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1.
BMC Cancer ; 18(1): 423, 2018 04 16.
Article in English | MEDLINE | ID: mdl-29661172

ABSTRACT

BACKGROUND: Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. METHODS: Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. RESULTS: MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. CONCLUSIONS: MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA Repair Enzymes/genetics , Phosphoric Monoester Hydrolases/genetics , A549 Cells , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA Damage/drug effects , DNA Repair Enzymes/deficiency , Gene Expression Regulation, Neoplastic/drug effects , Humans , Oxidation-Reduction , Oxidative Stress/drug effects , Phosphoric Monoester Hydrolases/deficiency , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Small Molecule Libraries/pharmacology
2.
BMC Microbiol ; 18(1): 9, 2018 01 24.
Article in English | MEDLINE | ID: mdl-29368646

ABSTRACT

BACKGROUND: Microbial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways. RESULTS: The "PHENOS" pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive. CONCLUSIONS: "PHENOS" is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos .


Subject(s)
Colony Count, Microbial/instrumentation , Colony Count, Microbial/methods , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Saccharomyces cerevisiae/growth & development , Agar , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Culture Media , Genomics/methods , Genotype , Image Processing, Computer-Assisted/methods , Phenotype , Saccharomyces cerevisiae/cytology , Software
3.
Proc Natl Acad Sci U S A ; 109(25): 9953-8, 2012 Jun 19.
Article in English | MEDLINE | ID: mdl-22670056

ABSTRACT

The DNA damage response comprises DNA repair, cell-cycle checkpoint control, and DNA damage-induced apoptosis that collectively promote genomic integrity and suppress tumorigenesis. Previously, we have shown that the Chk2 kinase functions independently of the Mre11 complex (Mre11, Rad50, and Nbs1) and ATM in apoptosis and suppresses tumorigenesis resulting from hypomorphic alleles of Mre11 or Nbs1. Based on this work, we have proposed that Chk2 limits the oncogenic potential of replication-associated DNA damage. Here we further address the role of Chk2 and damage-induced apoptosis in suppressing the oncogenic potential of chromosome breaks. We show that loss of Chk2 or a mutation in p53 (R172P), which selectively impairs its function in apoptosis, rescued the lethality of mice lacking Lig4, a ligase required for nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks in G0/G1. In contrast to Lig4(-/-)p53(-/-) mice, Lig4(-/-)Chk2(-/-) and Lig4(-/-)p53(R172P/R172P) mice were not prone to organ-specific, rapid tumorigenesis. Although the severe NHEJ deficiency of Lig4(-/-) was a less potent initiator of tumorigenesis in the p53(R172P/R172P) and Chk2(-/-) backgrounds, where p53 cell-cycle functions are largely intact, even mild defects in the intra-S and G2/M checkpoints caused by mutations in Nbs1 are sufficient to influence malignancy in p53(R172P/R172P) mice. We conclude that the oncogenic potential of double-strand breaks resulting from NHEJ deficiency is highly restricted by nonapoptotic functions of p53, such as the G1/S checkpoint or senescence, suggesting that the particular facets of the DNA damage response required for tumor suppression are dictated by the proliferative status of the tumor-initiating cell.


Subject(s)
Apoptosis , Cell Cycle , DNA Repair , Neoplasms, Experimental/pathology , Animals , DNA Damage , Genes, p53 , Mice , Mutation , Neoplasms, Experimental/genetics
4.
Mol Cell Biol ; 31(21): 4379-89, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21876003

ABSTRACT

The Mre11 complex is a central component of the DNA damage response, with roles in damage sensing, molecular bridging, and end resection. We have previously shown that in Saccharomyces cerevisiae, Ku70 (yKu70) deficiency reduces the ionizing radiation sensitivity of mre11Δ mutants. In this study, we show that yKu70 deficiency suppressed the camptothecin (CPT) and methyl methanesulfonate (MMS) sensitivity of nuclease-deficient mre11-3 and sae2Δ mutants in an Exo1-dependent manner. CPT-induced G(2)/M arrest, γ-H2AX persistence, and chromosome breaks were elevated in mre11-3 mutants. These outcomes were reduced by yKu70 deficiency. Given that the genotoxic effects of CPT are manifest during DNA replication, these data suggest that Ku limits Exo1-dependent double-strand break (DSB) resection during DNA replication, inhibiting the initial processing steps required for homology-directed repair. We propose that Mre11 nuclease- and Sae2-dependent DNA end processing, which initiates DSB resection prevents Ku from engaging DSBs, thus promoting Exo1-dependent resection. In agreement with this idea, we show that Ku affinity for binding to short single-stranded overhangs is much lower than for blunt DNA ends. Collectively, the data define a nonhomologous end joining (NHEJ)-independent, S-phase-specific function of the Ku heterodimer.


Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Replication , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/genetics , Endonucleases/genetics , Endonucleases/metabolism , Exodeoxyribonucleases/genetics , Flap Endonucleases/genetics , Flap Endonucleases/metabolism , Genes, Fungal , Models, Biological , Mutation , S Phase , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
5.
Mol Cell ; 33(2): 147-59, 2009 Jan 30.
Article in English | MEDLINE | ID: mdl-19187758

ABSTRACT

Oligomeric assembly of Brca1 C-terminal (BRCT) domain-containing mediator proteins occurs at sites of DNA damage. However, the functional significance and regulation of such assemblies are not well understood. In this study, we defined the molecular mechanism of DNA-damage-induced oligomerization of the S. cerevisiae BRCT protein Rad9. Our data suggest that Rad9's tandem BRCT domain mediates Rad9 oligomerization via its interaction with its own Mec1/Tel1-phosphorylated SQ/TQ cluster domain (SCD). Rad53 activation is unaffected by mutations that impair Rad9 oligomerization, but checkpoint maintenance is lost, indicating that oligomerization is required to sustain checkpoint signaling. Once activated, Rad53 phosphorylates the Rad9 BRCT domain, which attenuates the BRCT-SCD interaction. Failure to phosphorylate the Rad9 BRCT results in cytologically visible Rad9 foci. This suggests a feedback loop wherein Rad53 activity and Rad9 oligomerization are regulated to tune the DNA-damage response.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage/physiology , DNA, Fungal/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Checkpoint Kinase 2 , Genes, cdc , Intracellular Signaling Peptides and Proteins/metabolism , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction
6.
Methods Enzymol ; 409: 285-300, 2006.
Article in English | MEDLINE | ID: mdl-16793407

ABSTRACT

Single-stranded DNA (ssDNA) is an important intermediate in many DNA repair pathways. Here we describe protocols that permit the measurement of ssDNA that has arisen in the yeast genome in vivo, in response to telomere uncapping. Yeast strains defective in DNA damage response (DDR) genes can be used to infer the roles of the corresponding proteins in regulating ssDNA production and in responding to ssDNA. Using column based methods to purify yeast genomic DNA and quantitative amplification of single-stranded DNA (QAOS) it is possible to measure ssDNA at numerous single copy loci in the yeast genome. We describe how to measure ssDNA in synchronous cultures of cdc13-1 mutants, containing a temperature sensitive mutation in an essential telomere capping protein, and in asynchronous cultures of yku70Delta mutants also defective in telomere capping.


Subject(s)
DNA Damage/genetics , DNA Repair , DNA, Single-Stranded/metabolism , Saccharomyces cerevisiae/genetics , Telomere , Base Sequence , DNA Primers
7.
DNA Repair (Amst) ; 5(7): 840-51, 2006 Jul 13.
Article in English | MEDLINE | ID: mdl-16765654

ABSTRACT

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.


Subject(s)
DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Base Sequence , Cell Cycle , DNA Repair , DNA, Fungal/genetics , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/genetics , Genes, Fungal , Models, Biological , Multiprotein Complexes , Mutation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/chemistry , Telomere/genetics
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