RESUMEN
p21CDKN1A (WAF1/CIP1/SDI1), the cyclin-dependent kinase (CDK) inhibitor belonging to the Cip/Kip family, was first described as a potent inhibitor of cell proliferation and DNA replication, both in physiological conditions and after DNA damage. More recently, p21 has been recognized to play additional and fundamental roles in other important pathways, including regulation of transcription, apoptosis and DNA repair. Knock-out mouse studies combined with biochemical and functional analysis of cells in culture have indicated a tumor suppressor activity for p21. However, these lines of evidence have been complicated by other findings indicating that p21 can exhibit oncogenic properties. In fact, the evidence that p21 expression may lead to proliferation arrest, is counterbalanced by the rescue of tumor cells from drug-induced apoptosis, and by promoting a metastatic potential. For these reasons, p21 is considered a protein with a dual behavior, with potential benefits, as well as dangerous effects of its expression in malignant cells. Thus, the effectiveness of targeting p21 expression for antitumor therapy needs to be carefully evaluated accordingly. This review summarizes the functions and regulations of p21, and focuses on its involvement in human diseases (particularly cancer), and on the pharmacological approaches to target p21 expression (either positively or negatively) for anticancer therapy. Based on these approaches, the search for new molecules that are able to promote the tumor-suppressor activity, and/or to interfere with the oncogenic properties of p21, could be promising.
Asunto(s)
Antineoplásicos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Neoplasias/metabolismoRESUMEN
This paper is an Italian Expert Consensus Document on multidimensional treatment of obesity and eating disorders. The Document is based on a wide survey of expert opinion. It presents, in particular, considerations regarding how clinicians go about choosing the most appropriate site of treatment for a given patient suffering from obesity and/or eating disorders: outpatient, partial hospitalization, residential rehabilitation centre, inpatient hospitalization. In a majority of instances obesity and eating disorders are long-term diseases and require a multiprofessional team-approach. In determining an initial level of care or a change to a different level of care, it is essential to consider together the overall physical condition, medical complications, disabilities, psychiatric comorbidity, psychology, behaviour, family, social resources, environment, and available services. We first created a review manuscript, a skeleton algorithm and two rating scales, based on the published guidelines and the existing research literature. As the second point we highlighted a number of clinical questions that had to be addressed in the specific context of our National Health Service and available specialized care units. Then we submitted eleven progressive revisions of the Document to the experts up to the final synthesis that was approved by the group. Of course, from point to point, some of the individual experts would differ with the consensus view. The document can be viewed as an expert consultation and the clinical judgement must always be tailored to the particular needs of each clinical situation. We will continue to revise the Document periodically based on new research information and on reassessment of expert opinion to keep it up-to-date. The Document was not financially sponsored.
Asunto(s)
Atención Ambulatoria , Testimonio de Experto , Trastornos de Alimentación y de la Ingestión de Alimentos/diagnóstico , Trastornos de Alimentación y de la Ingestión de Alimentos/terapia , Hospitalización , Obesidad/diagnóstico , Obesidad/terapia , Grupo de Atención al Paciente , Tratamiento Domiciliario , Algoritmos , Atención Ambulatoria/normas , Anorexia Nerviosa/diagnóstico , Anorexia Nerviosa/terapia , Trastorno por Atracón/diagnóstico , Trastorno por Atracón/terapia , Bulimia Nerviosa/diagnóstico , Bulimia Nerviosa/terapia , Comorbilidad , Consenso , Centros de Día , Evaluación de la Discapacidad , Trastornos de Alimentación y de la Ingestión de Alimentos/fisiopatología , Trastornos de Alimentación y de la Ingestión de Alimentos/psicología , Trastornos de Alimentación y de la Ingestión de Alimentos/rehabilitación , Adhesión a Directriz , Humanos , Italia , Actividad Motora , Programas Nacionales de Salud , Estado Nutricional , Obesidad/fisiopatología , Obesidad/psicología , Obesidad/rehabilitación , Guías de Práctica Clínica como Asunto , Tratamiento Domiciliario/normas , Factores de Riesgo , Medio Social , CaminataRESUMEN
Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antiproliferative activity. We previously found that 4-hydroxy group in the trans conformation was absolutely required for the inhibition of cell proliferation. In the present work we have synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene, which contains two OH in 4' and 4 positions, with the aim of developing a compound with an antiproliferative potential higher than that of resveratrol, on the basis of the correlation between structure and activity previously observed. In comparison with resveratrol, 4,4'-dihydroxy-trans-stilbene inhibited cell clonogenic efficiency of fibroblasts nine times more although with a different mechanism. First, 4,4'-dihydroxy-trans-stilbene induced predominantly an accumulation of cells in G1 phase, whereas resveratrol perturbed the G1/S phase transition. Second, although both compounds were able to inhibit DNA polymerase (pol) delta in an in vitro assay, 4, 4'-dihydroxy-trans-stilbene did not affect pol alpha activity. Finally, 4,4'-dihydroxy-trans-stilbene increased p21(CDKN1A) and p53 protein levels, whereas resveratrol led to phosphorylation of the S-phase checkpoint protein Chk1. Taken together, our results demonstrated for the first time that the two hydroxyl groups on 4- and 4'- positions of the stilbenic backbone enhance the antiproliferative effect and introduce additional targets in the mechanism of action of resveratrol. In conclusion, 4,4'-dihydroxy-trans-stilbene has potent antiproliferative activities that differ from the effect of resveratrol shown in this system, suggesting that it warrants further development as a potential chemopreventive or therapeutic agent.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Polimerasa III/antagonistas & inhibidores , Fibroblastos/citología , Estilbenos/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Pulmón/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Conformación Proteica , Resveratrol , Estilbenos/química , Vitis , VinoRESUMEN
Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.
Asunto(s)
Antocianinas/farmacología , Antioxidantes/farmacología , Daño del ADN , terc-Butilhidroperóxido/antagonistas & inhibidores , terc-Butilhidroperóxido/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Rotura Cromosómica , Ensayo Cometa , Glutatión/metabolismo , Humanos , Peroxidación de Lípido , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Oxidantes/toxicidad , Oxidación-Reducción , Ratas , Células Tumorales CultivadasRESUMEN
The tumour suppressor gene TP53 plays an important role in the regulation of DNA repair, and particularly of nucleotide excision repair. The influence of p53 status on the efficiency of the principal steps of this repair pathway was investigated after UV-C irradiation in the human ovarian carcinoma cell line IGROV-1 (expressing wild-type p53) and in the derived clone IGROV-1/Pt1 (with p53 mutations at codons 270 and 282). Clonogenic survival after UV-C irradiation showed that IGROV-1/Pt1 cells were approximately 2-fold more resistant to DNA damage than parental cells. Modulation of p53 protein levels, cell cycle arrest and apoptosis were induced in UV-irradiated IGROV-1 cells, but not in the p53-mutant cell line. Exposure to UV or cisplatin induced down-regulation of p53-replication protein A (RPA) interaction in parental, but not in IGROV-1/Pt1 cells. However, persistent binding of p53 to RPA did not affect the early steps of DNA repair. In fact, both UV-induced DNA incision and the recruitment of proliferating cell nuclear antigen (PCNA) to DNA repair sites occurred to a comparable extent in p53-wild type and -mutant cell lines, although PCNA remained associated with chromatin for a longer period of time in IGROV-1/Pt1 cells. Global genome repair, as detected by immunoblot analysis of cyclobutane pyrimidine dimers, was not significantly different in the two cell lines at 3 h after UV irradiation. In contrast, lesion removal at 24 h was markedly reduced in IGROV-1/Pt1 cells, being approximately 25% of the initial amount of damage, as compared with approximately 50% repair in parental cells. These results indicate that the presence of mutant p53 protein and its persistent interaction with RPA do not affect the early steps of nucleotide excision repair in IGROV-1/Pt1 cells. Thus, repair defects in p53-mutant ovarian carcinoma cells may be attributed to late events, possibly related to a reduced removal/recycling of PCNA at repair sites.
Asunto(s)
Daño del ADN/efectos de la radiación , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Neoplasias Ováricas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Western Blotting , Supervivencia Celular/efectos de la radiación , Reparación del ADN/genética , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Mutación Puntual/genética , Proteína de Replicación A , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Gastric cancer is poorly-responsive to widely used antitumour drugs, the efficacy of which is thought to be related to the capacity of triggering apoptosis. This process requires a series of gene products including a functional p53 protein. We tested the effects of two DNA topoisomerase II poisons, etoposide and doxorubicin, on gastric cancer cell lines with different genetic lesions. We characterised MKN74 and MKN28 cells for p53 gene status and for the expression of p53 and p21 proteins, as well as of topoisomerase II alpha and beta isoforms. After drug treatments, the cells were analysed for drug cytotoxicity, colony forming ability, cell cycle distribution and presence of apoptotic features. Our findings demonstrated that both etoposide and doxorubicin have a potent anti-proliferative effect on gastric cancer cells. Cell death kinetics was different in the two cell lines, MKN74 cells being more sensitive than MKN28 to the drugs. MKN74 cells, although harboring a wt p53 gene, were unable to undergo a massive apoptosis following etoposide treatment. The response of this cell line might be related to the topoisomerase II beta isozyme, the expression of which proved to be undetectable.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Inhibidores de Topoisomerasa II , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , ADN-Topoisomerasas de Tipo II/biosíntesis , Doxorrubicina/farmacología , Etopósido/farmacología , Citometría de Flujo , Genes p53/genética , Humanos , Reacción en Cadena de la Polimerasa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Activación Transcripcional , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.
Asunto(s)
ADN Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Zidovudina/farmacología , Animales , Células Cultivadas , Histocitoquímica , Masculino , Potenciales de la Membrana/efectos de los fármacos , Microscopía de Fuerza Atómica , Mitocondrias/genética , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Músculo Esquelético/citología , Ratas , Ratas WistarRESUMEN
In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA--cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , ADN Ligasas/metabolismo , Replicación del ADN/efectos de los fármacos , Etopósido/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de Topoisomerasa II , Antineoplásicos Fitogénicos/metabolismo , Afidicolina/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Daño del ADN , ADN Ligasa (ATP) , Proteínas de Unión al ADN , Inhibidores Enzimáticos/farmacología , Etopósido/metabolismo , Células HeLa , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estaurosporina/farmacología , Proteínas Supresoras de Tumor , Rayos UltravioletaRESUMEN
Antifolates exert their antiproliferative activity through the inhibition of dihydrofolate reductase and, as a consequence, of thymidylate synthesis, thereby inducing nucleotide misincorporation and impairment of DNA synthesis. We investigated the processes involved in the repair of antifolate-induced damage and their relationship with cell death. Since misincorporated bases may be removed by DNA mismatch repair (MMR), the study was carried out on the MMR-proficient human cell lines HeLa and HCT116+chr3, and, in parallel, on the MMR-deficient cell lines HeLa cell-clone12, defective in the protein hPMS2, and HCT116, with an inactive hMLH1. After treatment with methotrexate (MTX), we observed that DNA repair synthesis occurs independently of the cellular MMR function. Clear signs of apoptosis such as nuclear shrinkage, chromatin condensation and degradation, DNA laddering, and poly (ADP-ribose) polymerase (PARP) proteolysis, were visible in both MMR(+) and MMR(-) cells. Remarkably, cell viability was lower and the apoptotic process was triggered more efficiently in the MMR-competent cells.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Disparidad de Par Base/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Metotrexato/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células HeLa , Humanos , Células Tumorales CultivadasRESUMEN
The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites.
Asunto(s)
Ciclinas/fisiología , Reparación del ADN/fisiología , Antígeno Nuclear de Célula en Proliferación/fisiología , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/deficiencia , ADN Ribosómico/genética , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Humanos , Pulmón/citología , Pulmón/efectos de la radiación , Familia de Multigenes , Tolerancia a Radiación/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Rayos UltravioletaRESUMEN
Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisometry modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4' position is not the sole determinant for antioxidant activity. In contrast, the presence of 4'-OH together with stereoisometry in the trans-conformation (4'-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases alpha and delta.
Asunto(s)
Antioxidantes/farmacología , Ciclo Celular/efectos de los fármacos , Estilbenos/farmacología , Animales , Células Cultivadas , ADN Polimerasa I/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Humanos , Peroxidación de Lípido , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Wistar , Proteína de Replicación A , Resveratrol , Estilbenos/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.
Asunto(s)
Folículo Piloso/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Apoptosis , Diferenciación Celular , División Celular , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Folículo Piloso/citología , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , MasculinoRESUMEN
In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.
Asunto(s)
Ciclinas/metabolismo , Proteínas Proto-Oncogénicas , beta Caroteno/farmacología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Fibroblastos , Técnica del Anticuerpo Fluorescente , Fase G1/efectos de los fármacos , Humanos , Liposomas/metabolismo , Fosforilación , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Desoxirribonucleasas/metabolismo , Etopósido/farmacología , Antineoplásicos Fitogénicos/farmacología , Caspasa 1/metabolismo , Caspasa 3 , Caspasas/metabolismo , Fragmentación del ADN , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.
Asunto(s)
Neoplasias del Colon/patología , Genes myc , Células Clonales , Neoplasias del Colon/genética , Medio de Cultivo Libre de Suero , Regulación Neoplásica de la Expresión Génica , Genes p53 , Humanos , Fenotipo , Transfección , Células Tumorales CultivadasRESUMEN
The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2 + M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas , Purinas/farmacología , Línea Celular , Ciclina B/metabolismo , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , ADN/biosíntesis , Fibroblastos/metabolismo , Fase G1 , Humanos , Cinetina , Fosforilación , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Proteína de Retinoblastoma/metabolismo , RoscovitinaRESUMEN
The involvement of the proliferating cell nuclear antigen (PCNA) in the process of DNA repair induced by alkylating agents or by oxidative damage was investigated in human quiescent fibroblasts by immunofluorescence and flow cytometry. Transition from soluble to the DNA-bound form of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA. Similar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA damage. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation. This mutagenic and carcinogenic product has been previously shown to form adducts with DNA. Therefore, the possibility that tert-butyl hydroperoxide could induce DNA damage through this pathway was investigated by incubating cells directly in the presence of malondialdehyde. Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA. The ability of oxidative and alkylating agents to induce the nuclear binding of PCNA was also assessed in proliferating cells. In these conditions, treatment with hydrogen peroxide or methylmethane sulfonate, resulted in an increase in nuclear-bound PCNA in the G1 and in the G2 + M compartments, but not in S phase. At longer times after treatment, PCNA immunostaining was reduced to basal levels, while an increase in nuclear binding of p21(waf1/cip1) protein was found in concomitance with cell-cycle arrest. These results indicate that agents inducing DNA base alterations in vivo, promote the nuclear binding of PCNA. These lines of evidence support the role of a PCNA-dependent reaction in the base excision repair system.
Asunto(s)
Alquilantes/farmacología , Reparación del ADN , Metilmetanosulfonato/farmacología , Metilnitronitrosoguanidina/farmacología , Estrés Oxidativo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , HumanosRESUMEN
We report the first Italian case of Nijmegen breakage syndrome (NBS). The proband is an immunodeficient, microcephalic, 11 year old boy with a "bird-like" face. He developed a T cell rich B cell lymphoma. Spontaneous chromosomal instability was detected in T and B lymphocytes and fibroblasts; chromosomes 7 and 14 were only sporadically involved in the rearrangements and no clonal abnormality was present. The patient appeared to be sensitive both to ionising radiation and to bleomycin, although his sensitivity did not reach the level of AT reference cells. After bleomycin treatment, inhibition of DNA synthesis was low when compared with normal cells, but higher than observed in an AT reference strain. Moreover, cell cycle analysis, after drug exposure, showed a progressive reduction in the percentage of S phase cells, but the G1 arrest, found in normal cells, was not observed. On clinical evaluation our patient shares features with NBS subjects, but cytogenetic and cell biological data do not completely overlap with those reported in Nijmegen breakage syndrome. The ethnic origin of our patient might account for these differences, as expression of different allelic forms at the NBS locus.
Asunto(s)
Aberraciones Cromosómicas , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Anomalías Craneofaciales , Tolerancia a Radiación , Antígenos de Superficie/análisis , Antimetabolitos Antineoplásicos/farmacología , Bleomicina/farmacología , Ciclo Celular , Células Cultivadas , Niño , Aberraciones Cromosómicas/inmunología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/inmunología , ADN/biosíntesis , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Humanos , Interleucina-2/biosíntesis , Italia , Leucocitos Mononucleares , Linfoma de Células B , Masculino , SíndromeAsunto(s)
Ciclo Celular , Ciclinas/análisis , Ciclinas/fisiología , Animales , Núcleo Celular/química , Ciclina A/análisis , Ciclina B/análisis , Ciclina D , Ciclina E/análisis , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/química , Citoplasma/química , Aparato de Golgi/química , Humanos , Microtúbulos/químicaRESUMEN
The proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase delta and epsilon, is involved in DNA replication and repair. This protein forms a homotrimeric structure which, encircling DNA, loads the polymerase on the DNA template. A role for PCNA in the cell cycle control is recognised on the basis of the interaction with cyclins, cyclin-dependent kinases (cdks) and the cdk-inhibitor p21 waf1/cip1/sdi1 protein. Association with the growth-arrest and DNA-damage inducible proteins gadd45 and MyD118, further demonstrates the role of PCNA as a component of the cell cycle control apparatus.