UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer.

Repair of UV induced DNA damage is of key importance to UV-induced skin carcinogenesis. Specific signal transduction pathways that regulate cell cycling, differentiation and apoptosis are found to be corrupted in skin cancers, e.g., the epidermal growth-stimulating Hedgehog pathway in basal cell carcinomas (BCCs). Mutations in genes coding for proteins in these pathways lead to persistent disturbances that are passed along to daughter cells, e.g., mutations in the gene for the Patched (PTCH) protein in the Hedgehog pathway. Thus far only the point mutations in the P53 gene from squamous cell carcinomas and BCCs, and in PTCH gene from BCC of xeroderma pigmentosum (XP) patients appear to be unambiguously attributable to solar UV radiation. Solar UVB radiation is most effective in causing these point mutations. Other forms of UV-induced genetic changes (e.g., deletions) may, however, contribute to skin carcinogenesis with different wavelength dependencies.     

Survival, excision repair and inhibition of DNA synthesis in normal human skin fibroblasts exposed to simulated sunlight.

The responses of normal human skin fibroblasts exposed to simulated sunlight produced by a solar simulator were examined. The parameters investigated were cellular survival, excision repair and the inhibition and recovery of DNA synthesis. The latter two effects were examined using the bromodeoxyuridine photolysis assay and the alkaline step elution assay respectively. The results of these experiments are consistent with the conclusion that the lesions induced by simulated sunlight represent a mixture of damage which elicits cellular responses and repair mechanisms similar to those manifested by cells irradiated with UVC and UVA radiation.     

Photoimmunology, DNA repair and photocarcinogenesis

In recent years major progress has been made in identifying the molecular mechanisms by which UV radiation modulates the immune system of the skin. From these studies it appears that the generation of DNA damage and the subsequent activation of DNA repair enzymes play a critical role in the generation of UV-B-induced immunosuppression. These studies have made use of cells from both nucleotide excision repair (NER)-deficient individuals and mice. Results obtained from these studies have important clinical implications for DNA-repair-deficient patients in particular and for effective photoprotection of human skin in general.     

Chemistry and biology of DNA repair

Numerous agents of endogenous and exogenous origin damage DNA in our genome. There are several DNA-repair pathways that recognize lesions in DNA and remove them through a number of diverse reaction sequences. Defects in DNA-repair proteins are associated with several human hereditary syndromes, which show a marked predisposition to cancer. Although DNA repair is essential for a healthy cell, DNA-repair enzymes counteract the efficiency of a number of important antitumor agents that exert their cytotoxic effects by damaging DNA. DNA-repair enzymes are therefore also targets for drug design. DNA-repair processes differ greatly in their nature and complexity. Whereas some pathways only require a single enzyme to restore the original DNA sequence, others operate through the coordinated action of 30 or more proteins. Our understanding of the genetic, biochemical, and structural basis of DNA repair and related processes has increased dramatically over the past decade. This review summarizes the latest developments in this field.     

Multiple pathways of DNA repair in bacteria and their roles in mutagenesis.

Abstract— In bacteria, three processes of DNA repair are known: photoreactivation, excision repair, and postreplication repair. Photoreactivation, the enzymatic splitting of cyclobutyl pyrimidine dimers in situ, is mediated by exposure of the enzyme-dimer complex to near-UV and visible light. This repair process appears to be error free. The excision repair of UV-induced DNA base damage has been divided into two major pathways on the basis of both physiological requirements and genetic control. The major pathway requires a functional pol A gene, does not require complete growth medium. and appears to be largely error-free and to produce short patches during repair. The second pathway requires complete growth medium and functional recA, recB, recC, lexA, uvrD, and polC genes, and appears to be mutagenic and to produce long patches during repair. There exists a second type of excision repair in which the modified base is removed by an N-glycosidase, and the chain is then nicked by an apurinic (apyrimidinic) acid endonuclease. Subsequent events are presumed to be similar to the above excision repair process. The postreplication repair system has been divided into at least four separate pathways. Three of these are dependent upon functional recB, lexA, and uvrD genes, respectively, and appear to be error free. A fourth pathway depends upon the above gene products, but is blocked by postirradiation treatment with chloramphenicol, and may be the UV-inducible, errorprone, mutagenic pathway of repair (“SOS repair”). A possible fifth pathway depends upon a functional recF gene, and is independent of the recB⁺-dependent pathway. Mutagenesis appears to be the result of error-prone DNA repair, and there is growing evidence that carcinogenesis is also the result of error-prone DNA repair.     

DNA polymerase I-mediated repair of 365 nm-induced single-strand breaks in the DNA of Escherichia coli.

Abstract. Irradiation of closed circular phage Λ DNA in vivo at 365 nm results in the induction of single-strand breaks and alkali-labile lesions at rates of 1.1 × 10^-14, and 0.2 × 10^-14/dalton/J/m², respectively. The sum of the induction rates is similar to the rate of induction of single-strand breaks plus alkali-labile lesions (1 × 10^-14/dalton/J/m²) observed in the E. coli genome. Postirradiation incubation of wild-type cells in buffer results in rapid repair of the breaks (up to 80% repaired in 10 min). No repair was observed in a DNA polymerase I-deficient mutant of E. coli.     

DNA damage and repair in Gorlin syndrome and normal fibroblasts after aminolevulinic acid photodynamic therapy: a comet assay study

Using normal, untransformed, human fibroblasts, the effectiveness of aminolevulinic (ALA)-mediated photodynamic therapy (PDT) was investigated in terms of both clonogenic survival and DNA damage. The response of normal fibroblasts was then compared with Gorlin syndrome-derived fibroblasts (basal cell nevus syndrome [BCNS]). In terms of clonogenic survival, no significant differences were observed between the two groups of cells. Using the alkaline comet assay, initial DNA damage after PDT was measured. Some DNA damage was detected at higher doses, but this was fully repaired within 24 h of treatment. The BCNS-derived cells showed levels of initial damage that did not differ significantly from normal lines.     

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.     

Direct repair of the exocyclic DNA adduct 1,N6-ethenoadenine by the DNA repair AlkB proteins

The exocyclic DNA base adduct 1,N6-ethenoadenine (epsilonA) is directly repaired by the AlkB proteins in vitro.     

recA-dependent DNA repair in UV-irradiated Escherichia coli

UV-radiation-induced lesions in DNA result in the formation of excision gaps, daughter-strand gaps (DSG) and double-strand breaks (DSB), which are repaired by several different mechanisms. Postreplication repair. The recA gene is a master gene that controls all of the pathways of postreplication repair. The repair of DSG proceeds by one pathway that is also recF dependent, and one pathway that is constitutive and independent of the recF and recBC genes. A small fraction of the recF recB-independent repair of DSG is dependent upon the umuC gene, and may define an error-prone pathway of postreplication repair. Unrepaired DSG can be converted to DSB, which are normally repaired by the RecBCD pathway. However, in the recBC sbcB background, these DSB are repaired by a recF-dependent process. The RecF pathways of postreplication repair appear to utilize DNA containing a single-stranded region (either a gap or a DSB with a single-stranded end), while the RecBCD pathway appears to utilize the blunt ends of duplex DNA to promote the recombinational repair of DSB. The polA gene (especially the 5'----3' exonuclease activity of DNA polymerase I) functions in pathways of postreplication repair (both for the repair of DSG and DSB) that are largely independent of the recF gene. Nucleotide excision repair. The repair of excision gaps is independent of the recA gene in cells with unreplicated chromosomes, but is recA dependent in cells with partially replicated chromosomes at the time of UV irradiation. This recA-dependent repair of excision gaps appears to be analogous to the recF- and recB-dependent pathways of postreplication repair, i.e. the RecF pathway repairs DNA gaps, and the RecBCD pathway repairs the DSB that arise at unrepaired gaps.     

UV protective effects of DNA repair enzymes and RNA lotion

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes ( Micrococcus luteus ) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.     

Chemical investigation of light induced DNA bipyrimidine damage and repair

In all organisms, genetic information is stored in DNA and RNA. Both of these macromolecules are damaged by many exogenous and endogenous events, with UV irradiation being one of the major sources of damage. The major photolesions formed are the cyclobutane pyrimidine dimers (CPD), pyrimidine-pyrimidone-(6-4)-photoproducts, Dewar valence isomers and, for dehydrated spore DNA, 5-(α-thyminyl)-5,6-dihydrothymine (SP). In order to be able to investigate how nature's repair and tolerance mechanisms protect the integrity of genetic information, oligonucleotides containing sequence and site-specific UV lesions are essential. This tutorial review provides an overview of synthetic procedures by which these oligonucleotides can be generated, either through phosphoramidite chemistry or direct irradiation of DNA. Moreover, a brief summary on their usage in analysing repair and tolerance processes as well as their biological effects is provided.     

Azole-bridged diplatinum anticancer compounds. Modulating DNA flexibility to escape repair mechanism and avoid cross resistance

Dinuclear azole-bridged Pt compounds bind to DNA helices, forming intrastrand crosslinks between adjacent guanines in a similar way to cisplatin. Their cytotoxic profile is, however, different from that of first and second generation Pt drugs in that they lack cross resistance in cisplatin-resistant cell lines. In contrast to cisplatin, which induces a large kink in DNA duplex, structural NMR studies and molecular dynamics simulations have shown that azole-bridged diplatinum compounds induce only small structural changes in double-stranded DNA. These structural differences have been invoked to explain the different cytotoxic profile of these compounds. Here, we show that in addition to the small structural changes in DNA, dinuclear Pt compounds also affect DNA minor groove flexibility in a different way than cisplatin. Free-energy calculations on azole-bridged diplatinum DNA adducts reveal that opening of the minor groove requires a higher free-energy cost (DeltaG ~ 7-15 kcal/mol) than in the corresponding cisplatin-DNA adduct (DeltaG ~ 0 kcal/mol). This could prevent minor groove binding proteins from binding to diplatinum-DNA adducts thus leading to a different cellular response than cisplatin and possibly decreasing the activity of excision repair enzymes. Although the development of drug resistance is a highly complex mechanism, our findings provide an additional rationale for the improved cytotoxic activity of these compounds in cell lines resistant to cisplatin.     

Glycosphingolipid antigens and cancer therapy

Specific types of glycosphingolipid (GSL), which are chemically detectable in normal cells, are more highly expressed in tumors. The high level of expression on the surfaces of tumor cells causes an antibody response to these GSLs, which can therefore be described as tumor-associated antigens. Some of these GSLs have been shown to be adhesion molecules involved in tumor cell metastasis, and to be modulators of signal transduction controlling tumor cell growth and motility. Tumor-associated GSL antigens have been used in the development of antitumor vaccines. GSLs and sphingolipids involved in adhesion and signaling are therefore targets for cancer therapy.     

Transition-state analysis of the DNA repair enzyme MutY

The transition state (TS) structure of MutY-catalyzed DNA hydrolysis was solved using multiple kinetic isotope effect (KIE) measurements. MutY is a base excision repair enzyme which cleaves adenine from 8-oxo-G:A mismatches in vivo, and also from G:A mismatches in vitro. TS analysis of G:A-DNA hydrolysis revealed a stepwise S(N)1 (D(N)*A(N)(double dagger)) mechanism proceeding through a highly reactive oxacarbenium ion intermediate which would have a lifetime in solution of <10(-10) s. C-N bond cleavage is reversible; the N-glycoside bond breaks and reforms repeatedly before irreversible water attack on the oxacarbenium ion. KIEs demonstrated that MutY uses general acid catalysis by protonating N7. It enforces a 3'-exo sugar ring conformation and other sugar ring distortions to stabilize the oxacarbenium ion. Combining the experimental TS structure with the previously reported crystal structure of an abortive Michaelis complex elucidates the step-by-step catalytic sequence.