DNA Synthesis Activity Research Articles

The Influence of Ghrelin on the Development of Dextran Sodium Sulfate-Induced Colitis in Rats.

Ghrelin has protective and therapeutic effects in the gut. The aim of present studies was to investigate the effect of treatment with ghrelin on the development of colitis evoked by dextran sodium sulfate (DSS). Methods. Studies have been performed on rats. Colitis was induced by adding 5% DSS to the drinking water for 5 days. During this period animals were treated intraperitoneally twice a day with saline or ghrelin given at the dose of 8 nmol/kg/dose. On the sixth day, animals were anesthetized and the severity of colitis was assessed. Results. Treatment with ghrelin during administration of DSS reduced the development of colitis. Morphological features of colonic mucosa exhibited a reduction in the area and deep of mucosal damage. Ghrelin reversed the colitis-induced decrease in blood flow, DNA synthesis, and superoxide dismutase activity in colonic mucosa. These effects were accompanied by a decrease in the colitis-evoked increase in mucosal concentration of interleukin-1β and malondialdehyde. Treatment with ghrelin reversed the DSS-induced reduction in body weight gain. Conclusions. Administration of ghrelin exhibits the preventive effect against the development of DSS-induced colitis. This effect seems to be related to ghrelin's anti-inflammatory and antioxidative properties.

High-resolution analysis of Mammalian DNA replication units.

Genomic DNA of a eukaryotic cell is replicated once during the S-phase of the cell cycle to precisely maintain the complete genetic information. In the course of S-phase, semiconservative DNA synthesis is sequentially initiated and performed at thousands of discrete patches of the DNA helix termed replicons. At any given moment of S-phase, multiple replicons are active in parallel in different parts of the genome. In the last decades, tools and methods to visualize DNA synthesis inside cells have been developed. Pulse labeling with nucleotides as well as detecting components of the replication machinery yielded an overall picture of multiple discrete sites of active DNA synthesis termed replication foci (RFi) and forming spatiotemporal patterns within the cell nucleus. Recent advances in fluorescence microscopy and digital imaging in combination with computational image analysis allow a comprehensive quantitative analysis of RFi and provide valuable insights into the organization of the genomic DNA replication process and also of the genome itself. In this chapter, we describe in detail protocols for the visualization and quantification of RFi at different levels of optical and physical resolution.

Recombinant neuregulin 1 does not activate cardiomyocyte DNA synthesis in normal or infarcted adult mice.

ObjectivesNeuregulin 1 signaling plays an important role in cardiac trabecular development, and in sustaining functional integrity in adult hearts. Treatment with neuregulin 1 enhances adult cardiomyocyte differentiation, survival and/or function in vitro and in vivo. It has also been suggested that recombinant neuregulin 1β1 (NRG1β1) induces cardiomyocyte proliferation in normal and injured adult hearts. Here we further explore the impact of neuregulin 1 signaling on adult cardiomyocyte cell cycle activity.Methods and ResultsAdult mice were subjected to 9 consecutive daily injections of recombinant NRG1β1 or vehicle, and cardiomyocyte DNA synthesis was quantitated via bromodeoxyuridine (BrdU) incorporation, which was delivered using mini-osmotic pumps over the entire duration of NRG1β1 treatment. NRG1β1 treatment inhibited baseline rates of cardiomyocyte DNA synthesis in normal mice (cardiomyocyte labelling index: 0.019±0.005% vs. 0.003±0.001%, saline vs. NRG1β1, P<0.05). Acute NRG1β1 treatment did result in activation of Erk1/2 and cardiac myosin regulatory light chain (down-stream mediators of neuregulin signalling), as well as activation of DNA synthesis in non-cardiomyocytes, validating the biological activity of the recombinant protein. In other studies, mice were subjected to permanent coronary artery occlusion, and cardiomyocyte DNA synthesis was monitored via tritiated thymidine incorporation which was delivered as a single injection 7 days post-infarction. Daily NRG1β1 treatment had no impact on cardiomyocyte DNA synthesis in the infarcted myocardium (cardiomyocyte labelling index: 0.039±0.011% vs. 0.027±0.021%, saline vs. NRG1β1, P>0.05).SummaryThese data indicate that NRG1β1 treatment does not increase cardiomyocyte DNA synthesis (and consequently does not increase the rate of cardiomyocyte renewal) in normal or infarcted adult mouse hearts. Thus, any improvement in cardiac structure and function observed following neuregulin treatment of injured hearts likely occurs independently of overt myocardial regeneration.

Antiviral effect of cidofovir on parvovirus B19 replication

Parvovirus B19 (B19V) is a human ssDNA virus responsible for a wide range of clinical manifestations, still lacking for a specific antiviral therapy. The identification of compounds active against B19V may add therapeutic options to the treatment of B19V infections, that now entirely relies on symptomatic treatments. In the search for compounds possibly inhibiting B19V replication, a particular focus was raised to cidofovir, an acyclic nucleoside phosphonate broadly active against dsDNA viruses. The present study was aimed at evaluating the effect of cidofovir against B19V in two model systems, the UT7/EpoS1 cell line and erythroid progenitor cells (EPC), generated from peripheral blood mononuclear cells. Experiments were carried out at different multiplicity of infections and cidofovir concentrations (0–500μM) during a course of infection. The effects of cidofovir on B19V replication were assessed by qPCR assays while influence of cidofovir on host cells was measured by cell proliferation and viability assays. Our findings demonstrated that cidofovir has a relevant inhibiting activity on B19V replication within infected UT7/EpoS1, and that the effect on B19V DNA amounts is dose-dependent allowing for the determination of EC50 and EC90 values (7.45–41.27μM, and 84.73–360.7μM, respectively). In EPCs, that constitute a cellular population close to the natural target cells in bone marrow, the inhibitory effect was demonstrated to a lesser extent, however provoking a significant reduction on B19V DNA amounts at 500μM (68.2–92.8%). To test infectivity of virus released from EPCs cultured in the presence of cidofovir, cell culture supernatants were used as inoculum for a further course of infection in UT7/EpoS1 cells, indicating a significant reduction in viral infectivity at 500μM cidofovir. Since the drug did not interfere with the overall cellular DNA synthesis and metabolic activity, the observed effect of cidofovir could be likely related to a specific inhibition of B19V replication.

EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune rheumatological disease thought to have substantial genetic contributions. Epidermal growth factor (EGF) can activate DNA synthesis and cellular proliferation. Early RA synovial fluid was characterized by significantly elevated levels stromal cell and macrophage-related cytokines including EGF. We therefore hypothesized that EGF polymorphisms may contribute to RA susceptibility in the Chinese population. We studied EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms in 520 patients with RA and 520 controls in a Chinese population. When the EGF rs11568835 GG homozygote genotype was used as the reference group, the AA genotype was associated with an increased risk for RA (AA versus GG, odds ratio [OR] = 3.64, 95% confidence interval [CI] = 1.19–11.17, p = 0.024), the GA or GA/AA genotype was not associated with the risk for RA (GA versus GG, OR = 0.99, 95% CI = 0.75–1.31, p = 0.931; GA + AA versus GG, OR = 1.06, 95% CI = 0.81–1.40, p = 0.659). EGF rs3756261 T/C was not associated with susceptibility to RA. These results provide the first positive evidence for an association between EGF rs11568835 G/A polymorphism and RA.

Evaluation of Anti-melanoma Activities of (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol, (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol from the Red Sea soft coral Sarcophyton glaucum

Three natural cembranoids from the Red Sea soft coral Sarcophyton glaucum namely (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol, (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol were evaluated for their inhibitory effects on mouse melanoma B16F10 cell growth. Results show that all the cembranoids strongly inhibit viability of melanoma cells particularly during 48 -72 hrs treatment and also inhibit de novo DNA synthesis and PARP activity and stimulate fragmentation of DNA. (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol was not cytotoxic to monkey kidney CV-1 cells at the concentration that produces the anti-melanoma effects which indicates that this compound may be a good candidate for further development. (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol were found to be cytotoxic to healthy cells.

Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5′dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polβ DNA synthesis activity is not necessary while 5′dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

Zinc in medicine and treatment

Zinc is an essential and the second most abundant trace element in humans. It is critical for the growth, development and differentiation of cells, as well as for RNA transcription, DNA synthesis, cell division and cell activation. Zinc deficiency affects mainly functions of the immune system, but other consequences include inferior sperm activity, skin lesions, growth retardation, impaired wound healing, anemia and gastrointestinal disorders. Zinc supplementation protects against the hepatotoxic effects of alcohol, enhances the transport of water and electrolytes across the intestinal mucosa and improves immune and anti-inflammatory responses. Zinc is also known as an essential mineral for normal mobilization of vitamin A from the liver to the plasma. Besides, it increases the promoter response to 1,25-dihydroxyvitamin D in osteoblasts. On the other hand, excessive amounts of free zinc in tissues are toxic and accelerated zinc accumulation of zinc is a potent killer of neurons and glial cells. Over 300 signaling molecules and transcription factors contain zinc as a cofactor. Free zinc in immune and tumor cells is regulated by 14 distinct zinc importers (ZIP) and transporters. An elevated amount of zinc transporters LIV-1, a subfamily of ZIP zinc transporters, appears in estrogen receptor–positive breast cancer and has been used as a reliable breast cancer marker. However, the fact that malignant cells are unable to accumulate zinc is an important factor in the development and progression of malignancy of prostate cancer.

The effect of epigallocatechin gallate on hepatocytes isolated from normal and partially hepatectomized rats.

Epigallocatechin gallate (EGCG) is an antioxidant found in green tea. In this study, male Wistar rats were subjected either to partial hepatectomy (PHx), or a sham operation (LAP). Twenty-four hours after surgery, hepatocytes were isolated and treated with various concentrations of EGCG for up to 72 h. We then measured markers of cell viability, oxidative stress, DNA synthesis, and caspase activity. Morphological criteria, cell viability tests, and albumin synthesis revealed toxicity starting at 10 μmol/L. DNA synthesis was higher in hepatocytes isolated from rats after PHx and inhibited by EGCG. Furthermore, EGCG increased the activity of caspases 3 and 7, seen more in hepatocytes from PHx rats. In conclusion, EGCG at a concentration of 10 μmol/L was toxic for hepatocytes isolated from both PHx and LAP rats.

Tipin Functions in the Protection against Topoisomerase I Inhibitor

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.

Somatic mutant alleles of POLE found in human cancers suppress proofreading and replication fidelity in vitro (LB121)

While it is now appreciated that tumor cells contain thousands of mutations, identifying their source(s) has remained problematic. Whole genome sequencing from colorectal and endometrial cancers recently identified mutations in DNA polymerase (Pol) epsilon, one of three main eukaryotic nuclear replication Pols. These mutations largely reside in the exonuclease domain, are associated with a hypermutated phenotype, and are predicted to promote tumorigenesis. However, the effects of these somatic mutations on enzyme function have not yet been studied. To address this, we introduced several of these mutations in recombinant human Pol ε and measured their effects on replication fidelity in vitro. While the mutants retained wild type DNA synthesis activity, 3'‐5' exonuclease proofreading was suppressed. Surprisingly, this suppression was variable, ranging from ~2‐fold to almost complete ablation, and resulted in increases in frameshifts and base pair substitutions in an in vitro forward mutation assay. Mutants also had increased ability to bypass abasic sites. Exonuclease inactivation of one POLE allele in human cells resulted in decreased sensitivity to H2O2 treatment. These results suggest that Pol ε mutant alleles may promote viability and mutagenesis, and possibly tumorigenesis, in part through bypass of oxidative stress‐induced DNA lesions.Grant Funding Source: NIH RR020152

Nisin/β-lactam adjunct therapy against Salmonella enterica serovar Typhimurium: a mechanistic approach

Multidrug resistance exhibited by Salmonella strains has proved to be a big hurdle in the development of an effective anti-Salmonella therapy. In this context, we had previously demonstrated strong synergism of nisin/ceftriaxone and nisin/cefotaxime combinations against Salmonella enterica serovar Typhimurium. However, the mechanism remained unexplored. The present study was therefore planned in order to evaluate the underlying mechanisms responsible for the synergistic effect of nisin in combination with these β-lactam antibiotics against serovar Typhimurium. A membrane permeabilization assay along with pulse labelling studies were performed to confirm the ability of the combinations to permeabilize the bacterial membrane and to verify their effects on macromolecule synthesis. Additionally, analysis of peroxidative liver damage was performed and levels of nitric oxide, antioxidant enzymes, tumour necrosis factor-α and nuclear factor-κB were also measured. 1-N-phenylnapthylamine (NPN) uptake assay results confirmed a permeabilization-dependent mechanism, as NPN was taken up by treated cells in a time- and concentration-dependent manner, indicating that the combination influenced membrane permeability. Likewise, dose- and time-dependent inhibition of DNA, RNA and protein synthesis in the presence of both the combinations was observed. Interestingly, synergistic results inferred from in vivo assays confirmed the immuno-modulatory effects of the combinations in the treated mice. Nisin/ceftriaxone and nisin/cefotaxime combinations exert their antibacterial activity against Salmonella by multiple modes of action that involve membrane permeabilization, inhibition of DNA, RNA and protein synthesis and direct immuno-modulatory activity.

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growth medium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett-Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150ngml(-1), respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53% metabolic activity and 24.8% DNA synthesis could be obtained, which suggested that IGF-I and IGF-II had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells.

Evaluation of the Anti-melanoma Activities of Sarcophine, (+)-7α,8β-Dihydroxydeepoxysarcophine and Sarcophytolide from the Red Sea Soft Coral Sarcophyton glaucum

Three natural cembranoids from the Red Sea soft coral Sarcophyton glaucum namely sarcophine (1), (+)-7alpha,8beta-dihydroxydeepoxysarcophine (2) and sarcophytolide (3) were evaluated for their potential inhibitory effects on growth of mouse melanoma B16F10 cells. Compounds (1) and (2) maximally inhibit viability of melanoma cells during 48 hr and 72 hr treatment at concentrations that show no cytotoxicity on monkey kidney CV-1 cells and also inhibit de novo DNA synthesis and PARP activity. Compound (3) produced cytotoxic effects at the same concentration range it produces its antitumor effects. These data suggest that (1) and (2), but not (3), have potential for further development as antitumor agents against melanoma.

A Missense Mutation in Rev7 Disrupts Formation of Polζ, Impairing Mouse Development and Repair of Genotoxic Agent-induced DNA Lesions

Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ζ (Polζ), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Polζ. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive γH2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Polζ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.

Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro

Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell-cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4(+) cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4(+) cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env(+) cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4(+) cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell-cell fusion can be performed by flow cytometry.

Complex Temperature Dependent Equilibria Dictate DNA Polymerase Exchange Processes during Synthesis

Most organisms encode for multiple DNA polymerases with similar substrate affinities, but vastly different fidelities. Proper genomic maintenance by the high fidelity (PolB1) and lesion bypass polymerases (PolY) from Sulfolobus solfataricus involves a complex solution equilibria of protein complexes and specific recognition of appropriate DNA substrates. Using isothermal titration calorimetry (ITC) and temperature dependent electrophoretic mobility shift assays (tEMSAs), we have found differences in oligomeric assemblies of Dpo1 and Dpo4 on DNA that include unusually strong temperature dependence changes in heat capacity (ΔCp), which switch from positive to negative values as temperature increases over a 60°C range. The thermodynamic data suggests that binding of PolB1 to DNA is favored over PolY by changes in solution multiequilibria (monomer-oligomer) with temperature that influence ΔCp values. We have also followed polymerase exchange events between PolB1 and PolY and themselves and with the sliding clamp, PCNA, during active DNA synthesis using ensemble kinetic and fluorescence resonance energy transfer (FRET) assays. Surprisingly, the assembled PolB1 holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. Exchange between polymerases is temperature and concentration dependent process that is orchestrated by several contacts with PCNA. Simultaneous binding of PolB1 and PolY1 to PCNA allows for dynamic exchange of polymerase active sites during replication and lesion bypass synthesis. Taken together, our results distinguish between specific thermodynamic parameters including temperature dependent coupled equilibria and structural complementary; kinetic processes; and protein contacts that direct binding for uninterrupted but dynamic DNA replication and repair processes at high temperatures.

Activity enhancement of G-quadruplex/hemin DNAzyme by spermine

Biogenic polyamines participate in regulating gene expression, activating DNA synthesis and facilitating DNA–protein interaction through interaction with DNA/RNA. The interaction of polyamines with G-quadruplexes (G4s) has been reported to modulate the structure of G4s. In this paper, we investigate the effects of polyamines on one of the properties of G4s, G4/hemin peroxidase. Three polyamines (spermine, spermidine and putrescine) are found to have positive effects on different G4/hemin DNAzymes, in which spermine exhibits the strongest enhancement efficiency. CD and UV/Vis spectral analysis suggests two reasons for the strong activity enhancement: first, spermine protects hemin from rapid degradation by H2O2; second, spermine condenses the G4 structures and provides a favorable microenvironment for the catalytic reaction. Since G4/hemin DNAzymes have been extensively applied in various chemical sensors and biosensors, this finding would be helpful for the design of G4/hemin based sensors and widen the application range of this kind of DNAzyme.

Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be "G2/M arrest." However, a B19V mutant infectious DNA (M20(mTAD2)) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G2/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol. 86:10748-10758, 2012). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2'-deoxyuridine (BrdU) pulse-labeling and DAPI (4',6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20(mTAD2) mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication.

Study of DNA synthesis and mitotic activity of hepatocytes and its relation to angiogenesis in hepatectomised tumour bearing mice

Partial hepatectomy (PH) alters serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. Furthermore, angiogenesis is mainly stimulated by vascular endothelial growth factor (VEGF) and is a fundamental requirement either in liver regeneration or in tumours growth. This study looks at the expression of VEGF, DNA synthesis (DNAs) and mitotic activity (MA) in hepatectomised (H) and hepatectomised-tumour bearing (HTB) mice throughout a 24 h period. Adult male mice were sacrificed every 4 h from 26 to 50 h post-hepatectomy. H mice show a circadian rhythm in VEGF expression with a maximum value of 2.6 ± 0.1 at 08/46 h of day/hours posthepatectomy (HD/HPH); in DNAs, the maximum value was 3.4 ± 0.3 at 16/30 (HD/HPH) and in MA it was 2.3 ± 0.01 at 12/50 (HD/HPH). In HTB animals the peak of VEGF expression appears at 16/30 (HD/HPH) with a maximum value of 3.7 ± 0.1, the peak of DNAs was at 00/38 (HD/HPH) with a value of 4.6 ± 0.3 and the maximum value of MA of 08/46 (HD/HPH) with a value of 3.01 ± 0.3. We can conclude that the presence of the tumour induces modifications in the intensity and the temporal distribution of the circadian curves of VEGF expression, DNAs and MA of hepatectomised animals.