L'activitat genètica en els micronuclis : es pot considerar realment perdut el DNA micronuclear?

Els micronuclis es produeixen per irregularitats en el procés de divisió cel·lular, generant-se aquestes estructures derivades del nucli, més petites i que poden contenir o bé cromosomes sencers o bé fragments cromosòmics derivats de trencaments no reparats. Aquesta recerca ha estudiat la naturalesa dels micronuclis. Els resultats indiquen que la seva pròpia naturalesa és la responsable de la destinació que pateixen, el què suposa un avenç important en el coneixement d'aquestes estructures utilitzades per detectar inestabilitats cromosòmiques.Los micronúcleos se producen por irregularidades en el proceso de división celular, generándose estas estructuras derivadas del núcleo, más pequeñas y que pueden contener o bien cromosomas enteros o bien fragmentos cromosómicos derivados de roturas no reparadas. Esta investigación ha estudiado la naturaleza de los micronúcleos. Los resultados indican que su propia naturaleza es la responsable del destino que sufren, lo que supone un avance importante en el conocimiento de estas estructuras utilizadas para detectar inestabilidades cromosómicas

Les lesions del DNA en el micronucli indueixen una resposta al dany localment deficient

Els micronuclis són un dels indicadors més emprats per estudiar la presència d' inestabilitat cromosòmica en humans. Els micronuclis poden contenir cromosomes sencers o fragments cromosòmics derivats d'una reparació anòmala del dany en el DNA. Partint de la relació existent entre la reparació del DNA i la presència de micronuclis, es va decidir estudiar l'habilitat de les lesions de DNA segrestades en els micronuclis per activar una resposta efectiva al dany. Mitjançant tècniques d'immunofluorescència es va poder veure que només una petita part de les lesions del DNA micronuclear reclutaven correctament proteïnes de detecció del dany. D'altra banda també es va demostrar que el DNA micronuclear podia ésser degradat i que aquesta degradació no afectava la proliferació de la cèl·lula. Així doncs, tant l'absència de reparació de les lesions del DNA micronuclear, com la degradació del mateix, es tradueixen en una pèrdua de material genètic que podria afavorir encara més el procés de tumorigènesi.Los micronúcleos son uno de los indicadores más empleados apra estudiar la presencia de inestabilidad cromosómica en humanos. Los micronúcleos pueden contener cromosomas enteros o fragmentos cromosómicos derivados de una reparación anómala del daño en el DNA. Partiendo de la relación existente entre la reparación del DNA y la presencia de micronúcleos, se decidió estudiar la habilidad de las lesiones de DNA secuestradas en los micronúcleos para activar una respuesta efectiva al daño. Mediante técnicas de inmunofluorescencia se pudo ver que sólo una pequeña parte de las lesiones del DNA micronuclear reclutaban correctamente proteínas de detección del daño. Por otro lado, también se demostró que el DNA micronuclear podía ser degradado y que esta degradación no afectaba la proliferación de la célula. Así, tanto la ausencia de reparación de las lesiones del DNA micronuclear, como la degradación del mismo, se traducen en una pérdida de material genético que podría favorecer todavía más el proceso de tumorigénesis

Dominantly inherited hereditary nonpolyposis colorectal cancer not caused by MMR genes

In the past two decades, multiple studies have been undertaken to elucidate the genetic cause of the predisposition to mismatch repair (MMR)-proficient nonpolyposis colorectal cancer (CRC). Here, we present the proposed candidate genes according to their involvement in specific pathways considered relevant in hereditary CRC and/or colorectal carcinogenesis. To date, only pathogenic variants inRPS20may be convincedly linked to hereditary CRC. Nevertheless, accumulated evidence supports the involvement in the CRC predisposition of other genes, includingMRE11,BARD1,POT1,BUB1B,POLE2,BRF1,IL12RB1,PTPN12, or the epigenetic alteration ofPTPRJ. The contribution of the identified candidate genes to familial/early onset MMR-proficient nonpolyposis CRC, if any, is extremely small, suggesting that other factors, such as the accumulation of low risk CRC alleles, shared environmental exposures, and/or gene-environmental interactions, may explain the missing heritability in CRC

ATM and DNA-PKcs make a complementary couple in DNA double strand break repair

The interplay between ATM and DNA-PKcs kinases during double strand breaks (DSBs) resolution is still a matter of debate. ATM and DNA-PKcs participate differently in the DNA damage response pathway (DDR), but important common aspects are indeed found: both of them are activated when faced with DSBs, they share common targets in the DDR and the absence of either kinase results in faulty DSB repair. Absence of ATM translates into timely repair that, nevertheless, is incomplete. On the other hand, DNA-PKcs absence translates into slower repair, which in turn gives rise to the accumulation of simple and complex reorganizations. These outcomes confirm that the function of both protein kinases is essential to guarantee genome integrity. Interestingly, V(D)J and CSR recombination events provide a powerful tool to study the interplay between both kinases in DSB repair. Although the physiological DSBs generated during V(D)J and CSR recombination are resolved by the non-homologous end-joining (NHEJ) repair pathway, ATMabsence during these events translates into chromosome translocations. These results suggest that NHEJ accuracy is threatened in the absence of ATM, which may play a role in avoiding illegitimate repair by favouring the joining of the correct DNA ends. Indeed, simultaneous DNA-PKcs and ATM deficiency during V(D)J and CSR recombination translates into a synergistic increase in potentially dangerous chromosomal translocations and deletions. Although the exact nature of their interaction remains elusive, the evidence indicates that ATM and DNA-PKcs play complementary roles that allow complete and legitimate DSB repair to be reached. Faithful repair can only be achieved by the presence and correct functioning of both kinases: while DNA-PKcs ensures fast rejoining, ATM guarantees complete repair

Role of H4K16 acetylation in 53BP1 recruitment to double-strand break sites in in vitro aged cells

Increased frequency of DNA double strand breaks (DSBs) with aging suggests an age-associated decline in DSB repair efficiency, which is also influenced by the epigenetic landscape. H4 acetylation at lysine 16 (H4K16Ac) has been related to DSB repair since deacetylation of this mark is required for efficient 53BP1 recruitment to DSBs. Although age-associated changes in H4K16Ac levels have been studied, their contribution to age-related DSB accumulation remains unknown. In vitro aged Human Dermal Fibroblasts (HDFs) display lower levels of H4K16A that correlate with reduced recruitment of 53BP1 to basal DSBs. Following DNA damage induction, early passage (EP) cells suffered from a transient H4K16 deacetylation that allowed proper 53BP1 recruitment to DSBs. In contrast, to reach this specific and optimum level, aged cells responded by increasing their overall lower H4K16Ac levels. Induced hyperacetylation of late passage (LP) cells using trichostatin A increased H4K16Ac levels but did not ameliorate 53BP1 recruitment. Instead, deacetylation induced by MOF silencing reduced H4K16Ac levels and compromised 53BP1 recruitment in both EP and LP cells. Age-associated decrease of H4K16Ac levels contributes to the repair defect displayed by in vitro aged cells. H4K16Ac responds to DNA damage in order to reach a specific, optimum level that allows proper 53BP1 recruitment. This response may be compromised with age, as LP cells depart from lower H4K16Ac levels. Variations in H4K16Ac following the activation of the DNA damage response and aging point at this histone mark as a key mediator between DNA repair and age-associated chromatin alterations

Identification of reference genes for RT-qPCR data normalisation in aging studies

Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (015), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in 015, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during 015, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies

Breaks invisible to the DNA damage response machinery accumulate in ATM-deficient cells

Altres ajuts: Fundació La Marató, Grant number TV32005-050110After irradiation, ATM defective cells accumulate unrepaired double strand breaks (DSBs) for several cell divisions. At the chromosome level, unresolved DSBs appear as chromosome breaks that can be efficiently scored by using telomeric and mFISH probes. H2AX is immediately activated by ATM in response to DNA damage and its phosphorylated form, γH2AX, flanks the DSB through several megabases. The γH2AX-labeling status of broken chromosome ends was analyzed in AT cells to check whether the DNA damage response was accurately taking place in these persistent DSBs. The results show that one quarter of the scored breaks are devoid of γH2AX foci in metaphase spreads from ATM-deficient cells, and this fraction is significantly higher than in normal cells (χ2 < 0.05). Accumulation of sensor and repair proteins at damaged sites is a key event in the cellular response to DSBs, so MRE11 labeling at broken ends was also analyzed. While all γH2AX foci scored at visible broken ends colocalize with MRE11 foci, all γH2AX-unlabeled breaks are also devoid of MRE11-labeling. The present results suggest that a significant subset of the AT long-lived DSBs may persist as " invisible" DSBs due to deficient detection by the DNA damage repair machinery. Eventually the properly signaled DSBs will be repaired while invisible breaks may indefinitely accumulate; most probably contributing to the AT cells' well known genomic instability

Identification of reference genes for RT-qPCR data normalisation in aging studies

Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies

The Inherited and Familial Component of Early-Onset Colorectal Cancer

Early-onset colorectal cancer (EOCRC), defined as that diagnosed before the age of 50, accounts for 10-12% of all new colorectal cancer (CRC) diagnoses. Epidemiological data indicate that EOCRC incidence is increasing, despite the observed heterogeneity among countries. Although the cause for such increase remains obscure, ≈13% (range: 9-26%) of EOCRC patients carry pathogenic germline variants in known cancer predisposition genes, including 2.5% of patients with germline pathogenic variants in hereditary cancer genes traditionally not associated with CRC predisposition. Approximately 28% of EOCRC patients have family history of the disease. This article recapitulates current evidence on the inherited syndromes that predispose to EOCRC and its familial component. The evidence gathered support that all patients diagnosed with an EOCRC should be referred to a specialized genetic counseling service and offered somatic and germline pancancer multigene panel testing. The identification of a germline pathogenic variant in a known hereditary cancer gene has relevant implications for the clinical management of the patient and his/her relatives, and it may guide surgical and therapeutic decisions. The relative high prevalence of hereditary cancer syndromes and familial component among EOCRC patients supports further research that helps understand the genetic background, either monogenic or polygenic, behind this increasingly common disease

Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

Altres ajuts: This work was funded by grants from Consejo de Seguridad Nuclear (CSN 2012-0001) and EURATOM (Dark.Risk GA323216). Laia Hernandez is supported by the Universitat Autònoma de Barcelona Ph.D. programme fellowshipIn order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70-85% of the AT viable cells (TUNEL-negative) carried ≥10 γ H2AX foci/cell, while only 12-27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γ H2AX-positive AT cells undergoing early apoptosis carried ≥10 γ H2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γ H2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γ H2AX foci might function as a predictive tool to assess radiation-induced apoptosis