A DNA repair pathway can regulate transcriptional noise to promote cell fate transitions.

Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean expression levels. This amplified expression noise originates from shorter-duration, higher-intensity transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we refer to as "discordant transcription through repair" ("DiThR," which is pronounced "dither"), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.

Osteogenic differentiation of skeletal muscle progenitor cells is activated by the DNA damage response.

Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.

Comparative gene expression analysis after exposure to 123I-iododeoxyuridine, γ- and α-radiation-potential biomarkers for the discrimination of radiation qualities.

Gene expression analysis was carried out in Jurkat cells in order to identify candidate genes showing significant gene expression alterations allowing robust discrimination of the Auger emitter 123I, incorporated into the DNA as 123I-iododeoxyuridine (123IUdR), from α- and γ-radiation. The γ-H2AX foci assay was used to determine equi-effect doses or activity, and gene expression analysis was carried out at similar levels of foci induction. Comparative gene expression analysis was performed employing whole human genome DNA microarrays. Candidate genes had to show significant expression changes and no altered gene regulation or opposite regulation after exposure to the radiation quality to be compared. The gene expression of all candidate genes was validated by quantitative real-time PCR. The functional categorization of significantly deregulated genes revealed that chromatin organization and apoptosis were generally affected. After exposure to 123IUdR, α-particles and γ-rays, at equi-effect doses/activity, 155, 316 and 982 genes were exclusively regulated, respectively. Applying the stringent requirements for candidate genes, four (PPP1R14C, TNFAIP8L1, DNAJC1 and PRTFDC1), one (KLF10) and one (TNFAIP8L1) gene(s) were identified, respectively allowing reliable discrimination between γ- and 123IUdR exposure, γ- and α-radiation, and α- and 123IUdR exposure, respectively. The Auger emitter 123I induced specific gene expression patterns in Jurkat cells when compared with γ- and α-irradiation, suggesting a unique cellular response after 123IUdR exposure. Gene expression analysis might be an effective tool for identifying biomarkers for discriminating different radiation qualities and, furthermore, might help to explain the varying biological effectiveness at the mechanistic level.

Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage.

Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2'-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 µM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy.

Lasting Adaptations in Social Behavior Produced by Social Disruption and Inhibition of Adult Neurogenesis.

UNLABELLED: Research on social instability has focused on its detrimental consequences, but most people are resilient and respond by invoking various coping strategies. To investigate cellular processes underlying such strategies, a dominance hierarchy of rats was formed and then destabilized. Regardless of social position, rats from disrupted hierarchies had fewer new neurons in the hippocampus compared with rats from control cages and those from stable hierarchies. Social disruption produced a preference for familiar over novel conspecifics, a change that did not involve global memory impairments or increased anxiety. Using the neuropeptide oxytocin as a tool to increase neurogenesis in the hippocampus of disrupted rats restored preference for novel conspecifics to predisruption levels. Conversely, reducing the number of new neurons by limited inhibition of adult neurogenesis in naive transgenic GFAP-thymidine kinase rats resulted in social behavior similar to disrupted rats. Together, these results provide novel mechanistic evidence that social disruption shapes behavior in a potentially adaptive way, possibly by reducing adult neurogenesis in the hippocampus. SIGNIFICANCE STATEMENT: To investigate cellular processes underlying adaptation to social instability, a dominance hierarchy of rats was formed and then destabilized. Regardless of social position, rats from disrupted hierarchies had fewer new neurons in the hippocampus compared with rats from control cages and those from stable hierarchies. Unexpectedly, these changes were accompanied by changes in social strategies without evidence of impairments in cognition or anxiety regulation. Restoring adult neurogenesis in disrupted rats using oxytocin and conditionally suppressing the production of new neurons in socially naive GFAP-thymidine kinase rats showed that loss of 6-week-old neurons may be responsible for adaptive changes in social behavior.

Effects of resveratrol and methoxyamine on the radiosensitivity of iododeoxyuridine in U87MG glioblastoma cell line.

The purpose of this study was to evaluate the combination effect of resveratrol and methoxyamine on radiosensitivity of iododeoxyuridine in spheroid culture of U87MG glioblastoma cell line using colony formation and alkaline comet assays. Spheroids on day-20 with 350 µm diameters were treated with 20 µM resveratrol and/or 6 mM methoxyamine and/or 1 µM iododeoxyuridine for one volume doubling time (67 h), and then irradiated with 2 Gy gamma-radiation ((60)Co) in different groups. After treatment, viability of the cells, colony forming ability and DNA damages were obtained by blue dye exclusion, colony formation and alkaline comet assay, respectively. Our results showed that methoxyamine and resveratrol could significantly reduce colony number and induce the DNA damages of glioblastoma spheroid cells treated with iododeoxyuridine in combination with gamma-rays. Therefore, methoxyamine as base excision repair inhibitor and resveratrol as hypoxia inducible factor 1-alpha inhibitor in combination with iododeoxyuridine as radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Role of resveratrol on the cytotoxic effects and DNA damages of iododeoxyuridine and megavoltage radiation in spheroid culture of U87MG glioblastoma cell line.

The purpose of this study was to evaluate the effect of resveratrol on cytogenetic damages of iododeoxyuridine (IUdR) and x-ray megavoltage radiation (6 MV) in spheroid model of U87MG glioblastoma cancer cell line using clonogenic and alkaline comet assay. Cells were cultured as spheroids (350 µm) that were treated with 20 µM resveratrol, 1 µM IUdR and 2 Gy of 6 MV x-ray. After treatment, viability of the cells, colony forming ability and the induced DNA damages were examined using trypan blue dye exclusion, colonogenic and alkaline comet assay, respectively. Our results showed that resveratrol could significantly reduce the colony number and induce the DNA damages of the cells treated with IUdR in combination with 6 MV x-ray radiation. That results indicated that resveratrol as an inhibitor of hypoxia inducible factor 1 alpha (HIF-1α) protein in combination with IUdR as a radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Impact of IUdR on Rat 9L glioma cell survival for 25-35 keV photon-activated auger electron therapy.

The goal of the current study was to measure the energy dependence of survival of rat 9L glioma cells labeled with iododeoxyuridine (IUdR) that underwent photon-activated Auger electron therapy using 25-35 keV monochromatic X rays, i.e., above and below the K-edge energy of iodine. Rat 9L glioma cells were selected because of their radioresistance, ability to be implanted for future in vivo studies and analogy to radioresistant human gliomas. Survival curves were measured for a 4 MV X-ray beam and synchrotron produced monochromatic 35, 30 and 25 keV X-ray beams. IUdR was incorporated into the DNA at levels of 0, 9 and 18% thymidine replacement for 4 MV and 35 keV and 0 and 18% thymidine replacement for 30 and 25 keV. For 10 combinations of beam energy and thymidine replacement, 62 data sets (3-13 per combination) provided 776 data points (47-148 per combination). Survival versus dose data taken for the same combination, but on different days, were merged by including the zero-dose points in the nonlinear, chi-squared data fitting using the linear-quadratic model and letting the best estimate to the zero-dose plating efficiency for each of the different days be a fitting parameter. When comparing two survival curves, the ratio of doses resulting in 10% survival gave sensitization enhancement ratios (SER10) from which contributions due to linear energy transfer (LET) (SER10,LET), IUdR radiosensitization (SER10,RS), the Auger effect (SER10,AE) and the total of all effects (SER10,T) were determined. At 4 MV and 35, 30 and 25 keV, SER10,LET values were 1.00, 1.08 ± 0.03, 1.22 ± 0.02 and 1.37 ± 0.02, respectively. At 4 MV SER10,RS values for 9 and 18% IUdR were 1.28 ± 0.02 and 1.40 ± 0.02, respectively. Assuming LET effects were independent of percentage IUdR and radiosensitization effects were independent of energy, SER10,AE values for 18% IUdR at 35, 30 and 25 keV were 1.35 ± 0.05, 1.06 ± 0.03 and 0.98 ± 0.03, respectively. The value for 9% IUdR at 35 keV was 1.01 ± 0.04. First, we found the radioresistant rat 9L glioma cell line exhibited an SER10 due to the Auger effect of 1.35 at (35 keV, 18% IUdR) and an SER10 due to the radiosensitizing effect of 1.40 at (4 MV, 18% IUdR), both significantly less than values for previously reported cell lines. These low individual values emphasize the benefit of their combined value (SER10 of approximately 1.9) for achieving clinical benefit. Second, as expected, we observed that energies below the K-edge of iodine (25 and 30 keV), for which there are L, M and higher shell photoelectric events creating Auger electrons, show no promise for Auger electron therapy. Third, to proceed with future in vivo studies, additional data from 35-65 keV are needed to determine the optimal X-ray energy for IUdR Auger electron therapy. Only then can there be an answer to the question, how well the energy dependence of in vitro survival data supports the potential for photon-activated Auger electron therapy with IUdR in cancer radiotherapy.

Quantitative analysis of the effects of iododeoxyuridine and ionising radiation treatment on the cell cycle dynamics of DNA mismatch repair deficient human colorectal cancer cells.

DNA mismatch repair (MMR) is involved in processing DNA damage following treatment with ionising radiation (IR) and various classes of chemotherapy drugs including iododeoxyuridine (IUdR), a known radiosensitiser. In this study, the authors have developed asynchronous probabilistic cell cycle models to assess the isolated effects of IUdR and IR and the combined effects of IUdR + IR treatments on MMR damage processing. The authors used both synchronous and asynchronous MMR-proficient/MMR-deficient cell populations and followed treated cells for up to two cell cycle times. They have observed and quantified differential cell cycle responses to MMR damage processing following IR and IUdR + IR treatments, principally in the duration of both G1 and G2/M cell cycle phases. The models presented in this work form the foundation for the development of an approach to maximise the therapeutic index for IR and IUdR + IR treatments in MMR-deficient (damage tolerant) cancers.

Identification of lineage-uncommitted, long-lived, label-retaining cells in healthy human esophagus and stomach, and in metaplastic esophagus.

BACKGROUND & AIMS: The existence of slowly cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients. METHODS: Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg/m(2) body surface area; maximum dose, 400 mg) over a 30-minute period; the IdU had a circulation half-life of 8 hours. Tissues were collected at 7, 11, 29, and 67 days after infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. RESULTS: No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this time point. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). CONCLUSIONS: LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia, is required.

Effect of 5-[(125)I]iodo-2'-deoxyuridine uptake on the proliferation and pluripotency of human embryonic stem cells.

PURPOSE: Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because, in principle, they can differentiate into any cell type found in the human body. In addition, studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state, as well as the consequences of IR exposure on the development of organisms. However, the effect of IR, in particular radionuclide uptake, on the pluripotency, proliferation and survival of hESC has not been extensively studied. METHODS: In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU), a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC. RESULTS: We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However, treatment with 0.1 µCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 µCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls. CONCLUSIONS: Our results provide important initial insights into the sensitivity of hESC to IR, and especially that produced by the decay of an internalized radionuclide.

Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues.

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 µM) and deoxyuridine (Km=578 µM), it prefers uridine (Km=92 µM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.

Chemical induction of endogenous retrovirus particles from the vero cell line of African green monkeys.

Endogenous retroviral sequences are present in high copy numbers in the genomes of all species and may be expressed as RNAs; however, the majority are defective for virus production. Although virus has been isolated from various Old World monkey and New World monkey species, there has been no report of endogenous retroviruses produced from African green monkey (AGM) tissues or cell lines. We have recently developed a stepwise approach for evaluating the presence of latent viruses by chemical induction (Khan et al., Biologicals 37:196-201, 2009). Based upon this strategy, optimum conditions were determined for investigating the presence of inducible, endogenous retroviruses in the AGM-derived Vero cell line. Low-level reverse transcriptase activity was produced with 5-azacytidine (AzaC) and with 5'-iodo-2'-deoxyuridine (IUdR); none was detected with sodium butyrate. Nucleotide sequence analysis of PCR-amplified fragments from the gag, pol, and env regions of RNAs, prepared from ultracentrifuged pellets of filtered supernatants, indicated that endogenous retrovirus particles related to simian endogenous type D betaretrovirus (SERV) sequences and baboon endogenous virus type C gammaretrovirus (BaEV) sequences were induced by AzaC, whereas SERV sequences were also induced by IUdR. Additionally, sequence heterogeneity was seen in the RNAs of SERV- and BaEV-related particles. Infectivity analysis of drug-treated AGM Vero cells showed no virus replication in cell lines known to be susceptible to type D simian retroviruses (SRVs) and to BaEV. The results indicated that multiple, inducible endogenous retrovirus loci are present in the AGM genome that can encode noninfectious, viruslike particles.

Antimicrobial activities of trimethoprim/sulfamethoxazole, 5-iodo-2'-deoxyuridine and rifampicin against Staphylococcus aureus.

Trimethoprim/sulfamethoxazole (SXT), alone and in combination with rifampicin (RIF), is a therapeutic option against Staphylococcus aureus, including strains expressing meticillin resistance. However, the antimicrobial activity of SXT is antagonised by thymidine, which can be present in infected and/or inflamed tissues such as the airways of cystic fibrosis (CF) patients. In this study, thymidine concentrations in CF sputa were determined and the antimicrobial activities of SXT, 5-iodo-2'-deoxyuridine (IdUrd) and RIF alone and in combination against S. aureus were analysed. Thymidine concentrations in the sputa of ten different CF patients varied from <100 µg/L to 38845 µg/L. The abolished antimicrobial activity of SXT against 22 S. aureus strains in the presence of thymidine was restored by combination with IdUrd. In contrast, SXT combined with RIF in the presence of thymidine did not show a synergistic effect and, furthermore, allowed the emergence of RIF-resistant bacteria. Adding RIF to the combination of SXT and IdUrd did not improve antimicrobial activity against S. aureus. In conclusion, the combination of SXT and RIF as a therapeutic option against S. aureus infections in chronic inflamed tissues should be judged critically.

Differential regulation of the variations induced by environmental richness in adult neurogenesis as a function of time: a dual birthdating analysis.

Adult hippocampal neurogenesis (AHN) augments after environmental enrichment (EE) and it has been related to some of the anxiolytic, antidepressant and neuroprotective effects of EE. Indeed, it has been suggested that EE specifically modulates hippocampal neurogenic cell populations over the course of time. Here we have used dual-birthdating to study two subpopulations of newborn neuron in mice (Mus musculus): those born at the beginning and at the end of enrichment. In this way, we demonstrate that while short-term cell survival is upregulated after an initial 1 week period of enrichment in 2 month old female mice, after long-term enrichment (2 months) neither cell proliferation nor the survival of the younger newly born cell populations are distinguishable from that observed in non-enriched control mice. In addition, we show that the survival of older newborn neurons alone (i.e. those born at the beginning of the enrichment) is higher than in controls, due to the significantly lower levels of cell death. Indeed, these parameters are rapidly adjusted to the sudden cessation of the EE conditions. These findings suggest both an early selective, long-lasting effect of EE on the neurons born in the initial stages of enrichment, and a quick response when the environment again becomes impoverished. Therefore, EE induces differential effects on distinct subpopulations of newborn neurons depending on the age of the immature cells and on the duration of the EE itself. The interaction of these two parameters constitutes a new, specific regulation of these neurogenic populations that might account for the long-term enrichment's behavioral effects.

Selective impairments to memory consolidation in chicks produced by 5'-iodo-2'-deoxyuridine.

The aim of the present work was to study the role of DNA synthesis in the formation of different types of memory in neonatal chicks. The nucleotide analogs 5'-iodo-2'-deoxyuridine (IdU) and 5'-bromo-2'-deoxyuridine (BrdU) were used; these are incorporated into DNA, impairing its function, and have amnestic actions in defined models of learning in mice. We studied the effects of 5'-iodo-2'-deoxyuridine of the formation of long-term memory in chicks during training in different models: passive avoidance, imprinting, taste aversion, and spatial learning in a maze. In the taste aversion model, i.p. administration of IdU (10 mg/kg 5 min before or 50 min after training) had an amnestic effect on testing 1-2 days after training. IdU-induced amnesia developed more than 6 h after training, while administration of IdU 2 h after training had no amnestic effect. 5'-Bromo-2'-deoxyuridine also had a similar amnestic action in the taste aversion model. In the passive avoidance, imprinting, and spatial maze learning models, administration of IdU at the same dose before and after training did not induce amnesia. These data lead to the suggestion that DNA synthesis in the brain may play a critical role in the mechanisms of memory consolidation in chicks in types of learning such as taste aversion.

Synergistic antimicrobial activities of folic acid antagonists and nucleoside analogs.

The antimicrobial activities of folic acid antagonists are supposed to be antagonized by elevated extracellular thymidine concentrations in damaged host tissues. Therefore, this study was aimed at screening for nucleoside analogs that impair bacterial thymidine utilization and analyzing the combined antimicrobial activities of nucleoside analogs and folic acid antagonists in the presence of thymidine. Our screening results revealed that different nucleoside analogs, in particular halogenated derivatives of 2'-deoxyuridine, substantially impaired the bacterial utilization of extracellular thymidine in Staphylococcus aureus. Time-kill methods showed that 5-iodo-2'-deoxyuridine enhanced the extent of killing of trimethoprim-sulfamethoxazole (SXT) at 24 h against S. aureus in the presence of thymidine (200 microg/liter). While SXT (40 mg/liter) alone did not kill bacteria in the presence of thymidine, its combination with the nucleoside analog at a concentration of 8 mumol/liter showed a bactericidal effect. Moreover, 5-iodo-2'-deoxyuridine combined with SXT in the presence of thymidine showed a broad spectrum of activity against several Gram-positive and Gram-negative bacteria. In conclusion, these data provide evidence that the in vitro antimicrobial activity of SXT in the presence of thymidine can be significantly improved by combination with a nucleoside analog.

Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells.

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.