cells. An appreciation of the significance of these changes requires describing the photochemical responses of DNA, mechanisms of DNA repair, and their mutagenic and carcinogenic consequences.

Figure 15.1

Age at onset of XP symptoms. Age at onset of cutaneous symptoms (generally sun sensitivity or pigmentation) was reported in 430 patients. Age at first skin cancer was reported in 186 patients and is compared with age distribution in 29,757 patients with (more...)

Sunlight-Induced Photoproducts In DNA

Action spectra for squamous carcinoma indicate that DNA is the target molecule; the absorption spectrum of DNA correlates well with lethality, mutation induction, and photoproduct formation.^20–24 The energy absorbed by DNA produces molecular changes, some of which involve single bases, others resulting in interactions between adjacent and nonadjacent bases, and still others between DNA and proteins. The relative proportions of DNA photoproducts will vary across the UV spectrum.

Dimerizations between adjacent pyrimidines are the most prevalent photoreactions resulting from direct absorption of UVR by DNA. The two major photoproducts are the cyclobutane pyrimidine dimer (CPD) and, at about 25% of the frequency, the [6-4] photoproduct [(6-4)PP] (Fig. 15.2). The distribution of these photoproducts in human chromatin depends on base sequence, secondary DNA structure, and DNA-protein interactions. Because cytosine more efficiently absorbs higher wavelengths of UVR than thymine, CPDs containing this base are formed more readily after UVB irradiation.²⁵ In conjunction with the [6-4]PPs, which are preferentially induced at thymine-cytosine dipyrimidines, cytosine CPDs may play a major role in UVB (solar) mutagenesis.²⁶ Recent data show that methylation at PyrCG sequences in the p53 gene enhances formation of CPDs at sites that are hotspots for mutations.²⁷ The [6-4]PP can further undergo a UVB-dependent conversion to its valence photoisomer, the Dewar pyrimidinone²⁸ (see Fig. 15.2). In addition to the major photoproducts, other less common lesions can form, including purine-purine and purine-pyrimidine photoadducts, photohydrations, and photo-oxidations.²⁹ Because the total yield of these photoproducts is only 3 to 4% of the yield of CPDs, their biologic role is considered minimal; however, their importance as premutagenic lesions in specific sites cannot be excluded.

Figure 15.2

Photochemical reactions in a dipyrimidine DNA sequence leading to the formation of CPDs (TpT1, TpT2) or a [6-4]PP (TpT4) and its photolytic derivative, the Dewar pyrimidone (TpT3). (Redrawn from Taylor and Cohrs).

UVA primarily produces damage indirectly through highly reactive chemical intermediates, oxygen and hydroxyl radicals, which in turn react with DNA to form base damage, strand breaks, and DNA-protein crosslinks. The importance of these photoproducts is not known, but evidence is accumulating to suggest that UVA may be an important pathogenic component of sunlight. Significant levels of cell killing and mutation induction have been observed in human epidermal cells after irradiation with UVA light.^20,22 These data are consistent with earlier studies that suggested that the lethal effects of UVA are not mediated by CPD damage,^30,31 and that free radical scavengers can mitigate the cytotoxicity.³² The biologic importance of UVA light is perhaps best illustrated by the recent demonstration that UVA causes significant levels of tumorigenesis in hairless mice.³³

Genetic Factors in Skin Carcinogenesis

Excision of UV Photoproducts

The idea that UV damage to DNA is an essential component of photocarcinogenesis arose from the discovery that cells from patients suffering from the inherited disorder XP are deficient in DNA repair.³⁴ Since these initial studies, the molecular basis of XP and related diseases has in large part been resolved and the basic mechanism and appropriate genes cloned and characterized. ^7,35

Two major pathways of excision repair, the nucleotide excision repair (NER) and base excision repair (BER) pathways, operate on different kinds of damage. The NER pathway, which is involved with XP, removes pyrimidine CPDs and large chemical adducts in DNA and replaces the damaged site with a newly synthesized polynucleotide patch approximately 29 bases in length (Fig. 15.3).^36,37 The BER pathway removes DNA bases that have undergone relatively small degrees of modification, such as photohydrations or photo-oxidations. BER involves excision of the damaged base by glycosylases followed by the action of apurinic/apyrimidinic endonucleases plus other enzymes and cofactors. The patch may be smaller than that resulting from NER; one pathway inserts only one to two bases and a second minor pathway inserts a larger patch close to the size of the NER patch. NER and BER are both complex processes involving multiple gene products that interact with damaged sites in different ways according to precise chemical form and location of the damage. Adjacent bases, DNA conformation, bound proteins, and transcriptional activity of both the gene and DNA strand containing the damage are among the many factors which can influence rates of repair.³⁶

Figure 15.3

Biochemical steps for nucleotide excision repair of CPDs in DNA prokaryotes showing biochemical details of events represented schematically in Figure 15.4. The XPA binds to photoproducts and excision occurs when UV-specific endonucleases make an incision (more...)

Excision repair requires a temporary relaxation of nucleosomal structure such that damaged regions are more accessible to exogenous nucleasesFigs. 15.4. The continuous excision of CPDs and insertion of the bases is associated with a very low net frequency of DNA strand breaks, no more than about 1 in 2 × 10⁸ daltons of DNA. Only about 1% of the CPDs produced in DNA by a dose of 10 J/m², which represents about a mean lethal dose for human cells, are therefore undergoing excision at any instant, but it takes only approximately 4 minutes to repair one lesion. Excision must therefore set up a dynamic balance between strand breakage and rejoining, and be rate limited by the enzymes involved in the early steps of repair.

Figure 15.4

Heuristic scheme for excision repair of damaged sites on DNA in mammalian chromatin. The first step involves mechanisms that recognize damage and dissociate nucleoproteins to make the DNA accessible to repair enzymes. This is followed by sequential incision (more...)

Excision repair is a heterogeneous process. There is considerable difference between CPDs and [6-4]PPs in their rates of excision from the overall genome of rodent and human fibroblasts and skin (Figs. 15.5 and 15.6),³⁸ even though the basic mechanism and patch sizes are essentially the same.³⁹ [6-4]PPs are the more rapidly excised, 50% being removed from human and rodent cells in 2 to 6 hours. CPDs are much more slowly removed; half are removed from human cell DNA in 12 to 24 hours,^40,41 but negligible amounts are removed from rodent DNA for even longer times. In part, the excision of CPDs may be delayed because the strong affinity of the excision system for [6-4]PPs initially sequesters available enzymes. There are also large variations in CPD excision among human subjects.⁴¹ The different rates of excision may reflect the fact that [6-4]PPs are considerably more distortive in DNA, and that they are preferentially located in the internucleosomal regions of DNA, which can lead to differences in the binding constant between the damage-recognition proteins and the DNA. CPDs are distributed more randomly but with a 10Å perodicity in the DNA wrapped around nucleosomes, due to a preference for formation of dimers on the side of the DNA opposite the DNA-protein contact surface.^42,43

Figure 15.5

Repair of [6-4]PPs and CPDs in mouse skin and cultured cells. Radioimmunoassays that specifically detect [6-4]PPs (A) or CPDs (B) were used to monitor the removal of these lesions from the DNA of irradiated mouse skin ( • ) and mouse cells in (more...)

Figure 15.6

Repair of [6-4]PPs and CPDs in human fibroblasts in culture. Radioimmunoassays that specifically detect [6-4]PPs (○, •) or CPDs (□, ▪) were used to monitor the removal of lesions from the DNA of normal (GM637) or XP revertant (more...)

When excision is considered on an individual gene basis, additional variation exists according to transcriptional activity. Both NER and BER occur as global genome repair (GGR) and transcription-coupled repair (TCR). CPDs are excised more rapidly from actively transcribed genes, especially the DNA strand used as the template for transcription.⁴⁴ An increased excision rate in active genes may also occur for [6-4]PPs, but this is less easily resolved against the greater overall rate of excision of these photoproducts in the genome as a whole. The difference in excision from active versus inactive genes occurs because a basal transcription factor, TFIIH, plays a major role in repair.⁴⁵ This factor regulates basal transcription by RNA polymerase II. Most of the genes that regulate TCR are associated with the human disorders XP, CS, and TTD. Two of the helicases in TFIIH correspond to the XPB and XPD genes, and others are known to play a role from their analogs in the yeast transcription factor b.⁴⁶ BER also can proceed preferentially in transcribing genes and appears to be influenced by such diverse gene products as XPG,⁴⁷ the mismatch repair gene MLH2,⁴⁸ and the breast cancer susceptibility gene BRCA1.⁴⁹ A detailed study of the promoter and first exons of the PGKI gene has indicated that excision is slow in regions of promoter binding but increases immediately after the ATG start site for transcription.⁵⁰

Mutagenicity of UV Photoproducts

Two molecular mechanisms are currently considered important in the initiation of carcinogenesis: activation of proto-oncogenes and inactivation of tumor-suppressor genes. Both sites of action are vulnerable to the lethal and mutagenic effects of UVR. A gene, such as the ras proto-oncogene, can be activated by a point mutation; p53 on the other hand is a tumor suppressor commonly inactivated by point mutations in human tumors.^51–52 Tumor progression is also influenced by UVR. Cell death due to the lethal effects of UV light may enhance the clonal expansion of surviving cells that may have been mutated or initiated, increasing the probability of tumor progression.⁵² Thus, the interplay of UV lethality and mutagenesis in human skin cells may determine the onset and progression of UV carcinogenesis, and the tumor suppressor p53 plays a major role in this determination.⁵²

The mutagenicity and tumorigenicity of a particular photoproduct may ultimately be influenced by its lethality. A lesion that blocks DNA polymerization may be due to termination of DNA synthesis.⁵¹ Although most photoproducts act as blocks to the replicative polymerases, α and δ, they can be bypassed during DNA synthesis to different degrees, depending on their structures by damage-specific polymerases, η and ζ.³⁵ This bypass allows polymerases to read through the noninformative sequence information: polymerase η preferentially inserts adenine in the nascent strand opposite the lesion (called the “A rule”) and hence can accurately replicate a thymine-containing CPD,^53,54 whereas polymerase ζ is mutagenic.⁵⁵ This mechanism has two important implications regarding the mutagenicity of different photoproducts. First, mutations will most often occur where cytosine is a component of the photoproduct since insertion of adenine opposite thymine is a correct and nonmutagenic event. Hence, most CPDs, because they form between two thymine bases, are nonmutagenic. Second, the more distortive a lesion is, the more likely it will block DNA synthesis and result in a lethal rather than mutagenic event. Since the [6-4]PP is considerably more distortive than the CPD (i.e., it causes a 47° as opposed to a 7° helical bend) it is more likely to be lethal rather than mutagenic. Because damage bypass and adenine insertion depend on a variety of conditions, both CPDs and [6-4]PPs contribute to tumorigenesis in a complex manner.

Site-specific determination of photoproduct induction in the lacI gene of Escherichia coli suggested a correlation between hotspots of [6-4]PP induction and of UV-induced mutations.⁵⁶ Analysis of sites of [6-4]PP induction suggested that this lesion was responsible for the major fraction of cytosine-to-thymine transition mutations in E. coli. Consistent with this observation, it was shown that the exclusive induction of CPDs by acetophenone and UVB light did not increase the induction of transition mutations in the lambda phage.⁵⁷ A similar relationship was observed in a study of photoreactivation in E. coli; whereas CPDs and [6-4]PPs were similarly cytotoxic, the latter were much more mutagenic.⁵⁸

The role of specific photoproducts in UV mutagenesis in human cells has been investigated with the use of shuttle vectors. In these systems, UV-irradiated simian virus (SV40)-based plasmids are transfected into human cells, where they are replicated by the host. The plasmids are subsequently recovered, amplified in bacteria, and analyzed for mutation induction by DNA sequencing. Sites of mutations can then be compared with sites of photoproduct induction in the target sequence. Results of these studies are similar to those obtained in E. coli: sites of transition mutations correlate with sites of increased [6-4]PP induction (Table 15.1). In particular, sites and frequencies of mutation hotspots in the lacI gene transfected into human cells were identical to those determined in E. coli.⁵⁹ In a shuttle vector system in which photoproduct induction and sites of mutation were examined in the supF gene, transfection into SV40-transformed human fibroblasts and monkey kidney cells indicated a similar correlation.⁶⁰ In the supF gene inserted into the mouse L-cell chromosome⁶¹ and in the endogenous APRT gene of CHO cells,⁶² most of the mutations consisted of cytosine-to-thymine transitions occurring at thymine-cytosine and cytosine-cytosine sequences. Due to the strand specificity of repair, there is a bias between mutations in the coding and the noncoding strands of expressed genes that differs according to the NER capacity of the cells.^63,64

Table 15.1

UVC-Induced Mutations Observed in Shuttle Vector pZ189 Replicated in XP or Normal Human Cells^a.

CPDs and [6-4]PPs can both form at sequences shown to be mutation hotspots in shuttle vectors, and the identity of the mutagenic lesion has been tested by photoreactivation of the supF sequence in plasmids before transfection.^65,66 Enzymatic photoreversal of CPDs reduced the mutation frequency in normal cells by 75% and in XP group A cells by 90%. Since co-transfection of monkey cells with a mixture of unirradiated supF plasmid and irradiated plasmid without the supF gene did not generate mutations, the role of an SOS-like system, as observed in E. coli, did not appear to be responsible for the results.⁶⁶ These results are not consistent with the model developed in E. coli and suggest that [6-4]PPs may be less mutagenic in human cells. A similar analysis with photoreactivation suggested that CPDs occurring at dipyrimidine sites containing at least one cytosine base were the predominant mutagenic lesions induced in human cells, and that [6-4]PPs at these sites accounted for only about 10% of the mutations.⁶⁵ However, this same study indicated that the frequencies of both CPDs and [6-4]PPs at individual dipyrimidine sites did not correlate with mutation frequency, suggesting that although UV-induced lesions are required for mutagenesis, mutation hotspots are determined by other factors.

A comparison of photoproduct yields, rates of repair, and mutations in the PGKI and p53 genes, however, has shown that regions of high UV-induced mutation can be caused by high photoproduct yield and/or low repair.^50,67–69 A combination of initial yields and rates of repair that leave a high net persistent load of photoproducts in a particular site appears to be directly related to the mutational yield. Using ligation-mediated polymerase chain reaction (LMPCR), which allows precise location of damaged bases, the photoproduct distribution in exons 1 and 2 of three ras proto-oncogenes was mapped, and no correlation between photoproduct frequency and mutation induction in codon 12 of H-ras and K-ras was found.⁷⁰ Further studies with LMPCR showed that the rate of excision repair of CPDs at specific nucleotides in the promoter and exon 1 of the PGK1 gene varied 15-fold with much reduced repair at transcription factor binding sites.⁵⁰ DNA repair at individual nucleotides in the p53 tumor suppressor gene was highly variable and sequence dependent, with slow repair observed at seven of eight of the positions associated with mutations.⁶⁷ UV-induced mutations in the p53 gene are a probable step in the formation of squamous cell carcinoma^51,52 and may arise at DNA repair “coldspots” rather than photoproduct “hotspots.”

Genetic Disorders of DNA Repair

The study of human sunlight-sensitive disorders and the selection of UV-sensitive hamster and mouse cells in culture have identified a large series of genetic loci that control the response of mammalian skin to damage (Table 15.2). These loci are all characterized by significant increases in sensitivity to UVC or UVB radiation and include the disorders XP (8 complementation groups),⁷ CS (2 complementation groups),^71,72 TTD (3 distinct phenotypic types),^73,74 and BCNS.⁷⁵ Many of these genes have also been defined in cell cultures as ERCC1-12 (excision repair cross complementing).⁷⁶

Table 15.2

Complementation Groups in XP and UV-Sensitive Chinese Hamster Ovary (CHO) Cells.

With the exception of BCNS, all of these disorders represent increased sensitivity to UVB and UVC wavelengths due to recessive mutations associated with a large family of genes that regulate human cell DNA repair. These disorders are not mutually exclusive because CS overlaps with XP groups B, D, and G, and TTD overlaps with groups B and D. Chromosome locations are known, and the genes have been cloned for most of these loci (see Table 15.2). Mutations in individual genes have pleiotropic effects on cellular sensitivity to UV light and DNA repair and are associated with a range of clinical syndromes involving skin, nervous system, and immunologic changes. BCNS, in contrast, is a dominant disorder involving a mutation in a tumor suppressor that regulates a signaling cascade involving the patched (PTC) gene.⁷⁷

Xeroderma Pigmentosum

XP is a rare autosomal recessive disease that occurs at a frequency of about 1:250,000 in the United States.⁷ Affected patients (homozygotes) have sun sensitivity resulting in progressive degenerative changes of the sun-exposed portions of the skin and eyes, often leading to neoplasia. Some XP patients have, in addition, progressive neurologic degeneration. Obligate heterozygotes (parents) are generally asymptomatic. The median age of onset is 1 to 2 years of age, with the skin rapidly taking on the appearance of that seen in individuals with many years of sun exposure. Pigmentation is patchy, and skin shows atrophy and telangiectasia, with development of basal and squamous cell carcinomas. The frequency of cancers is about 2,000 times that seen in the general population under 20 years of age, with an approximate 30-year reduction in lifespan.

Cells from patients with XP excise pyrimidine CPDs and [6-4]PPs at reduced rates of 0 to 90% of normal, except for the variant group, which has near-normal rates (Table 15.3). Reduced excision is correlated with low levels of repair replication. The reductions are similar in all tissues thus far investigated, including skin in vivo, peripheral lymphocytes, fibroblasts, liver cell cultures, and tumor cells. There are seven complementation groups among patients who are deficient in excision repair, and an eighth, the XP variant, has a defect in replication of damaged DNA (see Table 15.2).

Table 15.3

DNA Repair Characteristics of Human Cells.

Considerable genetic diversity exists within these disorders and the capacity for excision repair correlates in many cases with the ability to survive UV irradiation. Compared with normal cells, cells from XP groups A and D are very sensitive to the lethal effects of UV light and are unable to excise the two major types of UV damage, the CPD and the [6-4]PP (see Table 15.3). XP group A cells also have a reduced capacity to repair the Dewar pyrimidinone, an important lesion induced with increased efficiency by UVB light.^26,38,93 XP group E cells display an intermediate phenotype, both in their UV sensitivity and their capacity to excise UV damage, and a number of XPE cells lack the p48/p125 damage-specific binding protein.⁹⁴

Group C is one of the largest groups and is often referred to as the common or classic form of XP. The patients show only skin disorders, which vary considerably in severity, depending on the climate. Tumors of the tongue have been observed in several patients. Cells have low but heterogeneous levels of excision repair (10–20% of normal) and are less sensitive to killing by UV light and chemical carcinogens than cells in groups A and D. One characteristic of repair unique to this group is that the reduced repair is not widespread in their genome, such as in groups A and D, but is confined to certain genomic regions.⁹⁵ These cells insert repair patches into small regions of their genome at normal rates, probably corresponding to [6-4]PP and CPD repair⁹⁵ and excise thymine CPDs preferentially from transcriptionally active regions.⁹⁵ This raises the dilemma that high rates of cell killing, somatic mutation, and cancer from UV light in XP group C are associated with repair deficiencies in the nontranscribed regions of the genome. This, in turn, suggests that activating rather than silencing mutations may be important, or that mutations arise from unrepaired lesions in the nontranscribed strand of active genes.

Group E is a rare group that exhibits mild symptoms and residual levels of repair that are between 50 and 100% of normal. Some XPE cells lack a DNA-binding protein⁹⁴ and correspondingly carry mutations in the p48 gene, but this is not seen in all cases raising the possibility that not all cells classified in group E are correctly assigned. The role of this protein is still unclear but it is dependent on p53 and involved in the repair of nontranscribed regions of DNA.^86,87

XP variant cells do not have an excision repair defect but appear near normal in their ability to excise DNA damage (see Table 15.3), yet show a slight but significant sensitivity to UV light. This complementation group is now known to lack a DNA polymerase, HRAD30 (polymerase η) that is required for accurate replication of pyrimidine dimers.^96,97 In its absence, XP variant cells are very susceptible to UV-induced mutagenesis and associated with essentially the same symptoms as other XP patients. Carcinogenesis from UV damage in XP patients arises therefore from the loss of either NER capacity or polymerase η; both lead to an increase in the amount of DNA damage that becomes the substrate for error generation (such as mutations, gene rearrangements, and deletions) by polymerase ζ.

Cockayne Syndrome

CS is an autosomal recessive disease characterized by cachectic dwarfism, retinal abnormalities, microcephaly, deafness, neural defects, and retardation of growth and development after birth. Carcinomas of the skin as a result of hyperphotosensitivity are not seen in patients with CS, setting this disease apart from XP.

CS patients are distributed unevenly within the complementation groups with significantly more group A than B patients.⁷¹ Three patients from two families are known from the XP complementation group B, who also show CS symptoms.⁹⁸ CS symptoms have also been reported in a few XPD and XPG patients. The UV sensitivity of most CS cells lies in a narrow range, with a D³⁷ about half of normal, unlike XP cells, which exhibit a wide range of sensitivity. Characteristic cellular changes in CS include a failure of DNA and RNA synthesis to return to normal levels after UV irradiation.⁷² The excision of DNA photoproducts from total genomic DNA of CS cells is normal, but repair of transcriptionally active genes is reduced.⁹⁹ The CS gene products are involved in coupling excision repair to transcription, but their precise function is not yet clear. They may be involved in the ubiquitination and degradation of stalled RNA polII at damaged sites.

Cockayne syndrome and XP group C therefore make an interesting contrast. CS cells repair only transcriptionally inactive genes, whereas XP group C cells repair only transcriptionally active genes. They show a similar increased sensitivity to cell killing, indicating that all regions of the genome must be repaired for normal survival. But only XP group C shows elevated mutagenesis and carcinogenesis, indicating that defective repair of transcriptionally inactive genes is more important for these endpoints.

Trichothiodystrophy

TTD is a rare autosomal recessive disorder characterized by sulfur-deficient brittle hair and ichthyosis. Hair shafts split longitudinally into small fibers, and this brittleness is associated with levels of cysteine/cystine in hair proteins that are 15 to 50% of those in normal individuals. The condition is also accompanied by physical and mental retardation of varying severity. The patients often have an unusual facial appearance, with protruding ears and a receding chin. Mental abilities range from low-normal to severe retardation.⁷³ Three categories of the disease can be recognized on the basis of cellular responses to UV damage. The most severe has repair deficiencies and complementation properties that place them in XP group D; intermediate cases show reduced DNA repair but normal UV sensitivity; and the third is indistinguishable in UV response from normal cells.⁷⁴

Repair profiles of three fibroblast lines derived from patients with TTD have been characterized; each displays a unique phenotype.⁷⁴ One TTD cell line shows normal UV resistance and DNA repair properties; another shows an XP group D response to UV irradiation, with greatly reduced survival and repair and is associated with mutations in the XPD gene;¹⁰⁰ cells derived from a third patient show normal survival after UV irradiation, but the repair capacity, as evidenced by repair synthesis and repair incision, is significantly reduced. Although the excision rate of the [6-4]PP in this third TTD class is slightly reduced, CPD repair appears normal, suggesting that either the [6-4]PP is not lethal in these cells or the observed defect in its repair is not sufficient to affect survival. Specific TTD genes (A, B) may be components of transcription factor TFIIH,⁴⁶ and the symptoms of this disease indicate a role for this factor in development and hair growth.

Basal Cell Nevus Syndrome and Basal Cell Cancers

BCNS is an autosomal dominant disorder with high penetrance (>97%). The principal manifestations of this syndrome are multiple tumors (average 50 to 100), primarily on sun-exposed skin, that usually appear at puberty and during the second and third decade of life.⁷⁵ Other symptoms include palmar and plantar pitting and musculoskeletal abnormalities (scoliosis, bifurcated rib, spina bifida). The high incidence of developmental anomalies suggests that the normal allele of the BCNS gene may play a role in growth and development, in addition to the acceleration of sunlight-induced carcinogenesis. The gene associated with BCNS was identified as one previously identified in Drosophila as the patched (PTC) gene.⁷⁷ Mutations in PTC have been identified in sporadic basal cell cancers (BCC) and in patients with BCNS and with increased frequency in BCCs from XP patients.¹⁰¹

The most common gross genetic alteration in sporadic BCCs (68%) is loss of heterozygosity (LOH) at the PTC locus on chromosome 9q22.¹⁰² This occurs even in small BCCs (< 1cm diameter), during their initial development from the stem cells of the hair follicles, suggesting that LOH at the PTC locus may be an early event in BCC tumorigenesis.¹⁰² BCNS-associated BCCs retain the mutant germline PTC allele but lose the wild-type (WT) allele by loss of large chromosomal fragments at 9q (Bonifas et al, 1994) or by point mutation. In accordance with Knudson’s two-hit hypothesis, BCNS patients develop more BCCs, tens to hundreds, and at a younger average age than most sporadic cases.

The human PTC gene encodes a 1296 amino acid membrane protein predicted to have 12 transmembrane domains that regulate a cell proliferation, signal transduction pathway. PTC protein inhibits hedgehog (HH) gene expression by interacting with a seven transmembrane protein resembling a G protein-coupled receptor (Smoothened, SMO) protein. PTC inhibition of SMO is relieved by PTC binding to SHH or following mutational inactivation of PTC.⁷⁷ SMO signaling may activate transcription of HH targets, including PTC, through a signaling pathway leading to activation of transcription factor, Gli. Therefore, paradoxically, mutational inactivation of PTC and consequent loss of PTC protein activity results in increased PTC expression and the accumulation of high levels of PTC transcript.

Development of other tumors in BCNS, including medulloblastoma and ovarian and uterine fibromas, suggests that BCNS fits Knudson’s two-mutation model for carcinogenesis.⁷⁵ However, the number of such mutations required to induce cancers in individuals with BCNS is unknown and could be higher than two. Nevertheless, in BCNS, one of the mutations is inherited as an autosomal dominant gene in all somatic cells, whereas environmental agents such as UV or ionizing radiation can induce the remaining mutation(s). This hypothesis is supported by the observation that presymptomatic children with BCNS who were treated with radiation for medulloblastoma developed multiple basal cell carcinomas in the area that received radiation 6 months to 3 years later.

Fibroblasts from individuals with BCNS have not shown consistent increases in sensitivity to x-rays or UVC radiation. Although CPD repair is normal in these cells, excision of [6-4]PPs may be reduced, resembling that shown for TTD group 3 patients¹⁰³ (Rosenstein B, Mitchell D.L. unpublished observations). The reduced repair of the [6-4]PP may not be sufficient to affect survival after UVC irradiation, and in general, BCNS does not appear to have major abnormalities in UV repair.

Familial melanoma

Melanoma is induced by sun exposure, but the precise mechanism is much less clear than for nonmelanoma skin cancer.^4,104 The incidence of melanoma is increased in XP patients, indicating that UV exposure and DNA damage can be involved in melanoma induction.⁹ But the induction seems more closely related to acute burns rather than chronic accumulated exposure. Familial cases indicate that there is a major melanoma tumor suppressor on chromosome 9q21, which codes for a cell cycle regulator, p16, an inhibitor of the cyclin kinase 4 gene family.¹⁰⁴ Gene and heterozygosity losses in this chromosomal region are seen in familial and in nonfamilial cases, demonstrating its general importance.¹⁰⁵ Additional genes on chromosome 6, 8, and 10 are also involved.^105,106 The deletion of chromosome 9p21 can, in a significant number of cases, extend to additional loss over large regions of 9p and 9q.¹⁰⁵ Since the 9q arm also contains the genes for BCNS and for XPA, these additional losses may conceivably contribute to changes in photosensitivity and repair associated with melanoma progression.

Carcinogenesis

Carcinogenesis often appears to proceed by a multi-step process, the first being an initiation event with subsequent promotional events that can often occur much later. One view of carcinogenesis would correlate initiation with the induction of somatic mutations, and promotion with further alterations in gene expression and copy number. Carcinogenesis appears to involve the activity of a large number of genes. These include genes for detoxifying carcinogenic chemicals, the DNA repair gene family, some 50 or more dominantly acting proto-oncogenes activated by mutation, deletion, translocation, or amplification, and tumor suppressor genes whose loss may contribute to the development of cancer.^107,108

The sequence of events seen in colorectal cancer and retinoblastoma may provide a useful model for skin carcinogenesis.^107,108 Early events may correspond to activating mutations, and various stages of tumor development occur as a result of progressive chromosome loss or conversion of heterozygosity to homozygosity. On the basis of studies with XP, early events in the skin may correspond to UV-induced mutations. Not only are major genetic defects in repair related to cancer in XP, but variations in repair among individuals also show a correlation with basal cell carcinoma. Here, however, recent studies on ras activation lead to a dilemma. Several investigations have led to identification of activating mutations in the Ha-ras and N-ras oncogenes at codon 61, from solar UV exposure.^109–111 However, although over 75% of UV-induced mutations are C to T transitions at TC or CC CPD photoproduct sites,¹¹² Ha-ras and N-ras activation occurred in tumors at a TT site and are transversions not previously identified in model culture systems.¹¹² Clearly, detailed investigation of oncogene activation in a number of mouse and human systems is needed to clarify the relationship between UV-induced mutations and ras activation. A large proportion of human skin tumors contains mutations in the p53 tumor suppressor gene that are caused by UV photoproducts.^51–52 This demonstrates a direct causal role for UVB from sunlight in causing one of the mutagenic events in skin carcinogenesis and demonstrates that p53 mutations are early events that affect the balance of pathways of cell death versus mutation and proliferation.

Inactivation of tumor suppressor genes has been demonstrated in retinoblastoma,¹¹³ Wilms’ tumor,¹¹⁴ and acoustic neuromas, and allelic loss resulting in conversion from heterozygosity to homozygosity appears to be a common consequence of tumor progression.¹⁰⁸ The high levels of skin cancer in XP patients may result from increased levels of UV damage caused by defective repair, which lead to activating mutations, inactivating p53 mutations, and chromosome instability. Tumor suppressor genes could contribute to tumor advancement by incremental effects on cell growth and intercellular regulation and loss of the apoptotic pathway due to p53 heterozygosity.^51,52 The observation that promotion may involve alterations in cell-cell communication is consistent with this interpretation. Tumor promoters may be environmental factors that mimic the effect of regulatory genes. Analysis of the various stages of skin tumor development would seem to be especially promising at this time since so many stages are accessible, and the environmental causative factors are so well known. The large reduction in the induction time for cancer in XP patients (see Fig 15.1) indicates that UV damage and repair are involved in both the initiation and progression of skin cancers and are therefore critical factors throughout the carcinogenic process.

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