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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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Related in: MedlinePlus

Cytotoxicity assay performance.Estimate of Dead cell relative light units (RLU), Live cell RLU, and % Viability for all clones at 0-Gy, 0.5-Gy and 4-Gy irradiation. While dead cell RLUs do not show statistically significant changes, changes in live cell RLUs and % Viability at 4-Gy are statistically significant indicating that as a whole, clones show fewer live cells and decreased viability at 4-Gy, presumably due to cell cycle arrest and decreased cell division.
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pone.0120534.g001: Cytotoxicity assay performance.Estimate of Dead cell relative light units (RLU), Live cell RLU, and % Viability for all clones at 0-Gy, 0.5-Gy and 4-Gy irradiation. While dead cell RLUs do not show statistically significant changes, changes in live cell RLUs and % Viability at 4-Gy are statistically significant indicating that as a whole, clones show fewer live cells and decreased viability at 4-Gy, presumably due to cell cycle arrest and decreased cell division.

Mentions: During screening, we did not observe large differences in radiation induced cell death based on genotype, possibly due to the inherent radiation resistance of stem cells (Fig. 1). However, we did observe moderate changes in live cell numbers and % viability (Fig. 1) due to radiation (paired t-test of difference in % viability over all clones, 0-Gy vs 4-Gy, p < 0.00001). Such results can be explained by an arrest in cell growth induced by radiation. Mean viability (μwt) for wild type cells was around 0.6 for all treatment conditions (0-Gy, 0.5-Gy, and 4-Gy). Changes in viability (ΔV) from 0.5 and 4-Gy to 0-Gy (i.e., ΔV = V4Gy—V0Gy) Δ viability = % viability4Gy—% viability0Gy), were calculated for all clones as well as for 22 wild-type replicate samples. To identify genes associated with changes in viability, Z-scores were calculated for all clones relative to the wild type asz=ΔV-μwtσwtwhere μ = mean and σ = standard deviation. Thus, wild type samples represent the variability expected due to chance, and those clones that significantly deviate from this distribution are likely to be associated with a response to radiation. In fact, a histogram of the Z-scores shows a standard Normal distribution (black line in S1b Fig.). To assess significance, p-values were calculated based on a two-tailed distribution of Z-scores. Using a p-value cut-off p < 0.01, we identified a total of 28 unique genes associated with significant changes in viability: 5 associated with decreased viability at 0.5-Gy, 16 associated with increased viability at 0.5-Gy, and 7 associated with increased viability at 4-Gy (Table 1). Of the 5 clones that exhibited a decrease in viability at 0.5-Gy, two are associated with a response to oxidative stress: Nuclear respiratory factor 1 (Nrf1) and Peroredoxin 3 (Prdx3). Of those that exhibited an increase in viability at 0.5-Gy, several are involved in controlling the cell cycle: Minichromosome maintenance complex component 2 (Mcm2), CASP2 and RIPK1 domain containing adaptor with death domain (Cradd) and retinoblastoma binding protein 5 (Rbbp5). At 4-Gy, no genes were statistically significantly associated with loss of viability, but among the 7 genes associated with increased viability, 2 are cell-cycle proteins such as cell division cycle 25c (Cdc25c) and Ras-related protein Rab-8a (Rab8a).


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)

Cytotoxicity assay performance.Estimate of Dead cell relative light units (RLU), Live cell RLU, and % Viability for all clones at 0-Gy, 0.5-Gy and 4-Gy irradiation. While dead cell RLUs do not show statistically significant changes, changes in live cell RLUs and % Viability at 4-Gy are statistically significant indicating that as a whole, clones show fewer live cells and decreased viability at 4-Gy, presumably due to cell cycle arrest and decreased cell division.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120534.g001: Cytotoxicity assay performance.Estimate of Dead cell relative light units (RLU), Live cell RLU, and % Viability for all clones at 0-Gy, 0.5-Gy and 4-Gy irradiation. While dead cell RLUs do not show statistically significant changes, changes in live cell RLUs and % Viability at 4-Gy are statistically significant indicating that as a whole, clones show fewer live cells and decreased viability at 4-Gy, presumably due to cell cycle arrest and decreased cell division.
Mentions: During screening, we did not observe large differences in radiation induced cell death based on genotype, possibly due to the inherent radiation resistance of stem cells (Fig. 1). However, we did observe moderate changes in live cell numbers and % viability (Fig. 1) due to radiation (paired t-test of difference in % viability over all clones, 0-Gy vs 4-Gy, p < 0.00001). Such results can be explained by an arrest in cell growth induced by radiation. Mean viability (μwt) for wild type cells was around 0.6 for all treatment conditions (0-Gy, 0.5-Gy, and 4-Gy). Changes in viability (ΔV) from 0.5 and 4-Gy to 0-Gy (i.e., ΔV = V4Gy—V0Gy) Δ viability = % viability4Gy—% viability0Gy), were calculated for all clones as well as for 22 wild-type replicate samples. To identify genes associated with changes in viability, Z-scores were calculated for all clones relative to the wild type asz=ΔV-μwtσwtwhere μ = mean and σ = standard deviation. Thus, wild type samples represent the variability expected due to chance, and those clones that significantly deviate from this distribution are likely to be associated with a response to radiation. In fact, a histogram of the Z-scores shows a standard Normal distribution (black line in S1b Fig.). To assess significance, p-values were calculated based on a two-tailed distribution of Z-scores. Using a p-value cut-off p < 0.01, we identified a total of 28 unique genes associated with significant changes in viability: 5 associated with decreased viability at 0.5-Gy, 16 associated with increased viability at 0.5-Gy, and 7 associated with increased viability at 4-Gy (Table 1). Of the 5 clones that exhibited a decrease in viability at 0.5-Gy, two are associated with a response to oxidative stress: Nuclear respiratory factor 1 (Nrf1) and Peroredoxin 3 (Prdx3). Of those that exhibited an increase in viability at 0.5-Gy, several are involved in controlling the cell cycle: Minichromosome maintenance complex component 2 (Mcm2), CASP2 and RIPK1 domain containing adaptor with death domain (Cradd) and retinoblastoma binding protein 5 (Rbbp5). At 4-Gy, no genes were statistically significantly associated with loss of viability, but among the 7 genes associated with increased viability, 2 are cell-cycle proteins such as cell division cycle 25c (Cdc25c) and Ras-related protein Rab-8a (Rab8a).

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