Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

BACKGROUND: Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood. RESULTS: In this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G(1)-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C) are used. CONCLUSION: We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP

The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA

Artículo científico -- Universidad de Costa Rica. Instituto de Investigaciones en Salud, 2003. Este documento es privado debido a limitaciones de derechos de autor.Among bacterial protein toxins, the cytolethal distending toxins (CDTs) are unique in their ability to activate the DNA damage checkpoint responses, causing cell cycle arrest or apoptosis in intoxicated cells. We provide direct evidence that natural intoxication of cells with the Haemophilus ducreyi CDT (HdCDT) holotoxin induces DNA double-strand breaks similarly to ionizing radiation. Upon DNA damage, epithelial cells and fibroblasts promote the formation of actin stress fibres via activation of the small GTPase RhoA. This phenomenon is not toxin specific, but is part of the ATM-induced cellular responses to genotoxic stresses, including ionizing radiation. Activation of RhoA is associated with prolonged cell survival, as HdCDT-treated epithelial cells expressing a dominant-negative form of RhoA detach and consequently die faster than cells expressing a functional RhoA. Our data highlight several novel aspects of CDT biology: (i) we show that a member of the CDT family causes DNA double-strand breaks in naturally intoxicated cells, acting as a true genotoxic agent; and (ii) we disclose the existence of a novel signalling pathway for intracellularly triggered activation of the RhoA GTPase via the ATM kinase in response to DNA damage, possibly required to prolong cell survival.Universidad de Costa Rica. Instituto de Investigaciones en SaludUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA

Enforcing the Federal Water Resource Servitude on Submerged and Riparian Lands

The epidermal growth factor receptor (EGFR) is frequently overexpressed in colorectal cancer and is therefore an attractive target for treatment. (ZEGFR:1907)2 is a newly developed dimeric affibody molecule with high affinity to the extracellular part of EGFR. In this study, we evaluated the cytotoxic effects of (ZEGFR:1907)2 in combination with external radiation and the possible inhibitory effects in the EGFR signalling pathways in the colon cancer cell lines HT-29 and HCT116. The effects were compared with an EGFR antibody (cetuximab) and the tyrosine kinase inhibitors (erlotinib and sunitinib). These cell lines are genotypically different with respect to e.g. KRAS and BRAF mutational status, recently shown to be of clinical significance for therapeutic effects. Both cell lines express approximately 100,000-150,000 EGFRs per cell but differ in the radiation response (HCT116, SF2=0.28 and HT-29, SF2=0.70). Exposure to (ZEGFR:1907)2 produced a small, but significant, reduction in survival in HCT116 but did not affect HT-29 cells. Similar results were obtained after exposure to EGF and the EGFR antibody cetuximab. The EGFR tyrosine kinase targeting inhibitor erlotinib and the multi-tyrosine kinase inhibitor sunitinib reduced survival in both cell lines. However, none of the drugs had any significant radiosensitizing effects in combination with radiation. Akt and Erk are central proteins in the EGFR downstream signalling and in the cellular response to ionizing radiation. The activation of Akt (Ser 473) and Erk (Thr202/Tyr204) by radiation was both dose- and time-dependent. However the activation of EGFR was not clearly affected by radiation. Neither (ZEGFR:1907)2 nor any of the other drugs were able to completely inactivate Akt or Erk. On the contrary, erlotinib stimulated Akt phosphorylation in both cell lines and in HCT116 cells Erk was activated. Overall the results illustrate the complexity in response to radiation and drugs in cells with differential phenotypic status.Online ISSN:1791-2423</p

Removal of heat-sensitive clustered damaged DNA sites is independent of double-strand break repair

DNA double-strand breaks (DSBs) are the most deleterious lesions that can arise in cells after ionizing radiation or radiometric drug treatment. In addition to prompt DSBs, DSBs may also be produced during repair, evolving from a clustered DNA damaged site, which is composed of two or more distinct lesions that are located within two helical turns. A specific type of cluster damage is the heat-sensitive clustered site (HSCS), which transforms into DSBs upon treatment at elevated temperatures. The actual lesions or mechanisms that mediate the HSCS transformation into DSBs are unknown. However, there are two possibilities; either these lesions are transformed into DSBs due to DNA lesion instability, e.g., transfer of HSCS into single-strand breaks (SSBs), or they are formed due to local DNA structure instability, e.g., DNA melting, where two SSBs on opposite strands meet and transform into a DSB. The importance of these processes in living cells is not understood, but they significantly affect estimates of DSB repair capacity. In this study, we show that HSCS removal in human cells is not affected by defects in DSB repair or inhibition of DSB repair. Under conditions where rejoining of prompt DSBs was almost completely inhibited, heat-sensitive DSBs were successfully rejoined, without resulting in increased DSB levels, indicating that HSCS do not transfer into DSB in cells under physiological conditions. Furthermore, analysis by atomic force microscopy suggests that prolonged heating of chromosomal DNA can induce structural changes that facilitate transformation of HSCS into DSB. In conclusion, the HSCS do not generate additional DSBs at physiological temperatures in human cells, and the repair of HSCS is independent of DSB repair