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Supplementary Data from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Supplementary Table from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy.Significance:SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy.This article is highlighted in the In This Issue feature, p. 2007

Activating signal co-integrator complex (ASCC) supports diverse genome maintenance and gene expression processes. Its ASCC3 subunit is an unconventional nucleic acid helicase, harboring tandem Ski2-like NTPase/helicase cassettes crucial for ASCC functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. Here, we present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-ASC1/TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. ASC1 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning a C-terminal ASC1-homology domain next to the C-terminal helicase cassette of ASCC3, likely assisting the DNA exit. ASC1 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC for specific processes. Our findings define ASCC3-ASC1/TRIP4 as a tunable motor module of ASCC that encompasses two cooperating ATPase/helicase units functionally expanded by ASC1/TRIP4.

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. Significance: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007

Background and Objectives: The purpose of the present study was to elucidate the in-hospital and long-term outcomes of patients with head and neck cancer (HNC) bleeding and to analyze the risk factors for mortality. Materials and Methods: We included patients who presented to the emergency department (ED) with HNC bleeding. Variables of patients who survived and died were compared and associated factors were investigated by logistic regression and Cox’s proportional hazard model. Results: A total of 125 patients were enrolled in the present study. Fifty-nine (52.8%) patients experienced a recurrent bleeding event. The in-hospital mortality rate was 16%. The overall survival at 1, 3 and 5 years was 48%, 41% and 34%, respectively. The median survival time was 9.2 months. Multivariate logistic regression analyses revealed that risk factors for in-hospital mortality were inotropic support (OR = 10.41; Cl 1.81–59.84; p = 0.009), hypopharyngeal cancer (OR = 4.32; Cl 1.29–14.46; p = 0.018), and M stage (OR = 5.90; Cl 1.07–32.70; p = 0.042). Multivariate Cox regression analyses indicate that heart rate >110 (beats/min) (HR = 2.02; Cl 1.16–3.51; p = 0.013), inotropic support (HR = 3.25; Cl 1.20–8.82; p = 0.021), and hypopharygneal cancer (HR = 2.22; Cl 1.21–4.06; p = 0.010) were all significant independent predictors of poorer overall survival. Conclusions: HNC bleeding commonly represents the advanced disease stage. Recognition of associated factors aids in the risk stratification of patients with HNC bleeding.

International audience; Summary Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

Objective The mainstay of first-line treatment of parotid tumors is adequate surgical removal. The present study was conducted to compare the differences between parotidectomy with postauricular incision (PI) and modified Blair incision (MBI). Data Sources A systematic search of PubMed, Embase and the Cochrane Library was performed. Methods The data of interest and study characteristics were extracted from the included studies. Statistical analysis was performed with Comprehensive Meta-Analysis software (version 3; BioStat, Englewood, NJ). Dichotomous data and continuous data were analyzed by calculating the risk difference and the mean difference with the 95% confidence interval respectively. Results Four retrospective studies were included in the present meta-analysis. The pooled results revealed that the cosmetic satisfaction score was higher in the PI group (MD = 2.67; 95% CI, 2.12 to 3.23) and that intraoperative blood loss was lower in the PI group (MD = −55.35; 95% CI, −100.33 to −10.36). The operative duration (MD = −5.15; 95% CI, −24.06 to 13.75), tumor size (MD = −.07; 95% CI, −.27 to .13) and incidences of common postoperative complications were comparable between the two groups. Conclusions According to these findings, the use of PI in parotidectomies may be one of the options for improving cosmetic outcomes. This technique may be considered if oncological safety can be secured.

SummaryA critical question in genome stability is the nature of the chemical damage responsible for repair activation. We previously reported a novel pathway specifically activated during alkylation damage in human cells, where the E3 ubiquitin ligase RNF113A mediates the recruitment of the ASCC repair complex. Yet the mechanistic basis for the alkylation damage selectivity of this pathway remains unclear. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. This aberrant RNA methylation causes transcriptional repression in a manner dependent on the ASCC complex. We show that an alkylated pre-mRNA, or an RNA containing a single damaged base, is sufficient to activate RNF113A E3 activity in a phosphorylation-dependent manner. Together, our work identifies an unexpected role for RNA damage in eliciting a DNA repair response, and suggests that RNA may serve as the “canary in the coal mine” for sensing alkylation damage.

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family that binds to the mitochondrial outer membrane to stimulate mitochondrial fusion. In this study, we showed that NME3 knockdown delayed DNA repair without reducing the cellular levels of nucleotide triphosphates. Further analyses revealed that NME3 knockdown increased fragmentation of mitochondria, which in turn led to mitochondrial oxidative stress-mediated DNA single-strand breaks (SSBs) in nuclear DNA. Re-expression of wild-type NME3 or inhibition of mitochondrial fission markedly reduced SSBs and facilitated DNA repair in NME3 knockdown cells, while expression of N-terminal deleted mutant defective in mitochondrial binding had no rescue effect. We further showed that disruption of mitochondrial fusion by knockdown of NME4 or MFN1 also caused mitochondrial oxidative stress-mediated genome instability. In conclusion, the contribution of NME3 to redox-regulated genome stability lies in its function in mitochondrial fusion.

The booming of video on demand (VOD) provides numerous TV series and movies for users to watch anywhere at any time. As the amount of video contents available for viewing grows explosively, thumbnail image representation of video works as a surrogate and facilitates quick and easy video retrieval. Unlike previous work, which considers representativeness as the main criterion for thumbnail image selection, the work described in this paper incorporates attractiveness as an additional criterion. Our idea is based on the observation that thumbnail image for VOD services should not only convey the gist of the video but also intrigue the users. We propose a two-stage method that efficiently utilizes visual features to select a thumbnail image from each TV series. The effectiveness of the proposed method is verified by a subjective test. The results provide further insight into the user preference.

Cellular supply of dNTPs via RNR (ribonucleotide reductase) is crucial for DNA replication and repair. It has been shown that DNA-damage-site-specific recruitment of RNR is critical for DNA repair efficiency in quiescent cells. The catalytic function of RNR produces dNDPs. The subsequent step of dNTP formation requires the function of NDP kinase. There are ten isoforms of NDP kinase in human cells. In the present study, we identified NME3 as one specific NDP kinase that interacts directly with Tip60, a histone acetyltransferase, to form a complex with RNR. Our data reveal that NME3 recruitment to DNA damage sites depends on this interaction. Disruption of interaction of NME3 with Tip60 suppressed DNA repair in serum-deprived cells. Thus Tip60 interacts with RNR and NME3 to provide site-specific synthesis of dNTP for facilitating DNA repair in serum-deprived cells which contain low levels of dNTPs.

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.

99mTc-N4-guanine (99mTc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of 99mTc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using 99mTc-N4amG in tumor specific imaging.

SummaryThe synthesis of dTDP is unique because there is a requirement for thymidylate kinase (TMPK). All other dNDPs including dUDP are directly produced by ribonucleotide reductase (RNR). We report the binding of TMPK and RNR at sites of DNA damage. In tumor cells, when TMPK function is blocked, dUTP is incorporated during DNA double-strand break (DSB) repair. Disrupting RNR recruitment to damage sites or reducing the expression of the R2 subunit of RNR prevents the impairment of DNA repair by TMPK intervention, indicating that RNR contributes to dUTP incorporation during DSB repair. We identified a cell-permeable nontoxic inhibitor of TMPK that sensitizes tumor cells to doxorubicin in vitro and in vivo, suggesting its potential as a therapeutic option.

This study was aimed to develop a glycopeptide (GP) to be used as a carrier for anti-cancer drug delivery. GP was synthesized by conjugating glutamate peptide and chitosan using carbodiimide as a coupling agent. Elemental analysis and capillary electrophoresis confirmed the purity was >95%. GP was labeled with sodium pertechnetate (Na99m TcO4) for in vitro and in vivo studies. Rhenium-GP was synthesized to support the binding site of 99m Tc at the glutamate positions 3-5. In vitro cellular uptake of 99m Tc-GP was performed in breast cancer cells. Cytosol had 60% whereas nucleus had 40% uptake of 99m Tc-GP. When cancer cells were incubated with glutamate or aspartate, followed by 99m Tc-GP, there was decreased uptake in cells treated with glutamate but not aspartate. The findings indicated that cellular uptake of 99m Tc-GP was via glutamate transporters. In addition, 99m Tc-GP was able to measure uptake differences after cells treated with paclitaxel. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Biodistribution of 99m Tc-GP showed increased tumor-to-tissue ratios as a function of time. Planar images confirmed that 99m Tc-GP could assess tumor uptake changes after paclitaxel treatment. In vitro and in vivo studies indicated that GP could target tumor cells, thus, GP may be a useful carrier for anti-cancer drug delivery.

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells.

Activation of hedgehog (HH) pathway signaling is observed in many tumors. Due to a feedback loop, the HH receptor Patched (PTCH-1) is overexpressed in tumors with activated HH signaling. Therefore, we sought to radiolabel the PTCH-1 ligand sonic (SHH) for detection of cancer cells with canonical HH activity. Receptor binding of131I-SHH was increased in cell lines with high HH pathway activation. Our findings also show that PTCH-1 receptor expression is decreased upon treatment with HH signaling inhibitors, and receptor binding of131I-SHH is significantly decreased following treatment with cyclopamine.In vivoimaging and biodistribution studies revealed significant accumulation of131I-SHH within tumor tissue as compared to normal organs. Tumor-to-muscle ratios were approximately 8 : 1 at 5 hours, while tumor to blood and tumor to bone were 2 : 1 and 5 : 1, respectively. Significant uptake was also observed in liver and gastrointestinal tissue. These studies show that131I-SHH is capable ofin vivodetection of breast tumors with high HH signaling. We further demonstrate that the hedgehog receptor PTCH-1 is downregulated upon treatment with hedgehog inhibitors. Our data suggests that radiolabeled SHH derivatives may provide a method to determine response to SHH-targeted therapies.

Objective. This study was aimed to study tissue distribution and tumor imaging potential of68Ga-glycopeptide (GP) in tumor-bearing rodents by PET.Methods. GP was synthesized by conjugating glutamate peptide and chitosan. GP was labeled with68Ga chloride for in vitro and in vivo studies. Computer outlined region of interest (counts per pixel) of the tumor and muscle (at the symmetric site) was used to determine tumor-to-muscle count density ratios. To ascertain the feasibility of68Ga-GP in tumor imaging in large animals, PET/CT imaging of68Ga-GP and18F-FDG were conducted in New Zealand white rabbits bearing VX2 tumors. Standard uptake value of tumors were determined by PET up to 45 min. To determine blood clearance and half-life of68Ga-GP, blood samples were collected from 10 seconds to 20 min.Results. Radiochemical purity of68Ga-GP determined by instant thin-layer chromatography was >95%. Tumor uptake values (SUV) for68Ga-GP and18F-FDG in New Zealand white rabbits bearing VX2 tumors were 3.25 versus 7.04. PET images in tumor-bearing rats and rabbits confirmed that68Ga-GP could assess tumor uptake. From blood clearance curve, the half-life of68Ga-GP was 1.84 hr.ConclusionOur data indicate that it is feasible to use68Ga-GP to assess tumor angiogenesis.

This study was to develop a (99m)Tc-labeled alpha-methyl tyrosine (AMT) using L,L-ethylenedicysteine (EC) as a chelator and to evaluate its potential in breast tumor imaging in rodents.EC-AMT was synthesized by reacting EC and 3-bromopropyl AMT (N-BOC, ethyl ester) in ethanol/potassium carbonate solution. EC-AMT was labeled with (99m)Tc in the presence of tin (II) chloride. Rhenium-EC-AMT (Re-EC-AMT) was synthesized as a reference standard for (99m)Tc-EC-AMT. To assess the cellular uptake kinetics of (99m)Tc-EC-AMT, 13 762 rat breast cancer cells were incubated with (99m)Tc-EC-AMT for 0-2 h. To investigate the transport mechanism, the same cell line was used to conduct the competitive inhibition study using L-tyrosine. Tissue distribution of (99m)Tc-EC-AMT was determined in normal rats at 0.5-4 h. Planar imaging of breast tumor-bearing rats was performed at 30 and 90 min. The data were compared with those of (18)F-2-fluoro-2-deoxy-glucose. Blocking uptake study using unlabeled AMT was conducted to investigate the transport mechanism of (99m)Tc-EC-AMT in vivo.Structures of EC-AMT and Re-EC-AMT were confirmed by nuclear magnetic resonance, high performance liquid chromatography and mass spectra. In-vitro cellular uptake of (99m)Tc-EC-AMT in 13,762 cells was increased as compared with that of (99m)Tc-EC and could be inhibited by L-tyrosine. Biodistribution in normal rats showed high in-vivo stability of (99m)Tc-EC-AMT. Planar scintigraphy at 30 and 90 min showed that (99m)Tc-EC-AMT could clearly visualize tumors. (99m)Tc-EC-AMT uptake could be significantly blocked by unlabeled AMT in vivo.The results indicate that (99m)Tc-EC-AMT, a new amino acid transporter-based radiotracer, is suitable for breast tumor imaging.

The elevation of RRM2A subunit of ribonucleotide reductase (RNR) has been shown oncogenic and associated with human cancers. However, the molecular mechanism of RRM2A in cancer promotion is unclear. In this study, we investigated whether RNR-mediated dUTP formation is involved in promoting genome instability. Since dUTP is degraded by dUTPase, we analyzed the expression of RRM2A and dUTPase in clinical samples from colorectal and breast cancer patients. The results revealed that high RRM2A and low dUTPase in tumors are correlated with poorer survival. In cancer cell lines that express high level of RRM2A and dUTPase, knockdown of dUTPase increases genome instability. Conversely, overexpression of dUTPase reduces genome stress in cancer cells expressing high RRM2A and low dUTPase. In non-tumorigenic cells, overexpression of RRM2A increases replication stress and genome instability accompanied by higher levels of 53BP1 foci and increased breaks at AT-rich fragile sites bound by 53BP1. Thymidine supplement in growth medium or co-expression of dUTPase was able to reduce RRM2A-mediated replication stress and 53BP1 foci. We further provide evidence that RRM2A promotes replication stress and error prone-translesion synthesis dependent on uracil DNA glycosylase. These results suggest that up-regulation of RRM2A in tumor cells increases uracil-mediated replication stress to promote genome instability. Therefore, high RRM2A/low dUTPase context confers tumor genome evolution advantage, correlating with poorer prognosis. Citation Format: Chih-Wei Chen, Ning Tsao, Yun Yen, Christine E. Lehman, Yuh-Hwa Wang, Chian-Feng Chen, Tse-Hsiang Wu, Sui-Chih Tien, Ming-Hsiang Lee, Zee-Fen Chang. Ribonucleotide reductase promotes uracil-mediated genome instability in tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3017. doi:10.1158/1538-7445.AM2015-3017

Introduction and Purpose: Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. It is known that ribonucleotide reductase (RNR), a rate-limiting enzyme for dNTP synthesis, is required for DNA repair. The catalytic function of RNR produces dNDPs. There are eight isoforms of NDP kinase that catalyzes the subsequent step for dNTP formation in human cells. In this study, we aim to identify the isoform of NDP kinase required for DNA repair. Preliminary results: By RNA interference experiments, we identified NME3, one isoform of the nucleoside diphosphate kinases, critical for DNA repair. It has been shown that RNR forms a complex with Tip60, a histone acetyltransferase, by which RNR is recruited to DNA damage sites for repair. In this study, we found that NME3 directly interacts with Tip60. The effect of knockdown of NME3 on DNA repair was rescued by re-expression of wild-type but not the mutant type defective in Tip60 interaction. Overexpression of NME3 was able to facilitate DNA repair, which was intervened by disrupting the interaction of NME3 with Tip60. Moreover, exogenous addition of deoxynucleosides facilitates NME3-mediated DNA repair. Conclusion: Among 8 isoforms of NDP kinase, NME3 is identified to be critical for DNA repair, indicating its potential as a target for developing chemosensitization agent. Citation Format: Ning Tsao, Yu-Jyun Deng, Zee-Fen Chang, Wei Zhang. Identification of the NDP kinase critical for DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2015-3014

The wood-rotting fungus Pleurotus ostreatus NRRL 2366 was grown successfully in submerged shaker cultures in which it produced oxalic acid from simple carbohydrates as efficiently as did Aspergillus niger. P. ostreatus also produced oxalic acid from mixtures of sawdust and CaCO 3 , and from the solid residue from the acid hydrolysis of wood when the culture was supplemented with inorganic nutrients. A. niger produced oxalic acid from the liquid hydrolysate.

SummaryThe appropriate supply of dNTPs is critical for cell growth and genome integrity. Here, we investigated the interrelationship between dUTP pyrophosphatase (dUTPase) and ribonucleotide reductase (RNR) in the regulation of genome stability. Our results demonstrate that reducing the expression of dUTPase increases genome stress in cancer. Analysis of clinical samples reveals a significant correlation between the combination of low dUTPase and high R2, a subunit of RNR, and a poor prognosis in colorectal and breast cancer patients. Furthermore, overexpression of R2 in non-tumorigenic cells progressively increases genome stress, promoting transformation. These cells display alterations in replication fork progression, elevated genomic uracil, and breaks at AT-rich common fragile sites. Consistently, overexpression of dUTPase abolishes R2-induced genome instability. Thus, the expression level of dUTPase determines the role of high R2 in driving genome instability in cancer cells.

A strain of the common mushroom Agaricus campestris was grown on a mixture of composted sawdust and CaCO 3 . On incubation for 47 days, the organism produced 20.5 g of oxalic acid per 100 g of initial dry compost solids.