RESUMO
Long wavelength ultraviolet radiation (UVA, 320-400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA-protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions.
Assuntos
Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , DNA/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Proteômica/métodos , Raios Ultravioleta , Células Cultivadas , Ciprofloxacina/farmacologia , Expressão Gênica , Humanos , Oxirredução , Proteínas/metabolismo , Tioguanina/farmacologiaRESUMO
Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib-a BRAF inhibitor used to treat metastatic melanoma-are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.
Assuntos
Antibacterianos/toxicidade , Antineoplásicos/toxicidade , Reparo do DNA/efeitos dos fármacos , Fluoroquinolonas/toxicidade , Indóis/toxicidade , Fármacos Fotossensibilizantes/toxicidade , Carbonilação Proteica/efeitos dos fármacos , Sulfonamidas/toxicidade , Raios Ultravioleta/efeitos adversos , Linhagem Celular , Ciprofloxacina/toxicidade , Reparo do DNA/efeitos da radiação , Células HeLa , Humanos , Ofloxacino/toxicidade , Antígeno Nuclear de Célula em Proliferação/análise , Carbonilação Proteica/efeitos da radiação , Tioguanina/toxicidade , VemurafenibRESUMO
The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA-protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (G(SO3))--an oxidized 6-TG and a previously identified UVA photoproduct--replaces 6-TG, suggesting that G(SO3) is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide-oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA-protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA-protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas Nucleares/química , Tioguanina/química , Raios Ultravioleta , DNA/efeitos da radiação , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/análise , Eletroforese em Gel Bidimensional , Humanos , Proteína 2 Homóloga a MutS/análise , Proteínas Nucleares/análise , Oligopeptídeos/química , Processos Fotoquímicos , Antígeno Nuclear de Célula em Proliferação/análise , Células Tumorais Cultivadas , Proteína de Xeroderma Pigmentoso Grupo A/análiseRESUMO
Photochemotherapy-in which a photosensitizing drug is combined with ultraviolet or visible radiation-has proven therapeutic effectiveness. Existing approaches have drawbacks, however, and there is a clinical need to develop alternatives offering improved target cell selectivity. DNA substitution by 4-thiothymidine (S(4)TdR) sensitizes cells to killing by ultraviolet A (UVA) radiation. Here, we demonstrate that UVA photoactivation of DNA S(4)TdR does not generate reactive oxygen or cause direct DNA breakage and is only minimally mutagenic. In an organotypic human skin model, UVA penetration is sufficiently robust to kill S(4)TdR-photosensitized epidermal cells. We have investigated the DNA lesions responsible for toxicity. Although thymidine is the predominant UVA photoproduct of S(4)TdR in dilute solution, more complex lesions are formed when S(4)TdR-containing oligonucleotides are irradiated. One of these, a thietane/S(5)-(6-4)T:T, is structurally related to the (6-4) pyrimidine:pyrimidone [(6-4) Py:Py] photoproducts induced by UVB/C radiation. These lesions are detectable in DNA from S(4)TdR/UVA-treated cells and are excised from DNA more efficiently by keratinocytes than by leukaemia cells. UVA irradiation also induces DNA interstrand crosslinking of S(4)TdR-containing duplex oligonucleotides. Cells defective in repairing (6-4) Py:Py DNA adducts or processing DNA crosslinks are extremely sensitive to S(4)TdR/UVA indicating that these lesions contribute significantly to S(4)TdR/UVA cytotoxicity.
Assuntos
Dano ao DNA , Timidina/análogos & derivados , Raios Ultravioleta , Animais , Linhagem Celular , Cricetinae , DNA/química , DNA/metabolismo , Reparo do DNA , Humanos , Mutagênese , Oligonucleotídeos/química , Dímeros de Pirimidina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pele/anatomia & histologia , Pele/efeitos da radiação , Timidina/efeitos da radiaçãoRESUMO
The thiopurines azathioprine, 6-mercaptopurine and 6-thioguanine (6-TG) are important medications for cancer and inflammatory disorders. They are also widely prescribed as immunosuppressants in organ transplant patients. Their metabolism results in the incorporation of 6-TG into patients' DNA, and this increases skin sensitivity to incident UVA. Unlike the canonical DNA bases, which do not absorb UVA to a significant degree, DNA 6-TG is a strong UVA chromophore. It acts as a Type II UVA photosensitizer, and the combination of 6-TG and UVA treatment induces a synergistic toxicity in cultured human cells. Here, we review some of the damage that this interaction causes. Photochemical activation of DNA 6-TG triggers DNA and protein oxidation; it induces DNA breakage, DNA crosslinking, oxidation of DNA bases and the covalent attachment of proteins to DNA. Many of these photochemical DNA lesions are difficult for cells to deal with, and we review the evidence linking thiopurine immunosuppression with genome instability and the high incidence of skin cancer in organ transplant recipients.
Assuntos
Azatioprina/uso terapêutico , Imunossupressores/uso terapêutico , Mercaptopurina/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Neoplasias Cutâneas/etiologia , Tioguanina/uso terapêutico , Raios Ultravioleta , HumanosRESUMO
Photochemotherapy, in which ultraviolet radiation (UVR: 280-400 nm) or visible light is combined with a photosensitizing drug to produce a therapeutic effect that neither drug or radiation can achieve alone, is a proven therapeutic strategy for a number of non-malignant hyperproliferative skin conditions and various cancers. Examples are psoralen plus UVA (320-400 nm) radiation (PUVA) and photodynamic therapy (PDT). All existing photochemotherapies have drawbacks - for example the association of PUVA with the development of skin cancer, and pain that is often associated with PDT treatment of skin lesions. There is a clear need to develop alternative approaches that involve lower radiation doses and/or improved selectivity for target cells. In this review, we explore the possibility to address this need by exploiting thionucleoside-mediated DNA photosensitisation to low, non toxic doses of UVA radiation.
Assuntos
DNA/química , Fotoquimioterapia , Timidina/análogos & derivados , Raios Ultravioleta , Animais , Morte Celular/efeitos da radiação , Modelos Animais , Timidina/metabolismoRESUMO
The DNA of patients taking the immunosuppressant and anticancer drugs azathioprine or 6-mercaptopurine contains 6-thioguanine (6-TG). The skin of these patients is selectively sensitive to ultraviolet A radiation (UVA) and they suffer an extremely high incidence of sunlight-induced skin cancer with long-term treatment. DNA 6-TG interacts with UVA to generate reactive oxygen species, which oxidize 6-TG to guanine sulphonate (G(SO3)). We suggested that G(SO3) is formed via the reactive electrophilic intermediates, guanine sulphenate (G(SO)) and guanine sulphinate (G(SO2)). Here, G(SO2) is identified as a significant and stable UVA photoproduct of free 6-TG, its 2'-deoxyribonucleoside, and DNA 6-TG. Mild chemical oxidation converts 6-TG into G(SO2), which can be further oxidized to G(SO3)-a stable product that resists further reaction. In contrast, G(SO2) is converted back to 6-TG under mild conditions. This suggests that cellular antioxidant defences might counteract the UVA-mediated photooxidation of DNA 6-TG at this intermediate step and ameliorate its biological effects. In agreement with this possibility, the antioxidant ascorbate protected DNA 6-TG against UVA oxidation and prevented the formation of G(SO3).
Assuntos
Sulfonatos de Arila/química , Guanina/análogos & derivados , Tioguanina/efeitos da radiação , Raios Ultravioleta , Ácido Ascórbico/química , DNA/química , Guanina/química , Oxirredução , Processos Fotoquímicos , Sulfetos/química , Tioguanina/químicaRESUMO
hMTH1 protects against mutation during oxidative stress. It degrades 8-oxodGTP to exclude potentially mutagenic oxidized guanine from DNA. hMTH1 expression is linked to ageing. Its downregulation in cultured cells accelerates RAS-induced senescence, and its overexpression in hMTH1-Tg mice extends lifespan. In this study, we analysed the effects of a brief (5 weeks) high-fat diet challenge (HFD) in young (2 months old) and adult (7 months old) wild-type (WT) and hMTH1-Tg mice. We report that at 2 months, hMTH1 overexpression ameliorated HFD-induced weight gain, changes in liver metabolism related to mitochondrial dysfunction and oxidative stress. It prevented DNA damage as quantified by a comet assay. At 7 months old, these HFD-induced effects were less severe and hMTH1-Tg and WT mice responded similarly. hMTH1 overexpression conferred lifelong protection against micronucleus induction, however. Since the canonical activity of hMTH1 is mutation prevention, we conclude that hMTH1 protects young mice against HFD by reducing genome instability during the early period of rapid growth and maximal gene expression. hMTH1 protection is redundant in the largely non-growing, differentiated tissues of adult mice. In hMTH1-Tg mice, expression of a less heavily mutated genome throughout life provides a plausible explanation for their extended longevity.
Assuntos
Gorduras na Dieta , Longevidade , Animais , Dieta Hiperlipídica , Gorduras na Dieta/farmacologia , Longevidade/genética , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Estresse FisiológicoRESUMO
Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Dano ao DNA , Espécies Reativas de Oxigênio/metabolismo , Tioguanina/metabolismo , Transcrição Gênica , Raios Ultravioleta , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/efeitos da radiação , Linhagem Celular , DNA/química , DNA/metabolismo , Reparo do DNA , Humanos , RNA Polimerase II/metabolismo , Tioguanina/farmacologia , Tioguanina/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiaçãoRESUMO
The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.
Assuntos
Antimetabólitos Antineoplásicos/efeitos da radiação , Antimetabólitos Antineoplásicos/toxicidade , Dano ao DNA , Tioguanina/efeitos da radiação , Tioguanina/toxicidade , Raios Ultravioleta , Antimetabólitos Antineoplásicos/química , Sulfonatos de Arila/química , Sulfonatos de Arila/metabolismo , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , DNA/efeitos da radiação , Replicação do DNA , Relação Dose-Resposta à Radiação , Feminino , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Humanos , Oxidantes Fotoquímicos/metabolismo , Oxirredução/efeitos da radiação , Tioguanina/análogos & derivados , Tioguanina/química , Tioguanina/metabolismoRESUMO
2-Hydroxyadenine (2-OH-A), a product of DNA oxidation, is a potential source of mutations. We investigated how representative DNA polymerases from the A, B and Y families dealt with 2-OH-A in primer extension experiments. A template 2-OH-A reduced the rate of incorporation by DNA polymerase alpha (Pol alpha) and Klenow fragment (Kf(exo-)). Two Y family DNA polymerases, human polymerase eta (Pol eta) and the archeal Dpo4 polymerase were affected differently. Bypass by Pol eta was very inefficient whereas Dpo4 efficiently replicated 2-OH-A. Replication of a template 2-OH-A by both enzymes was mutagenic and caused base substitutions. Dpo4 additionally introduced single base deletions. Thermodynamic analysis showed that 2-OH-A forms stable base pairs with T, C and G, and to a lesser extent with A. Oligonucleotides containing 2-OH-A base pairs, including the preferred 2-OH-A:T, were recognized by the human MutSalpha mismatch repair (MMR). MutSalpha also recognized 2-OH-A located in a repeat sequence that mimics a frameshift intermediate.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Guanina/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Pareamento Incorreto de Bases , Sequência de Bases , Células Cultivadas , DNA/química , DNA/metabolismo , Reparo de Erro de Pareamento de DNA , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Temperatura , Termodinâmica , Células Tumorais CultivadasRESUMO
The thymidine analogue 4-thiothymidine (S(4)TdR) is a photosensitizer for UVA radiation. The UV absorbance spectrum of S(4)TdR and its incorporation into DNA suggests that it might act synergistically with nonlethal doses of UVA to selectively kill hyperproliferative or cancerous skin cells. We show here that nontoxic concentrations of S(4)TdR combine with nonlethal doses of UVA to kill proliferating cultured skin cells. Established cell lines with a high fraction of proliferating cells were more sensitive than primary keratinocytes or fibroblasts to apoptosis induction by S(4)TdR/UVA. Although S(4)TdR plus UVA treatment induces stabilization of p53, cell death, as measured by apoptosis or clonal survival, occurs to a similar extent in both p53 wild-type and p53-null backgrounds. Furthermore, different types of human papilloma virus E6 proteins, which protect against UVB-induced apoptosis, have little effect on killing by S(4)TdR/UVA. S(4)TdR/UVA offers a possible therapeutic intervention strategy that seems to be applicable to human papilloma virus-associated skin lesions.
Assuntos
Apoptose , Fibrossarcoma/terapia , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Neoplasias Cutâneas/terapia , Timidina/análogos & derivados , Raios Ultravioleta , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Western Blotting , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Terapia Combinada , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/virologia , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/radioterapia , Fibrossarcoma/virologia , Citometria de Fluxo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Queratinócitos/virologia , Camundongos , Pessoa de Meia-Idade , Octreotida/análogos & derivados , Pele/citologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/radioterapia , Neoplasias Cutâneas/virologia , Timidina/uso terapêutico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs.
Assuntos
Pareamento Incorreto de Bases , Reparo do DNA , Nucleotídeos de Desoxiguanina/metabolismo , Guanosina Monofosfato/metabolismo , Animais , Mamíferos , OxirreduçãoRESUMO
Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA , Reparo do DNA/genética , Desoxirribonucleotídeos/metabolismo , Instabilidade Genômica , Oxirredução , Animais , Sequência de Bases , Camundongos , Repetições de Microssatélites , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase alpha can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSalpha mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSalpha and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Guanina/análogos & derivados , Proteínas Proto-Oncogênicas/metabolismo , Adenosina/química , Pareamento Incorreto de Bases , Citosina/química , DNA/química , DNA Glicosilases/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Guanina/química , Guanina/metabolismo , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/metabolismo , Proteína 2 Homóloga a MutS , Sequências Repetitivas de Ácido NucleicoRESUMO
Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of < or =6 bp. Type B changes are more drastic and involve modifications of > or =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.
Assuntos
Pareamento Incorreto de Bases , Neoplasias Colorretais/genética , Reparo do DNA , Repetições de Dinucleotídeos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte , Linhagem Celular , Análise Mutacional de DNA/métodos , Proteínas de Ligação a DNA/genética , Fluorescência , Genes p53 , Humanos , Camundongos , Camundongos Knockout , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genéticaRESUMO
UVA accounts for about 95% of the solar ultraviolet (UV) radiation that reaches Earth and most likely contributes to human skin cancer risk. In contrast to UVB, which comprises the remaining 5% and is absorbed by DNA nucleobases to cause direct photodamage, UVA damages DNA indirectly. It does this largely through its interactions with cellular chromophores that act as photosensitisers to generate reactive oxygen species. Exogenously supplied chemicals, including some widely-prescribed medicines, may also act as photosensitisers and these drugs are associated with an increased risk of sun-related cancer. Because they amplify the effects of UVA on cells, they provide a means to investigate the mechanisms and effects of UVA-induced photodamage. Here, we describe some of the major lesions induced by two groups of UVA photosensitisers, the DNA thionucleotides and the fluoroquinolone antibiotics. In thionucleotides, replacement of the oxygen atoms of canonical nucleobases by sulfur converts them into strong UVA chromophores that can be incorporated into DNA. The fluoroquinolones are also UVA chromophores. They are not incorporated into DNA and induce a different range of DNA damages. We also draw attention to the potentially important contribution of photochemical protein damage to the cellular effects of photosensitised UVA. Proteins targeted for oxidation damage include DNA repair factors and we suggest that UVA-mediated protein damage may contribute to sunlight-induced cancer risk.
Assuntos
Antibacterianos/química , Dano ao DNA , DNA/efeitos da radiação , Fluoroquinolonas/química , Estresse Oxidativo , Neoplasias Cutâneas/metabolismo , Tionucleotídeos/química , Antibacterianos/uso terapêutico , Reparo do DNA , Fluoroquinolonas/uso terapêutico , Humanos , Oxirredução , Fármacos Fotossensibilizantes , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Tionucleotídeos/uso terapêutico , Raios Ultravioleta/efeitos adversosRESUMO
Potentially mutagenic DNA lesions induced by UVB (wavelengths 280-320 nm) are important risk factors for solar ultraviolet (UV) radiation-induced skin cancer. The carcinogenicity of the more abundant UVA (320-400 nm) is less well understood but is generally regarded to reflect its interaction with cellular chromophores that act as photosensitisers. The arylhydrocarbon receptor agonist 6-formylindolo[3,2-b] carbazole (FICZ), is a UVB photoproduct of tryptophan and a powerful UVA chromophore. Combined with UVA, FICZ generates reactive oxygen species (ROS) and induces oxidative DNA damage. Here we demonstrate that ROS generated by FICZ/UVA combinations also cause extensive protein damage in HaCaT human keratinocytes. We show that FICZ/UVA-induced oxidation significantly inhibits the removal of potentially mutagenic UVB-induced DNA photolesions by nucleotide excision repair (NER). DNA repair inhibition is due to FICZ/UVA-induced oxidation damage to the NER proteome and DNA excision repair is impaired in extracts prepared from FICZ/UVA-treated cells. NER protects against skin cancer. As a likely UVB photoproduct of intracellular tryptophan, FICZ represents a de facto endogenous UVA photosensitiser in sun-exposed skin. FICZ formation may increase the risk of solar UV-induced skin cancer by promoting photochemical damage to the NER proteome and thereby preventing the removal of UVB-induced DNA lesions.
Assuntos
Carbazóis/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Triptofano/metabolismo , Raios Ultravioleta/efeitos adversos , Dano ao DNA , Humanos , OxirreduçãoRESUMO
Microsatellite instability (MSI) in tumors is diagnostic for inactive DNA mismatch repair. It is widespread among some tumor types, such as colorectal or endometrial carcinoma, but is rarely found in leukemia. Therapy-related acute myeloid leukemia/myelodysplastic syndrome (tAML/MDS) is an exception, and MSI is frequent in tAML/MDS following cancer chemotherapy or organ transplantation. The development of MSI+ tumors is associated with an accumulation of insertion/deletion mutations in repetitive sequences. These events can cause inactivating frameshifts or loss of expression of key growth control proteins. We examined established MSI+ cell lines and tAML/MDS cases for frameshift-like mutations of repetitive sequences in several genes that have known, or suspected, relevance to leukemia. CASPASE-5, an acknowledged frameshift target in MSI+ gastrointestinal tract tumors, was frequently mutated in MSI+ cell lines (67%) and in tAML/MDS (29%). Frameshift-like mutations were also observed in the NF1 and FANCD2 genes that are associated with genetic conditions conferring a predisposition to leukemia. Both genes were frequent targets for mutation in MSI+ cell lines and colorectal carcinomas. FANCD2 mutations were also common in MSI+ tAML/MDS, although NF1 mutations were not observed. A novel FANCD2 polymorphism was also identified.