Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.Several dozen oxidatively modified, and mostly mutagenic, bases are induced in the genomes of aerobic organisms by endogenous and environmentally induced reactive oxygen species (ROS) (1, 2). For example, 5-hydroxyuracil (5-OHU), a predominant lesion generated by oxidative deamination of C, is mutagenic because of its mispairing with A (3). The bases in the single-stranded (ss) replicating DNA template are particularly prone to oxidation (4); the lack of their repair before replication could fix the mutations. The bulky base adducts if formed in the template strand would block replication and trigger DNA damage-response signaling. In contrast, oxidized bases with minor modifications, which are continuously formed in much higher abundance than the bulky adducts, would mostly allow replication. This raises the question of how these bases are marked for repair before replication to avoid mutagenic consequences. Oxidized base repair in mammalian genomes occurs primarily via the base excision repair (BER) pathway which is initiated with lesion base excision mediated by one of five major DNA glycosylases belonging to the Nth or Nei families, with distinct structural features and reaction mechanisms (1). Nei-like (NEIL) 1 and NEIL2 DNA glycosylases (5, 6) of the Nei family (which also contains the less characterized NEIL3; ref. 7) are distinct from NTH1 and OGG1 of the Nth family because the NEILs can excise damaged bases from ssDNA substrates (8). Furthermore, NEIL1 is activated during the S phase (5). Our earlier studies also showed that NEIL1 functionally interacts with many DNA replication proteins including sliding clamp proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN-1), and Werner RecQ helicase (WRN) via its disordered C-terminal segment (9–12). Importantly, mammalian ssDNA-binding replication protein A (RPA), essential for DNA replication and most other DNA transactions, inhibits NEIL1 or NEIL2 activity with primer-template DNA substrates mimicking the replication fork, presumably to prevent double-strand break formation (13). Although they collectively implicate NEIL1 in the repair of replicating DNA, those observations did not provide direct evidence for NEIL1’s role in prereplicative repair, nor did they address whether NEIL1 is unique for this function. In this report, we document that NEIL1 binds to the lesion base in an RPA-coated ssDNA template in vitro, without excising the lesion and cleaving the DNA strand, and blocks primer elongation by the replicative DNA polymerase δ (Polδ). This strongly suggests that the replication complex at the lesion site is stalled in vivo in the presence of NEIL1, which provides the signal for repair of lesions in the template strand before replication.     

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.DNA double-strand break repair (DSBR) is essential for cell survival and repair-deficient cells are highly sensitive to chromosome breakage. In Escherichia coli, a single unrepaired DNA DSB per replication cycle is lethal, illustrating the critical nature of the repair reaction (1). DSBR in E. coli is mediated by homologous recombination, which relies on the RecA protein to efficiently recognize DNA sequence identity between two molecules. RecA homologs are widely conserved from bacteriophages to mammals, where they are known as the Rad51 proteins (2). The RecA protein plays its central role by binding single-stranded DNA (ssDNA) to form a presynaptic filament that searches for a homologous double-stranded DNA (dsDNA) donor from which to repair. It then catalyzes a strand-exchange reaction to form a joint molecule (3), which is stabilized by the branch migration activities of the RecG and RuvAB proteins (4). The joint molecule is then resolved by cleavage at its four-way Holliday junction by the nuclease activity of RuvABC (5, 6).RecA binding at the site of a DSB is dependent on the activity of the RecBCD enzyme (Fig. 1A). RecBCD is a helicase-nuclease that binds to dsDNA ends, then separates and unwinds the two DNA strands using the helicase activities of the RecB and RecD subunits (see refs. 7 and 8 for recent reviews). RecD is the faster motor of the two and this consequently results in the formation of a ssDNA loop ahead of RecB (Loop 1 in Fig. 1A) (9). As the enzyme translocates along dsDNA, the 3′-terminated strand is continually passed through the Chi-scanning site thought to be located in the RecC protein (10). When a Chi sequence (the octamer 5′-GCTGGTGG-3′) enters this recognition domain, the RecD motor is disengaged and the 3′ strand continues to be unwound by RecB. Under in vitro conditions, where the concentration of magnesium exceeds that of ATP, the 3′ end (unwound by RecB) is rapidly digested before Chi recognition, whereas the 5′ end (unwound by RecD) is intermittently cleaved (11, 12). After Chi recognition the 3′ end is no longer cleaved but the nuclease domain of RecB continues to degrade the 5′ end as it exits the enzyme (11, 12). Under in vitro conditions where the concentration of ATP exceeds that of magnesium, unwinding takes place but the only site of cleavage detected is ∼5 nucleotides 3′ of the Chi sequence (13, 14). Because the RecB motor continues to operate while the RecD motor is disengaged, Loop 1 is converted to a second loop located between the RecB and RecC subunits or to a tail upon release of the Chi sequence from its recognition site. We therefore describe this single-stranded region as Loop/Tail 2 in Fig. 1A. After the whole of Loop 1 is converted to Loop/Tail 2, this second single-stranded region continues to grow as long as the RecB subunit unwinds the dsDNA. The RecBCD enzyme enables RecA protein to load on to Loop/Tail 2 to generate the presynaptic filament necessary to search for homology and initiate strand-exchange (15). Finally, the RecBCD enzyme stops translocation and disassembles as it dissociates from the DNA, releasing a DNA-free RecC subunit (16).Open in a separate windowFig. 1.DSBR in E. coli. (A and B) Schematic representation of DSB processing by the RecBCD complex. (A) Before Chi recognition, both the RecB and RecD motors progress along the DNA. RecD is the faster motor and as a result a loop of ssDNA (Loop 1) is formed ahead of the slower RecB motor. The 3′ ssDNA strand is scanned for the Chi sequence by the RecC protein. (B) After Chi recognition, RecBCD likely undergoes a conformational change so that only the RecB motor is engaged. The RecA protein is recruited by the RecB nuclease domain and loaded onto the ssDNA loop generated by RecB unwinding to promote RecA nucleoprotein filament formation. In this schematic representation, the Chi site is shown held in its recognition site. However, the Chi site will be released either by disassembly of the RecBCD complex or at some point before this and the second single-stranded region on the 3′ terminating strand will be converted from a loop to a tail. Therefore, this region is denoted Loop/Tail 2. The mathematical model described in SI Appendix does not depend on the ATP/magnesium dependent differential cleavage of DNA strands (7, 8), nor does it depend on the precise time that the 3′ end is released from the complex following Chi recognition. (C) The hairpin endonuclease SbcCD is used to cleave a 246-bp interrupted palindrome inserted in the lacZ gene of the E. coli chromosome. Cleavage of this DNA hairpin results in the generation of a site-specific DSB on only one of a pair of replicating sister chromosomes, thus leaving an intact sister chromosome to serve as a template for repair by homologous recombination.Our understanding of the action of RecBCD and RecA has been the result of more than 40 years of genetic analysis and biochemical investigation of these purified proteins in vitro. However, relatively little is known about their activities on the genomic scale. To investigate these reactions in vivo, we have used RecA chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) in an experimental system that allows us to introduce a single and fully repairable DSB into the chromosome of E. coli (1). Because DSBR by homologous recombination normally involves the repair of a broken chromosome by copying the information on an unbroken sister chromosome, our laboratory has previously developed a procedure for the cleavage of only one copy of two genetically identical sister chromosomes (1). We have made use of the observation that the hairpin nuclease SbcCD specifically cleaves only one of the two sister chromosomes following DNA replication through a 246-bp interrupted palindrome to generate a two-ended DSB (1). As shown in Fig. 1B, this break is fully repairable and we have shown that recombination-proficient cells suffer very little loss of fitness in repairing such breaks (17).Here we investigate in vivo and in a quantitative manner the first steps of DSBR: because the outcome of RecBCD action is understood to be the loading of RecA on DNA in a Chi-dependent manner, we use RecA-ChIP to reveal the consequences of RecBCD action on a genomic scale during DSBR. Analyses of most ChIP-Seq datasets focus on the identification of regions of significant enrichment of a given protein but do not take into account the underlying mechanisms giving rise to the binding (18). We reasoned that given the detailed mechanistic understanding of RecBCD in vitro, we could gain a deeper insight into its in vivo functions by developing a mathematical model of RecBCD action that would enable us to estimate the mechanistic parameters of the complex in live cells. Our ChIP data indicate that RecA is indeed loaded on to DNA in a Chi-dependent manner and we have used our mathematical model to infer the parameters of RecBCD action in vivo on a genomic scale. Furthermore, our analysis reveals that DSBR at lacZ induces DSBR in the terminus region of the chromosome, an unanticipated observation illuminated by the genomic scale of our data.     

The Role of the DNA Damage Response throughout the Papillomavirus Life Cycle

The DNA damage response (DDR) maintains genomic integrity through an elaborate network of signaling pathways that sense DNA damage and recruit effector factors to repair damaged DNA. DDR signaling pathways are usurped and manipulated by the replication programs of many viruses. Here, we review the papillomavirus (PV) life cycle, highlighting current knowledge of how PVs recruit and engage the DDR to facilitate productive infection.     

Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity

The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5′-ended strand to form 5′ mononucleotides and a long 3′ overhang. The crystal structure of λ exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5′-ended strand insert into the active site of one subunit of the trimer, while the 3′-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5′ phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg²⁺ ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5′ phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.     

From the Cover: PNAS Plus: Single-molecule visualization of RecQ helicase reveals DNA melting,nucleation, and assembly are required for processive DNA unwinding

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.DNA helicases are ubiquitous enzymes involved in many aspects of DNA metabolism, including DNA replication, repair, and recombination. These enzymes work by coupling the hydrolysis of nucleoside triphosphates (NTPs) to unwinding of double-stranded DNA (dsDNA) to produce single-stranded DNA (ssDNA) (1). This activity allows the cell’s machinery to access the information stored within the bases of the double helix. RecQ helicase from Escherichia coli is the founding member of the RecQ family of helicases (2). These enzymes belong to the superfamily 2 (SF2) group of helicases, yet share greater sequence homology with their own family members, and they play important roles in the maintenance of genomic integrity by DNA recombination and repair (1, 3). Mutations in the human RecQ-like helicases, Bloom (BLM), Werner (WRN), and RecQ4 proteins, lead to Bloom’s, Werner’s, and Rothmund–Thomson syndromes, respectively. These genetic disorders are characterized by genomic instability and an increased incidence of cancers (4).E. coli RecQ is a 3′ → 5′ helicase that functions in DNA-break repair by homologous recombination (2, 5, 6). RecQ and RecJ, a 5′ → 3′ exonuclease, process ssDNA gaps or dsDNA breaks into ssDNA for recombinational repair by RecA (7–10). In addition, RecQ ensures recombination fidelity in vivo by removing inappropriately paired joint molecules to prevent illegitimate recombination and also by disrupting joint molecule intermediates to facilitate repair by synthesis-dependent strand annealing, preventing chromosomal crossing over (7, 9, 11, 12). RecQ also functions with topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate DNA molecules and to separate converged replication forks (13, 14). RecQ and Topo III provide an alternative to the RuvABC pathway for disengaging double Holliday junctions and do so without producing chromosomal crossovers (15).In vitro, RecQ can unwind a multitude of DNA substrates and does not require a ssDNA tail, or even a dsDNA end, to initiate unwinding; consequently, it is distinctive in being able to unwind covalently closed circular plasmid DNA (16, 17). The winged-helix domain of RecQ is important for the recognition of this broad array of DNA substrates (18). This domain binds to dsDNA yet it adopts a flexible conformation that allows it to adapt to many DNA structures. A curious feature of RecQ is that maximal unwinding requires nearly stoichiometric amounts of protein relative to the DNA (one protein per ∼10 bp), which can be partially mitigated (one protein per ∼30 bp) by including the ssDNA binding protein, SSB (5, 6, 16, 19). This behavior is compatible with the low processivity of ssDNA translocation (∼30–100 nucleotides) by RecQ (20–22) and other RecQ members (23). Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9, 16, 24–26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27–29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.Fluorescence techniques coupled with single-molecule microscopy have emerged as a powerful method for studying the unwinding mechanism of helicases (30–35). These assays measure individual enzymes directly or indirectly through their actions on individual DNA molecules, thus alleviating many of the drawbacks of ensemble experiments. In particular, total internal reflection fluorescence (TIRF) microscopy permits the detection of individual fluorophores with high sensitivity in the presence of a high background (30). To visualize the activity of DNA binding proteins and helicases, TIRF microscopy can be coupled with microfluidic techniques to facilitate visualization of molecules and exchange of solution components (36–39).Ensemble assays have elucidated many features of DNA unwinding by the RecQ helicase family, but the need to average over a heterogeneous and unsynchronized population of enzymes has precluded a thorough understanding of this diverse and universally important helicase family. To permit a more insightful analysis, we directly visualized unwinding of individual molecules of DNA by RecQ helicase. Unwinding was monitored using fluorescent SSB to visualize generation of ssDNA. On single DNA molecules, we could see tracks of the fluorescent SSB binding to ssDNA produced by the helicase activity of RecQ. We show that RecQ initiates DNA unwinding via melting of duplex DNA at internal sites. Once initiated, DNA unwinding propagates either uni- or bidirectionally via the cooperative action of multiple RecQ molecules at the junction of ssDNA with dsDNA. Collectively, these observations define a stable oligomeric complex of subunits involved in processive helicase action, which is concordant with both biochemical and biological function of RecQ helicases and other helicases.     

Ca2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages

In migrating eukaryotic cells, phosphatidylinositol 3-kinase (PI3K), filamentous actin (F-actin), and monomeric Rho GTPases are key components of a complex positive-feedback system that maintains and amplifies a phosphatidylinositol-3,4,5-trisphosphate signal at the leading edge of the cell. This lipid signal is required for cell polarization and movement. In leukocytes and Dictyostelium, activation or inhibition of any one of these components leads to the activation or inhibition, respectively, of the others via undefined feedback interactions. The role of Ca(2+) signals in migrating leukocytes is controversial, and there has been no indication that Ca(2+) participates in positive feedback. Here, we demonstrate that an extracellular Ca(2+) influx is required for positive feedback at the leading edge of spontaneously polarized macrophages. Inhibition of extracellular Ca(2+) influx leads to loss of leading-edge PI3K activity, disassembly of F-actin, cessation of ruffling, and decay of chemoattractant signals. Conversely, increasing cytosolic Ca(2+) enhances membrane ruffling, PI3K activity, and F-actin accumulation. Overall, these findings demonstrate that an extracellular Ca(2+) influx is an essential component, together with PI3K and F-actin, of the positive-feedback cycle that maintains leading-edge structure and ruffling activity and that supports the chemoattractant response. Strikingly, the Ca(2+)-sensitive enzyme protein kinase Calpha (PKCalpha) is enriched at the leading edge, and its enrichment is sensitive to blockade of Ca(2+) influx, to inhibition of PI3K activity, and to F-actin depolymerization. These findings support the working hypothesis that a local, leading-edge Ca(2+) signal recruits PKCalpha as a central player in the positive-feedback loop.     

Clinicopathological analysis of CD133+ and NCAM+ human hepatic stem/progenitor cells in damaged livers and hepatocellular carcinomas

Aim:  Hepatic stem cells are capable of dramatically changing and differentiating to form mature hepatocytes in acute and chronically damaged livers; however, the clinicopathological characteristics of these heterogeneous cell populations have not been sufficiently analyzed.
Methods:  In this study, cells in tissue sections from 12 cases of acute damaged livers and 31 cases of hepatocellular carcinomas (HCC), and the surrounding chronically damaged liver tissues, were analyzed by immunohistochemistry using the previously reported hepatic stem/progenitor cell marker CD133 (AC133) and the neural cell adhesion molecule (NCAM) marker.
Results:  In both the acute and chronically damaged livers, CD133⁺ cells and NCAM⁺ cells were present in ductular reactions (DR), which include hepatic stem/progenitor cells, and became more apparent in proportion to the degree of fibrosis or histological damage. Analysis of their distribution and morphological similarities revealed that the NCAM⁺ cell population included cells that were closer to, and morphologically more similar to, hepatocytes than were CD133⁺ cells. Analysis of HCC using these markers revealed that 9.7% of HCC expressed NCAM (two cases had abundant NCAM⁺ cells), while CD133⁺ HCC were not detected.
Conclusion:  These results suggest that CD133 and NCAM can be employed to enrich for hepatic stem/progenitor cells and that DR can be distinguished in greater detail using these markers. NCAM⁺ HCC were detected, but their function remains unresolved. Expression of CD133, a potent stem cell marker, may be extremely rare in the common human HCC examined.     

Zalypsis has in vitro activity in acute myeloid blasts and leukemic progenitor cells through the induction of a DNA damage response

Background

Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin.

Design and Methods

The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid.

Results

Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34⁺ CD38⁻ Lin⁻) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX).

Conclusions

The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients’ samples provides the rationale for the investigation of this compound in clinical trials.     

Umbilical vein endothelial cells are an important source of c-kit and stem cell factor which regulate the proliferation of haemopoietic progenitor cells

The expression and production of c-kit and its ligand, stem cell factor (SCF), in cord blood and neonates were studied. Serum SCF levels were significantly higher in cord blood, neonates aged 1–30 d, and in 4-month-old infants than in the maternal serum ( P  < 0.01). SCF levels decreased in children from 7 months to 15 years of age ( P  < 0.01). The serum soluble c-kit levels were significantly higher in cord ( P  < 0.01) and neonatal blood ( P  < 0.05) than in the maternal blood. SCF and c-kit levels in placental tissue homogenates and the culture media of decidual cells and trophoblasts were low. To determine the sites of high SCF and c-kit production in cord blood and in early neonates, SCF and c-kit mRNA expression was analysed in various tissues by polymerase chain reaction. High SCF mRNA expression was observed in human umbilical vein endothelial cells (HUVEC). Moderate c-kit mRNA expression was detected in HUVEC, the bone marrow, and cord blood. These findings suggest that endothelial cells mainly produce the SCF in cord blood and in early neonates. To confirm the role of endothelial cells in haemopoiesis, colony-forming assays were performed in the presence of HUVEC culture media, which induced the formation of high numbers of granulocyte and erythroid colonies in cord blood. IL-3, IL-6 and SCF levels were elevated in the media. Our findings suggest that endothelial cells have an important role in the maintenance and proliferation of progenitor cells in neonatal blood via the interaction of c-kit and SCF with other factors. The ex vivo expansion of cord progenitor cells in the presence of endothelial cells needs to be investigated further.     

The effects of oxidative DNA damage and mutations in the p53 protein on cells of the colonic mucosa with and without the fecal stream: an experimental study in rats

Abstract

Objective. The aim of this study was to investigate the levels of oxidative DNA damage and p53 mutations in an experimental model of diversion colitis. Material and methods. Sixty rats were divided into three groups with 20 animals in accordance with the sacrifice was carried out 6, 12 and 18 weeks. For each group, 15 animals were subjected to diversion of the fecal stream through colostomy in the left proximal colon and distal mucous fistula (experimental group), and five to a laparotomy without deviation of the fecal stream (control group). The presence of colitis was evaluated by inflammatory grading scale. Mutations in the p53 protein were evaluated by immunohistochemistry with primary antibody with cross-reactivity for rats. The oxidative DNA damage was measured using the comet assay. To statistical analysis were used the Student's t, Mann-Whitney and Kruskal-Wallis test adopting a significance level of 5% (p < 0.05). Results. Colon segments without fecal stream showed greater degree of inflammation when compared to animals with preserved fecal stream (p = 0.01). The levels of oxidative stress were significantly higher in segments without fecal stream (p < 0.0001) and increased with the time of fecal diversion (p = 0.007). The levels of oxidative DNA damage are directly related to tissue degree of inflammation. There were no mutations in the p53 protein in the segments without fecal stream regardless of time of exclusion considered. Conclusion. Despite higher levels of oxidative damage to nuclear DNA on segments without fecal stream that developed colitis mutations in the p53 protein were not detected.     

The radiotherapy-sensitization effect of cantharidin: Mechanisms involving cell cycle regulation,enhanced DNA damage,and inhibited DNA damage repair

Background

Cantharidin is an inhibitor of protein phosphatase 2?A (PP2A), and has been frequently used in clinical practice. In our previous study, we proved that cantharidin could arrest cell cycle in G2/M phase. Since cells at G2/M phase are sensitive to radiotherapy, in the present study, we investigated the radiotherapy-sesitization effect of cantharidin and the potential mechanisms involved.