Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases in Mycobacteria.

Class I ribonucleotide reductases consisting of α and ß subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by di-manganese ions in the ß subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the ß subunits with no external support from another subunit. Class 1b ribonucleotide reductase complex is an essential enzyme complex in the human pathogen Mycobacterium tuberculosis and its structural details are largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidised and reduced forms, and similarly those of NrdF2:NrdI complexes. These structures provide detailed atomic view of the mechanism of free radical generation in the ß subunit in this pathogen. We observe a well-formed channel in NrdI from the surface leading to the buried FMN moiety and propose that oxygen molecule accesses FMN through it. The oxygen molecule is further converted to a superoxide ion upon electron transfer at the FMN moiety. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. The oxidation of Mn(II) in NrdF2I to high valent oxidation state (either Mn(III) or Mn(IV) assisted by the reduced FMN site was evidently confirmed by EPR studies. SEC-MALS and low resolution cryo-EM map indicate unusual stoichiometry of 2:1 in the M. tuberculosis NrdF2I complex. A density close to Tyr 110 at a distance <2.3 Å is observed, which we interpret as OH group. Overall, the study therefore provides important clues on the initiation of free radical generation in the ß subunit of the ribonucleotide reductase complex in M. tuberculosis.

Deferasirox's Anti-Chemoresistance and Anti-Metastatic Effect on Non-Small Cell Lung Carcinoma.

Clinically approved iron chelators, originally designed to address iron overload disorders, have emerged as potential anticancer agents. Deferasirox (Def), a tridentate iron chelator, has demonstrated antiproliferative effects in cancer. Background/Objectives: This study aims to elucidate the mechanism of action of Def and its impact on non-small cell lung carcinoma (NSCLC). Methods: NSCLC A549 cells were treated with Def to assess cytotoxicity, the effect on nuclear and mitochondrial pathways, and iron-containing proteins and genes to evaluate anti-metastasis and chemoresistance. A lung carcinoma mouse model was used for in vivo studies. Results: Our findings revealed that Def induced cytotoxicity, effectively chelated intracellular iron, and triggered apoptosis through the increase in phosphatidylserine externalization and caspase 3 activity. Additionally, Def caused G0/G1 cell cycle arrest by downregulating the ribonucleotide reductase catalytic subunit. Furthermore, Def perturbed mitochondrial function by promoting the production of reactive oxygen species and the inhibition of glutathione as a measurement of ferroptosis activation. Def demonstrated inhibitory effects on cell migration in scratch assays, which was supported by the upregulation of n-myc downstream-regulated gene 1 and downregulation of the epidermal growth factor receptor protein. Also, Def downregulated one of the main markers of chemoresistance, the ABCB1 gene. In vivo experiments using a lung carcinoma mouse model showed that Def treatment did not affect the animal's body weight and showed a significant decrease in tumor growth. Conclusions: This investigation lays the groundwork for unraveling Def action's molecular targets and mechanisms in lung carcinoma, particularly within iron-related pathways, pointing out its anti-metastasis and anti-chemoresistance effect.

Unveiling the Connection: Viral Infections and Genes in dNTP Metabolism.

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism.

Circular RNA RRM2 alleviates metabolic dysfunction-associated steatotic liver disease by targeting miR-142-5p to increase NRG1 expression.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver condition worldwide, demanding further investigation into its pathogenesis. Circular RNAs (circRNAs) are emerging as pivotal regulators in MASLD processes, yet their pathological implications in MASLD remain poorly understood. This study focused on elucidating the role of circular RNA ribonucleotide reductase subunit M2 (circRRM2) in MASLD progression. In this study, we used both in vitro and in vivo MASLD models using long-chain-free fatty acid (FFA)-treated hepatocytes and high-fat diet (HFD)-induced MASLD in mice, respectively. We determined the expression patterns of circRRM2, microRNA-142-5p (miR-142-5p), and neuregulin 1 (NRG1) in livers of MASLD-afflicted mice and MASLD hepatocytes by RT-qPCR. Dual-luciferase reporter assays verified the binding relationships among circRRM2, miR-142-5p, and NRG1. We conducted further analyses of their roles in MASLD hepatocytes and modulated circRRM2, miR-142-5p, and NRG1 expression in vitro by transfection. Our findings were validated in vivo. The results demonstrated reduced levels of circRRM2 and NRG1, along with elevated miR-142-5p expression in MASLD livers and hepatocytes. Overexpression of circRRM2 downregulated lipogenesis-related genes and decreased triglycerides accumulation in livers of MASLD mice. MiR-142-5p, which interacts with circRRM2, effectively counteracted the effects of circRRM2 in MASLD hepatocytes. Furthermore, NRG1 was identified as a miR-142-5p target, and its overexpression mitigated the regulatory impact of miR-142-5p on MASLD hepatocytes. In conclusion, circRRM2, via its role as a miR-142-5p sponge, upregulating NRG1, possibly influenced triglycerides accumulation in both in vitro and in vivo MASLD models.NEW & NOTEWORTHY CircRRM2 expression was downregulated in free fatty acid (FFA)-challenged hepatocytes and high-fat diet (HFD) fed mice. Overexpressed circular RNA ribonucleotide reductase subunit M2 (circRRM2) attenuated metabolic dysfunction-associated steatotic liver disease (MASLD) development by suppressing FFA-induced triglycerides accumulation. CircRRM2 targeted microRNA-142-5p (miR-142-5p), which served as an upstream inhibitor of neuregulin 1 (NRG1) and collaboratively regulated MASLD progression.

EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.

RNA interference targeting WSSV ribonucleotide reductase 2 provides long-term protection against infection in Litopenaeus vannamei.

Many studies have demonstrated that long double-stranded RNAs (dsRNAs) targeting essential genes of white spot syndrome virus (WSSV) can induce a sequence-specific antiviral RNA interference (RNAi) response in shrimp, thereby offering protection against WSSV infection. However, further experimental data on the required dose of dsRNAs and the duration of protection from a single administration are necessary to establish RNAi-mediated methods as effective and practical antiviral measures. In this study, we evaluated the protective efficacy and the duration of protection provided by a single administration of various doses of long dsRNA targeting WSSV ribonucleotide reductase 2 (rr2) in white-leg shrimp Litopenaeus vannamei. The protective efficacy of long dsRNA targeting WSSV rr2 was not diminished by the reduction of the dose to 100 ng g-1 of body weight, suggesting that a relatively low dose can effectively induce an RNAi response in shrimp. Furthermore, shrimp were well-protected against WSSV challenges for up to 4 wk post-administration of the rr2-targeting long dsRNA, although the protective effect almost disappeared at 6 wk post-administration. These results suggest that long dsRNAs can provide protection against WSSV for at least 1 mo, and monthly administration of long dsRNAs could serve as a long-term protective strategy for shrimp against WSSV.

Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding.

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.

Preparation and characterization of MnIIMnIII complexes with relevance to class Ib ribonucleotide reductases.

The Mn2 complex [MnII2(TPDP)(O2CPh)2](BPh4) (1, TPDP = 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol, Ph =phenyl) was prepared and subsequently characterized via single-crystal X-ray diffraction, X-ray absorption, electronic absorption, and infrared spectroscopies, and mass spectrometry. 1 was prepared in order to explore its properties as a structural and functional mimic of class Ib ribonucleotide reductases (RNRs). 1 reacted with superoxide anion (O2•-) to generate a peroxido-MnIIMnIII complex, 2. The electronic absorption and electron paramagnetic resonance (EPR) spectra of 2 were similar to previously published peroxido-MnIIMnIII species. Furthermore, X-ray near edge absorption structure (XANES) studies indicated the conversion of a MnII2 core in 1 to a MnIIMnIII state in 2. Treatment of 2 with para-toluenesulfonic acid (p-TsOH) resulted in the conversion to a new MnIIMnIII species, 3, rather than causing O-O bond scission, as previously encountered. 3 was characterized using electronic absorption, EPR, and X-ray absorption spectroscopies. Unlike other reported peroxido-MnIIMnIII species, 3 was capable of oxidative O-H activation, mirroring the generation of tyrosyl radical in class Ib RNRs, however without accessing the MnIIIMnIV state.

Targeting of G protein-coupled receptor 39 alleviates angiotensin II-induced renal damage by reducing ribonucleotide reductase M2.

Renal fibrosis, apoptosis and autophagy are the main pathological manifestations of angiotensin II (Ang II)-induced renal injury. G protein-coupled receptor 39 (GPR39) is highly expressed in various tissues including the kidney, but its role in the kidney is entirely unclear. This study was performed to investigate the underlying mechanism by which knockdown of GPR39 alleviated Ang II-induced renal injury. In vivo, GPR39 knockout (KO) mice were constructed and infused with Ang II for 4 weeks, followed by renal function tests. In vitro, Ang II-induced cells were treated with si-GPR39 for 48 h. Fibrosis, apoptosis and autophagy were detected in both cells and mice. The underlying mechanism was sought by mRNA transcriptome sequencing and validated in vitro. GPR39 was upregulated in renal tissues of mice with Ang II-mediated renal injury. Knockdown of GPR39 ameliorated renal fibrosis, apoptosis, and autophagy, and decreased the expression of ribonucleotide reductase M2 (RRM2). In vitro, knockdown of GPR39 was also identified to improve the Ang II-induced cell fibrosis, apoptosis, and autophagy. mRNA transcriptome results showed that knockout of GPR39 reduced the expression of RRM2 in Ang II-induced kidney tissue. Activation of RRM2 could reverse the therapeutic effect of GPR39 knockout, and the inhibitor of RRM2 could improve the cell fibrosis, apoptosis and autophagy caused by GPR39 agonist. These results indicated that targeting of GPR39 could alleviate Ang II-induced renal fibrosis, apoptosis, and autophagy via reduction of RRM2 expression, and GPR39 may serve as a potential target for Ang II-induced renal injury.

Inhibition of ribonucleotide reductase subunit M2 enhances the radiosensitivity of metastatic pancreatic neuroendocrine tumor.

Ribonucleotide Reductase (RNR) is a rate-limiting enzyme in the production of deoxyribonucleoside triphosphates (dNTPs), which are essential substrates for DNA repair after radiation damage. We explored the radiosensitization property of RNR and investigated a selective RRM2 inhibitor, 3-AP, as a radiosensitizer in the treatment of metastatic pNETs. We investigated the role of RNR subunit, RRM2, in pancreatic neuroendocrine (pNET) cells and responses to radiation in vitro. We also evaluated the selective RRM2 subunit inhibitor, 3-AP, as a radiosensitizer to treat pNET metastases in vivo. Knockdown of RNR subunits demonstrated that RRM1 and RRM2 subunits, but not p53R3, play significant roles in cell proliferation. RRM2 inhibition activated DDR pathways through phosphorylation of ATM and DNA-PK protein kinases but not ATR. RRM2 inhibition also induced Chk1 and Chk2 phosphorylation, resulting in G1/S phase cell cycle arrest. RRM2 inhibition sensitized pNET cells to radiotherapy and induced apoptosis in vitro. In vivo, we utilized pNET subcutaneous and lung metastasis models to examine the rationale for RNR-targeted therapy and 3-AP as a radiosensitizer in treating pNETs. Combination treatment significantly increased apoptosis of BON (human pNET) xenografts and significantly reduced the burden of lung metastases. Together, our results demonstrate that selective RRM2 inhibition induced radiosensitivity of metastatic pNETs both in vitro and in vivo. Therefore, treatment with the selective RRM2 inhibitor, 3-AP, is a promising radiosensitizer in the therapeutic armamentarium for metastatic pNETs.

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating ß subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[ß], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any ß metallocofactor highlights the adaptability of the 10-stranded αß barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

Pharmacogenomic study of gemcitabine efficacy in patients with metastatic pancreatic cancer: A multicenter, prospective, observational cohort study (GENESECT study).

BACKGROUND: Genetic polymorphisms of molecules are known to cause individual differences in the therapeutic efficacy of anticancer drugs. However, to date, germline mutations (but not somatic mutations) for anticancer drugs have not been adequately studied. The objective of this study was to investigate the association between germline polymorphisms of gemcitabine metabolic and transporter genes with carbohydrate antigen 19-9 (CA 19-9) response (decrease ≥50% from the pretreatment level at 8 weeks) and overall survival (OS) in patients with metastatic pancreatic cancer who receive gemcitabine-based chemotherapy. METHODS: This multicenter, prospective, observational study enrolled patients with metastatic pancreatic cancer patients who were receiving gemcitabine monotherapy or gemcitabine plus nanoparticle albumin-bound paclitaxel combination chemotherapy. Thirteen polymorphisms that may be involved in gemcitabine responsiveness were genotyped, and univariate and multivariate logistic regression analyses were used to determine the association of these genotypes with CA 19-9 response and OS. The significance level was set at 5%. RESULTS: In total, 180 patients from 11 hospitals in Japan were registered, and 159 patients whose CA 19-9 response could be assessed were included in the final analysis. Patients who had a CA 19-9 response had significantly longer OS (372 vs. 241 days; p = .007). RRM1 2464A>G and RRM2 175T>G polymorphisms suggested a weak association with CA 19-9 response and OS, but it was not statistically significant. COX-2 -765G>C polymorphism did not significantly correlate with CA 19-9 response but was significantly associated with OS (hazard ratio, 2.031; p = .019). CONCLUSIONS: Genetic polymorphisms from the pharmacokinetics of gemcitabine did not indicate a significant association with efficacy, but COX-2 polymorphisms involved in tumor cell proliferation might affect OS.

Reduced ribonucleotide reductase RRM2 subunit expression increases DNA damage and mitochondria dysfunction in woody breast chickens.

OBJECTIVE: The aim of this study was to investigate the roles of ribonucleotide reductase subunit M2 (RRM2; subunit of ribonucleotide reductase) in severe woody breast (WB) and normal breast muscles. ANIMALS: 40 8-week-old male Ross-708 broiler chickens. METHODS: Quantitative PCR was performed to determine gene expression, and commercial ELISA/assay kits were used to obtain several enzymatic activities. RESULTS: Results showed that RRM2 activity (P = .0002) and RRM2 (P = .05) and hydroxymethylbilane synthase expression (impaired oxygen transport and metabolism, P = .002) were reduced in WB, while caveolin-3 (defected membrane integrity, P = .09), endoglin (increased fibrosis, P = .06), and secreted protein acidic rich in cysteine (metabolic dysregulation, P = .09) expression tended to increase in WB. WB tended to have increased levels of homocysteine (P = .06), aspartate aminotransferase mitochondria (P = .02), pyruvate kinase (P = .04), DNA damage (P = .06), creatine kinase (P = .05), and triglyceride (P = .002) but decreased ATPase activity (P = .01), all indicating mitochondria dysfunction and tissue damage. CLINICAL RELEVANCE: In this study, differences in various enzyme activities and increased DNA damage suggest that RRM2-mediated mitochondrial abnormalities may play a role in WB myopathy.

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

3-AP inhibits the growth of human osteosarcoma by decreasing the activity of the iron-dependent pathway.

3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has broad-spectrum antitumor activity. However, its role in osteosarcoma (OS) remains unclear. Therefore, this study explored the effects of 3-AP on OS in vitro and in vivo using three human OS cell lines (MG-63, U2-OS, and 143B) and a nude mice model generated by transplanting 143B cells. The cells and mice were treated with DMSO (control) or gradient concentrations of 3-AP. Then, various assays (e.g., cell counting kit-8, flow cytometry, immunohistochemistry, and western blotting) were performed to assess cell viability and apoptosis levels, as well as γH2A.X (DNA damage correlation), ribonucleotide reductase catalytic subunit M1 and M2 (RRM1 and RRM2, respectively) protein levels (iron-dependent correlation). 3-AP time- and dose-dependably suppressed growth and induced apoptosis in all three OS cell lines, and ferric ammonium citrate (FAC) blocked these effects. Moreover, 3-AP decreased RRM2 and total ribonucleotide reductase (RRM1 plus RRM2) protein expression but significantly increased γH2A.X expression; treatment did not affect RRM1 expression. Again, FAC treatment attenuated these effects. In vivo, the number of apoptotic cells in the tumor slices increased in the 3-AP-treated mice compared to the control mice. 3-AP treatment also decreased Ki-67 and p21 expression, suggesting inhibited OS growth. Furthermore, the expression of RRM1, RRM2, and transferrin receptor protein 1 (i.e., Tfr1) indicated that 3-AP inhibited OS growth via an iron-dependent pathway. In conclusion, 3-AP exhibits anticancer activity in OS by decreasing the activity of iron-dependent pathways, which could be a promising therapeutic strategy for OS.

Clinical use of biomarkers in the field of cytotoxic nucleoside analogues.

OBJECTIVES: Cytotoxic nucleosides (gemcitabine, cytarabine…) are used for the treatment of various malignancies. Their activity is dependent on the interaction with several proteins and enzymes of nucleotide metabolism. It has for a long time been hypothesized that the clinical activity of nucleoside analogues can be predicted by studying corresponding genes or gene products in clinical samples. METHODS: In this short review, I will present old and new published data from our group and others about the prediction of activity of these drugs concentrating on gene-candidate approaches, and discuss biological and technical limitations of these. RESULTS: A large number of studies have been conducted in various clinical settings (drugs, disease, patient cohort…) evaluating DNA, mRNA or protein-related markers. Although some individual parameters and associations thereof have been validated, only a very few numbers have been implemented in pretreatment evaluations of patients. CONCLUSION: There is still much to do in the field of outcome-prediction with nucleoside analogues. The use of multiparametric methods could increase the success rate but at the cost of a poorer understanding of molecular mechanisms.

Atypical genotoxicity of carcinogenic nickel(II): Linkage to dNTP biosynthesis, DNA-incorporated rNMPs, and impaired repair of TOP1-DNA crosslinks.

Cancer is a genetic disease requiring multiple mutations for its development. However, many carcinogens are DNA-unreactive and nonmutagenic and consequently described as nongenotoxic. One of such carcinogens is nickel, a global environmental pollutant abundantly emitted by burning of coal. We investigated activation of DNA damage responses by Ni and identified this metal as a replication stressor. Genotoxic stress markers indicated the accumulation of ssDNA and stalled replication forks, and Ni-treated cells were dependent on ATR for suppression of DNA damage and long-term survival. Replication stress by Ni resulted from destabilization of RRM1 and RRM2 subunits of ribonucleotide reductase and the resulting deficiency in dNTPs. Ni also increased DNA incorporation of rNMPs (detected by a specific fluorescent assay) and strongly enhanced their genotoxicity as a result of repressed repair of TOP1-DNA protein crosslinks (TOP1-DPC). The DPC-trap assay found severely impaired SUMOylation and K48-polyubiquitination of DNA-crosslinked TOP1 due to downregulation of specific enzymes. Our findings identified Ni as the human carcinogen inducing genome instability via DNA-embedded ribonucleotides and accumulation of TOP1-DPC which are carcinogenic abnormalities with poor detectability by the standard mutagenicity tests. The discovered mechanisms for Ni could also play a role in genotoxicity of other protein-reactive carcinogens.

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

RRM2­mediated Wnt/ß­catenin signaling pathway activation in lung adenocarcinoma: A potential prognostic biomarker.

The present study aimed to investigate the role and mechanism of action of ribonucleotide reductase M2 (RRM2) in lung adenocarcinoma and its potential as a therapeutic target. Data of patients with lung adenocarcinoma from The Cancer Genome Atlas database were collected and analyzed to evaluate the potential of RRM2 as a biomarker. The expression of RRM2 was evaluated in the A549 cell line and its cisplatin-resistant A549/DDP cell line derivative by western blot and reverse transcription-quantitative PCR. The study also investigated cell proliferation and the mechanism by which RRM2 controls cellular cisplatin resistance using CCK-8 and colony-formation assays. In addition, cell migration was assessed using Transwell assays, and the cell cycle and apoptosis were examined using flow cytometry. RRM2 was highly expressed in lung adenocarcinoma and was associated with the clinical TMN stage. Functional enrichment analysis showed that RRM2 was enriched in the cell cycle. Immune cell infiltration analysis identified 12 types of immune cell that exhibited differences between patients expressing different levels of RRM2. Cellular assays revealed higher levels of RRM2 expression in A549/DDP cells than A549 cells, and its expression was induced by cisplatin. RRM2 knockdown decreased cell proliferation and migration, accelerated apoptosis and caused cell cycle arrest in the S-phase, increasing the sensitivity of A549 and A549/DDP cells to cisplatin through the Wnt/ß-catenin signaling pathway. Overexpression of ß-catenin reduced the effects of RRM2 knockdown on A549 cells. Lung adenocarcinoma growth may be influenced by RRM2 through the Wnt/ß-catenin signaling pathway, suggesting a potential pathway for cancer progression.