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Renal diseases impose considerable health and economic burdens on health systems worldwide, and there is a lack of efficient methods for the prevention and treatment due to their complexity and heterogeneity. Kidneys are organs with a high demand for energy produced by mitochondria, in which Rrm2b has critical functions as reported. The Rrm2b kidney-specific knockout mice we generated exhibited age-dependent exacerbated features, including mitochondrial dysfunction and increased oxidative stress; additionally, resulted in severe disruption of mitochondria-related metabolism. Rrm2b is vital not only to supply dNTPs for DNA replication and repair, but also to maintain structural integrity and metabolic homeostasis in mitochondria. Thence, Rrm2b deletion might induce chronic kidney defects in mice. This model can facilitate exploration of novel mechanisms and targeted therapies in the kidney diseases and has important translational and clinical implications.

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the "end-replication problem" and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1 ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells.

Objective: To explore the role of the special AT rich sequence binding protein-1 (SATB1) and ribonucleotide reductase M2 (RRM2) in enhancing malignant progression of non-Hodgkin lymphoma (NHL). 
 Methods: A total of 42 NHL and 42 chronic lymphadenitis patients were recruited. The protein expressions of SATB1 and RRM2 in cervical lymph nodes were determined by Western blot. After overexpression of SATB1, siSATB1 or siRRM2, the mRNA levels of SATB1 and RRM2 in cells were analyzed via RT-PCR, the cell proliferation was evaluated via MTT and EdU assays, while the migration and invasion of cells were assessed by transwell assays.
 Results: Compared with chronic lymphadenitis, the expressions of SATB1 and RRM2 in NHL patients were up-regulated. There was positive correlation between SATB1 and RRM2 in NHL patients. RRM2 mRNA level was up-regulated after transfection of SATB1 and down-regulated after transfection of siSATB1. Overexpression of SATB1 increased tumor cell proliferation, migration and invasion, while knockdown of RRM2 reversed those phenomena.
 Conclusion: SATB1 functions as an oncogene and promotes tumor cell proliferation, migration and invasion by up-regulation of RRM2 in NHL.

p53R2 is a p53-inducible human ribonucleotide reductase subunit involved in critical cellular mechanisms, such as DNA repair, cell cycle arrest, and mitochondrial homeostasis. Molecular investigations and animal studies have revealed functional regulations of p53R2 and its disease relevance. The relationship between p53R2 expression and disease progression in different cancers has been evaluated, and researchers have discovered novel transcription factors and cellular mechanisms that control p53R2 in a p53-independent manner. In addition, p53R2-Mediated mechanisms that affect mitochondria, inflammation, and cancer have been addressed. The role of p53R2 in mitochondria diseases and in cancer is discussed. Finally, p53R2 is taken as a potential target for cancer treatment. This review summarizes the general background, novel regulatory findings, and medical prospect of p53R2., (Copyright © 2016. Published by Elsevier B.V.)

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., (Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

Synthesis of deoxynucleoside triphosphates (dNTPs) is essential for both DNA replication and repair and a key step in this process is catalyzed by ribonucleotide reductases (RNRs), which reduce ribonucleotides (rNDPs) to their deoxy forms. Tight regulation of RNR is crucial for maintaining the correct levels of all four dNTPs, which is important for minimizing the mutation rate and avoiding genome instability. Although allosteric control of RNR was the first discovered mechanism involved in regulation of the enzyme, other controls have emerged in recent years. These include regulation of expression of RNR genes, proteolysis of RNR subunits, control of the cellular localization of the small RNR subunit, and regulation of RNR activity by small protein inhibitors. This review will focus on these additional mechanisms of control responsible for providing a balanced supply of dNTPs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria.

Unlabelled: Ribonucleotide reductase (RR)M2B is an enzyme belonging to the ribonucleotide reductase enzyme family, which is essential for DNA synthesis and repair. RRM2B plays an important role in tumor progression and metastasis; however, little is known about the expression and underlying molecular mechanisms of RRM2B in hepatocellular carcinoma (HCC). In the present study, we report that down-regulation of RRM2B in HCC is negatively associated with intrahepatic metastasis, regardless of p53 status. Moreover, the ectopic overexpression of RRM2B decreased HCC cell migration and invasion in vitro, whereas silencing RRM2B expression resulted in increased migration and invasion in vitro and intrahepatic and lung metastasis in vivo. Additionally, knockdown of RRM2B by short hairpin RNA (shRNA) in HCC cells was associated with epithelial-mesenchymal transition (EMT), including the down-regulation of E-cadherin, and the concomitant up-regulation of N-cadherin and slug. A further experiment showed that RRM2B inhibited cell migration and spreading through regulation of the early growth response protein 1 (Egr-1)/phosphatase and tensin homolog (PTEN)/Akt1 pathway. Consistently, we also detected a significant correlation between RRM2B and E-cadherin protein expression in HCC tissues. Furthermore, Egr-1 also directly bound to the RRM2B promoter and repressed RRM2B transcription, thereby establishing a negative regulatory feedback loop., Conclusion: These findings indicate that RRM2B suppresses cell migration and spreading by way of modulation of the Egr-1/PTEN/Akt1 pathway., (© 2014 by the American Association for the Study of Liver Diseases.)

Although the deregulated expression of p53R2, a p53-inducible protein and homologue of the R2 subunit of ribonucleotide reductase, has been detected in several human cancers, p53R2 roles in cancer progression and malignancy still remains controversial. In this article, we present a viable hypothesis about the roles of p53R2 in cancer progression and therapy resistance based on the roles of cytoplasmic p21 and mutant p53. Since p53R2 can up-regulate p21 and p21, it in turn has a dual role in cell cycle. Hence, p53R2 can play a dual role in cell cycle progression. In addition, because p53 is the main regulator of p53R2, the mutant p53 may induce the expression of p53R2 in some cancer cells based on the "keep of function" phenomenon. Therefore, depending on the locations of p21 and the new abilities of mutant p53, p53R2 has dual role in cell cycle progression. Since the DNA damaging therapies induce p53R2 expression through the induction of p53, p53R2 can be the main therapy resistance mediator in cancers with cytoplasmic p21., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Ribonucleotide reductase (RR) is a unique enzyme, because it is responsible for reducing ribonucleotides to their corresponding deoxyribonucleotides, which are the building blocks required for DNA replication and repair. Dysregulated RR activity is associated with genomic instability, malignant transformation and cancer development. The use of RR inhibitors, either as a single agent or combined with other therapies, has proven to be a promising approach for treating solid tumors and hematological malignancies., Areas Covered: This review covers recent publications in the area of RR, which include: i) the structure, function and regulation of RR; ii) the roles of RR in cancer development; iii) the classification, mechanisms and clinical application of RR inhibitors for cancer therapy and iv) strategies for developing novel RR inhibitors in the future., Expert Opinion: Exploring the possible nonenzymatic roles of RR subunit proteins in carcinogenesis may lead to new rationales for developing novel anticancer drugs. Updated information about the structure and holoenzyme models of RR will help in identifying potential sites in the protein that could be targets for novel RR inhibitors. Determining RR activity and subunit levels in clinical samples will provide a rational platform for developing personalized cancer therapies that use RR inhibitors.

Alternative splicing regulators have emerged as new players in cancer development, modulating the activities of many tumor suppressors and oncogenes and regulating the signaling pathways. However, little is known about the mechanisms by which these oncogenic splicing factors lead to cellular transformation. We have shown previously that the splicing factor serine and arginine splicing factor 1 (SRSF1; SF2/ASF) is a proto-oncogene, which is amplified in breast cancer and transforms immortal cells when overexpressed. In this study, we performed a structure-function analysis of SRSF1 and found that the RNA recognition motif 1 (RRM1) domain is required for its oncogenic activity. Deletion of RRM1 eliminated the splicing activity of SRSF1 on some of its endogenous targets. Moreover, we found that SRSF1 elevates the expression of B-Raf and activates the mitogen-activated protein kinase kinase (MEK) extracellular signal-regulated kinase (ERK) pathway and that RRM1 is required for this activation as well. B-Raf-MEK-ERK activation by SRSF1 contributes to transformation as pharmacological inhibition of MEK1 inhibits SRSF1-mediated transformation. In conclusion, RRM1 of SRSF1 is both required (and when tethered to the RS domain) also sufficient to activate the Raf-MEK-ERK pathway and to promote cellular transformation.

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: Ribonucleotide reductase (RNR) supplies deoxyribonucleotide diphosphates demanded by cells to repair radiation-induced DNA damage. Here, we investigate the impact of pretherapy RNR M1, M2, and M2b (p53R3) subunit level upon human cervical cancer radiochemosensitivity., Materials/methods: Immunohistochemistry was performed on a tissue array comprised of 18 paired benign uterine cervix and stage IB2 cervical cancers to evaluate the relationship between cytosolic RNR M1, M2, and M2b staining intensity and radiochemotherapy cancer response. Patients underwent surgical hysterectomy (n = 8), or daily radiation (45 Gy), coadministered once-weekly cisplatin (40 mg/m), then low-dose rate brachytherapy (30 Gy) followed by adjuvant hysterectomy (n = 10). Radiochemotherapy response was determined by Response Evaluation Criteria In Solid Tumors version 1.0 criteria during brachytherapy. Cancer relapse rates and disease-free survival were calculated., Results: M1, M2, and M2b antibody staining intensity was low (0-1+) in benign uterine cervical tissue. M1 and M2b immunoreactivity was 2+ or 3+ in most (13/18) cervical cancers. M2 immunoreactivity was 3+ in nearly all (16/18) cervical cancers. Cervical cancers overexpressing M1 and M2b had an increased hazard for incomplete radiochemotherapy response, relapse, and shortened disease-free survival., Conclusions: Ribonucleotide reductase subunit levels may predict human cervical cancer radiochemosensitivity and subsequent posttherapy cancer outcome. Further validation testing of RNR subunits as biomarkers for radiochemotherapy response is warranted.

p53R2 is a newly identified small subunit of ribonucleotide reductase and plays a pivotal role in the supply of dNTPs for genomic DNA repair and mitochondrial DNA synthesis, but little is known about its functions in zebrafish. Herein, we obtained the cDNA of zebrafish p53R2 that shares 72.8% and 72.5% amino acid identities with human p53R2 and zebrafish R2, respectively. Residues crucial for enzymatic activity are highly conserved among p53R2 proteins from different species. p53R2 in zebrafish was maternally expressed, its transcripts were detected in developing embryos and all adult tissues examined. A 250-bp minimal promoter upstream of the translational initiation site was identified to drive basal expression of p53R2 in a p53-independent manner. Expression of p53R2 was induced by DNA-damaging reagents CPT or MMS, but suppressed by p53-knockdown in zebrafish embryos. Moreover, p53R2 was mainly distributed in the cytoplasm of cells under normal condition and upon DNA damage. Furthermore, overexpression of p53R2 attenuated apoptosis of embryonic cells caused by CPT or MMS treatment and protected developing embryos from death. Therefore, functions of p53R2 in zebrafish are closely associated with its activity in DNA repair and synthesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Ribonucleotide reductase (RNR) catalyzes the rate-limiting de novo synthesis of 2'-deoxyribonucleotides from the corresponding ribonucleotides and thereby provides balanced deoxyribonucleotide pools required for error-free DNA replication and repair. The essential role of RNR in DNA synthesis and the use of DNA as genetic material has made it an important target for the development of anticancer and antiviral agents. The most well known feature of the universal RNR reaction in all kingdoms of life is the involvement of protein free radicals. Redox-active cysteines, thiyl radicals, and thiol redox proteins of the thioredoxin superfamily play major roles in the catalytic mechanism. The involvement of cysteine residues in catalysis is common to all three classes of RNR. Taking account of the recent progress in this field of research, this review focuses on the use of thiols in the redox mechanism of RNR enzymes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint-the replicative potential of spontaneous enzyme mutants-has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.

Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during dNTP synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R1(2)R2(2) heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Co-immunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5'-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, alpha and beta, for activity. Humans possess two beta subunits: one involved in S phase DNA replication (beta) and a second in mitochondrial DNA replication (beta' or p53R2) and potentially DNA repair. Gemcitabine (F(2)C) is used clinically as an anticancer agent, and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with alpha (specific activity of 400 nmol min(-1) mg(-1)) and beta' (0.6 Y./beta'2 and a specific activity of 420 nmol min(-1) mg(-1)) establishes that F(2)CDP is a substoichiometric inactivator of RNR. Incubation of this alpha/beta' with [1'-(3)H]-F(2)CDP or [5-(3)H]-F(2)CDP and reisolation of the protein by Sephadex G-50 chromatography resulted in recovery 0.5 equiv of covalently bound sugar and 0.03 equiv of tightly associated cytosine to alpha2. SDS-PAGE analysis (loaded without boiling) of the inactivated RNR showed that 60% of alpha migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1'-(3)H]-F(2)CDP with active site mutants C444S/A, C218S/A, and E431Q/D-alpha and the C-terminal tail C787S/A and C790S/A mutants reveals that no sugar label is bound to the active site mutants of alpha and that, in the case of C218S-alpha, alpha migrates as a 90 kDa protein. Analysis of the inactivated wt-alpha/beta' RNR by size exclusion chromatography indicates a quaternary structure of alpha6beta'6. A mechanism of inactivation common with halpha/beta is presented.

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents., ((c) 2009 UICC.)

Cytomegaloviruses (CMVs) code for immunoevasins, glycoproteins that are specifically dedicated to interfere with the presentation of antigenic peptides to CD8 T cells. Nonetheless, the biological outcome is not an immune evasion of the virus, since CD8 T cells can control CMV infection even when immunoevasins are expressed. Here, we compare the processing of a protective and a nonprotective epitope derived from the same viral protein, the antiapoptotic protein M45 in the murine model. The data provide evidence to conclude that protection against CMVs critically depends on antigenic peptides generated in an amount sufficient to exhaust the inhibitory capacity of immunoevasins.

The virescent3 (v3) and stripe1 (st1) mutants in rice (Oryza sativa) produce chlorotic leaves in a growth stage-dependent manner under field conditions. They are temperature-conditional mutants that produce bleached leaves at a constant 20 degrees C or 30 degrees C but almost green leaves under diurnal 30 degrees C/20 degrees C conditions. Here, we show V3 and St1, which encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1, and RNRS1, respectively. RNR regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. RNRL1 and RNRS1 are highly expressed in the shoot base and in young leaves, and the expression of the genes that function in plastid transcription/translation and in photosynthesis is altered in v3 and st1 mutants, indicating that a threshold activity of RNR is required for chloroplast biogenesis in developing leaves. There are additional RNR homologs in rice, RNRL2 and RNRS2, and eukaryotic RNRs comprise alpha(2)beta(2) heterodimers. In yeast, RNRL1 interacts with RNRS1 (RNRL1:RNRS1) and RNRL2:RNRS2, but no interaction occurs between other combinations of the large and small subunits. The interacting activities are RNRL1:RNRS1 > RNRL1:rnrs1(st1) > rnrl1(v3):RNRS1 > rnrl1(v3):rnrs1(st1), which correlate with the degree of chlorosis for each genotype. This suggests that missense mutations in rnrl1(v3) and rnrs1(st1) attenuate the first alphabeta dimerization. Moreover, wild-type plants exposed to a low concentration of an RNR inhibitor, hydroxyurea, produce chlorotic leaves without growth retardation, reminiscent of v3 and st1 mutants. We thus propose that upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves, leading to continuous plant growth.

Herpes simplex virus (HSV) inhibits apoptosis induced by external stimuli in epithelial cells. In contrast, apoptosis is the primary outcome in HSV-infected lymphocytes. Here, we show that HSV type 2 (HSV-2) gene expression appears to be necessary for the induction of apoptosis in Jurkat cells, a T-cell leukemia line. HSV-2 ICP10 gene expression is sufficient to induce apoptosis in Jurkat cells, while its expression protects epithelial HEp-2 cells from apoptosis triggered by cycloheximide and tumor necrosis factor alpha. Thus, the effect of HSV-2 gene expression on the cellular apoptotic pathway appears to depend on the specific cell type.

Qualitative and quantitative changes in mitochondrial DNA (mtDNA) have been shown to be common causes of inherited neurodegenerative and muscular diseases, and have also been implicated in ageing. These diseases can be caused by primary mtDNA mutations, or by defects in nuclear-encoded mtDNA maintenance proteins that cause secondary mtDNA mutagenesis or instability. Furthermore, it has been proposed that mtDNA copy number affects cellular tolerance to environmental stress. However, the mechanisms that regulate mtDNA copy number and the tissue-specific consequences of mtDNA mutations are largely unknown. As post-mitotic tissues differ greatly from proliferating cultured cells in their need for mtDNA maintenance, and as most mitochondrial diseases affect post-mitotic cell types, the mouse is an important model in which to study mtDNA defects. Here, we review recently developed mouse models, and their contribution to our knowledge of mtDNA maintenance and its role in disease.

We have previously shown that intrastriatal injection of Delta RR, the growth-compromised herpes simplex virus type 2 (HSV-2) vector for the antiapoptotic protein ICP10PK, prevents apoptosis caused by the excitotoxin N-methyl-D-aspartate (NMDA) in a mouse model of glutamatergic neuronal cell death (Golembewski et al. [2007] Exp. Neurol. 203:381-393). Because apoptosis regulation is stimulus and cell type specific, our studies were designed to examine the mechanism of Delta RR-mediated neuroprotection in striatal neurons. Organotypic striatal cultures (OSC) that retain much of the synaptic circuitry of the intact striatum were infected with Delta RR or a growth-compromised HSV-2 vector that lacks ICP10PK (Delta PK) and examined for neuroprotection-associated signaling. The mutated ICP10 proteins (p175 and p95) were expressed in 70-80% of neurons from Delta RR- and Delta PK-infected cultures, respectively, as determined by double-immunofluorescent staining with antibodies to ICP10 and NeuN or GAD65. Delta RR- but not Delta PK-treated OSC were protected from NMDA-induced apoptosis, as verified by ethidium homodimer staining, TUNEL, caspase-3 activation, and poly(AD-ribose) polymerase (PARP) cleavage. Neuroprotection was through ICP10PK-mediated activation of the survival pathways MEK/ERK and PI3-K/Akt, up-regulation of the antiapoptotic proteins Bag-1 and Bcl-2, and phosphorylation (inactivation) of the proapoptotic protein Bad. It was blocked by the MEK inhibitor U0126 or the PI3-K inhibitor LY294002, suggesting that either pathway can prevent NMDA-induced apoptosis. The data indicate that Delta RR-delivered ICP10PK stimulates redundant survival pathways that override proapoptotic cascades. Delta RR is a promising gene therapy platform against glutamatergic cell death., ((c) 2007 Wiley-Liss, Inc.)

Cytotoxic deoxynucleoside analogues are widely used in the treatment of haematological malignancies and solid tumours. Their metabolism and mechanisms of action are relatively well known, but with ongoing technological development, a continuous flow of scientific data is constantly adding new knowledge to this field. Thus, what was already a well-developed area some years ago has continued its expansion and become a better understood part of medical sciences. In order to keep abreast of the latest advances on cellular and clinical resistance to deoxynucleoside analogues, we have reviewed the recent literature and provide here an update on the subject. We have particularly focused on changes in gene products involved in the metabolic pathway of these drugs, such as membrane transporters, kinases, deaminases and 5'-nucleotidases. We also gave an overview on the chemical and biological development of modified deoxynucleoside analogues such as conjugates and pronucleotides.

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.

The herpes simplex virus type 2 (HSV-2) protein ICP10PK has anti-apoptotic activity in virus-infected hippocampal cultures through activation of the Ras/Raf-1/MEK/ERK pathway. To exclude the possible contribution of other viral proteins to cell fate determination, we examined the survival of primary hippocampal cultures and neuronally differentiated PC12 cells transfected with ICP10PK from apoptosis caused by nerve growth factor (NGF) withdrawal. NGF deprivation caused apoptosis in cultures mock-transfected or transfected with the kinase-negative ICP10 mutant p139(TM), but not in ICP10PK-transfected cultures. In one clone (PC47), ICP10PK inhibited caspase-3 activation through up-regulation/stabilization of adenylate cyclase (AC), activation of PKA and MEK, and the convergence of the two pathways on extracellular signal-regulated kinase activation. The anti-apoptotic proteins Bag-1 and Bcl-2 were stabilized and the pro-apoptotic protein Bad was phosphorylated (inactivated). In another clone (PC70), ICP10PK inhibited apoptosis through MEK-dependent up-regulation of the anti-apoptotic protein XIAP (that inhibits the activity of processed caspase-3) and down-regulation of the apoptogenic protein Smac/DIABLO. This may be cell-type specific, but the baculovirus p35 protein did not potentiate the neuroprotective activity of ICP10PK in PC12 cells, suggesting that ICP10PK inhibits both caspase activation and activity. The data indicate that ICP10PK inhibits apoptosis independent of other viral proteins and is a promising neuronal gene therapy platform.

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5'-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1-2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b-/- mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis.

Ribonucleotide reductase (RNR), which consists of R1 and R2 subunits, catalyzes a key step of deoxyribonucleoside triphosphate (dNTP) synthesis for DNA replication and repair. The R2 subunit is controlled in a cell cycle-specific manner for timely DNA synthesis and is negatively regulated by p53 in response to DNA damage. Herein we demonstrate that the presence of excess R2 subunits in p53(-/-) HCT-116 human colon cancer cells protects against DNA damage and replication stress. siRNA-mediated stable knockdown (>80%) of excess R2 subunits has no effect on proliferative growth but results in enhanced accumulation of gamma-H2Ax and delayed recovery from DNA lesions inflicted by exposure to cisplatin and Triapine. This accentuated induction of gamma-H2Ax in R2-knockdown cells is attributed to reduced ability to repair damaged DNA and overcome replication blockage. The lack of excess R2 subunits consequently augments chk1 activation and cdc25A degradation, causing impeded cell progression through the S phase and enhanced apoptosis in response to DNA damage and replication stress. In contrast, the level of R1 subunits appears to be limiting, since depletion of the R1 subunit directly activates the S phase checkpoint due to replication stress associated with impaired RNR activity. These findings suggest that excess R2 subunits facilitate DNA damage repair and recovery from replication stress through coordination with the S phase checkpoint in the absence of functional p53. Thus, the level of the R2 subunit constitutes an important determinant of the chemosensitivity of cancer cells and serves as a potential target for enhancement of DNA-damage based therapy.

Ribonucleotide reductase maintains cellular deoxyribonucleotide pools and is thus tightly regulated during the cell cycle to ensure high fidelity in DNA replication. The Sml1 protein inhibits ribonucleotide reductase activity by binding to the R1 subunit. At the completion of each turnover cycle, the active site of R1 becomes oxidized and subsequently regenerated by a cysteine pair (CX2C) at its C-terminal domain (R1-CTD). Here we show that R1-CTD acts in trans to reduce the active site of its neighboring monomer. Both Sml1 and R1-CTD interact with the N-terminal domain of R1 (R1-NTD), which involves a conserved two-residue sequence motif in the R1-NTD. Mutations at these two positions enhancing the Sml1-R1 interaction cause SML1-dependent lethality. These results point to a model whereby Sml1 competes with R1-CTD for association with R1-NTD to hinder the accessibility of the CX2C motif to the active site for R1 regeneration.

Replication-selective oncolytic herpes simplex virus (HSV) has shown considerable promise as an antitumor agent. Although the current oncolytic HSVs were exclusively constructed from HSV-1, HSV-2 has several unique features that could be exploited to convert the virus to an oncolytic agent. The N-terminus of the HSV-2 ICP10 gene product contains a well-defined serine/threonine protein kinase (PK) domain, which can activate the Ras/MEK/MAPK mitogenic pathway and thus facilitate efficient HSV-2 replication. Because the Ras signaling pathway is a key regulator of normal cell growth and malignant transformation, it is aberrantly activated in many human tumors. Here we report that a mutant HSV-2 (FusOn-H2), constructed by replacing the PK domain of ICP10 with the gene encoding the green fluorescent protein, can selectively replicate in and thus lyse tumor cells. Moreover, infection of FusOn-H2 led to syncytia formation in tumor cells, providing an additional tumor-destroying mechanism. A single moderate-dose injection of FusOn-H2 into established breast cancer xenografts completely eradicated the tumors in more than 80% of the animals, leading to their long-term survival. We conclude that this HSV-2 mutant is a safe and potent oncolytic agent useful for the treatment of malignant solid tumors such as breast cancer.

Ribonucleoside diphosphate reductase (EC 1.17.4.1) (RR) is a potential target for antineoplastic agents due to its crucial role in DNA replication and repair. The expression and activity of RR subunits are highly regulated to maintain an optimal dNTP pool, which is required to maintain genetic fidelity. The human RR small subunit M2B (p53R2) is thought to contribute to DNA repair in response to DNA damage. However, it is not clear whether M2B is involved in providing dNTPs for DNA replication under physiological growth conditions. Serum starvation synchronized studies showed that a rapid increase of M2B was associated with cyclin E, which is responsible for regulation of G(1)/S-phase transition. A living cell sorting study that used KB cells in normal growth, further confirmed that M2B increased to maximum levels at the G(1)/S-phase transition, and decreased with DNA synthesis. Confocal studies revealed that M2B redistributed from the cytoplasm to the nucleus earlier than hRRM2 in response to DNA replication. Nuclear accumulation of M2B is associated with dynamic changes in dNTP at early periods of serum addition. By using M2B-shRNA expression vectors, inhibition of M2B may result in growth retardation in KB cells. We conclude that M2B may translocate from the cytoplasm into the nucleus and allow dNTPs to initiate DNA synthesis in KB cells under physiological conditions. Thus, our findings suggested that M2B might play an important role for initiating DNA replication of KB cells in normal growth.

Herpes simplex viruses types 1 (HSV-1) and 2 (HSV-2) are associated with a wide range of diseases related to infection of epithelial or neuronal tissues. The two viruses evidence distinct pathogenesis aspects, which are likely mediated by distinct viral genes. One such gene is UL39, which codes for the large subunit of ribonucleotide reductase (R1, also known as ICP6 and ICP10 for HSV-1 and HSV-2 respectively). The HSV-2 R1 has serine-threonine protein kinase (PK) activity, which is located within the first 411 amino acids (ICP10PK). ICP10PK is a constitutively activated growth factor receptor (GFR) that signals through the Ras/MEK/ERK pathway. It has transforming activity in immortalized cells, mitogenic (but not transforming) activity in normal diploid cells, and anti-apoptotic (survival) activity in post mitotic neurons in the central nervous system (CNS). In addition to the Ras/MEK/ERK, ICP10PK also activates the PI3-K/Akt pathway, upregulates the Ras family member Rap-1 and adenylate cyclase and activates the B-Raf kinase activity. ICP10 PK appears to have a cellular origin. Its conservation is most likely to reflect the ability to impart an evolutionary advantage, particularly in the face of pro-apoptotic viral genes. Indeed, activation of the Ras/MEK/ERK pathway by ICP10PK is required for virus growth.

The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other gamma-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6-7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5'-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyurea-treated cells in the presence of the vitamin B12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.

Background: G207 is an oncolytic herpes virus whose replicative cycle requires cellular ribonucleotide reductase (RR) for viral DNA synthesis. We attempt to enhance viral cytotoxicity in carcinoembryonic antigen (CEA)-producing colorectal cancer (CRC) cells through CEA-driven RR production., Methods: CEA enzyme-linked immunosorbent assay was performed on LS174T and HCT-8 human CRC cells. The CEA enhancer-promoter (CEA E-P) was functionally assessed by luciferase assay. CEA E-P was cloned upstream of UL39, the gene encoding the large subunit of RR. Cells were transfected with CEA E-P/UL39 and infected with G207 for cytotoxicity assays. LS174T, with or without CEA E-P/UL39, were implanted into athymic mouse flanks (n = 28) and treated with G207., Results: CEA levels were 7-fold higher in LS174T versus HCT-8 ( P <.00001). CEA E-P increased luciferase expression 7.5-fold in LS174T ( P <.01), with no increase in HCT-8. G207 cytotoxicity of'CEA E-P/UL39-transfected LS174T cells increased 69% by day 10 versus nontransfected cells ( P <.001), with no significant increase in HCT-8. Combining CEA E-P/UL39 with G207 in LS174T flank tumors resulted in a 65% decrease in tumor volume versus G207, phosphate-buffered saline, or'CEA E-P/UL39 alone ( P <.0001)., Conclusions: CEA-driven RR production by CEA-secreting CRC cells significantly improves oncolytic viral cytotoxicity and specificity in vitro, and reduces tumor burden in vivo.

Ribonucleotide reductase (RNR) is the enzyme responsible for the conversion of ribonucleotides to 2'-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA. In the recent years, many new crystal structures have been obtained of the protein subunits of all three classes of RNR. This review will focus upon recent structural and spectroscopic studies, which have offered deeper insight to the mechanistic properties as well as evolutionary relationship and diversity among the different classes of RNR. Although the three different classes of RNR enzymes depend on different metal cofactors for the catalytic activity, all three classes have a conserved cysteine residue at the active site located on the tip of a protein loop in the centre of an alpha/beta-barrel structural motif. This cysteine residue is believed to be converted into a thiyl radical that initiates the substrate turnover in all three classes of RNR. The functional and structural similarities suggest that the present-day RNRs have all evolved from a common ancestral reductase. Nevertheless, the different cofactors found in the three classes of RNR make the RNR proteins into interesting model systems for quite diverse protein families, such as diiron-oxygen proteins, cobalamin-dependent proteins, and SAM-dependent iron-sulfur proteins. There are also significant variations within each of the three classes of RNR. With new structures available of the R2 protein of class I RNR, we have made a comparison of the diiron centres in R2 from mouse and Escherichia coli. The R2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms, and new radical forms are different from non-radical forms. In mouse R2, the binding of iron(II) or cobalt(II) to the four metal sites shows high cooperativity. A unique situation is found in RNR from baker's yeast, which is made up of heterodimers, in contrast to homodimers, which is the normal case for class I RNR. Since the reduction of ribonucleotides is the rate-limiting step of DNA synthesis, RNR is an important target for cell growth control, and the recent finding of a p53-induced isoform of the R2 protein in mammalian cells has increased the interest for the role of RNR during the different phases of the cell cycle.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a DeltanrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O(2)-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O(2) restriction. However, survival of the DeltanrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F(1) mice infected with the DeltanrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

Ribonucleotide reductase (RR) is responsible for the de novo conversion of the ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. RR consists of two subunits, hRRM1 and hRRM2. p53R2 is a new RR family member. Because the majority of human tumors possess mutant p53, it is important to know the molecular mechanism by which mutant p53 regulates RR and to what extent. In this study, we investigated the expression and function of p53R2 and hRRM2 after UV treatment in human prostate cancer PC3 cells, which possess mutant p53 with a truncated COOH-terminal, and in human oropharyngeal cancer KB cells, which possess wild-type p53. p53R2 (analyzed by Western blot and standardized relative to Coomassie Blue-stained band) was down-regulated in PC3 cells and up-regulated in KB cells after UV exposure. In contrast, hRRM2 was up-regulated by UV in both PC3 cells and KB cells. hRRM2 and p53R2 mRNA levels were assessed by Northern blot, and the results paralleled that of the Western blot. Coimmunoprecipitation assays using agarose-conjugated goat antihuman RRM1 antibody confirmed that the p53R2 binding to hRRM1 decreased in PC3 cells but increased in KB cells after UV treatment. hRRM2 binding to hRRM1 increased in both cell lines under the same conditions. These results suggest that PC3 cells are deficient in both transcription of p53R2 and binding to hRRM1 in response to UV irradiation. Confocal microscopy further confirmed that these findings were not due to translocation of hRRM2 and p53R2 from the cytoplasm to the nucleus. RR activity was measured following UV treatment and shown to increase in PC3 cells. It was unchanged in proportional of KB cells. The RR activity is consistent with the expression of hRRM2 seen in the Western blots. Thus, we hypothesize that hRRM2 complements p53R2 to form RR holoenzyme and maintain RR activity in PC3 cells after UV treatment. To further confirm this hypothesis, we examined the effect of RRM2 inhibitors on cells exposed to UV. In PC3 cells, hydroxyurea inhibited hRRM2 and resulted in increased sensitivity to UV irradiation. We also examined the effect of UV treatment on the colony-forming ability of cells transfected with hRRM2 as well as p53R2 sense or antisense expression vectors. Expression of antisense hRRM2 in PC3 cells led to decreased hRRM2 expression and resulted in greater sensitivity to UV than observed in wild-type PC3 cells. Taken together, we conclude that UV-induced activation of p53R2 transcription and binding of p53R2 to hRRM1 to form RR holoenzyme are impaired in the p53-mutant cell line PC3.

The introduction of systemic chemotherapy improved significantly the prognosis of osteosarcoma. Despite this success, approximately 30-40% of patients will relapse. Cytotoxic drugs have been shown to induce apoptosis in the target cells independent of their primary effects. The underlying molecular mechanisms and the intracellular mediators, however, are still largely unknown. Therefore, the purpose of our study was to identify drug-regulated genes in osteosarcoma cells useful as prognostic factors and for the development of new therapeutic strategies. Using suppressive subtractive hybridization (SSH) the gene expression pattern of untreated Saos-2 cells was compared to cells treated with cisplatin, methotrexate and doxorubicin, respectively. We identified 8 genes that are regulated >2-fold in drug-treated osteosarcoma cell lines. Expression of ferritin light chain, rhoA, inosine monophosphatdgehydrogenase II, ribonucleotide reductase M2, pro2000 and pro1859 were increased after drug treatment, whereas prohibitin and alpha-actinin expressions were significantly downregulated. Differential expression of the identified genes was verified by Northern blot analysis of 3 different osteosarcoma cell lines. In addition, the effects on chemosensitivity of 4 selected genes was analyzed by overexpression of recombinant constructs in Saos-2 cells and subsequent quantification of drug-induced apoptosis. Overexpression of prohibitin and rhoA reduced significantly drug sensitivity to approximately 52% and 59% indicating a crucial role in the modulation of drug-induced cell death., (Copyright 2003 Wiley-Liss, Inc.)

Ribonucleotide reductase is an enzyme that is essential for DNA synthesis and repair. It is composed of 2 dimeric proteins called R1 and R2 that are both necessary for enzymatic activity that reduces ribonucleotides to deoxyribonucleotides. This is the rate-limiting reaction that provides a supply of precursors for DNA synthesis therefore it is essential for cell proliferation. The importance of understanding the complex regulation of ribonucleotide reductase is emphasized by observations that mechanisms controlling its expression and activity may be altered during malignant cell proliferation which leads to drug resistance, making it a useful target to develop chemotherapeutic compounds in the treatment of cancer. Expression studies with the R1 and R2 genes have provided evidence for a direct role for the components of ribonucleotide reductase in determining malignant potential. Ribonucleotide reductase is regulated by transcriptional activation of gene expression and post-transcriptional mechanisms that alter mRNA message stability. Post-transcriptional regulation of mRNA turnover plays an important role in modulating mRNA steady state levels and therefore directly influences gene expression. The 3'-untranslated region (UTR) of R1 and R2 messages contain sequences that are important in regulating gene expression through changes in message stability. Studies have found that mRNA message stability is mediated by growth factors, cytokines and tumor promoters. Several studies have elucidated signal transduction pathways of tumor promoters, TGF-beta and oxidation/reduction agents. This report reviews how knowledge of these signaling pathways is revealing new insights into how ribonucleotide reductase mRNA binding proteins are important in regulating cellular proliferation, drug resistance and malignancy.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) can trigger or block apoptosis in a cell type-dependent manner. We have recently shown that the protein kinase activity of the large subunit of the HSV-2 ribonucleotide reductase (R1) protein (ICP10 PK) blocks apoptosis in cultured hippocampal neurons by activating the extracellular signal-regulated kinase (ERK) survival pathway (Perkins et al., J. Virol. 76:1435-1449, 2002). The present studies were designed to better elucidate the mechanism of ICP10 PK-induced neuroprotection and determine whether HSV-1 has similar activity. The data indicate that apoptosis inhibition by ICP10 PK involves a c-Raf-1-dependent mechanism and induction of the antiapoptotic protein Bag-1 by the activated ERK survival pathway. Also associated with neuroprotection by ICP10 PK are increased activation/stability of the transcription factor CREB and stabilization of the antiapoptotic protein Bcl-2. HSV-1 and the ICP10 PK-deleted HSV-2 mutant ICP10DeltaPK activate JNK, c-Jun, and ATF-2, induce the proapoptotic protein BAD, and trigger apoptosis in hippocampal neurons. c-Jun activation and apoptosis are inhibited in hippocampal cultures infected with HSV-1 in the presence of the JNK inhibitor SP600125, suggesting that JNK/c-Jun activation is required for HSV-1-induced apoptosis. Ectopically delivered ICP10 PK (but not its PK-negative mutant p139) inhibits apoptosis triggered by HSV-1 or ICP10DeltaPK. Collectively, the data indicate that ICP10 PK-induced activation of the ERK survival pathway results in Bag-1 upregulation and overrides the proapoptotic JNK/c-Jun signal induced by other viral proteins.

Bacteroides fragilis, a component of the normal intestinal flora, is an obligate anaerobe capable of long-term survival in the presence of air. Survival is attributed to an elaborate oxidative stress response that controls the induction of more than 28 peptides, but there is limited knowledge concerning the identities of these peptides. In this report, RNA fingerprinting by arbitrarily primed PCR identified five new genes whose expression increased following exposure to O2. Nucleotide sequence analysis of the cloned genes indicated that they encoded an outer membrane protein, an aspartate decarboxylase, an efflux pump, heat shock protein HtpG, and an NrdA ortholog constituting the large subunit of a class Ia ribonucleotide reductase (RRase). Attention was focused on the nrdA gene since class I RRases are obligate aerobic enzymes catalyzing the reduction of ribonucleoside 5'-diphosphates by a mechanism that requires molecular oxygen for activity. Sequence analysis of the nrd locus showed that two genes, nrdA and nrdB, are located in the same orientation in a 4.5-kb region. Northern hybridization and primer extension experiments confirmed induction of the genes by O2 and suggested they are an operon. The B. fragilis nrdA and nrdB genes were overexpressed in Escherichia coli, and CDP reductase assays confirmed that they encoded an active enzyme. The enzyme activity was inhibited by hydroxyurea, and ATP was shown to be a positive effector of CDP reductase activity, while dATP was an inhibitor, indicating that the enzyme was a class Ia RRase. A nrdA mutant was viable under anaerobic conditions but had decreased survival following exposure to O2, and it could not rapidly resume growth after O2 treatment. The results presented indicate that during aerobic conditions B. fragilis NrdAB may have a role in maintaining deoxyribonucleotide pools for DNA repair and growth recovery.

Herpes simplex virus type 1 (HSV-1) and HSV-2 trigger or counteract apoptosis by a cell-specific mechanism. Our studies are based on previous findings that the protein kinase (PK) domain of the large subunit of HSV-2 ribonucleotide reductase (ICP10) activates the Ras/MEK/MAPK pathway (Smith et al., J. Virol. 74:10417, 2000). Because survival pathways can modulate apoptosis, we used cells that are stably or transiently transfected with ICP10 PK, an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) and the MEK-specific inhibitor U0126 to examine the role of ICP10 PK in apoptosis. Apoptosis was induced by staurosporine or D-mannitol in human (HEK293) cells or HEK293 cells stably transfected with the ICP10 PK-negative mutant p139 (JHL15), as determined by morphology, DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage. HEK293 cells stably transfected with ICP10 (JHLa1) were protected from apoptosis. ICP10 but not p139 protected neuronally differentiated PC12 cells from death due to nerve growth factor withdrawal, and apoptosis (determined by TUNEL) and caspase-3 activation were seen in primary hippocampal cultures infected with ICP10DeltaPK but not with HSV-2 or a revertant virus [HSV-2(R)]. The data indicate that ICP10 has antiapoptotic activity under both paradigms and that it requires a functional PK activity. The apoptotic cells in primary hippocampal cultures were neurons, as determined by double immunofluorescence with fluorescein-labeled dUTP (TUNEL) and phycoerythrin-labeled antibodies specific for neuronal proteins (TuJ1 and NF-160). Protection from apoptosis was associated with MEK/MAPK activation, as evidenced by (i) increased levels of activated (phosphorylated) MAPK in HSV-2- but not ICP10DeltaPK-infected cultures and (ii) inhibition of MAPK activation by the MEK-specific inhibitor U0126. MEK and MAPK were activated by infection with UV-inactivated but not antibody-neutralized HSV-2, suggesting that activation requires cellular penetration but is independent of de novo viral protein synthesis.

Earlier studies have shown that the retinoblastoma protein (pRb) is involved in cell-cycle regulation under conditions of moderate hypoxia, which is the term we use to denote oxygen concentrations just above the lower level giving full respiration, ie. 1300 ppm O2. We have studied the cell cycle regulatory influence of varying levels of ribonucleotide reductase under moderate hypoxia in human cancer cells with either functional (T47D and T47DHU-res) or non-functional pRb due to expression of HPV18 E7 (HeLa S3). In this study we adapted a cell-line to hydroxyurea (T47DHU-res) resulting in an increased level of ribonucleotide reductase tyrosyl-radical that is not cell-cycle dependent. Under moderate hypoxic stress, pRb becomes dephosphorylated and rebound in the cell nucleus in all phases of the cell cycle, in contrast to its function under aerobic conditions where it binds only during early G1. We have shown in this paper that, upon reoxygenation, dephosphorylation and binding to the nucleus of pRb is reversible in T47D cells, although phosphorylation is delayed by more than 12 hours. As a result of the dephosphorylation of pRb, T47D cells are between 12 and 24 hours slower in reinitiating their S-phase progression than HeLa S3 cells (not containing functional pRb) following reoxygenation. However, with an increased level of ribonucleotide reductase tyrosyl-radical in the T47DHU-res cells, the S-phase progression after reoxygenation is reinitiated earlier, although the pRb status of these cells is the same as in T47D cells.

A recently identified ribonucleotide reductase (RR), p53R2, is directly regulated by p53 for supplying nucleotides to repair damaged DNA. We examined the role of this p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint, comparing it to R2-dependent DNA synthesis. The elevation of DNA synthesis activity through RR in response to gamma-irradiation was closely correlated with the level of expression of p53R2 but not of R2. The p53R2 product accumulated in nuclei, whereas R2 levels in cytoplasm decreased. We found a point mutation of p53R2 in cancer cell line HCT116, which resulted in loss of RR activity. In those cells, DNA damage-inducible apoptotic cell death was enhanced through transcriptional activation of p53AIP1. The results suggest that p53R2-dependent DNA synthesis plays a pivotal role in cell survival by repairing damaged DNA in the nucleus and that dysfunction of this pathway might result in activation of p53-dependent apoptosis to eliminate dangerous cells.