Summary of Study ST001658
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001063. The data can be accessed directly via it's Project DOI: 10.21228/M89695 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001658 |
| Study Title | Control of Topoisomerase II Activity and Chemotherapeutic Inhibition by TCA Cycle Metabolites |
| Study Summary | Topoisomerase II (topo II) is essential for disentangling newly-replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapies to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies. |
| Institute | Johns Hopkins University |
| Last Name | Mosher |
| First Name | Eric |
| Address | 725 North Wolfe Street, Biophysics 307 |
| emosher2@jhmi.edu | |
| Phone | 410-952-9154 |
| Submit Date | 2021-01-21 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-02-17 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001063 |
| Project DOI: | doi: 10.21228/M89695 |
| Project Title: | Control of Topoisomerase II Activity and Chemotherapeutic Inhibition by TCA Cycle Metabolites |
| Project Summary: | Topoisomerase II (topo II) is essential for disentangling newly-replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapies to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies. |
| Institute: | Johns Hopkins University |
| Last Name: | Mosher |
| First Name: | Eric |
| Address: | 725 North Wolfe Street, Biophysics 307 |
| Email: | emosher2@jhmi.edu |
| Phone: | 410-952-9154 |
Subject:
| Subject ID: | SU001735 |
| Subject Type: | Yeast |
| Subject Species: | Saccharomyces cerevisiae |
| Taxonomy ID: | 4932 |
| Cell Strain Details: | BY4741 |
Factors:
Subject type: Yeast; Subject species: Saccharomyces cerevisiae (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA151945 | F4 | HPLC fraction 4 |
| SA151946 | F5 | HPLC fraction 5 |
| SA151947 | F6 | HPLC fraction 6 |
| Showing results 1 to 3 of 3 |
Collection:
| Collection ID: | CO001728 |
| Collection Summary: | Yeast cultures were grown in SC-sulfate media and were harvested at OD600 0.4-0.8 by vacuum filtration then washed with 100mM ammonium acetate - TEA (pH 8.0). Metabolites were extracted by the addition of pre-chilled 10mL 90% methanol, 10mM ammonium acetate - TEA (pH 8.0). Cell debris was removed by centrifugation at 4000g for 10-15 minutes. Extracts were subsequently passed through 3 kDa MWCO filters. Filtrate was diluted 2-fold with water and lyophilized. Lyophilized metabolite extracts were resuspended with water and pH was adjusted to 10 with TEA. The extract was then passed through a mixed-mode anion exchange solid phase extraction cartridge and fractionated by reverse-phase HPLC on a C18 column. One column volume of water plus 0.1% formic acid and eight column volumes of methanol were passed through the column. Fractions that eluted during the aqueous phase were then lyophilized. |
| Sample Type: | Yeast cells |
Treatment:
| Treatment ID: | TR001748 |
| Treatment Summary: | Active fractions were run on a normal-phase HPLC on an amide column and fractions were collected. Fractions 4, 5, and 6 were subjected to metabolomics analyses. |
Sample Preparation:
| Sampleprep ID: | SP001741 |
| Sampleprep Summary: | Fractions of interest were lyophilized then reconstituted in water plus 0.1% formic acid such that samples reached a final concentration of 20 mg/mL. |
Combined analysis:
| Analysis ID | AN002706 | AN002707 | AN002708 | AN002709 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Normal phase | Normal phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Thermo Hypersil Gold aQ (150 x 2.1mm,1.9um) | Thermo Hypersil Gold aQ (150 x 2.1mm,1.9um) | PolyLC Polyhydroxyethyl A (200 x 2.1mm,5um) | PolyLC Polyhydroxyethyl A (200 x 2.1mm,5um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | Peak Area | Peak Area | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH001997 |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Thermo Hypersil Gold aQ (150 x 2.1mm,1.9um) |
| Column Temperature: | 30 |
| Flow Gradient: | 0-2min 0% B, 2-13min 0-60% B, 13-15min 60-90% B, 15-16min 90-0% B, 16-20min 0% B |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 1mM ammonium acetate |
| Solvent B: | 20% water/80% acetonitrile; 0.1% formic acid; 1 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001998 |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | PolyLC Polyhydroxyethyl A (200 x 2.1mm,5um) |
| Column Temperature: | 30 |
| Flow Gradient: | 0-2min 80% B, 2-13min 80-40% B, 13-15min 40-10% B, 15-16min 10-80% B, 16-20min 80% B |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 1mM ammonium acetate |
| Solvent B: | 20% water/80% acetonitrile; 0.1% formic acid; 1 mM ammonium acetate |
| Chromatography Type: | Normal phase |
MS:
| MS ID: | MS002503 |
| Analysis ID: | AN002706 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002504 |
| Analysis ID: | AN002707 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS002505 |
| Analysis ID: | AN002708 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002506 |
| Analysis ID: | AN002709 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software. |
| Ion Mode: | NEGATIVE |
