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Functional genomics screening utilizing mutant mouse embryonic stem cells identifies novel radiation-response genes.

Loesch K, Galaviz S, Hamoui Z, Clanton R, Akabani G, Deveau M, DeJesus M, Ioerger T, Sacchettini JC, Wallis D - PLoS ONE (2015)

Bottom Line: We focused on a cancer-relevant subset of over 500 mutant ESC lines.After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4).Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

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Cell growth as a function of time for wild type (WT) and (A) Dpagt1-mutant ESCs, (B) Klf8-mutant ESCs, (C) Dstn-mutant ESCs, (D) Iqgap1-mutant ESCs, (E) Map2k6-mutant ESCs, (F) Vcan-mutant ESCs, (G) Rbpms-mutant ESCs.Cells were irradiated with a dose of 2-Gy just before incubation using x-rays. Error bars indicate SEM.
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pone.0120534.g003: Cell growth as a function of time for wild type (WT) and (A) Dpagt1-mutant ESCs, (B) Klf8-mutant ESCs, (C) Dstn-mutant ESCs, (D) Iqgap1-mutant ESCs, (E) Map2k6-mutant ESCs, (F) Vcan-mutant ESCs, (G) Rbpms-mutant ESCs.Cells were irradiated with a dose of 2-Gy just before incubation using x-rays. Error bars indicate SEM.

Mentions: Seven of the validated mutant clones were selected for further analysis. Cell growth studies were carried out using a Real Time Cell Analyzer (RTCA, ACEA Biosciences, Inc., California) to assess their radiation response as a function of time. The power inherent in this particular assay is it allows us to follow the cell index (CI) continuously over several days such that we are allowed a more complete overview than the previous time course assay where we only were able to obtain 4 discreet time points. The results are presented in Fig. 3 and show the cell growth as a function of time after irradiation at 2 and 0-Gy (control/sham) for a period of 120 hours. The data indicate that gene traps even without radiation exposure result in changes in both the maximum CI and the time in which it is achieved. For example, WT cells achieve a maximum CI of 2 within 40 hours and then declined to a CI of approximately 0.5 at 100 hours. Map2k6 clones only reach a CI of 1.5 and take about 20 hours to achieve. However, Vcan reached a CI of 3.5 within 48 hours. Irradiated WT ESCs became arrested with no significant change in cell population (CI ~ 1) as a function of time. The irradiated mutants behave differently and such data can be utilized to predict how using a therapeutic drug to inhibit these genes would result in increased (or decreased) response to radiation therapy. For example, we have 2 targets that when knocked down show enhanced sensitivity to 2-Gy radiation (in comparison to WT). Dstn is the best target. Dstn has a lower CI than WT even w/o radiation. ES cells with this gene trap show CI that do not recover after irradiation and our other assays (time and dose response) support this finding as we saw a change in viability of-10% at 2-Gy in our dose response assays and-7% at 4-Gy after 24 hour (S2 Table). Map2k6 also shows enhanced sensitivity to 2-Gy over time. The RTCA data is further supported by our dose response data showing a change of-10% viability at 2-Gy. Further, we have identified clones that show enhanced resistance to radiation. Iqgap and Vcan clones both have cell indexes higher than WT and when exposed to radiation, the clones initially show a slight decrease in CI, but then the CI rebounds to a level even higher than the unirradiated WT clones. This rebound occurs at a late time point, past 48 hours which was the last time point we checked in the secondary assays. Vcan clones show significant resistance to radiation as the CI persists over 2 at 120 hours.


Functional genomics screening utilizing mutant mouse embryonic stem cells identifies novel radiation-response genes.

Loesch K, Galaviz S, Hamoui Z, Clanton R, Akabani G, Deveau M, DeJesus M, Ioerger T, Sacchettini JC, Wallis D - PLoS ONE (2015)

Cell growth as a function of time for wild type (WT) and (A) Dpagt1-mutant ESCs, (B) Klf8-mutant ESCs, (C) Dstn-mutant ESCs, (D) Iqgap1-mutant ESCs, (E) Map2k6-mutant ESCs, (F) Vcan-mutant ESCs, (G) Rbpms-mutant ESCs.Cells were irradiated with a dose of 2-Gy just before incubation using x-rays. Error bars indicate SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390347&req=5

pone.0120534.g003: Cell growth as a function of time for wild type (WT) and (A) Dpagt1-mutant ESCs, (B) Klf8-mutant ESCs, (C) Dstn-mutant ESCs, (D) Iqgap1-mutant ESCs, (E) Map2k6-mutant ESCs, (F) Vcan-mutant ESCs, (G) Rbpms-mutant ESCs.Cells were irradiated with a dose of 2-Gy just before incubation using x-rays. Error bars indicate SEM.
Mentions: Seven of the validated mutant clones were selected for further analysis. Cell growth studies were carried out using a Real Time Cell Analyzer (RTCA, ACEA Biosciences, Inc., California) to assess their radiation response as a function of time. The power inherent in this particular assay is it allows us to follow the cell index (CI) continuously over several days such that we are allowed a more complete overview than the previous time course assay where we only were able to obtain 4 discreet time points. The results are presented in Fig. 3 and show the cell growth as a function of time after irradiation at 2 and 0-Gy (control/sham) for a period of 120 hours. The data indicate that gene traps even without radiation exposure result in changes in both the maximum CI and the time in which it is achieved. For example, WT cells achieve a maximum CI of 2 within 40 hours and then declined to a CI of approximately 0.5 at 100 hours. Map2k6 clones only reach a CI of 1.5 and take about 20 hours to achieve. However, Vcan reached a CI of 3.5 within 48 hours. Irradiated WT ESCs became arrested with no significant change in cell population (CI ~ 1) as a function of time. The irradiated mutants behave differently and such data can be utilized to predict how using a therapeutic drug to inhibit these genes would result in increased (or decreased) response to radiation therapy. For example, we have 2 targets that when knocked down show enhanced sensitivity to 2-Gy radiation (in comparison to WT). Dstn is the best target. Dstn has a lower CI than WT even w/o radiation. ES cells with this gene trap show CI that do not recover after irradiation and our other assays (time and dose response) support this finding as we saw a change in viability of-10% at 2-Gy in our dose response assays and-7% at 4-Gy after 24 hour (S2 Table). Map2k6 also shows enhanced sensitivity to 2-Gy over time. The RTCA data is further supported by our dose response data showing a change of-10% viability at 2-Gy. Further, we have identified clones that show enhanced resistance to radiation. Iqgap and Vcan clones both have cell indexes higher than WT and when exposed to radiation, the clones initially show a slight decrease in CI, but then the CI rebounds to a level even higher than the unirradiated WT clones. This rebound occurs at a late time point, past 48 hours which was the last time point we checked in the secondary assays. Vcan clones show significant resistance to radiation as the CI persists over 2 at 120 hours.

Bottom Line: We focused on a cancer-relevant subset of over 500 mutant ESC lines.After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4).Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

Show MeSH
Related in: MedlinePlus