RESUMEN
This review uses the National Cancer Institute (NCI) COMPARE program to establish an extensive list of heterocyclic iminoquinones and quinones with similarities in differential growth inhibition patterns across the 60-cell line panel of the NCI Developmental Therapeutics Program (DTP). Many natural products and synthetic analogues are revealed as potential NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates, through correlations to dipyridoimidazo[5,4-f]benzimidazoleiminoquinone (DPIQ), and as potential thioredoxin reductase (TrxR) inhibitors, through correlations to benzo[1,2,4]triazin-7-ones and pleurotin. The strong correlation to NQO1 infers the enzyme has a major influence on the amount of the active compound with benzo[e]perimidines, phenoxazinones, benz[f]pyrido[1,2-a]indole-6,11-quinones, seriniquinones, kalasinamide, indolequinones, and furano[2,3-b]naphthoquinones, hypothesised as prodrugs. Compounds with very strong correlations to known TrxR inhibitors had inverse correlations to the expression of both reductase enzymes, NQO1 and TrxR, including naphtho[2,3-b][1,4]oxazepane-6,11-diones, benzo[a]carbazole-1,4-diones, pyranonaphthoquinones (including kalafungin, nanaomycin A, and analogues of griseusin A), and discorhabdin C. Quinoline-5,8-dione scaffolds based on streptonigrin and lavendamycin can correlate to either reductase. Inhibitors of TrxR are not necessarily (imino)quinones, e.g., parthenolides, while oxidising moieties are essential for correlations to NQO1, as with the mitosenes. Herein, an overview of synthetic methods and biological activity of each family of heterocyclic imino(quinone) is provided.
Asunto(s)
Antineoplásicos , Indolquinonas , Neoplasias , Estados Unidos , National Cancer Institute (U.S.) , Quinonas/química , Oxidorreductasas , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/químicaRESUMEN
1,5-Dideoxy-1,5-imino-l-fucitol (1-deoxyfuconojirimycin, DFJ) is an iminosugar that inhibits fucosidases. Herein, N-alkyl DFJs have been synthesised and tested against the α-fucosidases of T. maritima (bacterial origin) and B. taurus (bovine origin). The N-alkyl derivatives were inactive against the bacterial fucosidase, while inhibiting the bovine enzyme. Docking of inhibitors to homology models, generated for the bovine and human fucosidases, was carried out. N-Decyl-DFJ was toxic to cancer cell lines and was more potent than the other N-alkyl DFJs studied.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Alcoholes del Azúcar/química , alfa-L-Fucosidasa/antagonistas & inhibidores , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Concentración 50 Inhibidora , Melfalán/síntesis química , Melfalán/metabolismo , Melfalán/farmacología , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Alcoholes del Azúcar/metabolismo , alfa-L-Fucosidasa/metabolismoRESUMEN
Cell viability studies for benzo[1,2,4]triazin-7-ones and 1,2,4-benzotriazinyl (Blatter-type) radical precursors are described with comparisons made with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). All of the stable free radicals were several orders of magnitude less cytotoxic than the benzo[1,2,4]triazin-7-ones. The synthesis and evaluation of two new pyrid-2-yl benzo[1,2,4]triazin-7-ones are described, where altering the 1,3-substitution from phenyl to pyrid-2-yl increased cytotoxicity against most cancer cell lines, as indicated using National Cancer Institute (NCI) one-dose testing. COMPARE analysis of five-dose testing data from the NCI showed very strong correlations to the naturally occurring anti-cancer compound pleurotin. COMPARE is program, which analyzes similarities in cytotoxicity data of compounds, and enables quantitative expression as Pearson correlation coefficients. Compounds were also evaluated using the independent MTT assay, which was compared with SRB assay data generated at the NCI.
Asunto(s)
Antineoplásicos/farmacología , Derivados del Benceno/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Triazinas/farmacología , Antineoplásicos/síntesis química , Derivados del Benceno/síntesis química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Radicales Libres , Células HT29 , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Humanos , Células MCF-7 , Relación Estructura-Actividad , Triazinas/síntesis químicaRESUMEN
The thioredoxin (Trx)-thioredoxin reductase (TrxR) system plays a key role in maintaining the cellular redox balance with Trx being over-expressed in a number of cancers. Inhibition of TrxR is an important strategy for anti-cancer drug discovery. The natural product pleurotin is a well-known irreversible inhibitor of TrxR. The cytotoxicity data for benzo[1,2,4]triazin-7-ones showed very strong correlation (Pearson correlation coefficients â¼0.8) to pleurotin using National Cancer Institute COMPARE analysis. A new 3-CF3 substituted benzo[1,2,4]triazin-7-one gave submicromolar inhibition of TrxR, although the parent compound 1,3-diphenylbenzo[1,2,4]triazin-7-one was more cytotoxic against cancer cell lines. Benzo[1,2,4]triazin-7-ones exhibited different types of reversible inhibition of TrxR, and cyclic voltammetry showed characteristic quasi-reversible redox processes. Cell viability studies indicated strong dependence of cytotoxicity on substitution at the 6-position of the 1,3-diphenylbenzo[1,2,4]triazin-7-one ring.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Triazinas/farmacología , Antineoplásicos/química , Línea Celular Transformada , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triazinas/químicaRESUMEN
Synthesis and cytotoxicity of imidazo[5,4-f]benzimidazolequinones and iminoquinone derivatives is described, enabling structure-activity relationships to be obtained. The most promising compound (an iminoquinone derivative) has undergone National Cancer Institute (NCI) 60 cell line (single and five dose) screening, and using the NCI COMPARE program, has shown correlation to NQO1 activity and to other NQO1 substrates. Common structural features suggest that the iminoquinone moiety is significant with regard to NQO1 specificity. Computational docking into the active site of NQO1 was performed, and the first comprehensive mitomycin C (MMC)-NQO1 docking study is presented. Small distances for hydride reduction and high binding affinities are characteristic of MMC and of iminoquinones showing correlations with NQO1 via COMPARE analysis. Docking also indicated that the presence of a substituent capable of hydrogen bonding to the His194 residue is important in influencing the orientation of the substrate in the NQO1 active site, leading to more efficient reduction.
Asunto(s)
Bencimidazoles/toxicidad , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Quinonas/química , Programas Informáticos , Antineoplásicos/química , Antineoplásicos/toxicidad , Sitios de Unión , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Modelos Moleculares , NAD(P)H Deshidrogenasa (Quinona)/química , Quinonas/toxicidad , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: While locally advanced prostate cancer is initially treatable with androgen ablation, eventually cells develop a castrate-resistant phenotype. Currently, there are no effective treatments for this form of the disease with Docetaxel only providing a small survival advantage. In this study, the effects of novel derivatives of titanocene dichloride on prostate cancer cell lines has been investigated. METHODS: Cellular effects were assessed using the crystal violet assay and the clonogenic survival assay. Cell cycle and apoptosis were assessed by propidium iodide staining. DNA damage was analyzed by comet assay and Western analysis. DNA damage response inhibition was achieved by pre-incubation with an ATM/ATR inhibitor; CGK733 and DNA-PK inhibitor; DMNB. RESULTS: These analogs caused a reduction in cell number. In particular titanocene Y and C had significant effects in all cell lines. A reduction in clonogenic survival was found in response to titanocene Y in three cell lines while the PC-3 cells exhibited increased resistance.Further analysis showed no effect on cell cycle however, the analogs were found to induce apoptosis in a dose-dependent manner in all cell lines. These analogs associate with DNA, induce DNA damage and a differential damage response. Inhibition of key regulators of this DNA damage response sensitized the PC-3 cell line to titanocene-induced apoptosis and significantly reduced the clonogenic capacity of the cells. CONCLUSION: These results demonstrate the mechanism of action of these novel titanocene dichloride analogs and their potential use in castrate-independent advanced prostate cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Daño del ADN , Compuestos Organometálicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Violeta de Genciana/química , Humanos , Masculino , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Titanio/químicaRESUMEN
Anionic aromatic ipso-substitution has allowed an aziridine ring to be fused onto pyrrolo[1,2-a]benzimidazole. This diazole analogue of aziridinomitosene, and N-[(aziridinyl)methyl]-1H-benzimidazole are shown to be significantly more cytotoxic towards the human breast cancer cell lines MCF-7 and HCC1937 than towards a human normal fibroblast cell line (GM00637). The aziridinyl fused pyrrolo[1,2-a]benzimidazole is less cytotoxic than the non-ring fused aziridinyl analogue towards all three cell lines. The BRCA1-deficient HCC1937 cells are more sensitive to mitomycin C (MMC) compared to GM00637 and MCF-7 cells. The evidence provided indicates that different pathways may mediate cellular response to benzimidazole-containing aziridine compounds compared to MMC.
Asunto(s)
Antineoplásicos/farmacología , Aziridinas/química , Bencimidazoles/farmacología , Neoplasias de la Mama/patología , Mama/efectos de los fármacos , Antineoplásicos/síntesis química , Antineoplásicos/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Mama/citología , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.
Asunto(s)
Daño del ADN , Mitosis , Proteína de Replicación A/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Mitosis/efectos de la radiación , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Radiación Ionizante , Proteína de Replicación A/química , Serina/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Genomic DNA is constantly damaged by exposure to exogenous and endogenous agents. Bulky adducts such as UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA present a barrier to DNA synthesis by the major eukaryotic replicative polymerases including DNA polymerase delta. Translesion synthesis (TLS) carried out by specialized DNA polymerases is an evolutionarily conserved mechanism of DNA damage tolerance. The Y family of DNA polymerases, including DNA polymerase eta (Pol eta), the subject of this chapter, play a key role in TLS. Mutations in the human POLH gene encoding Pol eta underlie the genetic disease xeroderma pigmentosum variant (XPV), characterized by sun sensitivity, elevated incidence of skin cancer, and at the cellular level, by delayed replication and hypermutability after UV-irradiation. Pol eta is a low fidelity enzyme when copying undamaged DNA, but can carry out error-free TLS at sites of UV-induced dithymine CPDs. The active site of Pol eta has an open conformation that can accommodate CPDs, as well as cisplatin-induced intrastrand DNA crosslinks. Pol eta is recruited to sites of replication arrest in a tightly regulated process through interaction with PCNA. Pol eta-deficient cells show strong activation of downstream DNA damage responses including ATR signaling, and accumulate strand breaks as a result of replication fork collapse. Thus, Pol eta plays an important role in preventing genome instability after UV- and cisplatin-induced DNA damage. Inhibition of DNA damage tolerance pathways in tumors might also represent an approach to potentiate the effects of DNA damaging agents such as cisplatin.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , HumanosRESUMEN
DNA lesions arising from both exogenous and endogenous sources occur frequently in DNA. During DNA replication, the presence of unrepaired DNA damage in the template can arrest replication fork progression, leading to fork collapse, double-strand break formation, and to genome instability. To facilitate completion of replication and prevent the generation of strand breaks, DNA damage tolerance (DDT) pathways play a key role in allowing replication to proceed in the presence of lesions in the template. The two main DDT pathways are translesion synthesis (TLS), which involves the recruitment of specialized TLS polymerases to the site of replication arrest to bypass lesions, and homology-directed damage tolerance, which includes the template switching and fork reversal pathways. With some exceptions, lesion bypass by TLS polymerases is a source of mutagenesis, potentially contributing to the development of cancer. The capacity of TLS polymerases to bypass replication-blocking lesions induced by anti-cancer drugs such as cisplatin can also contribute to tumor chemoresistance. On the other hand, during homology-directed DDT the nascent sister strand is transiently utilised as a template for replication, allowing for error-free lesion bypass. Given the role of DNA damage tolerance pathways in replication, mutagenesis and chemoresistance, a more complete understanding of these pathways can provide avenues for therapeutic exploitation. A number of small molecule inhibitors of TLS polymerase activity have been identified that show synergy with conventional chemotherapeutic agents in killing cancer cells. In this review, we will summarize the major DDT pathways, explore the relationship between damage tolerance and carcinogenesis, and discuss the potential of targeting TLS polymerases as a therapeutic approach.
RESUMEN
Bu(3)SnH/1,1'-azobis(cyclohexanecarbonitrile) (ACN)-mediated five, six, and seven-membered double alkyl radical cyclizations onto imidazo[5,4-f]benzimidazole and imidazo[4,5-f]benzimidazole are described. The quinone derivatives evaluated show selective toxicity towards human cervical (HeLa) and prostate (DU145) cancer cell lines (with negligible toxicity towards a normal human cell line, GM00637). Only the Fremy oxidation of the 6-aminoimidazo[5,4-f]benzimidazole gave iminoquinone, which showed high specificity towards the prostate cancer cell line (DU145).
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Antineoplásicos/síntesis química , Bencimidazoles/síntesis química , Línea Celular Tumoral , Humanos , Concentración 50 InhibidoraRESUMEN
A series of new aromatic monoesters of alpha-aminoaralkylphosphonic acids were synthesized by selective hydrolysis of corresponding aromatic diesters of alpha-aminoaralkylphosphonic acids. New potential inhibitors of aminopeptidase N/CD13, an enzyme important in tumour angiogenesis, were developed. Some derivatives of the homophenylalanine and norleucine related monoaryl phosphonates displayed higher inhibition potency than corresponding alpha-aminoaralkylphosphonic acids toward aminopeptidase N/CD13. The effect of one of the new inhibitors on the growth of human PANC-1 and HT-1080 cell lines was examined, either alone or in combination with TNF-alpha.
Asunto(s)
Inhibidores de la Angiogénesis/química , Antígenos CD13/antagonistas & inhibidores , Ácidos Fosfínicos/química , Inhibidores de Proteasas/química , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Antígenos CD13/metabolismo , Línea Celular Tumoral , Humanos , Ácidos Fosfínicos/síntesis química , Ácidos Fosfínicos/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A diazole is N-substituted with 1-trityl-2-methylaziridine and demethylated and oxidised with NBS under acidic conditions to give a benzimidazolequinone; this novel anti-tumour agent is marginally more cytotoxic than mitomycin C (MMC) towards the normal human fibroblast cell line GM00637, while the MMC-hypersensitive human Fanconi anaemia (FA) cell line, PD20i, lacking the FANCD2 protein, is also hypersensitive to the benzimidazolequinone, with expression of FANCD2 protein decreasing sensitivity to both MMC and the benzimidazolequinone.
Asunto(s)
Aziridinas/síntesis química , Aziridinas/toxicidad , Bencimidazoles/síntesis química , Bencimidazoles/toxicidad , Anemia de Fanconi/patología , Quinolinas/síntesis química , Quinolinas/toxicidad , Piel/citología , Piel/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/biosíntesis , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Estructura Molecular , Estereoisomerismo , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
Isosorbide was functionalized with furoxan for the first time to give adducts that release nitric oxide up to 7.5 times faster than the commercial vasodilator, isosorbide-5-mononitrate (Is5N). The synthesis was facilitated by MeMgCl-mediated selective acetylation of isosorbide or selective deacetylation of isosorbide-2,5-diacetate, which was rationalized in terms of a more stable 5-alkoxide magnesium salt using DFT. Isosorbide-furoxans are safer to handle than Is5N due to greater thermal stability.
RESUMEN
Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/deficiencia , Proteína de Replicación A/metabolismo , Rayos Ultravioleta , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN/biosíntesis , ADN/efectos de la radiación , Daño del ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Fibroblastos/citología , Regulación Enzimológica de la Expresión Génica , Histonas/metabolismo , Humanos , Cinética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de la radiación , Proteínas Quinasas/metabolismo , Tetraciclina/metabolismoRESUMEN
Skin cancer, the most common cancer in the general population, is strongly associated with exposure to the ultraviolet component of sunlight. To investigate the relationship between DNA damage processing and skin tumour development, we determined the POLH status of a cohort of skin cancer patients. The human POLH gene encodes DNA polymerase eta (poleta), which normally carries out accurate translesion synthesis past the major UV-induced photoproduct, the dithymine cyclobutane dimer. In the absence of active poleta in xeroderma pigmentosum variant (XPV) patients, mutations accumulate at sites of UV-induced DNA damage, providing the initiating step in skin carcinogenesis. Forty patients diagnosed with skin cancer were genotyped for polymorphisms in the POLH protein-coding sequence, using glycosylase-mediated polymorphism detection (GMPD) and direct DNA sequencing of POLH PCR products derived from white blood cell genomic DNA. All individuals carried the wild-type POLH sequence. No POLH mutations were identified in genomic DNA from skin tumours derived from 15 of these patients. As determined by RT-PCR, POLH mRNA was expressed in all normal and skin tumour tissue examined. Poleta protein was also detectable by Western blotting, in two matched normal and skin tumour extracts. An alternatively spliced form of POLH mRNA, lacking exon 2, was more readily detected in skin tissue than in white blood cells from the same patient. Real-time PCR was used to quantify POLH expression in matched normal and skin tumour-derived mRNA from a series of patients diagnosed with either basal or squamous cell carcinoma. Compared to matched normal skin tissue from the same patient, 1 of 7 SCC, and 4 of 10 BCC tumours examined showed at least a 2-fold reduction in POLH expression, while 1 of 7 SCC, and 3 of 10 BCC tumours showed at least a 2-fold increase in POLH expression. Differences in gene expression, rather than sequence changes may be the main mechanism by which POLH status varies between normal and skin tumours in the population under investigation. Knowledge of the POLH status in skin tumours could contribute to an understanding of the role of this gene in the development of the most common cancer in the general population.
Asunto(s)
Carcinoma de Células Escamosas/genética , ADN Polimerasa Dirigida por ADN/genética , Expresión Génica , Neoplasias Cutáneas/genética , Anciano , Anciano de 80 o más Años , Empalme Alternativo , Secuencia de Bases , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Polimorfismo Genético , ARN Mensajero , Análisis de Secuencia de ADNRESUMEN
Nuclear substructures associated with apoptosis in HeLa cells have been examined using light-microscopic morphometry, trichrome staining, spectral imaging and transmission electron microscopy. This detailed analysis reveals several sites where alterations in the normal cellular ultrastructure occur during apoptotic progression. To correlate these ultrastructural changes with the underlying molecular processes, we have characterized and quantified apoptotic cell morphology with and without inhibition of two caspases, which are key effectors of the apoptotic program. Using this analysis, early apoptotic events included: (a) the segregation of nucleolar components, a diminished granular component, and a reduction in number but increase in size of fibrillar centers, (b) an increase in the number of cytoplasmic ribosomes and (c) a very minimal increase in the amount of peripherally condensed DNA. Apoptosis progressed with: (a) an increase in the number of perichromatin granules and perichromatin fibrils, (b) a reduction in number of interchromatin granule centers concomitant with an increase in their size, (c) partial digestion and circumferential condensation of the DNA at the nuclear membrane and (d) rounding of the cytoplasm with an increase in organellar density and shrinkage in cell size. Endstage apoptotic cells showed: (a) one (or two) very large pools of incompletely digested DNA, (b) one (or two) very large interchromatin granule centers, (c) an increased number of perichromatin granules which were distanced from DNA and often closely apposed to the nucleolus, (d) formation of unusually condensed, highly coiled perinucleolar bodies and (e) blebbing of highly dense cytoplasm. In HeLa cells treated with UV and inhibitors of caspase 1 and 3, the length of time from early apoptosis to the formation of apoptotic bodies was greatly extended. Inhibiting caspase activity: (a) prevented the pooling of DNA, (b) retarded the formation of large interchromatin granule centers, (c) increased the number of perichromatin granules, (d) produced disassembly of the nucleolus, (e) prevented the formation of highly coiled perinucleolar bodies, and (f) caused vacuolization in the cell center and a unipolar blebbing of the cytoplasm. Spectral imaging in conjunction with serial section electron microscopy confirmed the staining specificities of the condensed DNA, of the large condensed interchromatin granule centers, and of the nucleoli. The results indicate that the interface between the components of the chromatin domain and the interchromatin space is a critical site of caspase activity in apoptosis, and precedes other events such as internucleosomal DNA degradation.
Asunto(s)
Apoptosis/efectos de la radiación , Compuestos Azo/metabolismo , Eosina Amarillenta-(YS)/metabolismo , Células HeLa/efectos de la radiación , Células HeLa/ultraestructura , Verde de Metilo/metabolismo , Rayos Ultravioleta , Humanos , Microscopía Electrónica/métodos , Análisis Espectral/métodosRESUMEN
Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.
Asunto(s)
Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Platino (Metal)/farmacología , Carboplatino/farmacología , Carboplatino/toxicidad , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular , Cisplatino/farmacología , Cisplatino/toxicidad , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , Sinergismo Farmacológico , Expresión Génica , Humanos , Fosforilación , Platino (Metal)/toxicidad , Proteína de Replicación A/metabolismoRESUMEN
Anthracyclines, including doxorubicin, are widely used in the treatment of leukemia. While the effects of doxorubicin on hematopoietic cells have been characterized, less is known about the response of human mesenchymal stem cells (hMSCs) in the bone marrow stroma to anthracyclines. We characterized the effect of doxorubicin on key DNA damage responses in hMSCs, and compared doxorubicin sensitivity and DNA damage response activation between isolated hMSCs and the chronic myelogenous leukemia cell line, K562. Phosphorylation of H2AX, Chk1, and RPA2 was more strongly activated in K562 cells than in hMSCs, at equivalent doses of doxorubicin. hMSCs were relatively resistant to doxorubicin such that, following exposure to 15 µM doxorubicin, the level of cleaved caspase-3 detected by western blotting was lower in hMSCs compared to K562 cells. Flow cytometric analysis of cell cycle progression demonstrated that exposure to doxorubicin induced G2/M phase arrest in hMSCs, while 48 h after exposure, 15.6 % of cells were apoptotic, as determined from the percentage of cells having sub-G1 DNA content. We also show that the doxorubicin sensitivity of hMSCs isolated from a healthy donor was comparable to that of hMSCs isolated from a chronic lymphocytic leukemia patient. Overall, our results demonstrate that high doses of doxorubicin induce the DNA damage response in hMSCs, and that cultured hMSCs are relatively resistant to doxorubicin.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Daño del ADN , Doxorrubicina/efectos adversos , Células Madre Mesenquimatosas/metabolismo , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Doxorrubicina/farmacología , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Histonas/metabolismo , Humanos , Pirofosfatasa Inorgánica/metabolismo , Células K562 , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Masculino , Células Madre Mesenquimatosas/patología , Proteínas Mitocondriales/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismoRESUMEN
DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-Irradiation arrested hMSCs in the G 1 phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.