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Isolation of ORCTL3 in a novel genetic screen for tumor-specific apoptosis inducers.

Irshad S, Mahul-Mellier AL, Kassouf N, Lemarie A, Grimm S - Cell Death Differ. (2009)

Bottom Line: Although ORCTL3 is a member of the organic cation transporter gene family, our data indicate that this gene induces apoptosis independently of its putative transporter activity.Rather, various lines of evidence suggest that ORCTL3 brings about apoptosis by an endoplasmic reticulum stress-mediated mechanism.Finally, we detected ORCTL3 to be downregulated in human kidney tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine and Toxicology, Imperial College London, Hammersmith Campus, London, UK.

ABSTRACT
We have established a systematic high-throughput screen for genes that cause cell death specifically in transformed tumor cells. In a first round of screening, cDNAs that induce apoptosis in a transformed human cell line are detected. Positive genes are subsequently tested in a synthetic lethal screen in normal cells versus their isogenic counterparts that have been transformed by a particular oncogene. In this way, the organic cation transporter-like 3 (ORCTL3) gene was found to be inactive in normal rat kidney (NRK) cells, but to induce apoptosis in NRK cells transformed by oncogenic H-ras. ORCTL3 also causes cell death in v-src-transformed cells and in various human tumor cell lines but not in normal cells or untransformed cell lines. Although ORCTL3 is a member of the organic cation transporter gene family, our data indicate that this gene induces apoptosis independently of its putative transporter activity. Rather, various lines of evidence suggest that ORCTL3 brings about apoptosis by an endoplasmic reticulum stress-mediated mechanism. Finally, we detected ORCTL3 to be downregulated in human kidney tumors.

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Effect of ORCTL3 on apoptosis in transformed versus normal cells. A, ORCTL3 induces apoptosis in various transformed tumor cells. Expression plasmids for ORCTL3, luciferase as a negative control, and caspase-2 as a positive control, were transfected into the respective cell lines and apoptosis was quantified. The data for the human tumor cell lines HEK293T and PC3 are based on flow cytometry analysis and normalized to the transfection efficiency as determined by the percentage of transfected (GFP-positive) cells. The experiments were repeated three times independently. The data for the tumour cell lines Hela, LnCap and BHK represents the ratio of morphologically apoptotic, GFP-positive (transfected) cells to all GFP-positive (transfected) cells. Cells were co-transfected at a ratio of 1:1 with a vector for GFP and an expression vector for ORCTL3, Caspase-2, or luciferase. Each data point represents the mean of triplicate counts, +/- the standard deviation. Background apoptosis levels observed in luciferase-transfected cells were subtracted (p<0.01 for ORCTL3 relative to luc-transfected cells in BHK and Hela, p<0.05 in all other cell types). B, ORCTL3 fails to provoke apoptosis in primary and untransformed cells. Primary HUVEC cells were transfected with an efficient electroporation method (Amaxa). Normal canine kidney cells (MDCK) were co-transfected with GFP and expression vectors (ORCTL3, Caspase-2 or luciferase). Apoptosis in these cells was determined by phenotype quantification as in A (p-value non significant in both cells types, n=3). Background apoptosis level of luciferase-transfected cells was subtracted. Apoptosis in primary renal cells (RPT) was quantified by the Vybrant® Apotosis Assay Kit (p>0.05, n=3). Data are presented relative to the transfection efficiency determined by the percentage of GFP transfected cells. As the proapoptotic Bax gene was a weak cell death inducer in these cells they were treated with 1.5 mM H2O2 for 6 hours as a positive control.
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Figure 3: Effect of ORCTL3 on apoptosis in transformed versus normal cells. A, ORCTL3 induces apoptosis in various transformed tumor cells. Expression plasmids for ORCTL3, luciferase as a negative control, and caspase-2 as a positive control, were transfected into the respective cell lines and apoptosis was quantified. The data for the human tumor cell lines HEK293T and PC3 are based on flow cytometry analysis and normalized to the transfection efficiency as determined by the percentage of transfected (GFP-positive) cells. The experiments were repeated three times independently. The data for the tumour cell lines Hela, LnCap and BHK represents the ratio of morphologically apoptotic, GFP-positive (transfected) cells to all GFP-positive (transfected) cells. Cells were co-transfected at a ratio of 1:1 with a vector for GFP and an expression vector for ORCTL3, Caspase-2, or luciferase. Each data point represents the mean of triplicate counts, +/- the standard deviation. Background apoptosis levels observed in luciferase-transfected cells were subtracted (p<0.01 for ORCTL3 relative to luc-transfected cells in BHK and Hela, p<0.05 in all other cell types). B, ORCTL3 fails to provoke apoptosis in primary and untransformed cells. Primary HUVEC cells were transfected with an efficient electroporation method (Amaxa). Normal canine kidney cells (MDCK) were co-transfected with GFP and expression vectors (ORCTL3, Caspase-2 or luciferase). Apoptosis in these cells was determined by phenotype quantification as in A (p-value non significant in both cells types, n=3). Background apoptosis level of luciferase-transfected cells was subtracted. Apoptosis in primary renal cells (RPT) was quantified by the Vybrant® Apotosis Assay Kit (p>0.05, n=3). Data are presented relative to the transfection efficiency determined by the percentage of GFP transfected cells. As the proapoptotic Bax gene was a weak cell death inducer in these cells they were treated with 1.5 mM H2O2 for 6 hours as a positive control.

Mentions: As our sequencing of the endogenous H-ras gene (variants 1, 2, 3) from HEK293T cells did not reveal activating mutations at codons 12, 13 or 61 (data not shown), we speculated that apoptosis induction by ORCTL3 is not limited to H-ras transformed cells. Hence, we assessed cell death by ORCTL3 in NRK cells transformed by v-src (supplementary fig. 1C,D) and likewise observed apoptosis in these cells (Fig. 2D,E). We then compared the ability of ORCTL3 to induce apoptosis in a number of transformed tumor cells and untransformed cells. Figure 3A shows that ORCTL3 can induce apoptosis in the human tumorigenic kidney cell line HEK293T, the human cervical Hela cell line, and in human LnCap and PC3 prostate cancer cells. ORCTL3 is also able to cause apoptosis in transformed baby hamster kidney (BHK) cells, but not in untransformed Madin-Darby canine kidney (MDCK) cells (Fig. 3B). Moreover, ORCTL3 was likewise inactive for apoptosis induction in human umbilical vein endothelial cells (HUVEC) and primary human renal proximal tubule (RPT) cells.


Isolation of ORCTL3 in a novel genetic screen for tumor-specific apoptosis inducers.

Irshad S, Mahul-Mellier AL, Kassouf N, Lemarie A, Grimm S - Cell Death Differ. (2009)

Effect of ORCTL3 on apoptosis in transformed versus normal cells. A, ORCTL3 induces apoptosis in various transformed tumor cells. Expression plasmids for ORCTL3, luciferase as a negative control, and caspase-2 as a positive control, were transfected into the respective cell lines and apoptosis was quantified. The data for the human tumor cell lines HEK293T and PC3 are based on flow cytometry analysis and normalized to the transfection efficiency as determined by the percentage of transfected (GFP-positive) cells. The experiments were repeated three times independently. The data for the tumour cell lines Hela, LnCap and BHK represents the ratio of morphologically apoptotic, GFP-positive (transfected) cells to all GFP-positive (transfected) cells. Cells were co-transfected at a ratio of 1:1 with a vector for GFP and an expression vector for ORCTL3, Caspase-2, or luciferase. Each data point represents the mean of triplicate counts, +/- the standard deviation. Background apoptosis levels observed in luciferase-transfected cells were subtracted (p<0.01 for ORCTL3 relative to luc-transfected cells in BHK and Hela, p<0.05 in all other cell types). B, ORCTL3 fails to provoke apoptosis in primary and untransformed cells. Primary HUVEC cells were transfected with an efficient electroporation method (Amaxa). Normal canine kidney cells (MDCK) were co-transfected with GFP and expression vectors (ORCTL3, Caspase-2 or luciferase). Apoptosis in these cells was determined by phenotype quantification as in A (p-value non significant in both cells types, n=3). Background apoptosis level of luciferase-transfected cells was subtracted. Apoptosis in primary renal cells (RPT) was quantified by the Vybrant® Apotosis Assay Kit (p>0.05, n=3). Data are presented relative to the transfection efficiency determined by the percentage of GFP transfected cells. As the proapoptotic Bax gene was a weak cell death inducer in these cells they were treated with 1.5 mM H2O2 for 6 hours as a positive control.
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Figure 3: Effect of ORCTL3 on apoptosis in transformed versus normal cells. A, ORCTL3 induces apoptosis in various transformed tumor cells. Expression plasmids for ORCTL3, luciferase as a negative control, and caspase-2 as a positive control, were transfected into the respective cell lines and apoptosis was quantified. The data for the human tumor cell lines HEK293T and PC3 are based on flow cytometry analysis and normalized to the transfection efficiency as determined by the percentage of transfected (GFP-positive) cells. The experiments were repeated three times independently. The data for the tumour cell lines Hela, LnCap and BHK represents the ratio of morphologically apoptotic, GFP-positive (transfected) cells to all GFP-positive (transfected) cells. Cells were co-transfected at a ratio of 1:1 with a vector for GFP and an expression vector for ORCTL3, Caspase-2, or luciferase. Each data point represents the mean of triplicate counts, +/- the standard deviation. Background apoptosis levels observed in luciferase-transfected cells were subtracted (p<0.01 for ORCTL3 relative to luc-transfected cells in BHK and Hela, p<0.05 in all other cell types). B, ORCTL3 fails to provoke apoptosis in primary and untransformed cells. Primary HUVEC cells were transfected with an efficient electroporation method (Amaxa). Normal canine kidney cells (MDCK) were co-transfected with GFP and expression vectors (ORCTL3, Caspase-2 or luciferase). Apoptosis in these cells was determined by phenotype quantification as in A (p-value non significant in both cells types, n=3). Background apoptosis level of luciferase-transfected cells was subtracted. Apoptosis in primary renal cells (RPT) was quantified by the Vybrant® Apotosis Assay Kit (p>0.05, n=3). Data are presented relative to the transfection efficiency determined by the percentage of GFP transfected cells. As the proapoptotic Bax gene was a weak cell death inducer in these cells they were treated with 1.5 mM H2O2 for 6 hours as a positive control.
Mentions: As our sequencing of the endogenous H-ras gene (variants 1, 2, 3) from HEK293T cells did not reveal activating mutations at codons 12, 13 or 61 (data not shown), we speculated that apoptosis induction by ORCTL3 is not limited to H-ras transformed cells. Hence, we assessed cell death by ORCTL3 in NRK cells transformed by v-src (supplementary fig. 1C,D) and likewise observed apoptosis in these cells (Fig. 2D,E). We then compared the ability of ORCTL3 to induce apoptosis in a number of transformed tumor cells and untransformed cells. Figure 3A shows that ORCTL3 can induce apoptosis in the human tumorigenic kidney cell line HEK293T, the human cervical Hela cell line, and in human LnCap and PC3 prostate cancer cells. ORCTL3 is also able to cause apoptosis in transformed baby hamster kidney (BHK) cells, but not in untransformed Madin-Darby canine kidney (MDCK) cells (Fig. 3B). Moreover, ORCTL3 was likewise inactive for apoptosis induction in human umbilical vein endothelial cells (HUVEC) and primary human renal proximal tubule (RPT) cells.

Bottom Line: Although ORCTL3 is a member of the organic cation transporter gene family, our data indicate that this gene induces apoptosis independently of its putative transporter activity.Rather, various lines of evidence suggest that ORCTL3 brings about apoptosis by an endoplasmic reticulum stress-mediated mechanism.Finally, we detected ORCTL3 to be downregulated in human kidney tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine and Toxicology, Imperial College London, Hammersmith Campus, London, UK.

ABSTRACT
We have established a systematic high-throughput screen for genes that cause cell death specifically in transformed tumor cells. In a first round of screening, cDNAs that induce apoptosis in a transformed human cell line are detected. Positive genes are subsequently tested in a synthetic lethal screen in normal cells versus their isogenic counterparts that have been transformed by a particular oncogene. In this way, the organic cation transporter-like 3 (ORCTL3) gene was found to be inactive in normal rat kidney (NRK) cells, but to induce apoptosis in NRK cells transformed by oncogenic H-ras. ORCTL3 also causes cell death in v-src-transformed cells and in various human tumor cell lines but not in normal cells or untransformed cell lines. Although ORCTL3 is a member of the organic cation transporter gene family, our data indicate that this gene induces apoptosis independently of its putative transporter activity. Rather, various lines of evidence suggest that ORCTL3 brings about apoptosis by an endoplasmic reticulum stress-mediated mechanism. Finally, we detected ORCTL3 to be downregulated in human kidney tumors.

Show MeSH
Related in: MedlinePlus