Large Subunit Of Ribonucleotide Reductase Research Articles

An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae.

P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB1 ). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB1 , the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB1 , and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217-227, 2017. © 2017 Wiley Periodicals, Inc.

A ribonucleotide reductase inhibitor with deoxyribonucleoside-reversible cytotoxicity

Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL-60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR-targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.

Expression of RRM1 in gastric adenocarcinoma and its relationship with clinical pathological characte-ristics

Objective To investigate the expression of human ribonucleotide reductase large subunit M1 (RRM1) in the patients with gastric adenocarcinoma and its clinical significance. Methods The expre-ssions of RRM1 were detected in 88 cases of gastric adenocarcinoma and 20 cases of normal gastric tissues by immunohistochemical method. The correlations between the expression of RRM1 and clinicopathological characteristics were analyzed. Results The positive expression rate of RRM1 in gastric adenocarcinoma was 83.0% (73/88). And RRM1 was negative in the entire normal gastric tissues. The positive expression of RRM1 was not associated with patient's age (χ2=0.352, P=0.553), gender (χ2=0.493, P=0.482), depth of tumor infiltration depth (χ2=0.007, P=0.933), lymph nodes metastasis (χ2=0.121, P=0.728) and distant metastasis (P=0.415). But it was related to the degree of tumor differentiation (χ2=7.740, P=0.021) and clinical stage (χ2=5.733, P=0.017). Conclusion The expression of RRM1 is positive expression in most gastric adenocarcinoma, and is associated with the degree of tumor differentiation and clinical stage. It is of great significance to detect the expression of RRM1 in gastric adenocarcinoma for confirming the diagnosis and judging malignant degree of the tumor. It also may have potential value in choosing the best therapeutic scheme and estimating prognosis. Key words: Stomach neoplasms; Immunohistochemistry; Ribonucleotide reductase large subunit M1

The interplay between human herpes simplex virus infection and the apoptosis and necroptosis cell death pathways.

Human herpes simplex virus (HSV) is a ubiquitous human pathogen that establishes a lifelong latent infection and is associated with mucocutaneous lesions. In multicellular organisms, cell death is a crucial host defense mechanism that eliminates pathogen-infected cells. Apoptosis is a well-defined form of programmed cell death executed by a group of cysteine proteases, called caspases. Studies have shown that HSV has evolved strategies to counteract caspase activation and apoptosis by encoding anti-apoptotic viral proteins such as gD, gJ, Us3, LAT, and the ribonucleotide reductase large subunit (R1). Recently, necroptosis has been identified as a regulated form of necrosis that can be invoked in the absence of caspase activity. Receptor-interacting kinase 3 (RIP3 or RIPK3) has emerged as a central signaling molecule in necroptosis; it is activated via interaction with other RIP homotypic interaction motif (RHIM)-containing proteins such as RIP1 (or RIPK1). There is increasing evidence that HSV R1 manipulates necroptosis via the RHIM-dependent inactivation or activation ofRIP3 in a species-specific manner. This review summarizes the current understanding of the interplay between HSV infection and cell death pathways, with an emphasis on apoptosis and necroptosis.

Abstract B13: Injection of the viroimmunotherapeutic oncolytic herpes virus rRp450 increases survival of orthotopic mouse models of Group 3 medulloblastoma and atypical teratoid/rhabdoid tumors

Abstract Background: Both Group 3 medulloblastomas and atypical teratoid/ rhabdoid tumors (AT/RTs) are highly aggressive pediatric brain tumors which have a tendency to metastasize early and are known for their poor outcomes. Current standards of care using chemotherapy and irradiation are associated with significant morbidities, giving impetus for the development of new treatments. Oncolytic virotherapy, the utilization of live attenuated viruses to selectively infect and induce destruction of neoplastic cells, is actively being pursued in pre-clinical and early phase clinical trials. rRp450 was derived from the KOS strain and attenuated by an insertion mutation in the UL39 gene, encoding the large subunit of ribonucleotide reductase, and is in clinical trials (NCT01071941). The mechanism of action of this novel cancer therapy is multi-faceted, and involves direct lysis of infected malignant cells, disruption of the tumor blood supply, and stimulation of the immune system to more effectively mount an anti-tumor response. The safety and tolerability of these oncolytic viral agents has been demonstrated in numerous clinical trials. Which cancer targets will ultimately be responsive to these agents is unknown. Methods: We assessed the permissivity and sensitivity of two Group 3 medulloblastoma and two AT/RT cell lines to the oncolytic herpes virus rRp450 in vitro. We implanted tumor cells into the brains of athymic nude mice and administered a single intratumoral dose of 1 million plaque-forming units of rRp450 using the same injection coordinates as tumor cell implantation. We monitored animals for survival and performed immunohistochemical staining against HSV-1 on post-mortem tumor specimens. Results: All four pediatric brain tumor cell lines were permissive to virus replication and sensitive to in vitro killing by rRp450, albeit to different degrees. There was not a direct correlation between permissivity to virus replication and cell killing. A one-time dose of rRp450 injected into tumor-bearing animals resulted in a significant survival advantage in all four tumor models, including the least permissive model, suggesting antitumor factors in addition to direct cell lysis. Post-mortem tissue sections showed no tumor or virus present in long-term surviving animals, whereas animals that died during the study had persistent tumor and persistent virus. Conclusions: These data suggest that the oncolytic herpes virus rRp450 may have therapeutic benefit against medulloblastoma and AT/RTs. Citation Format: Adam W. Studebaker, Brian J. Hutzen, Kellie B. Haworth, Christopher R. Pierson, Timothy P. Cripe. Injection of the viroimmunotherapeutic oncolytic herpes virus rRp450 increases survival of orthotopic mouse models of Group 3 medulloblastoma and atypical teratoid/rhabdoid tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B13.

Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.

Functions of Herpesvirus-Encoded Homologs of the Cellular Ribonucleotide Reductase Large Subunit

Deoxyribonucleotides (dNTPs) are important for the efficient growth of DNA viruses. Therefore, many DNA viruses have strategies for the upregulation of cellular dNTP levels. Both α- and γ-herpesviruses encode functional homologs of cellular dNTP anabolic enzymes, including the class I ribonucleotide reductase (RNR) large (R1) and small (R2) subunits, whereas β-herpesviruses modulate host cells to induce genes that increase dNTP levels. Interestingly, β-herpesviruses still express the nonfunctional RNR R1 subunit. However, it is not clear why β-herpesviruses still carry inactive R1 homologs. Recently, the R1 homologs of herpesviruses have been shown to inhibit innate immune signaling pathways. In particular, both functional and nonfunctional R1 homologs target receptor-interacting protein kinase 1 (RIP1) and inhibit RIP1-mediated signaling pathways to promote viral replication. Here, we summarize recent findings on the activity of herpesviral R1 homologs and discuss their roles in the regulation of innate immune signaling pathways.

Ribonucleotide reductase large subunit M1 plays a different role in the invasion and metastasis of papillary thyroid carcinoma and undifferentiated thyroid carcinoma.

Ribonucleotide reductase (RR) has been reported to be associated with several types of cancer while the expression and role of RR in thyroid carcinoma (TC) has not been investigated. Here, we first examined the expression level of three RR subunit proteins (RRM1, RRM2, and RRM2B) in papillary thyroid carcinoma (PTC) and undifferentiated thyroid carcinoma (UTC) patient samples by immunohistochemistry. The results showed that RRM1 was higher expressed in 95.2% cancer tissues compared with their adjacent normal tissues in 146 PTC samples. The expression level of RRM1 was positively correlated with T stage, lymph node metastasis (LNM), extrathyroidal invasion (ETI), and TNM stage in PTC patients. However, in 12 UTC samples, RRM1 expression was negatively expressed in six cases. To further determine the biological role of RRM1 in TC, ectopic expression or siRNA-mediated knockdown of RRM1 were carried out in the high-differentiated thyroid carcinoma cell line TPC-1 and the poor-differentiated thyroid carcinoma cell line SW579, respectively. In TPC-1 and SW579 cells, overexpression and siRNA knockdown of RRM1 demonstrated that RRM1 promoted DNA synthesis and proliferation in both cell lines as shown by EdU incorporation and cell viability assays. However, RRM1 enhanced cell migration and invasion in TPC-1 cells but inhibited that in SW579 cells as shown by wound healing and transwell assays. Moreover, we also found that RRM1 promoted PTEN expression and reduced Akt phosphorylation in a RR-activity-independent manner in the low-differentiated TC cells but not in the high-differentiated TC cells. In contrast, RRM2 expression was higher expressed in both PTC and UTC patient samples, consisting with its oncogenic role in other cancers. Therefore, we suggest that RRM1 promotes thyroid carcinoma proliferation as a component of RR but may play a different role in the invasion and metastasis of differently differentiated thyroid carcinomas through a non-RR pathway, which could be meaningful to precision treatment of thyroid carcinoma with RR inhibitors.

Potent effect of adenoviral vector expressing short hairpin RNA targeting ribonucleotide reductase large subunit M1 on cell viability and chemotherapeutic sensitivity to gemcitabine in non-small cell lung cancer cells

BackgroundRibonucleotide reductase large subunit (RRM1) is the main enzyme responsible for synthesis of the deoxyribonucleotides used during DNA synthesis. It is also a cellular target for gemcitabine (GEM). Overexpression of RRM1 is reportedly associated with resistance to GEM and the poor prognosis for many types of malignant tumours. Aim of the present study is to establish gene therapy against RRM1-overexpressing tumours. MethodAn adenoviral vector that encoded a short hairpin siRNA targeting the RRM1 gene (Ad-shRRM1) was constructed. Two RRM1-overexpressing non-small cell lung cancer (NSCLC) lines, MAC10 and RERF-LC-MA, were used. Finally, a human tumour xenograft model in nude mice was prepared by subcutaneously implanting tumours derived from RERF-LC-MA cells. ResultsAd-shRRM1 effectively downregulated RRM1 mRNA and protein in both types of NSCLC cells and significantly reduced the percentage of viable cells as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (p<0.005). Caspase 3/7 analysis revealed that transfection with Ad-RRM1 increased the percentage of apoptotic cells in culture containing either type of RRM1-overexpressing cell (p<0.001). Treatment with Ad-shRRM1 exerted a potent antitumour effect against the RRM1-overexpressing RERF-LC-MA xenografts (p<0.05). Furthermore, Ad-shRRM1-mediated inhibition of RRM1 specifically increased sensitivity to gemcitabine of each type of RRM1-overexpressing tumour cell. Combination treatment with Ad-shRRM1 and GEM exerted significantly greater inhibition on cell proliferation than Ad-shRRM1 or GEM treatment alone. ConclusionRRM1 appeared to be a promising target for gene therapy, and Ad-shRRM1 had strong antitumour effects, specifically anti-proliferative and pro-apoptotic effects, against NSCLC cells that overexpressed RRM1. Combination therapy with Ad-shRRM1 and GEM may become a new treatment option for patients with NSCLC.

RRM1 expression is associated with the outcome of gemcitabine-based treatment of non-small cell lung cancer patients--a short report.

RRM1 is the large subunit of ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in the production of deoxyribonucleotides (dNTPs) and is essential for DNA synthesis and repair. Through a meta-analysis of observational studies, we evaluated whether RRM1 expression levels are associated with the clinical outcome of gemcitabine-containing treatment regimens in patients with advanced non-small cell lung cancer (NSCLC). A literature search was conducted using the PubMed, Embase, Web of Science, Wanfang and Chinese National Knowledge Infrastructure databases from inception to September 2014. A meta-analysis was conducted to pool eligible studies, and pooled analyses were performed using fixed effects models. A total of 12 studies encompassing 593 NSCLC patients met our search criteria and were, therefore, included. Pooled analyses revealed that patients with low/negative RRM1 expression levels exhibited significantly higher response rates (OR: 0.35, 95% CI 0.24-0.51) and better survival rates (OR: 0.41, 95% CI 0.23-0.75) than those with high/positive RRM1 expression levels. Subgroup analyses did not reveal any significant heterogeneity in outcome regarding the RRM1 assessment methods used or the ethnicities of patient populations studied. The meta-analysis reported here indicates that RRM1 expression is associated with the response rate and overall survival rate of advanced NSCLC patients treated with gemcitabine-based chemotherapy. Additional phase III randomized trials are required to confirm our current findings.

Manipulation of apoptosis and necroptosis signaling by herpesviruses.

Like apoptosis, necroptosis is an innate immune mechanism that eliminates pathogen-infected cells. Receptor-interacting protein kinase (RIP)3 (also called RIPK3) mediates necrotic death by phosphorylating an executioner protein, MLKL, leading to plasma membrane leakage. The pathway is triggered against viruses that block caspase 8. In murine CMV, the viral inhibitor of caspase 8 activation prevents extrinsic apoptosis but also has the potential to unleash necroptosis. This virus encodes the viral inhibitor of RIP activation to prevent RIP homotypic interaction motif (RHIM)-dependent signal transduction and necroptosis. Recent investigations reveal a similar mechanism at play in the human alpha-herpesviruses, herpes simplex virus (HSV)1 and HSV2, where RHIM competitor function and caspase 8 suppression are carried out by the virus-encoded large subunit of ribonucleotide reductase (R1). In human cells, R1 inhibition of caspase 8 prevents TNF-induced apoptosis, but sensitizes to TNF-induced necroptosis. The RHIM and caspase 8 interaction domains of R1 collaborate to prevent RIP3-dependent steps and enable both herpesviruses to deflect host cell death machinery that would cut short infection. In mouse cells, HSV1 infection by itself triggers necroptosis by driving RIP3 protein kinase activity. HSV1 R1 contributes to the activation of RIP3 adaptor function in mice, a popular host animal for experimental infection. Based on these studies, infection of RIP3-kinase inactive mice should be explored in models of pathogenesis and latency. The necrotic death pathway that is suppressed during infection in the natural host becomes a cross-species barrier to infection in a non-natural host.

Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Non-enzymatic action of RRM1 protein upregulates PTEN leading to inhibition of colorectal cancer metastasis.

Ribonucleotide reductase large subunit M1 (RRM1) forms a holoenzyme with small subunits to provide deoxyribonucleotides for DNA synthesis and cell proliferation. Here, we reported a non-RR role of the catalytic subunit protein RRM1 and related pathway in inhibiting colorectal cancer (CRC) metastasis. Ectopic overexpression of the wild-type RRM1, and importantly, its Y738F mutant that lacks RR enzymatic activity, prevented the migration and invasion of CRC cells by promoting phosphatase and tensin homolog on chromosome 10 (PTEN) transactivation. Furthermore, overexpression of the wild-type and RR-inactive mutant RRM1 similarly reduced the phosphorylation of Akt and increased the E-cadherin expression in CRC cells, which were blocked by PTEN knockdown attenuation. Examination of clinical CRC specimens demonstrated that both RRM1 protein expression and RR activity were elevated in most cancer tissues compared to the paired normal tissues. However, while RR activity did not change significantly in different cancer stages, the RRM1 protein level was significantly increased at stages T1-3 but decreased at stage T4, in parallel with the PTEN expression level and negatively correlated with invasion and liver metastasis. Thus, we propose that RRM1 protein can inhibit CRC invasion and metastasis at the advanced stage by regulating PTEN transactivation and its downstream pathways in addition to forming an RR holoenzyme for supporting cancer proliferation. Understanding of the seemingly contrary dual roles of RRM1 protein may further help to explain the complex mechanisms by which this key enzyme and its components are involved in cancer development.

Predictive biomarkers for chemotherapies in pancreatic cancer

Pancreatic cancer is associated with high rates of mortality especially due to advanced stages of diagnosis. Adjuvant chemotherapy role is thus essential in the attempt to downstage the tumoral grade. In cases of locally advanced tumors, this step could be followed by curative surgical resection. Characterization of predictor biomarkers for adjuvant chemotherapy has drawn increasing interest in molecular biology research of pancreatic cancer. Personalized treatment is one of the solutions proposed for advancing towards a better outcome. In this paper we discuss available predictor biomarkers with focus upon gemcitabine and FOLFIRINOX (5-fluoro uracil (5-FU), irinotecan and oxaliplatin) chemotherapy regimens, present approaches in advanced pancreatic cancer. Many data are current available in relation with gemcitabine chemotherapy regimen, where biomarkers like ribonucleotide reductase large subunit (RRM1), human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), human antigen R (HuR), and secreted protein acidic rich in cysteine (SPARC) were proposed. Regarding the response to FOLFIRINOX components, other markers such as thymidylate synthethase (TS), topoisomerase I, and excision repair cross-complementation 1 (ERCC1) or KRAS mutation status were also investigated.

Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense.

The receptor-interacting kinase-3 (RIP3) and its downstream substrate mixed lineage kinase domain-like protein (MLKL) have emerged as the key cellular components in programmed necrotic cell death. Receptors for the cytokines of tumor necrosis factor (TNF) family and Toll-like receptors (TLR) 3 and 4 are able to activate RIP3 through receptor-interacting kinase-1 and Toll/IL-1 receptor domain-containing adapter inducing IFN-β, respectively. This form of cell death has been implicated in the host-defense system. However, the molecular mechanisms that drive the activation of RIP3 by a variety of pathogens, other than the above-mentioned receptors, are largely unknown. Here, we report that human herpes simplex virus 1 (HSV-1) infection triggers RIP3-dependent necrosis. This process requires MLKL but is independent of TNF receptor, TLR3, cylindromatosis, and host RIP homotypic interaction motif-containing protein DNA-dependent activator of IFN regulatory factor. After HSV-1 infection, the viral ribonucleotide reductase large subunit (ICP6) interacts with RIP3. The formation of the ICP6-RIP3 complex requires the RHIM domains of both proteins. An HSV-1 ICP6 deletion mutant failed to cause effective necrosis of HSV-1-infected cells. Furthermore, ectopic expression of ICP6, but not RHIM mutant ICP6, directly activated RIP3/MLKL-mediated necrosis. Mice lacking RIP3 exhibited severely impaired control of HSV-1 replication and pathogenesis. Therefore, this study reveals a previously uncharacterized host antipathogen mechanism.

Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.

RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions

The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions’ samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative.

Generation of an attenuated Tiantan vaccinia virus by deletion of the ribonucleotide reductase large subunit

Attenuation of the virulence of vaccinia Tiantan virus (VTT) underlies the strategy adopted for mass vaccination campaigns. This strategy provides advantages of safety and efficacy over traditional vaccines and is aimed at minimization of adverse health effects. In this study, a mutant form of the virus, MVTT was derived from VTT by deletion of the ribonucleotide reductase large subunit (R1) (TI4L). Compared to wild-type parental (VTT) and revertant (VTT-rev) viruses, virulence of the mutant MVTT was reduced by 100-fold based on body weight reduction and by 3,200-fold based on determination of the intracranial 50% lethal infectious dose. However, the immunogenicity of MVTT was equivalent to that of the parental VTT. We also demonstrated that the TI4L gene is not required for efficient replication. These data support the conclusion that MVTT can be used as a replicating virus vector or as a platform for the development of vaccines against infectious diseases and for cancer therapy.

Murine Gammaherpesvirus 68 Encodes a Second PML-Modifying Protein

The ORF75c tegument protein of murine gammaherpesvirus 68 (MHV68) promotes the degradation of the antiviral promyelocytic leukemia (PML) protein. Surprisingly, MHV68 expressing a degradation-deficient ORF75c replicated in cell culture and in mice similar to the wild-type virus. However, in cells infected with this mutant virus, PML formed novel track-like structures that are induced by ORF61, the viral ribonucleotide reductase large subunit. These findings may explain why ORF75c mutant viruses unable to degrade PML had no demonstrable phenotype after infection.

Genetic polymorphisms of SLC28A3, SLC29A1 and RRM1 predict clinical outcome in patients with metastatic breast cancer receiving gemcitabine plus paclitaxel chemotherapy

BackgroundPaclitaxel and gemcitabine (PG) combination chemotherapy is effective as a maintenance chemotherapeutic regimen in metastatic breast cancer (MBC) patients because it increases progression-free survival (PFS), which increases overall survival (OS). The primary purpose of our study was to investigate the association between genetic polymorphisms in the genes involved in PG pathways and clinical outcomes in MBC patients treated with PG chemotherapy. MethodsA total of 324 MBC patients were enrolled in this prospective multicenter trial of PG as the first-line chemotherapy. Eighty-five of the 324 patients from two institutes were available for analysis of single nucleotide polymorphisms (SNPs). Germline DNA was extracted from peripheral blood mononuclear cells. Thirty-eight SNPs in 15 candidate genes selected from pathways that may influence the metabolism and transport of, or sensitivity, to PG were analysed. ResultsThe median PFS and OS of all 324 patients were 8.7months (95% confidence interval [CI]: 7.5–9.6months) and 26.9months (95% CI: 23.6–30.1months), respectively. An SNP in SLC28A3 (rs7867504, C/T) was associated with OS (CC or CT versus TT: 37 versus 21months, p=0.027, hazard ratio [HR] 2.6, 95% CI: 1.1–6.3). SLC29A1 GA haplotype had a significantly shorter OS (p=0.030, HR 3.391, 95% CI: 1.13–10.19). RRM1 (ribonucleotide reductase large subunit M1) SNP (rs9937), and haplotypes ATAA and ATGA were significantly associated with neurotoxicity. ConclusionGenetic polymorphisms in SLC28A3, SLC29A1 and RRM1 can influence the clinical outcome of MBC patients treated with PG chemotherapy. Further studies on the functional mechanisms relating to these germline polymorphisms in these genes are warranted.