RESUMO
It is believed that normal cells with an unaffected DNA damage response (DDR) and DNA damage repair machinery, could be less prone to DNA damaging treatment than cancer cells. However, the anticancer drug, etoposide, which is a topoisomerase II inhibitor, can generate DNA double strand breaks affecting not only replication but also transcription and therefore can induce DNA damage in non-replicating cells. Indeed, we showed that etoposide could influence transcription and was able to activate DDR in resting human T cells by inducing phosphorylation of ATM and its substrates, H2AX and p53. This led to activation of PUMA, caspases and to apoptotic cell death. Lymphoblastoid leukemic Jurkat cells, as cycling cells, were more sensitive to etoposide considering the level of DNA damage, DDR and apoptosis. Next, we used ATM inhibitor, KU 55933, which has been shown previously to be a radio/chemo-sensitizing agent. Pretreatment of resting T cells with KU 55933 blocked phosphorylation of ATM, H2AX and p53, which, in turn, prevented PUMA expression, caspase activation and apoptosis. On the other hand, KU 55933 incremented apoptosis of Jurkat cells. However, etoposide-induced DNA damage in resting T cells was not influenced by KU 55933 as revealed by the FADU assay. Altogether our results show that KU 55933 blocks DDR and apoptosis induced by etoposide in normal resting T cells, but increased cytotoxic effect on proliferating leukemic Jurkat cells. We discuss the possible beneficial and adverse effects of drugs affecting the DDR in cancer cells that are currently in preclinical anticancer trials.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Etoposídeo/toxicidade , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Células Jurkat , Morfolinas/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pironas/farmacologia , Linfócitos T/efeitos dos fármacos , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA- and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells committed to die, a portion of the cellular DEK pool is extensively posttranslationally modified by phosphorylation and poly(ADP-ribosyl)ation. Through interference with DEK expression, we further show that DEK promotes the repair of DNA lesions and protects cells from genotoxic agents that typically trigger poly(ADP-ribose) polymerase activation. The posttranslational modification of DEK during apoptosis is accompanied by the removal of the protein from chromatin and its release into the extracellular space. Released modified DEK is recognized by autoantibodies present in the synovial fluids of patients affected by juvenile rheumatoid arthritis/juvenile idiopathic arthritis. These findings point to a crucial role of poly(ADP-ribosyl)ation in shaping DEK's autoantigenic properties and in its function as a promoter of cell survival.
Assuntos
Apoptose/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Mutagênicos/toxicidade , Proteínas Oncogênicas/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Animais , Anticorpos Monoclonais , Artrite Juvenil/imunologia , Autoanticorpos/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Células HeLa , Humanos , Células Jurkat , Camundongos , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Poly(ADP-ribosyl)ation is a posttranslational modification of proteins in eukaryotic cells catalysed by a family of NAD+ ADP-ribosyl transferases, the poly(ADP-ribose) polymerases (PARPs). PARP-encoding genes now constitute a superfamily of at least 18 members encoding proteins that share homology with the catalytic domain of the founding member, PARP-1. Poly(ADP-ribose) metabolism is of central importance in a wide variety of biological processes including maintenance of genomic stability, DNA repair, transcriptional regulation, centromere function, modulation of telomere length, regulation of proteasomal protein degradation, regulation of endosomal vesicle trafficking and apoptosis. The life cycle of poly(ADP-ribose) is discussed in the following section. In addition, an overview of the genes and proteins involved in poly(ADP-ribose) metabolism and their possible cellular function is provided.
Assuntos
Centrômero/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Telômero/metabolismo , Animais , Morte Celular/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição GênicaRESUMO
Nickel, cadmium, cobalt, and arsenic compounds are well-known carcinogens to humans and experimental animals. Even though their DNA-damaging potentials are rather weak, they interfere with the nucleotide and base excision repair at low, noncytotoxic concentrations. For example, both water-soluble Ni(II) and particulate black NiO greatly reduced the repair of DNA adducts induced by benzo[a]pyrene, an important environmental pollutant. Furthermore, Ni(II), As(III), and Co(II) interfered with cell cycle progression and cell cycle control in response to ultraviolet C radiation. As potential molecular targets, interactions with so-called zinc finger proteins involved in DNA repair and/or DNA damage signaling were investigated. We observed an inactivation of the bacterial formamidopyrimidine-DNA glycosylase (Fpg), the mammalian xeroderma pigmentosum group A protein (XPA), and the poly(adenosine diphosphate-ribose)polymerase (PARP). Although all proteins were inhibited by Cd(II) and Cu(II), XPA and PARP but not Fpg were inhibited by Co(II) and Ni(II). As(III) deserves special attention, as it inactivated only PARP, but did so at very low concentrations starting from 10 nM. Because DNA is permanently damaged by endogenous and environmental factors, functioning processing of DNA lesions is an important prerequisite for maintaining genomic integrity; its inactivation by metal compounds may therefore constitute an important mechanism of metal-related carcinogenicity.
Assuntos
Ciclo Celular/efeitos dos fármacos , Reparo do DNA , Metais Pesados/efeitos adversos , Dedos de Zinco , Animais , Proteínas de Ligação a DNA/farmacologia , DNA-Formamidopirimidina Glicosilase , Humanos , N-Glicosil Hidrolases/farmacologia , Poli(ADP-Ribose) Polimerases/farmacologia , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
Metal ions are essential components of biological systems; nevertheless, even essential elements may have toxic or carcinogenic properties. Thus, besides As(III) and Cd(II), also Ni(II) and Co(II) have been shown previously to disturb different types of DNA repair systems at low, non-cytotoxic concentrations. Since some metals exert high affinities for SH groups, we investigated whether zinc finger structures in DNA-binding motifs of DNA repair proteins are potential targets for toxic metal ions. The bacterial formamidopyrimidine-DNA glycosylase (Fpg protein) involved in base excision repair was inhibited by Cd(II), Cu(II) and Hg(II) with increasing efficiencies, whereas Co(II), As(III), Pb(II) and Ni(II) had no effect. Furthermore, Cd(II) still disturbed enzyme function when bound to metallothionein. Strong inhibition was also observed in the presence of phenylselenyl chloride, followed by selenocystine, while selenomethionine was not inhibitory. Regarding the mammalian XPA protein involved in the recognition of DNA lesions during nucleotide excision repair, its DNA-binding capacity was diminished by Cd(II), Cu(II), Ni(II) and Co(II), while Hg(II), Pb(II) and As(III) were ineffective. Finally, the H(2)O(2)-induced activation of the poly(ADP-ribose)polymerase (PARP) involved in DNA strand break detection and apoptosis was greatly reduced by Cd(II), Co(II), Ni(II) and As(III). Similarly, the disruption of correct p53 folding and DNA binding by Cd(II), Ni(II) and Co(II) has been shown by other authors. Therefore, zinc-dependent proteins involved in DNA repair and cell-cycle control may represent sensitive targets for some toxic metals such as Cd(II), Ni(II), Co(II) and Cu(II), as well as for some selenium compounds. Relevant mechanisms of inhibition appear to be the displacement of zinc by other transition metals as well as redox reactions leading to thiol/disulfide interchange.
Assuntos
Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Metais Pesados/toxicidade , Dedos de Zinco/efeitos dos fármacos , Zinco/toxicidade , Cátions Bivalentes , Reparo do DNA/fisiologia , Humanos , N-Glicosil Hidrolases/metabolismo , Dobramento de Proteína , Proteínas de Ligação a RNA/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A , Dedos de Zinco/genéticaRESUMO
One of the immediate eukaryotic cellular responses to DNA breakage is the covalent post-translational modification of nuclear proteins with poly(ADP-ribose) from NAD+ as precursor, mostly catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Recently several other polypeptides have been shown to catalyse poly(ADP-ribose) formation. Poly(ADP-ribosyl)ation is involved in a variety of physiological and pathophysiological phenomena. Physiological functions include its participation in DNA-base excision repair, DNA-damage signalling, regulation of genomic stability, and regulation of transcription and proteasomal function, supporting the previously observed correlation of cellular poly(ADP-ribosyl)ation capacity with mammalian life. The pathophysiology effects are mediated through PARP-1 overactivity, which can cause cell suicide by NAD+ depletion. It is apparent that the latter effect underlies the pathogenesis of a wide range of disease states including type-1 diabetes, ischaemic infarcts in various organs, and septic or haemorrhagic shock. Therefore pharmacological modulation of poly(ADP-ribosyl)ation may prove to be an exciting option for various highly prevalent, disabling and even lethal diseases.
Assuntos
Reparo do DNA , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cisteína Endopeptidases/genética , Dano ao DNA , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/fisiopatologia , Regulação da Expressão Gênica , Humanos , Infarto/enzimologia , Infarto/fisiopatologia , Modelos Biológicos , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , Transcrição GênicaRESUMO
Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation or oxidants. The resulting formation of protein-coupled poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage, probably via its contribution to DNA base-excision repair. Furthermore, based on recent results there is a role emerging for PARP-1 as a negative regulator of genomic instability in cells under genotoxic stress. Regarding possible applications for clinical cancer therapy with DNA-damaging agents, it appears that both inhibition and up-regulation of the poly(ADP-ribosyl)ation response in the malignant cells to be eradicated are promising strategies to improve the outcome of such therapy, albeit for different reasons.
Assuntos
Aberrações Cromossômicas/genética , Dano ao DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Ativação Enzimática , Genoma , HumanosRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) encoded by the PARP-1 gene, is a ubiquitous and abundant DNA-binding protein involved in the cellular response to various genotoxic agents. In a previous study we showed that maximal oligonucleotide-stimulated poly(ADP-ribosyl)ation was significantly higher in permeabilised lymphoblastoid cell lines from a French population of centenarians compared with controls aged 20-70 years, supporting the notion that longevity is associated with a genetically determined, high poly(ADP-ribosyl)ation capacity. Here, we describe four new genetic polymorphisms, three of which represent silent nucleotide variants (C402T, T1011C, G1215A), and one of which leads to a valine762-to-alanine exchange (T2444C). We undertook an association study between two of these polymorphisms and human longevity or poly(ADP-ribosyl)ation capacity in permeabilised lymphoblastoid cells. By analysing 648 DNA samples from a French population (324 centenarians and 324 controls) by fluorescent-allele-specific PCR, we showed the absence of any significant enrichment of any of the genotypes in the study of centenarians versus controls. Furthermore, we studied genotype distributions from individuals who had previously been tested for poly(ADP-ribosyl)ation capacity. None of the genotype combinations at any polymorphic site studied could be related to a high or low level of poly(ADP-ribosyl)ation capacity. Together, these results strongly suggest that the longevity-related differences in the poly(ADP-ribosyl)ation capacity of human lymphoblastoid cell lines cannot be explained by genetic polymorphisms in the PARP-1 coding sequence and that other mechanisms have to be considered as potential regulators of specific poly(ADP-ribosyl)ation capacity.
Assuntos
Longevidade/genética , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Polimorfismo Genético , Idoso , Idoso de 80 Anos ou mais , Sítios de Ligação , Feminino , França , Genótipo , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Poli(ADP-Ribose) Polimerases/química , Reação em Cadeia da Polimerase , Estrutura Secundária de ProteínaRESUMO
Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is catalytically activated by DNA strand interruptions. It catalyses the covalent modification of proteins with ADP-ribose polymers, using NAD(+) as precursor. Here, we have studied the DNA damage-induced formation of poly(ADP-ribose) in intact human peripheral blood lymphocytes (PBL) by in-situ immunofluorescence detection. The response of PBL to bleomycin (BLM), which is known to induce DNA single and double strand breaks, was investigated with regard to polymer formation. For this purpose, a quantitative approach was developed to assess more accurately the immunostaining of polymer formation by computerised image analysis. As an application of this new method, we have determined the polymer formation following BLM treatment in quiescent human PBL versus mitogen activated cells. Quiescent human PBL showed a similar basal immunostaining for the polymer compared to phytohemagglutinin (PHA)-activated cells, expressed as relative mean pixel intensity (RMPI) (1.3+/-0.8 and 2.2+/-0.9, respectively; P<0.3). After BLM treatment, there was a clear-cut enhancement of polymer immunostaining, with PHA-activated cells showing significantly higher RMPI than non-activated cells (9.2+/-1.4 and 4.2+/-1.0, respectively; P<0.005). As expected, in the presence of the ADP-ribosylation inhibitor 3-aminobenzamide (3-AB), the RMPI of immunostained polymer was decreased in both quiescent and PHA-activated PBL to 1.2+/-0.7 and 1.5+/-0.9, respectively. Our findings reveal (i) that mitogen-stimulated, intact lymphocytes show enhanced polymer formation following BLM treatment, and (ii) that our new quantitative immunofluorescence assay coupled with computerised image analysis is reliable and sensitive enough to detect changes in polymer formation rate.
Assuntos
Bleomicina/farmacologia , Substâncias Intercalantes/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Poli Adenosina Difosfato Ribose/biossíntese , Adulto , Antibióticos Antineoplásicos/farmacologia , Dano ao DNA , Feminino , Imunofluorescência/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Fito-Hemaglutininas/farmacologiaRESUMO
One of the earliest responses to DNA damage in eukaryotic cells is activation of poly(ADP-ribose) polymerase-1 (PARP-1), a DNA strand break-dependent nuclear enzyme which covalently modifies proteins with poly(ADP-ribose). Here, we show that conditional over-expression of PARP-1 in stably transfected hamster cells, which causes cellular over-accumulation of poly(ADP-ribose) by several-fold, strongly suppresses alkylation-induced sister-chromatid exchange (SCE), while cytotoxicity of alkylation treatment is slightly enhanced. Viewed together with the known potentiation of SCE by abrogation of PARP-1 activity, our results provide evidence that PARP-1 activity is an important regulator of alkylation-induced SCE formation, imposing a control that is strictly negative and commensurate with the level of enzyme activity.
Assuntos
DNA/metabolismo , Isoenzimas/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Troca de Cromátide Irmã , Alquilação , Animais , Linhagem Celular , Cricetinae , Dano ao DNA , Dexametasona/farmacologia , Metilnitronitrosoguanidina/toxicidadeRESUMO
Poly(ADP-ribosyl)ation is a DNA strandbreak-driven posttranslational modification of nuclear proteins that is catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), with NAD+ serving as substrate. Recently, additional PARP isoforms were described that seem to account for a minor fraction of cellular poly(ADP-ribose) synthesis. We have previously described a correlation between poly(ADP-ribosyl)ation capacity of mononuclear leukocytes of various mammalian species and species-specific life span. Likewise, lymphoblastoid cell lines derived from human centenarians display a higher poly(ADP-ribosyl)ation capacity than do controls. At the functional level, recent data show that PARP-1 is a key regulator of alkylation-induced sister-chromatid exchange, imposing a negative control commensurate with the enzyme activity. PARP-1 activity may therefore be responsible for tuning the rate of genomic instability events that are provoked by the constant attack of endogenous and exogenous genotoxins to a level appropriate for the longevity potential of a given organism or species.
Assuntos
Senescência Celular/fisiologia , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Sobrevivência Celular , HumanosRESUMO
Beta-D-glucosyl-ifosfamide mustard (D 19575, glc-IPM, INN = glufosfamide) is a new agent for cancer chemotherapy. Its mode of action, which is only partly understood, was investigated at the DNA level. In the breast carcinoma cell line MCF7 glufosfamide inhibited both the synthesis of DNA and protein in a dose-dependent manner, as shown by the decreased incorporation of [3H-methyl]-thymidine into DNA and [14C]-methionine into protein of these cells. Treatment of MCF7 cells with 50 microM glufosfamide was sufficient to trigger poly(ADP-ribose) polymerase (PARP) activation, as revealed by immunofluorescence analysis. Both CHO-9 cells, which are O6-methylguanine-DNA methyltransferase (MGMT)-deficient, and an isogenic derivative, which has a high level of MGMT, showed the same cytotoxic response to beta-D-glc-IPM, indicating that the O6 position of guanine is not the critical target for cytotoxicity. By contrast, a sharp decrease in survival of cross-link repair deficient CL-V5 B cells was observed already at concentrations of 0.1 mM beta-D-glc-IPM, whereas the wild-type V79 cells showed a 90% reduction in survival only after treatment with 0.5 mM of this compound. The therapeutically inactive beta-L-enantiomer of glufosfamide also showed genotoxic effects in the same assays but at much higher doses. This was probably due to small amounts of ifosfamide mustard formed under the conditions of incubation. The results indicate that the DNA crosslinks are the most critical cytotoxic lesions induced by beta-D-glc-IPM.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Mostardas de Fosforamida/farmacologia , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Células CHO , Cricetinae , Dano ao DNA , Reparo do DNA , Glucose/análogos & derivados , Humanos , Ifosfamida/análogos & derivados , Poli(ADP-Ribose) Polimerases/metabolismo , Células Tumorais CultivadasRESUMO
Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by the 113-kDa enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and, to a lesser extent, by several other recently described polypeptides. The catalytic function of PARP-1 is directly stimulated by DNA strand breaks, thus making poly(ADP-ribosyl)ation one of the immediate cellular responses to oxidative and other types of DNA damage. Poly(ADP-ribosyl)ation plays an important role in the recovery of proliferating cells from certain types of DNA damage, and this has been linked mechanistically with an involvement in DNA base-excision repair. Furthermore PARP-1 activity is necessary to maintain genomic stability under conditions of genotoxic stress and is actually a key regulator of alkylation-induced sister-chromatid exchange formation, imposing a control that is strictly negative and commensurate with the enzyme activity level. Finally, there is a positive correlation between the poly(ADP-ribosyl)ation capacity of mononuclear leukocytes of various mammalian species and species-specific life span. Likewise, lymphoblastoid cell lines derived from human centenarians display a higher poly(ADP-ribosyl)ation capacity than controls. In conclusion, PARP-1 may be viewed as a factor that is responsible for downregulating the rate of genomic instability events, which are provoked by the constant attack by endogenous and exogenous DNA-damaging agents, in such a way as to tune them to a level which is just appropriate for the life span potential of a given species.
Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Genoma , Humanos , Longevidade/genética , Longevidade/fisiologia , Mamíferos , TelômeroRESUMO
Poly(ADP-ribose) polymerase (PARP4) catalyzes the formation of ADP-ribose polymers covalently attached to proteins by using NAD+ as substrate. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. We characterized a dominant negative PARP mutant, i.e. the DNA-binding domain of this enzyme, whose overexpression in cells leads to increased genetic instability following DNA damage. In order to study whether PARP activity is also implicated in the process of tumorigenesis, we generated stably transfected HeLa cell clones with constitutive overexpression of dominant negative PARP and investigated tumor formation of these clones in nude mice. We found that inhibition of PARP activity dramatically reduces tumor forming ability of HeLa cells. Moreover, we provide strong evidence that the observed reduction in tumor forming ability is due to increased tumor cell apoptosis in vivo. Viewed together, our data and those from other groups show that inhibition of PARP enzyme activity interferes with DNA base excision repair and leads to increased genetic instability and recombination but, on the other hand, can sensitize cells to apoptotic stimuli and by this mechanism may prevent tumor formation.
Assuntos
Apoptose/genética , Neoplasias Experimentais/patologia , Poli(ADP-Ribose) Polimerases/genética , Animais , Células HeLa , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/enzimologia , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
Alkylation treatment of HeLa cells results in the rapid induction of apoptosis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP) into the 29-and 85-kDa fragments (Kumari S. R., Mendoza-Alvarez, H. & Alvarez-Gonzalez, R. (1998) Cancer Res. 58, 5075-5078). Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.
Assuntos
Apoptose , Nucléolo Celular/metabolismo , Fragmentos de Peptídeos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Alquilação , Dano ao DNA , Células HeLa , Humanos , Peso Molecular , Fatores de TempoRESUMO
UV-radiation is a major risk factor for non-melanoma skin cancer causing specific mutations in the p53 tumor suppressor gene and other genetic aberrations. We here propose that elevated temperature, as found in sunburn areas, may contribute to skin carcinogenesis as well. Continuous exposure of immortal human HaCaT skin keratinocytes (possessing UV-type p53 mutations) to 40 degrees C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors. Growth at 40 degrees C was correlated with the appearance of PARP, an enzyme activated by DNA strand breaks and the level corresponded to that seen after 5 Gy gamma-radiation. Concomitantly, comparative genomic hybridization (CGH) analyis demonstrated that chromosomal gains and losses were present in cells maintained at 40 degrees C while largely absent at 37 degrees C. Besides individual chromosomal aberrations, all tumor-derived cells showed gain of chromosomal material on 11q with the smallest common region being 11q13.2 to q14.1. Cyclin D1, a candidate gene of that region was overexpressed in all tumor-derived cells but cyclinD1/cdk4/cdk6 kinase activity was not increased. Thus, these data demonstrate that long-term thermal stress is a potential carcinogenic factor in this relevant skin cancer model, mediating its effect through induction of genetic instability which results in selection of tumorigenic cells characterized by gain of 11q.
Assuntos
Transformação Celular Neoplásica , Temperatura Alta , Queratinócitos/patologia , Aneuploidia , Animais , Linhagem Celular Transformada , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Ciclina D1/biossíntese , Ciclina D1/genética , Dano ao DNA , Indução Enzimática , Raios gama , Humanos , Camundongos , Hibridização de Ácido Nucleico , Poli(ADP-Ribose) Polimerases/biossíntese , Poli(ADP-Ribose) Polimerases/genética , Pele/citologia , Neoplasias Cutâneas/etiologiaRESUMO
In most eukaryotic cells, the catalytic activation of poly(ADP-ribose) polymerase (PARP) represents one of the earliest cellular responses to the infliction of DNA damage. To study the biological function(s) of poly(ADP-ribosyl)ation, we have established stable transfectants (COM3 cells) of the SV40-transformed Chinese hamster cell line C060 which conditionally overexpress the PARP DNA-binding domain upon addition of dexamethasone. We could demonstrate that DNA-binding domain overexpression, which leads to trans-dominant inhibition of poly(ADP-ribosyl)ation, potentiates the cytotoxicity of alkylation treatment and of gamma-radiation. Likewise, carcinogen-induced gene amplification, viewed as a manifestation of genomic instability, was potentiated by the overexpression of the PARP DNA-binding domain. Recently, we studied the effect of trans-dominant PARP inhibition on mutagenesis by employing a shuttle-vector assay in which mutagen-exposed plasmid pYZ289 is electroporated into COM3 cells. We could show that dexamethasone-induced overexpression of the PARP DNA-binding domain in COM3 cells potentiates the mutagenicity of the alkylating agent N-methyl-N-nitrosourea, while no effect of dexamethasone treatment on mutation frequency was recorded in control cells lacking the PARP DNA-binding domain transgene. Taken together, our results further substantiate the role of poly(ADP-ribosyl)ation in the maintenance of genomic integrity and stability under conditions of genotoxic stress.
Assuntos
Mutagênese Insercional , Inibidores de Poli(ADP-Ribose) Polimerases , Alquilantes/farmacologia , Alquilação , Animais , Células CHO , Linhagem Celular Transformada , Cricetinae , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Humanos , Metilnitrosoureia/farmacologiaRESUMO
P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several factors shown to induce mdr1 overexpression (UV irradiation, epidermal growth factor, tumour necrosis factor alpha, doxorubicin) have been associated with the generation of reactive oxygen species (ROS). In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the mdr1b gene were used as a model system to investigate whether ROS might participate in the regulation of intrinsic mdr1b overexpression. Addition of H2O2 to the culture medium resulted in a significant increase in mdrlb mRNA and P-glycoprotein after 3 days of culture, with maximal (approximately 2-fold) induction being observed with 0.5-1 mM H2O2. Furthermore, H2O2 led to activation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, indicating that ROS reached the nuclear compartment. Thus, extracellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdrlb mRNA and P-glycoprotein expression. Conversely, antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycoprotein overexpression. Intracellular steady-state levels of the mdrl substrate rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreated with H2O2 or aminotriazole and decreased in antioxidant-treated cells. The induction of mdr1b mRNA and of functionally active mdr1-type P-glycoproteins by elevation in intracellular ROS levels and the repression of intrinsic mdrlb mRNA and P-glycoprotein overexpression by antioxidant compounds support the conclusion that the expression of the mdr1b P-glycoprotein is regulated in a redox-sensitive manner.