Fiber Assay Research Articles

Abstract A16: Boosting of in vivo hollow fiber assay by using luciferase-labeled tumor cell lines

Abstract In drug development, predicting in vivo inhibitory drug activity from cellular assay data is difficult due to the complexity of the living system, resulting in the sacrifice of many mice in vain. In order to prescreen compounds for their in vivo activity more efficiently, the hollow fiber assay (HFA) was developed in 1995 by Hollingshead et al. The HFA assay allows the simultaneous evaluation of multiple cell lines implanted in separated drug-(but not cell-) permeable fibers within a single mouse. We herein report on the advancement of this technology by use of luciferase-labeled tumor cell lines. The limits of detection are boosted and disturbing (non-labeled) stroma cells attached to the fibers are excluded enabling a clear separation of drug effects on the tumor cells. The effect of Gemcitabine and Paclitaxel on growth of luciferase-labeled MDA-MB-231 and A549 cells in HFA was analyzed by MTT in comparison to luciferase activity analysis. The general findings between both read-outs were comparable. The growth of MDA-MB-231 cells was inhibited by both, Paclitaxel and Gemcitabine, whereas inhibition of A549 cell growth was only observed under Paclitaxel treatment. T/C calculation for the MTT analysis predicted a partial response in all cases. In contrast, the luciferase activity analysis clearly distinguished between a complete response for MDA-MB-231 cell growth when treated with Paclitaxel and Gemcitabine and a partial response for Paclitaxel on A549 cell growth. These results correspond to the in vivo xenograft studies: T/C for Gemcitabine using MDA-MB-231 cells was 2-fold lower (16.2%) than T/C in A549 tumors using Paclitaxel (36.3%). In addition, results are shown applying this technology for up to 42 days instead of the typical 14 days, enhancing the possibility to analyze slow-acting drugs. In summary, the HFA assay for in vivo drug screening with luciferase-labeled tumor cells remarkably increases the power and predictability for drug efficacy in xenograft studies. Citation Format: Cynthia Schaefer-Obodozie, Ulrike Leisegang, Susanne Ruf, Bianca Giesen, Christoph Schaechtele, Holger Weber. Boosting of in vivo hollow fiber assay by using luciferase-labeled tumor cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A16.

Anticancer Activity and Autophagy Involvement of Self-Assembled Arene–Ruthenium Metallacycles

A suite of six metallacycles (1–6) was generated via coordination-driven self-assembly using the three dicarboxylate-bridged arene–Ru precursors [Ru2(μ-η4-OO∩OO)(η6-p-iPrC6H4Me)2][CF3SO3]2; (OO∩OO = oxalate (A1), 2,5-dihydroxy-1,4-benzoquinonato (dobq) (A2), 5,8-dihydroxy-1,4-naphthoquinonato (donq) (A3); CF3SO3 = triflate) with one of two dipyridyl ligands (L1 and L2). The metallacycles were isolated in excellent yield (86–92%) as triflate salts and characterized by proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR) and electrospray ionization–mass spectrometry (ESI-MS) to confirm their structural assignments. Single-crystal X-ray crystal analysis of 1 revealed that two L1 ligands bridged two A1 acceptors to form a rectangular architecture. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to evaluate the in vitro cytotoxicities relative to two chemotherapeutic agents: namely, cisplatin and doxorubicin. Metallacycles 3 and 6 potently inhibited the growth of HCT-15 human colon and AGS human gastric cancer cells. The hollow fiber (HF) assay was performed to investigate the in vivo antitumor activities of metallacycles 3 and 6. Metallacycle 6 was more effective in inhibiting HCT-15 cells than metallacycle 3 in both in vitro and in vivo studies. Moreover, 3 and 6 induced autophagic activity in HCT-15 cells. These results suggested that the autophagic response elicited by metallacycles 3 and 6 could mediate the anticancer effects observed in human colorectal cancer cells.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50μM) and clodronate (125, 250 and 500μM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72h with zoledronate, ibandronate, alendronate and clodronate. Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5μM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Abstract 3036: The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing

Abstract Regulation of dormant origin firing under conditions of replication stress is poorly understood. The Bloom syndrome DNA helicase BLM functions in maintaining replication fork stability, with sumoylation regulating this function, and BLM deficiency is associated with increased dormant origin firing. We found here that BLM sumoylation levels increase in response to replication stalling by hydroxyurea (HU) and to proteasome inhibition; depletion of the SUMO-targeted ubiquitin ligase RNF4 also causes increased levels of BLM sumoylation. RNF4 directly interacts with BLM and can ubiquitylate sumoylated BLM in vitro. These data indicate that sumoylated BLM is an RNF4 substrate. RNF4 is recruited to DNA repair foci in response to replication stalling. RNF4 depletion causes the accumulation of increased numbers of HU-induced BLM foci, whereas the numbers of RPA, RAD51, and gamma-H2AX foci are unaffected by fork stalling in RNF4-depleted cells. Sister chromatid exchanges are normal in RNF4-depleted cells. These data indicate that RNF4 can regulate BLM retention at stalled forks without affecting repair by homologous recombination. RNF4 depletion reduces cell proliferation and survival of untreated cells, and it confers hypersensitivity to replication stalling by HU. Studies of cell-cycle progression using bromodeoxyuridine incorporation and flow cytometry show that RNF4 depletion causes a delay of re-entry into S phase in HU-treated cells and the replication delay is partially rescued by BLM mutation. The delay is not caused by modification of checkpoint kinase activities. Analysis of replication dynamics using the DNA fiber assay show that RNF4 depletion causes an increase of permanently stalled replication forks and a decrease in activation of dormant origins following recovery from HU-induced replication stress. Co-depletion of RNF4 and BLM partially rescues these defects. Replication dynamics is unaffected by RNF4 depletion in untreated cells. These data indicate that RNF4 regulates BLM's functions in replication fork stability and dormant origin firing, without affecting BLM's roles in homologous recombination. We propose that sumoylation of BLM at stalled replication forks leads to RNF4-mediated ubiquitylation and subsequent proteasome-dependent degradation of BLM, which relieves BLM's active inhibition of dormant origin firing. Citation Format: Nathan A. Ellis, Wei-Chih Yang, Mary Yagle, Jianmei Zhu, Jing Huang, Michael Seidman, Michael J. Matunis. The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2015-3036

A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells.

It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44-52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers.

Genetic Variability for Vitamin B9 and Total Dietary Fiber in Lentil (Lens culinaris L.) Cultivars

The present study was conducted to determine the genetic variability of two major health-promoting bioactive components in lentil (lens culinaris L.) cultivars. The total dietary fiber content in lentil genotypes was analyzed using a combination of enzymatic and gravimetric methods utilizing the Total Dietary Fiber Assay Kit (Sigma Chemicals, St. Louis, USA). The total dietary fiber varied significantly in the range of 10 ± 1 to 21 ± 1 g/100 g lentil dry seed weight with an average of 15.0 g/100 g lentil dry seed weight (p < 0.1). Further, a significant genotypic difference was also recorded in the lentil genotypes (p < 0.1) for folate content which was estimated using the trienzyme extraction and high-performance liquid chromatography method with an average value of 222 µg/100 g lentil dry seed weight. The range of variability for folate content was 114 ± 3 to 330 ± 7 µg/100 g dry seed weight, except in one of the genotype IG-163, where the folate content was recorded as high as 448 ± 7 µg/100 g dry seed weight. The mean total dietary fiber and folate content of Mediterranean landraces of lentil was higher (18 g/100 g and 247 µg/100 g lentil dry seed weight) as compared to the other genotypes (15 g/100 g and 198 µg/100 g lentil dry seed weight), respectively.

Direct radiocarbon dating of Spanish moss (Tillandsia usneoides) from early fiber-tempered pottery in the southeastern U.S.

Growing archaeological interest in illuminating human-scale events and experiences in the ancient past has led to increased scrutiny of chronological assumptions based on old and (by today's standards) imprecise radiocarbon databases. One result has been a greater awareness of the need for more numerous and higher quality assays that can be directly linked to the specific events in question. In this paper, a method is described and tested for extracting and directly dating the organic fiber temper characteristic of North America's oldest pottery technology. Multiple extraction techniques are considered, as are a number of chemical pretreatment options. Six pairs of assays on Orange and Stallings vessels from Florida and Georgia are used to demonstrate the veracity of radiocarbon age estimates from fiber temper. In each of these cases, the fiber assay meets or exceeds recently proposed chronometric hygiene standards.

Antitumour efficacy of Piper tuberculatum and piplartine based on the hollow fiber assay.

Piper tuberculatum, popularly known in Brazil as "jaborandi falso" and "pimenta darta", is widely used in folk medicine for the treatment of several diseases. In this study, the in vivo hollow fiber assay was used to investigate the antitumour efficacy of the crude extract and piplartine obtained from P. tuberculatum roots. Human glioblastoma (SF-295) and colon carcinoma (HCT-8) cell lines were used. In vitro cytotoxicity was assayed by the MTT assay. In the hollow fiber assay, nude mice implanted with tumour cells in hollow fibers were treated for four consecutive days via the intraperitoneal route, and tumour cell populations were assessed by the MTT assay. Both the crude extract and piplartine displayed cytotoxicity. In the hollow fiber assay, tumour growth inhibition rates were 24.6-54.8 % for the crude extract and 33.7-62.2 % for piplartine. No signal of toxicity was noticed. In conclusion, the crude extract and piplartine obtained from P. tuberculatum roots displayed in vitro and in vivo anticancer efficacy.

Potent cytotoxic arylnaphthalene lignan lactones from Phyllanthus poilanei

Two new and six known arylnaphthalene lignan lactones were discovered from Phyllanthus poilanei Beille (Phyllanthaceae) collected in South Vietnam. The structures of the new compounds were determined by interpretation of their spectroscopic data, with the structure of one of these new compounds, phyllanthusmin D, being confirmed by single-crystal X-ray diffraction analysis. Several of these compounds were cytotoxic toward the HT-29 human colon cancer cells, with phyllanthusmin D found to be potently cytotoxic in vitro showing an IC50 value of 170 nM and active when tested in an in vivo hollow fiber assay. This compound showed a different mechanism of action when compared with etoposide. It mediated its cytotoxicity toward the HT-29 cells by induction of tumor cell apoptosis through activation of caspase-3 with no inhibitory activity against DNA topoisomerase IIα.

Design, synthesis, and biological evaluation of analogues of the phyllanthusmin class of arylnaphthalene lignan natural products

Recently a series of arylnaphthalene lignans was isolated from Phyllanthus poilanei by the Kinghorn lab. These compounds bear a strong structural resemblance to etoposide, a semi-synthetic DNA topoisomerase II (topo II) poison, and the natural product podophyllotoxin. Although the newly isolated compounds do not appear to act on topo II, they have demonstrated potent in vitro antiproliferative activity against HT-29 human colon cancer cells. Moreover, one of these compounds, phyllanthusmin D, also showed promising activity in a hollow fiber assay using HT-29 cells in mice. In an effort to understand the basic structure-activity requirements of this class of natural product and to remedy the limited aqueous solubility of these molecules, a series of structural analogues were synthesized and tested for biological activity. These studies have focused primarily on the role of the saccharide moiety in the activity of this class, although variation of the aryl substituents has also been examined.

Potent cytotoxic arylnaphthalene lignan lactones from Phyllanthus poilanei.

Two new (1 and 2) and four known arylnaphthalene lignan lactones (3–6) were isolated from different plant parts of Phyllanthus poilanei collected in Vietnam, with two further known analogues (7 and 8) being prepared from phyllanthusmin C (4). The structures of the new compounds were determined by interpretation of their spectroscopic data and by chemical methods, and the structure of phyllanthusmin D (1) was confirmed by single-crystal X-ray diffraction analysis. Several of these arylnaphthalene lignan lactones were cytotoxic toward HT-29 human colon cancer cells, with compounds 1 and 7-O-[(2,3,4-tri-O-acetyl)-α-l-arabinopyranosyl)]diphyllin (7) found to be the most potent, exhibiting IC50 values of 170 and 110 nM, respectively. Compound 1 showed activity when tested in an in vivo hollow fiber assay using HT-29 cells implanted in immunodeficient NCr nu/nu mice. Mechanistic studies showed that this compound mediated its cytotoxic effects by inducing tumor cell apoptosis through activation of caspase-3, but it did not inhibit DNA topoisomerase IIα activity.

Optimisation of tetrahydroisoquinoline-based chimeric microtubule disruptors.

Tetrahydroisoquinoline (THIQ)-based "chimeric" microtubule disruptors were optimised through modification of the N-benzyl motif, in concert with changes at C3 and C7, resulting in the identification of compounds with improved in vitro antiproliferative activities (e.g. 15: GI50 20 nM in DU-145). The broad anticancer activity of these novel structures was confirmed in the NCI 60-cell line assay, with 12 e,f displaying MGM values in the 40 nM region. In addition, their profiles as inhibitors of tubulin polymerisation and colchicine binding to tubulin were confirmed. Compound 15, for example, inhibited tubulin polymerisation with an IC50 of 1.8 μM, close to that of the clinical drug combretastatin A-4, and also proved effective at blocking colchicine binding. Additionally, compound 20 b was identified as the only phenol in the series to date showing both better in vitro antiproliferative properties than its corresponding sulfamate and excellent antitubulin data (IC50=.6 μM). Compound 12 f was selected for in vivo evaluation at the NCI in the hollow fibre assay and showed very good activity and wide tissue distribution, illustrating the value of this template for further development.

Preclinical Efficacy Testing for Stomach and Liver Cancers

PurposeHollow fiber assays offer an early in vivo method of anticancer drug screening. The assays have been optimized for human cancers originating from the lung, breast, colon, ovary, and brain, but not from the stomach and liver. The current study focused on optimization of hollow fiber assays for gastric and hepatocellular carcinoma cell lines.Materials and MethodsGastric (SNU-16, SNU-484, SNU-668) and hepatocellular (HepG2, SK-Hep-1, Hep3B) carcinoma cell lines in hollow fibers were transplanted subcutaneously and intraperitoneally into mice, which were subsequently treated with a standard anticancer agent, paclitaxel. The hollow fiber activity of paclitaxel in each cell line was compared with the xenograft activity.ResultsUsing optimized inoculation densities and schedules, treatment with paclitaxel was effective in gastric carcinoma cell lines, SNU-16 and SNU-484, but not in SNU-668. In the hollow fiber assays, paclitaxel was effective in hepatocellular carcinoma cell lines, HepG2 and SK-Hep-1, but not in Hep3B. Consistent with the results of the hollow fiber assay, SNU-16 and SNU-484, but not SNU-668, showed tumor regression, and HepG2 and SK-Hep-1, but not Hep3B, showed effective tumor responses following treatment with paclitaxel in xenograft models. When EW7197, a novel compound, and flavopiridol were tested in SNU-16 cells under optimized conditions, the hollow fiber activity showed good correlation with the xenograft activity of each compound.ConclusionOur protocols may be useful for screening candidate small molecules that may exhibit activity against stomach and liver cancers, both of which are common in Korea.

Differences among pulse grains and cereal grains in dietary fiber (259.5)

Pulses, i.e., dry beans, dry peas, chickpeas, and lentils, are grain legumes that serve as staple food crops for populations around the world. Since ancient times, pulses have been combined with cereal grains, primarily wheat, rice, or corn, or tubers such as potato or cassava to provide a balanced source of protein and carbohydrate and a nutritionally adequate diet when combined with seed oils, fruit and vegetables. In this setting, the established ratio of cereal grains to pulses to meet nutritional requirements is 2:1; however, over the last 60 years both production and consumption patterns, globally, have changed to 8:1. One consequence of these changes has been on a marked but largely unappreciated shift in dietary carbohydrate quality. This presentation focuses on differences among pulse crops in their amount and type of dietary fiber and how this relates to the amounts and types of dietary fiber provided by various cereal grains. Data will be reported using the AOAC 2011.25 Integrated Total Dietary Fiber Assay to quantify dietary fiber and oligosaccharide content. These data will be considered in the context of the pandemic of overweight and obesity that has swept the globe over the last 60 years since there has been virtually no scrutiny of the potential association between the decline in pulse consumption and the global rise in obesity and its associated metabolic disorders.Grant Funding Source: PHS R01‐ CA172375 and American Pulse Association

Anti-tumor pharmacological evaluation of extracts from stellera chamaejasme L based on hollow fiber assay

BackgroundStellera chamaejasme L, a traditional Chinese herb, has long been used for treatment of various tumors in the Chinese population. In our previous study, we paid an attention to the cytotoxic and proapoptotic effects of Stellera chamaejasme L extracts (ESC, ESC-1 and ESC-2, the latter two were isolated from ESC) on 4 various tumor cells (NCI-H157, NCI-H460, BEL-7402 and SK-HEP-1) in vitro. ESCs showed significantly inhibitory effects on the 4 tumor cells. ESC-2 had the strongest inhibitory effect and the broadest sensitive cell spectrum. ESC-2 and ESC acted in a similar way against tumor cells, which suggested anti-tumor active fraction of ESC might exist in ESC-2. Here, we further observe the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts in vivo.MethodsIn this study, we used hollow fiber tumor model to evaluate the inhibitory and proapoptotic effects of Stellera chamaejasme L extracts. Apoptotic rates of the cancer cells retrieved from the hollow fibers were measured with flow cytometric analysis, caspase 3, 8, 9 enzyme activities were detected by colorimetric assay, Fas, Fas-L, TNF-R1 and TNF-α expression were determined with elisa assay and radioimmunoassay respectively.ResultsThe results showed that ESC, ESC-2 all had inhibitory effects on 4 tumor cells. According to the effect strength, dose and antitumor spectrum, the order of antitumor effects of ESCs was: ESC-2 > ESC > ESC-1. NCI-H460 cells were the most sensitive to ESCs. ESC, ESC-2 increased greatly the apoptotic rate and caspase 3, 8 enzyme activities in NCI-H460. ESCs had no significant effects on expression of Fas, Fas-L protein, but TNF-α/TNFR1 protein expression in NCI-H460 cells changed significantly after ESC and ESC-2 treatment.ConclusionESC-2 had the similar antitumor effect to that of ESC in vivo and further confirmed that ESC-2 may be the main antitumor active fraction of ESC, which was consistent with our previous results in vitro.

Methods for the detection of genome instability derived from replication stress in primary mouse embryonic fibroblasts.

Replication stress, with its subsequent genome instability, is a hallmark of cancer from its earliest stages of development. Here, we describe assays that are sufficiently sensitive to detect intrinsic replicative stress and its consequences in primary mouse embryonic fibroblasts. First, we explain the non-denatured DNA fiber assay, a powerful tool to directly measure DNA replication kinetics via the dual-labeling of active replication forks. Then, we describe the cytokinesis-block micronucleus assay, which can be combined with detection of 53BP1 nuclear bodies to measure the levels of replication-associated genome instability carried over into G1 phase of the cell cycle.

Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη(KD) cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype.

Overview of safety pharmacology.

Safety pharmacology entails the assessment of the potential risks of novel pharmaceuticals for human use. As detailed in the ICH S7A guidelines, safety pharmacology for drug discovery involves a core battery of studies on three vital systems: central nervous (CNS), cardiovascular (CV), and respiratory. Primary CNS studies are aimed at defining compound effects on general behavior, locomotion, neuromuscular coordination, seizure threshold, and vigilance. The primary CV test battery includes an evaluation of proarrhythmic risk using in vitro tests (hERG channel and Purkinje fiber assays) and in vivo measurements in conscious animals via telemetry. Comprehensive cardiac risk assessment also includes full hemodynamic evaluation in a large, anesthetized animal. Basic respiratory function can be examined in conscious animals using whole-body plethysmography. This allows for an assessment of whether the sensitivity to respiratory-depressant effects can be enhanced by exposure to increased CO2 . Other safety pharmacology topics detailed in this unit are the timing of such studies, ethical and animal welfare issues, and statistical evaluation.

Patient-Specific Stem Cells and Cardiovascular Drug Discovery

Patient-Specific Stem Cells and Cardiovascular Drug Discovery

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(β-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.