Induction Of DNA Synthesis Research Articles

L-Glutamine mitigates bile acid-induced inhibition of growth factor activity in rat hepatocyte cultures

Bile acid-induced hepatotoxicity is inevitable in Cholestasis pathogenesis and L-Glutamine (L-Gln) has been reported to prevent total parenteral nutrition (TPN)-induced cholestasis in premature neonates. While mechanisms remain unknown, we hypothesize that bile acids impair growth factor (GF) function in hepatocytes which L-glutamine prevents through NAPDH oxidase (NOX) modulation. Glycochenodeoxycholic acid (GCDC, 0–100 µM) when added to primary hepatocyte cultures significantly (p < 0.01) decreased the FBS-induced BrdU incorporation, however inhibition of Fibroblast Growth factor (FGF)- or Hepatocyte growth factor (HGF)-induced DNA synthesis was more pronounced (p < 0.001). L-Gln markedly attenuated GCDC-mediated inhibition of DNA synthesis in both FBS and GF-treated cells. GCDC significantly increased the NADPH oxidase activity and NOX-1 protein expression that were markedly reduced by L-Gln and protein kinase c (PKC) inhibitor, LY-333531. Apocynin (APCN) and diphenyliodonium (DPI) significantly blocked the GCDC-mediated inhibition of GF-induced DNA synthesis. This study demonstrates that bile acid-induced hepatotoxicity involves dysfunction of certain growth factors via protein kinase c (PKC)- mediated NOX modulation which can be corrected, at least partly, by L-glutamine.

In vitro and in vivo antiproliferative activity on lung cancer of two acylhydrazone based zinc(II) complexes

Two acylhydrazone based zinc(II) complexes [Zn(HL)2Cl2(CH3OH)2] (Zn1) and [ZnL(AC)]2 (Zn2) were synthesized from 3-(1-(salicyloylhydrazono)ethyl) pyridine (HL). Single crystal X-ray structure analyses showed that complexes Zn1 and Zn2 have a zero-dimensional monomer or dimer structure. Antiproliferative activity studies revealed that Zn1 and Zn2 are both more effective against A549 cells than cisplatin. The results of the reactive oxygen species (ROS) generation assay on A549 cells showed that both Zn1 and Zn2 induced apoptosis through ROS accumulation. The apoptosis-inducing and cell cycle arrest effects of Zn1 and Zn2 on A549 cells indicated that the antitumor effect was achieved through apoptosis induction and inhibition of DNA synthesis by blocking the G0/G1 phase of the cell cycle. What’s more, the results of wound-healing assay showed that Zn1 and Zn2 could inhibit the migration of A549 cells. Western blot analysis further demonstrated that Zn1 and Zn2 induced cell apoptosis through the mitochondrial pathway, in which process, the expression level of cytochrome C, cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 9 proteins increased while pro-caspase 3 and pro-caspase 9 expression decreased. In vivo anticancer evaluation demonstrated that both Zn1 and Zn2 complexes effectively inhibited tumor growth without causing significant toxicity in systemic organs.

FGF9, a Potent Mitogen, Is a New Ligand for Integrin αvβ3, and the FGF9 Mutant Defective in Integrin Binding Acts as an Antagonist.

FGF9 is a potent mitogen and survival factor, but FGF9 protein levels are generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in cancer. However, the mechanism of FGF9 action has not been fully established. Previous studies showed that FGF1 and FGF2 directly bind to integrin αvβ3, and this interaction is critical for signaling functions (FGF-integrin crosstalk). FGF1 and FGF2 mutants defective in integrin binding were defective in signaling, whereas the mutants still bound to FGFR suppressed angiogenesis and tumor growth, indicating that they act as antagonists. We hypothesize that FGF9 requires direct integrin binding for signaling. Here, we show that docking simulation of the interaction between FGF9 and αvβ3 predicted that FGF9 binds to the classical ligand-binding site of αvβ3. We show that FGF9 bound to integrin αvβ3 and generated FGF9 mutants in the predicted integrin-binding interface. An FGF9 mutant (R108E) was defective in integrin binding, activating FRS2α and ERK1/2, inducing DNA synthesis, cancer cell migration, and invasion in vitro. R108E suppressed DNA synthesis and activation of FRS2α and ERK1/2 induced by WT FGF9 (dominant-negative effect). These findings indicate that FGF9 requires direct integrin binding for signaling and that R108E has potential as an antagonist to FGF9 signaling.

Multiple DNA repair pathways contribute to MMS-induced post-replicative DNA synthesis in S. pombe .

Replication stress can induce DNA synthesis outside of replicative S-phase. We have previously demonstrated that fission yeast cells stimulate DNA synthesis in G2-phase but not in M-phase in response to DNA alkylating agent MMS. In this study, we show that various DNA repair pathways, including translesion synthesis and break-induced replication contribute to post-replicative DNA synthesis. Checkpoint kinases, various repair and resection proteins, and multiple polymerases are also involved.

Rat prostate tumors induce DNA synthesis in remote organs

Advanced cancers induce systemic responses. However, if such systemic changes occur already when aggressive tumors are small, have not been thoroughly characterized. Here, we examined how localized prostate cancers of different sizes and metastatic potential affected DNA synthesis in the rest of the prostate and in various remote organs. Non-metastatic Dunning R-3327 G (G) tumor cells, metastatic MatLyLu (MLL) tumor cells, or vehicle were injected into the prostate of immunocompetent rats. All animals received daily injections of Bromodeoxyuridine (BrdU), to label cells/daughter cells with active DNA synthesis. Equal sized G- and MLL-tumors, similarly increased BrdU-labeling in the prostate, lymph nodes and liver compared to tumor-free controls. Prior to metastasis, MLL-tumors also increased BrdU-labeling in bone marrow and lungs compared to animals with G-tumors or controls. In animals with MLL-tumors, BrdU-labeling in prostate, lungs, brown adipose tissue and skeletal muscles increased in a tumor-size-dependent way. Furthermore, MLL-tumors induced increased signs of DNA damage (γH2AX staining) and accumulation of CD68 + macrophages in the lungs. In conclusion, small localized prostate cancers increased DNA synthesis in several remote tissues in a tumor type- and size-dependent way. This may suggest the possibility for early diagnosis of aggressive prostate cancer by examining tumor-induced effects in other tissues.

The Nuclear Proteins TP73 and CUL4A Confer Resistance to Cytarabine by Induction of Translesion DNA Synthesis via Mono-ubiquitination of PCNA.

Resistance to cytarabine is a key problem in the treatment of acute myeloid leukemia (AML). To understand the molecular biology of resistance to cytarabine, a viability-based chemosensitizer screen was utilized. We screened synthetic lethal targets using 437 different small interfering RNAs (siRNAs) directed against factors involved in DNA repair mechanisms and cytarabine as the chemical compound. Three hits were identified: CUL4A, TP73, and RFC2. We show here that the ubiquitin ligase CULLIN 4A (CUL4A) and the tumor-suppressive transcription factor p73 contribute to drug resistance by modulating DNA damage response. P73 confers resistance to cytarabine therapy by transactivation of REV3L, encoding the catalytic subunit of translesion DNA polymerase ζ, and CUL4A probably by influencing proliferating cell nuclear antigen (PCNA) and the polymerase switch towards error-prone translesion DNA polymerases. Abrogation of the polymerase ζ by siRNA causes identical effects as siRNAs against CUL4A or TP73 and resensitizes cells towards cytarabine therapy in vitro. As CUL4A needs to be activated by neddylation to facilitate the degradation of several proteins including PCNA, we propose a novel explanation for the synergism between cytarabine and the neddylation inhibitor pevonedistat by inhibition of translesion synthesis. In keeping with this, in AML patients treated with cytarabine, we found high expression of CUL4A and TP73 to be associated with poor prognosis.

Three metal complexes with a pyridyl Schiff base: cytotoxicity, migration and mechanism of apoptosis.

Three metal complexes [CuL(NO3)]n (1), [Cd(HL)(NO3)2]n (2) and [EuL(HCOOH)(H2O)(NO3)2] (3) were synthesized with a pyridyl Schiff ligand L (N'-[(1E)-pyridin-2-ylmethylidene]pyridine-4-carbohydrazide). A crystallographic study revealed that complexes 1 and 2 have a chain structure, and complex 3 is a zero-dimensional monomer. In vitro cytotoxicity studies showed that complex 2 had the best antiproliferative activity against SMMC-7721 cells and complex 3 had the best antiproliferative activity against MDA-MB-231 cells with single-digit IC50 values, both exceeding those of the control drug cisplatin by far. The cell invasion and migration ability through the transwell assay and wound-healing assay showed that the selected complexes could inhibit the invasion and migration of cancer cells. The Hoechst staining assay and ROS generation assay with SMMC-7721 cells indicated that the cytotoxic effects of complex 2 involved apoptosis induction through ROS accumulation. The apoptosis-inducing and cell cycle arrest effects of complex 2 on SMMC-7721 cells indicated that the antitumor effect was achieved through apoptosis induction and inhibition of DNA synthesis by blocking the G0/G1 phase of the cell cycle. In addition, complex 2 showed significant inhibition against B. dysentery with an inhibition circle diameter of 24 mm.

755 Rat engineered heart tissue is a novel in vitro model to evaluate cardiomyocyte proliferation and fibroblast activation after injury

Abstract Aims Adult mammals, including humans, fail to regenerate the majority of the lost cardiomyocytes (CMs) that are replaced with scar tissue after injury. This lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs 7 days after birth. An in vitro model that recapitulates these changes has not been developed yet. Using rat engineered heart tissues (rEHTs) we have developed a custom-made cryoinjury system to test the hypothesis that maturation of CMs in EHTs regulates the proliferative response of CMs after injury. Methods rEHT were generated using neonatal rat heart cells. A discrete lesion was produced on the mid-section of mature (Day 18) and immature (Day 6) EHTs using a custom-made system based on liquid nitrogen and a 23G needle and medium was supplemented with EdU for 48 h. Results Cryoinjury in mature EHTs produces a localized injury, preserving their residual contractile activity that does not recover over time. We observed a significant increase of EdU+CMs post injury (6.3 ± 1.9% vs. 10.1 ± 1.6%) without significant changes in Ki67+ and pH3+ CMs suggesting that cryoinjury in mature rEHTs induces DNA synthesis but not CM proliferation. Injury in mature EHTs induced also significant proliferation and activation of fibroblasts with collagen deposition. Interestingly, cryoinjury performed in immature EHTs stimulated a significant proliferative response in CMs Conclusions Similar to adult rodents, we show that cryoinjury induces DNA synthesis in CMs without proliferative response and contractile recovery. On the other hand, cryoinjury in immature EHTs leads to CMs proliferation. Moreover, mature EHT fibroblast response to injury retraces the activation progression of cardiac fibroblast after infarction characterized by proliferation, increase of activation markers, increase of collagen deposition suggesting EHTs as a novel model to investigate the biology of cardiac regeneration upon injury.

Abstract 11992: Rat Engineered Heart Tissue is a Novel in vitro Model to Evaluate Cardiomyocyte Proliferation and Fibroblast Activation After Injury

Introduction: Adult mammals, including humans, fail to regenerate the majority of the lost cardiomyocytes (CMs) that are replaced with scar tissue after injury. This lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs 7 days after birth. An in vitro model that recapitulates these changes has not been developed yet. Hypothesis: Using rat engineered heart tissues (rEHT) we have developed a custom-made cryoinjury system to test the hypothesis that maturation of CMs in EHTs regulates the proliferative response of CMs after injury. Methods: rEHT were generated using neonatal rat heart cells. A discrete lesion was produced on the mid-section of mature (day 18) and immature (day 6) EHTs using a custom-made system based on liquid nitrogen and a 23G needle and medium was supplemented with EdU for 48h. Results: Cryoinjury in mature EHTs produces a localized injury, preserving their residual contractile activity that does not recover over time. We observed a significant increase of EdU + CMs post injury (6.3 ± 1.9% vs. 10.1 ± 1.6 %) without significant changes in Ki67 + and pH3 + CMs suggesting that cryoinjury in mature rEHTs induces DNA synthesis but not CM proliferation. Injury in mature EHTs induced also significant proliferation and activation of fibroblasts with collagen deposition. Interestingly, cryoinjury performed in immature EHTs stimulated a significant proliferative response in CMs. Discussion: Similar to adult rodents, we show that cryoinjury induces DNA synthesis in CMs without proliferative response and contractile recovery. On the other hand, cryoinjury in immature EHTs leads to CMs proliferation. Moreover, mature EHT fibroblast response to injury retraces the activation progression of cardiac fibroblast after infarction characterized by proliferation, increase of activation markers, increase of collagen deposition suggesting EHTs as a novel model to investigate the biology of cardiac regeneration upon injury.

Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats.

We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams' medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10-6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10-6 M), RTK (AG538, 10-6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10-6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10-6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

A plant DNA virus replicates in the salivary glands of its insect vector via recruitment of host DNA synthesis machinery

Whereas most of the arthropod-borne animal viruses replicate in their vectors, this is less common for plant viruses. So far, only some plant RNA viruses have been demonstrated to replicate in insect vectors and plant hosts. How plant viruses evolved to replicate in the animal kingdom remains largely unknown. Geminiviruses comprise a large family of plant-infecting, single-stranded DNA viruses that cause serious crop losses worldwide. Here, we report evidence and insight into the replication of the geminivirus tomato yellow leaf curl virus (TYLCV) in the whitefly (Bemisia tabaci) vector and that replication is mainly in the salivary glands. We found that TYLCV induces DNA synthesis machinery, proliferating cell nuclear antigen (PCNA) and DNA polymerase δ (Polδ), to establish a replication-competent environment in whiteflies. TYLCV replication-associated protein (Rep) interacts with whitefly PCNA, which recruits DNA Polδ for virus replication. In contrast, another geminivirus, papaya leaf curl China virus (PaLCuCNV), does not replicate in the whitefly vector. PaLCuCNV does not induce DNA-synthesis machinery, and the Rep does not interact with whitefly PCNA. Our findings reveal important mechanisms by which a plant DNA virus replicates across the kingdom barrier in an insect and may help to explain the global spread of this devastating pathogen.

Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats

Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 μM sodium phenobarbital (NaPB), 3–100 μM fluquinconazole (FQZ), or 3–300 μM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 μM 5-bromo-2´-deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall, these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.

Extra Virgin Olive Oil Minor Compounds Modulate Mitogenic Action of Oleic Acid on Colon Cancer Cell Line.

Experimental and clinical findings suggest that olive oil has a protective effect, whereas oleic acid consumption induces colorectal cancer (CRC). Considering this apparent contradiction and that olive oil is a complex mix of fatty acids, mainly oleic acid and minor compounds such as phenolic compounds, lignans, hydrocarbons, and triterpenes, we study its effects on intestinal epithelial cell growth. Our results show that oleic acid (1-100 μM) but not elaidic acid induced DNA synthesis and Caco-2 cell growth (2-fold higher than cells without growth factors, p < 0.05). These effects were inhibited by 5-lipoxygenase inhibitors as well as the leukotriene antagonist (p < 0.05), suggesting the implication of this pathway in this mitogenic action. Hydroxytyrosol, oleuropein, pinoresinol, squalene, and maslinic acid (0.1-10 μM) reverted DNA synthesis and Caco-2 cell growth induced by oleic acid. These effects were not the consequence of the cell cycle arrest or the impairment of cell viability with the exception of hydroxytyrosol and maslinic acid that induced cell detachment and apoptosis (35.6 ± 2.3 and 43.2 ± 2.4%, respectively) at the higher concentration assayed. Oleuropein effects can be related with hydroxytyrosol release as a consequence of oleuropein hydrolysis by Caco-2 cells (up to 25%). Furthermore, hydroxytyrosol modulates the arachidonic acid cascade, and this event can be associated with its antimitogenic action. In conclusion, oleic acid and oleic acid in the presence of olive oil representative minor components have opposite effects, suggesting that the consumption of seed oils, high oleic acid seed oils, or olive oil will probably have different effects on CRC.

RAD51AP1 Is an Essential Mediator of Alternative Lengthening of Telomeres.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.

Impaired Insulin‐like Growth Factor‐1 function in X‐linked Adrenoleukodystrophy patients

IntroductionX‐linked Adrenoleukodystrophy (X‐ALD), a common peroxisomal disorder is characterized by elevated very long chain fatty acids (VLCFA) levels in tissues and body fluids of patients. X‐ALD patients present with adrenal insufficiency, demyelination and progressive mental retardation and IGF‐1 has been reported to alleviate demyelination and neurodegeneration in an animal model of X‐ALD. We examined the effect of VLCFA on IGF‐1 function in human skin fibroblasts.MethodsHuman skin fibroblasts grown from skin explants were cultured in growth medium. Cell cultures were treated with varying concentrations of IGF‐1 and/or VLCFA (Lignoceric acid; C24:0, hexadecanoic acid; C26:0 ) for 24–72 hrs. DNA synthesis into cultured fibroblasts was measured and total RNA was extracted for RT‐PCR analysis of IGF‐1 receptor and GAPDH. Cell homogenates were prepared for western blotting and protein measurements. Levels of IGF‐1 and VLCFA were measured in blood samples of 10 patients with confirmed diagnosis of X‐ALD.ResultsBoth VLCFAs used in this study significantly (p &lt;0.01) reduced the IGF‐1–induced DNA synthesis in cultured skin fibroblasts. Lignoceric acid as well as hexadecanoic acid markedly reduced the gene expression of IGF‐1 receptor. IGF‐1 receptor protein was also significantly reduced following 48 hrs of treatment with VLCFAs. X‐ALD patients were observed to have significantly lower levels of plasma IGF‐1 as compared to healthy controls.ConclusionsThis study suggests that abnormally elevated VLCFA in X‐ALD patients might impair IGF‐1 function through downregulation of IGF‐1 receptor thereby providing a new insight into molecular mechanisms of X‐ALD pathogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aedes aegypti antiviral adaptive response against DENV-2

Priming is the conceptual term defining memory phenomenon in innate immune response. Numerous examples of enhanced secondary immune response have been described in diverse taxa of invertebrates; which naturally lacks memory response. In mosquitoes, a previous non-lethal challenge with some specific pathogens modify their immune response against the same microorganism; developing an improved antimicrobial reaction. In this work, we explore the ability of Aedes aegypti to mount a higher antiviral response upon a second oral DENV challenge. When previously challenged with inactive virus, we observed that the posterior infection showed a diminished number of DENV infectious particles in midguts and carcasses. In challenged tissues, we detected higher de novo midgut DNA synthesis than control group, as determined by DNA incorporation of 5-bromo-2-deoxyuridine. We demonstrated that inactive DENV particle are capable to induce DNA synthesis levels comparable to infective DENV. We considered the Drosophila melanogaster hindsight and Delta-Notch mosquitoes orthologues as potential de novo DNA synthesis pathway components (as observed in fly oocyte development and midgut tissue renewal). We showed that Aedes aegypti hindsight transcript relative expression levels were higher than control during DENV infection and inactive DENV particle alimentation. Also, Aedes aegypti second challenge with active DENV induced higher hindsight, Delta and Notch transcriptions in the primed mosquitoes (compared with the primary infection levels). Considering that the mosquito de novo DNA synthesis is concomitant to viral particle reduction, this finding opens a new perspective on the mechanisms underlying the vector antiviral immune response and the effector molecules involved.

Mesenchyme-derived factors enhance preneoplastic growth by non-genotoxic carcinogens in rat liver

Many frequently prescribed drugs are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action and health risks for humans remain to be elucidated. Here, we investigated the impact of two model NGC, the anti-epileptic drug phenobarbital (PB) and the contraceptive cyproterone acetate (CPA), on intrahepatic epithelial–mesenchymal crosstalk and on growth of first stages of hepatocarcinogenesis. Unaltered hepatocytes (HC) and preneoplastic HC (HCPREN) were isolated from rat liver for primary culture. DNA replication of HC and HCPREN was increased by in vitro treatment with 10 µM CPA, but not 1 mM PB. Next, mesenchymal cells (MC) obtained from liver of rats treated with either PB (50 mg/kg bw/day) or CPA (100 mg/kg bw/day), were cultured. Supernatants from both types of MC raised DNA synthesis of HC and HCPREN. This indicates that PB induces replication of HC and HCPREN only indirectly, via growth factors secreted by MC. CPA, however, acts on HC and HCPREN directly as well as indirectly via mesenchymal factors. Transcriptomics and bio-informatics revealed that PB and CPA induce extensive changes in the expression profile of MC affecting many growth factors and pathways. MC from PB-treated rats produced and secreted enhanced levels of HBEGF and GDF15, factors found to suppress apoptosis and/or induce DNA synthesis in cultured HC and HCPREN. MC from CPA-treated animals showed enhanced expression and secretion of HGF, which strongly raised DNA replication of HC and HCPREN. In conclusion, our findings reveal profound effects of two prototypical NGC on the hepatic mesenchyme. The resulting release of factors, which suppress apoptosis and/or enhance cell replication preferentially in cancer prestages, appears to be crucial for tumor promotion by NGC in the liver.

The Influence of E1A C-Terminus on Adenovirus Replicative Cycle.

Adenovirus Early 1A proteins (E1A) are crucial for initiation of the viral life cycle after infection. The E1A gene is encoded at the left end of the viral genome and consists of two exons, the first encoding 185 amino acids in the 289 residues adenovirus 5 E1A, while the second exon encodes 104 residues. The second exon-encoded region of E1A is conserved across all E1A isoforms except for the 55 residues protein, which has a unique C-terminus due to a frame shift following splicing into the second exon. This region of E1A contributes to a variety of processes including the regulation of viral and cellular gene expression, immortalization and transformation. Here we evaluated the contributions that different regions of the second exon of E1A make to the viral life cycle using deletion mutants. The region of E1A encoded by the second exon was found to be important for overall virus growth, induction of viral and cellular gene expression, viral genome replication and deregulation of the cell cycle. Efficient viral replication was found to require exon 2 and the nuclear localization signal, as loss of either resulted in severe growth deficiency. Induction of cellular DNA synthesis was also deficient with any deletion of E1A within the C-terminus even if these deletions were outside of conserved region 4. Overall, our study provides the first comprehensive insight into the contributions of the C-terminus of E1A to the replicative fitness of human adenovirus 5 in arrested lung fibroblasts.

A high throughput method to study the physiology of E2:ERα signaling in breast cancer cells.

17β-estradiol (E2) regulates diverse physiological effects including cell proliferation through the estrogen receptor α (ERα), which as a transcription factor drives gene transcription and as an extra-nuclear localized receptor triggers the membrane-dependent activation of diverse kinase cascades. E2 also modifies ERα intracellular levels via diverse intracellular mechanisms. In this way, the E2-acivated ERα integrates signaling cascades with the modulation of receptor intracellular concentration and with the induction of DNA synthesis and ultimately drives cell proliferation. In turn, E2 signaling deregulation can cause many diseases including breast cancer (BC). Recently, we performed a Western blotting (WB)-based screen to identify novel pathways affecting ERα intracellular levels and BC cell proliferation. However, because WB lacks high throughput potential, a high-content method to detect all aspects of E2:ERα signaling (nuclear and extra-nuclear receptor activity, ERα levels, E2-induced DNA synthesis) is desirable. Here, we set up a rapid way to measure E2:ERα signaling in 96-well plate format. To demonstrate its robustness, we also challenged 4OH-tamoxifen resistant (Tam-Res) BC cells with a library of anti-cancer drugs and identified methotrexate (MTX) as a molecule inducing ERα degradation and preventing BC cell proliferation. Overall, our research provides a high-content technique to study the physiology of E2:ERα signaling in cells and further suggests a possible anti-ERα and anti-proliferative use for MTX in Tam-Res BCs.

NOX-mediated impairment of PDGF-induced DNA synthesis in peripheral blood lymphocytes of children with idiopathic nephrotic syndrome.

BackgroundCellular oxidative stress, inflammatory responses, and immunogenic events are involved in pathogenesis of idiopathic nephrotic syndrome (INS); however, the exact mechanism remains unknown. We examined NADPH oxidase (NOX) activity and platelet-derived growth factor (PDGF)-induced DNA synthesis in peripheral blood lymphocytes (PBL) of patients with INS.MethodsPBL from 15 patients with INS and 15 age- and gender-matched controls were isolated, and enzyme activities of NOX, catalase, and superoxide dismutase (SOD) were measured along with the assay of malondialdehyde levels and bromo-deoxyuridine incorporation. Protein expression of NOX-1 was measured using western blot analysis.ResultsPatients with INS had significantly (P<0.01) higher NOX activity and increased protein expression of NOX-1 in PBL as compared with controls. Catalase and SOD activities were markedly lower with lipid peroxide levels significantly (P<0.01) increased in patients with INS. Ex vivo DNA synthesis in PDGF-stimulated PBL was significantly (P<0.01) reduced in patients with INS; however, diphenyliodonium, an inhibitor of NOX, markedly corrected impairment in growth factor-induced BrdU incorporation.ConclusionsThese results show that NOX activation might have a role in regulation of lymphocytic activity in patients with INS through the impairment of PDGF mitogenic function and might contribute toward pathogenesis of nephrotic syndrome.