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Indexing TNF-alpha gene expression using a gene-targeted reporter cell line.

Yan Z, Lei-Butters D, Engelhardt JF, Leno GH - BMC Biol. (2009)

Bottom Line: In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site.As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-alpha) gene.Drugs known to induce TNF-alpha expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA. ziying-yan@uiowa.edu

ABSTRACT

Background: Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-alpha) gene. Drugs known to induce TNF-alpha expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.

Results: TNF-alpha-targeted reporter activity reflected endogenous TNF-alpha mRNA expression, whereas randomly integrated TNF-alpha reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce TNF-alpha gene transcription, was only effective at inducing reporter expression from TNF-alpha gene-targeted cells.

Conclusion: We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.

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Anthracycline treatment of TNF-α targeted and non-targeted R-Luc reporter cell lines. (a) Dose-response profile of R-Luc activity in the targeted (Tg) cell line treated with four closely related anthracycline antibiotics. Cells treated with Epirubicin were grown on a separate plate. Luciferase activity was assessed using biophotonic imaging on the IVIS. (b) Dose-response profile of cell viability in the Tg line following anthracycline treatment. The same culture plates that are shown in 'a' are also shown in 'b'. Fluorescence was assessed using biophotonic imaging on the IVIS. (c) Relative R-Luc activity in targeted (Tg) and non-targeted (nTg4) cell lines following treatment with a fixed concentration of the different anthracyclines (1 μM). (d) Anthracycline-induced cytotoxicity in the Tg and nTg4 cell lines was similar. Values represent the mean (+/-SEM, N = 4).
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Figure 4: Anthracycline treatment of TNF-α targeted and non-targeted R-Luc reporter cell lines. (a) Dose-response profile of R-Luc activity in the targeted (Tg) cell line treated with four closely related anthracycline antibiotics. Cells treated with Epirubicin were grown on a separate plate. Luciferase activity was assessed using biophotonic imaging on the IVIS. (b) Dose-response profile of cell viability in the Tg line following anthracycline treatment. The same culture plates that are shown in 'a' are also shown in 'b'. Fluorescence was assessed using biophotonic imaging on the IVIS. (c) Relative R-Luc activity in targeted (Tg) and non-targeted (nTg4) cell lines following treatment with a fixed concentration of the different anthracyclines (1 μM). (d) Anthracycline-induced cytotoxicity in the Tg and nTg4 cell lines was similar. Values represent the mean (+/-SEM, N = 4).

Mentions: Anthracycline antibiotics are also known activators of TNF-α promoter transcription. Dose-response studies with four closely related anthracycline antibiotics demonstrated a pronounced up-regulation of R-Luc activity in the Tg cell line at a drug concentration of 1 μM (Figure 4a). Anthracycline exposure did not appear to significantly reduce cell viability in the Tg line at this drug concentration (Figure 4b). Therefore, both Tg and nTg4 cell lines were treated with anthracyclines at 1 μM drug and assayed for R-Luc activity. Differential R-Luc activity was evident between these cell lines, most notably following Idarubicin treatment (Figure 4c). Indeed, Idarubicin induced R-Luc activity ~300-fold in the Tg line, but only ~50-fold in the nTg4 line – representing a 6-fold difference in induction between the Tg and the nTg4 cell lines. Differential induction between Tg and nTg4 cell lines was also observed following treatment with daunorubicin, doxorubicin and epirubicin. Again, these differences were not due to differences in anthracycline-induced cell death in the Tg and nTg4 cell lines (Figure 4d). Rather, we attribute the differences in R-Luc reporter activity to unique genetic and/or epigenetic features of the endogenous TNF-α gene locus. We also conclude that targeted reporter cell lines may be superior tools for screening drugs that modulate the transcriptional activity of target genes. The use of such cell lines with biophotonic imaging as presented here (Figure 4a and 4b) may be extremely useful for multi-parameter HTS to identify novel therapeutics.


Indexing TNF-alpha gene expression using a gene-targeted reporter cell line.

Yan Z, Lei-Butters D, Engelhardt JF, Leno GH - BMC Biol. (2009)

Anthracycline treatment of TNF-α targeted and non-targeted R-Luc reporter cell lines. (a) Dose-response profile of R-Luc activity in the targeted (Tg) cell line treated with four closely related anthracycline antibiotics. Cells treated with Epirubicin were grown on a separate plate. Luciferase activity was assessed using biophotonic imaging on the IVIS. (b) Dose-response profile of cell viability in the Tg line following anthracycline treatment. The same culture plates that are shown in 'a' are also shown in 'b'. Fluorescence was assessed using biophotonic imaging on the IVIS. (c) Relative R-Luc activity in targeted (Tg) and non-targeted (nTg4) cell lines following treatment with a fixed concentration of the different anthracyclines (1 μM). (d) Anthracycline-induced cytotoxicity in the Tg and nTg4 cell lines was similar. Values represent the mean (+/-SEM, N = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657777&req=5

Figure 4: Anthracycline treatment of TNF-α targeted and non-targeted R-Luc reporter cell lines. (a) Dose-response profile of R-Luc activity in the targeted (Tg) cell line treated with four closely related anthracycline antibiotics. Cells treated with Epirubicin were grown on a separate plate. Luciferase activity was assessed using biophotonic imaging on the IVIS. (b) Dose-response profile of cell viability in the Tg line following anthracycline treatment. The same culture plates that are shown in 'a' are also shown in 'b'. Fluorescence was assessed using biophotonic imaging on the IVIS. (c) Relative R-Luc activity in targeted (Tg) and non-targeted (nTg4) cell lines following treatment with a fixed concentration of the different anthracyclines (1 μM). (d) Anthracycline-induced cytotoxicity in the Tg and nTg4 cell lines was similar. Values represent the mean (+/-SEM, N = 4).
Mentions: Anthracycline antibiotics are also known activators of TNF-α promoter transcription. Dose-response studies with four closely related anthracycline antibiotics demonstrated a pronounced up-regulation of R-Luc activity in the Tg cell line at a drug concentration of 1 μM (Figure 4a). Anthracycline exposure did not appear to significantly reduce cell viability in the Tg line at this drug concentration (Figure 4b). Therefore, both Tg and nTg4 cell lines were treated with anthracyclines at 1 μM drug and assayed for R-Luc activity. Differential R-Luc activity was evident between these cell lines, most notably following Idarubicin treatment (Figure 4c). Indeed, Idarubicin induced R-Luc activity ~300-fold in the Tg line, but only ~50-fold in the nTg4 line – representing a 6-fold difference in induction between the Tg and the nTg4 cell lines. Differential induction between Tg and nTg4 cell lines was also observed following treatment with daunorubicin, doxorubicin and epirubicin. Again, these differences were not due to differences in anthracycline-induced cell death in the Tg and nTg4 cell lines (Figure 4d). Rather, we attribute the differences in R-Luc reporter activity to unique genetic and/or epigenetic features of the endogenous TNF-α gene locus. We also conclude that targeted reporter cell lines may be superior tools for screening drugs that modulate the transcriptional activity of target genes. The use of such cell lines with biophotonic imaging as presented here (Figure 4a and 4b) may be extremely useful for multi-parameter HTS to identify novel therapeutics.

Bottom Line: In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site.As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-alpha) gene.Drugs known to induce TNF-alpha expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA. ziying-yan@uiowa.edu

ABSTRACT

Background: Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-alpha) gene. Drugs known to induce TNF-alpha expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.

Results: TNF-alpha-targeted reporter activity reflected endogenous TNF-alpha mRNA expression, whereas randomly integrated TNF-alpha reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce TNF-alpha gene transcription, was only effective at inducing reporter expression from TNF-alpha gene-targeted cells.

Conclusion: We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.

Show MeSH
Related in: MedlinePlus