Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA

The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling.Fil: Speroni, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Mansilla, Sabrina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Soria, Ramiro Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Atuação dos agentes comunitários de saúde na estratégia de saúde da família: percepções dos trabalhadores

Doi: 10.5902/2179769211210 Aim: to identify the perception of the Community Health Agent regarding their work in the context of the Family Health Strategy. Method: qualitative research, developed in six units of FHS bound to the Health Secretary of Santa Maria- RS. The participants of the study were 33 Community Health Agents. The data were collected through semi-structured interviews and submitted to thematic content analysis. Results: the professional intervention of the agent is connected to its role as a community mediator and educator in regards to health, able to identify the social necessities and representing a spokesperson of the community. Conclusions: the work of the Community Health Agent is related to the potential to bind the users to the actions of the health team, co-responsibilization, and the possibility to prompt processes of change. The strategies of the Community Health Agents aim to enable the participation of the community and to strenghten the Unified Health System.Objetivo: conocer la percepción de Agentes Comunitarios de Salud acerca de su proceso de trabajo en la Estrategia de Salud Familiar. Método: investigación cualitativa desarrollada en seis unidades ESF vinculadas a la Secretaría Municipal de Salud de Santa Maria-RS. Participaron 33 Agentes Comunitarios de Salud. La recolección de datos se realizó mediante entrevistas semi-estructuradas y los datos sometidos a análisis de contenido temático. Resultados: la actuación profesional del Agente se articula con su papel como mediador comunitario y educador de salud de la comunidad, capaz de identificar las necesidades sociales, y ejercer de portavoz de la comunidad. Conclusiones: el trabajo de Agentes Comunitarios de Salud se relaciona a su potencial de vincular a los usuarios a las acciones del equipo de salud, la corresponsabilidad y la posibilidad de inducir procesos de cambio. Sus estrategias visan la participación de la comunidad y el fortalecimiento del Sistema Único de Salud.Doi: 10.5902/2179769211210Objetivo: conhecer a percepção do Agente Comunitário de Saúde acerca de seu processo de trabalho no âmbito da Estratégia de Saúde da Família. Método: pesquisa qualitativa desenvolvida em seis Unidades de ESF vinculadas à Secretaria Municipal de Saúde de Santa Maria – RS. Foram participantes do estudo 33 Agentes Comunitários de Saúde. A coleta de dados foi realizada por meio de entrevistas semiestruturadas e os dados submetidos à análise de conteúdo temática. Resultados: a atuação profissional do agente articula-se ao seu papel como mediador comunitário e educador em saúde, capaz de identificar as necessidades sociais, representando um porta-voz da comunidade. Conclusões: O trabalho do agente comunitário de saúde relaciona-se ao seu potencial para vincular os usuários às ações da equipe de saúde, a corresponsabilização e a possibilidade de induzir processos de mudança, sendo que suas estratégias visam a participação da comunidade e o fortalecimento do Sistema Único de Saúde

p21 regulates the cell cycle...or the other way around?

For many years, the upregulation of the p53- p21 pathway was supposed to represent the universal response to genotoxic stress. i n triguingly, many genotoxic agents (hydroxy - urea, aphidicolin, hypoxia, cisplatinun and U v irradiation) do not upregulate p21 despite p53 activation. 1 Remarkably, in the August 15th issue of Cell Cycle , Ciznadija and colleagues 2 showed that g i R (gamma ionizing radiation), a D n A damaging agent which was previously characterized as a bona fide inducer of p21, fails to promote such accumulation in s phase. Remarkably, this happens in the context of high p53 levels demonstrating that also in the context of g i R, as well as signals downstream of p53 specifically prevent p21 accumulation during s phase.Fil: Speroni, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

p21 differentially regulates DNA replication and DNA-repair-associated processes after UV irradiation

Although p21 upregulation is required to block cell-cycle progression following many types of genotoxic insult, UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated with DNA repair. While the role of p21 in nucleotide excision repair (NER) remains controversial, recent reports have explored its effect on translesion DNA synthesis (TLS), a process that avoids replication blockage during S phase. Herein, we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK-binding domain of p21 is required for cell-cycle arrest in unstressed cells, neither the CDK-binding nor the PCNA-binding domain of p21 is able to block early and late steps of NER. Intriguingly, through its PCNA-binding domain, p21 inhibits the interaction of the TLS polymerase, pol eta (pol eta), with PCNA and impairs the assembly of pol eta foci after UV. Moreover, this obstruction correlates with accumulation of phosphorylated H2AX and increased apoptosis. By showing that p21 is a negative regulator of PCNA-pol eta interaction, our data unveil a link between efficient TLS and UV-induced degradation of p21.Fil: Soria, Ramiro Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires; ArgentinaFil: Speroni, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires; ArgentinaFil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Prives, Carol. Columbia University; Estados UnidosFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires; Argentin

The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models

p53 sensitizes cells to the antiproliferative effect of DhL.

<p>(A) Synchronized HeLa cells treated with 0–20 µM of DhL were lysed at the indicated times. Immunoblot analyses of proteins p53 and its transcriptional target p21 are shown. Lower plots: densitometric analysis of p21 and p53 normalized to β-actin. Relative intensities of the bands are expressing percentages relative to the control (defined as 100%). The immunoblots shown are representative of 3 independent experiments with similar result. * p≤0.05, ** p≤0.01 *** p≤0.001 for the treated group (20 µM DhL) vs. control group (0 µM DhL). (B) Unsynchronized HCT116 p53+/+ and HCT116 p53−/− cells were treated with 0, 5, 10, or 20 µM DhL, lysed at the indicate times and proteins p53 and p21 were analyzed by immunoblot. β-actin was employed as a loading control (upper panel) or counted at the indicated times (lower panel). The immunoblot shown are representative of 3 independent experiments with similar result. Data represent the mean ± SEM of 3 experiments, * p≤0.05, ** p≤0.01 for HCT116 p53+/+ vs. HCT116 p53−/−.</p