Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution

1989 ◽  
Vol 13 (3) ◽  
pp. 211-217 ◽  
Author(s):  
Jeffrey R. Hincks ◽  
Roger A. Coulombe
Keyword(s):  
Rapid Detection ◽  
Mammalian Cells ◽  
Gravity Flow ◽  
Alkaline Elution ◽  
Cross Links

DNA-Protein Cross-Links: New Insights into their Formation and Repair in Irradiated Mammalian Cells

1986 ◽  
pp. 181-192 ◽  
Author(s):  
Nancy L. Oleinick ◽  
Song-mao Chiu ◽  
Libby R. Friedman ◽  
Liang-yan Xue ◽  
Narayani Ramakrishnan
Keyword(s):  
Mammalian Cells ◽  
Cross Links

Measurement of DNA damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay

1980 ◽  
Vol 106 (1) ◽  
pp. 169-174 ◽  
Author(s):  
Leonard C. Erickson ◽  
Rainhardt Osieka ◽  
Nancy A. Sharkey ◽  
Kurt W. Kohn
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Alkaline Elution ◽  
Dna Assay

scholarly journals Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

2020 ◽  
Vol 11 ◽  
Author(s):  
Luping Xu ◽  
Xingjian Bai ◽  
Shivendra Tenguria ◽  
Yi Liu ◽  
Rishi Drolia ◽  
...  
Keyword(s):  
Shelf Life ◽  
Mammalian Cell ◽  
Rapid Detection ◽  
Mammalian Cells ◽  
Host Cells ◽  
Detection Time ◽  
Primary Methods ◽  
Widespread Application ◽  
Lateral Flow Assays ◽  
Set Up

Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1–10 CFU/25 g was 16–21 h using a traditional enrichment set up but the detection time was shortened to 10–12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.

scholarly journals Schizosaccharomyces pombe Checkpoint Response to DNA Interstrand Cross-Links

2003 ◽  
Vol 23 (13) ◽  
pp. 4728-4737 ◽  
Author(s):  
Sarah Lambert ◽  
Sarah J. Mason ◽  
Louise J. Barber ◽  
John A. Hartley ◽  
Jackie A. Pearce ◽  
...  
Keyword(s):  
Dna Damage ◽  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Excision Repair ◽  
S Phase ◽  
Repair Gene ◽  
Phase Arrest ◽  
S Phase Arrest ◽  
Interstrand Cross Links ◽  
Cross Links

ABSTRACT Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the “checkpoint Rad” family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G2 arrest following ICL induction. In cells able to bypass the G2 block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G2 arrest-deficient mutants. This likely reflects the fraction of cells escaping the G2 damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.

scholarly journals Repair of DNA–Protein Cross-links in Mammalian Cells

Cell Cycle ◽  
2006 ◽  
Vol 5 (13) ◽  
pp. 1366-1370 ◽  
Author(s):  
Joyce T. Reardon ◽  
Yuan Cheng ◽  
Aziz Sancar
Keyword(s):  
Mammalian Cells ◽  
Cross Links

scholarly journals The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

2004 ◽  
Vol 24 (13) ◽  
pp. 5776-5787 ◽  
Author(s):  
Laura J. Niedernhofer ◽  
Hanny Odijk ◽  
Magda Budzowska ◽  
Ellen van Drunen ◽  
Alex Maas ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Wild Type ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Normal Metabolism ◽  
Cross Links

ABSTRACT Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1 −/− cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1 −/− cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1 −/− and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

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