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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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ORCTL3 localises to the cell membrane, ER, and Golgi, but not to mitochondria. Hela cells were plated on coverslips, transfected with an ORCTL3-ECFP expression vector and incubated with zVAD. Cells were processed as descried under Materials and Methods. The overlay of the fluorescence microscopic image of CFP and the histochemical staining indicated the localisation of the fusion protein.
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Figure 5: ORCTL3 localises to the cell membrane, ER, and Golgi, but not to mitochondria. Hela cells were plated on coverslips, transfected with an ORCTL3-ECFP expression vector and incubated with zVAD. Cells were processed as descried under Materials and Methods. The overlay of the fluorescence microscopic image of CFP and the histochemical staining indicated the localisation of the fusion protein.

Mentions: We studied the spatial distribution of the ECFP fusion protein of ORCTL3 with specific staining of organelles in Hela cells. This fusion protein was found at the endoplasmic reticulum (ER), the Golgi and the plasma membrane (Fig. 5), indicating that the ORCTL3 protein transport conforms to the known internal route of plasma membrane proteins. Importantly, we have not found an overlap with the signal from the mitochondria-specific probe mitotracker Red CMXRos. Since another tumour-specific apoptosis inducer, the viral apoptin gene is differentially localized in normal versus tumour cells (3), we determined the localization of the ORCTL3-ECFP fusion protein in WT and ras-transformed NRK cells but found no difference in its cellular locale between these cell types (not shown). As ORCTL3 could cause cell death at intracellular sites during transit, we applied the pan-caspase inhibitor zVAD to block apoptosis and expected that those cells that accrue contain ORCTL3 at the relevant locale for apoptosis induction. Figure 6A shows that upon cell death inhibition the percentage of cells with a prominent intracellular accumulation of the ORCTL3-ECFP protein increased while the percentage of apoptotic cells decreased proportionally (see also supplementary figure 3). Hence, we speculated that ORCTL3 exerts its apoptosis activity at the ER or the Golgi. A fusion construct with an ER retention signal at the C-terminus of ORCTL3 led to higher apoptosis induction than wild type ORCTL3, while a fusion construct with a Golgi retention signal caused less apoptosis (Fig. 6B). Consequently, we speculated that ORCTL3 is inducing the signal for apoptosis at the ER, an organelle with a well-recognized role in apoptosis. In support of this hypothesis we found that co-transfection of the ER stress inhibitor BiP/GRP78 caused a reduction in apoptosis (Fig 6C). ER stress is a complex cellular response that leads to the upregulation of both pro- and anti-apoptotic genes. We determined the protein levels of the ER stress factors ATF4/CREB2, as a pro-, and BiP as an anti-apoptotic protein in ORCTL3 transfected cells. In these experiments we used the ER stressor tunicamycin or ionomycin as controls whose apoptotic effects are influenced by the expression level of BiP (23, 24). Our results show that in cells that undergo apoptosis upon ORCTL3 expression (Hela and HEK293T cells), ATF4 was upregulated to an extent comparable with control-treated cells. BiP, on the other hand, did not increase when compared to tunicamycin-or ionomycin-treated cells (Fig. 6D,E). In contrast, HUVEC cells that do not succumb to apoptosis upon ORCTL3 transfection failed to show any signs of ER stress, with neither BiP nor ATF4 being upregulated (Fig. 6F). Transfection efficiency in these experiments, as determined by GFP transfected cells, was 40% in Hela cells, 60% in HEK293T and 65% in HUVEC cells (not shown). Treatment with the ER stress inducer tunicamycin could also increase BiP in HUVEC cells (Fig. 6F) and did not indicate that Hela cells are more susceptible to ER stress than HUVEC cells (supplementary fig. 4).


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)

ORCTL3 localises to the cell membrane, ER, and Golgi, but not to mitochondria. Hela cells were plated on coverslips, transfected with an ORCTL3-ECFP expression vector and incubated with zVAD. Cells were processed as descried under Materials and Methods. The overlay of the fluorescence microscopic image of CFP and the histochemical staining indicated the localisation of the fusion protein.
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Related In: Results  -  Collection

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Figure 5: ORCTL3 localises to the cell membrane, ER, and Golgi, but not to mitochondria. Hela cells were plated on coverslips, transfected with an ORCTL3-ECFP expression vector and incubated with zVAD. Cells were processed as descried under Materials and Methods. The overlay of the fluorescence microscopic image of CFP and the histochemical staining indicated the localisation of the fusion protein.
Mentions: We studied the spatial distribution of the ECFP fusion protein of ORCTL3 with specific staining of organelles in Hela cells. This fusion protein was found at the endoplasmic reticulum (ER), the Golgi and the plasma membrane (Fig. 5), indicating that the ORCTL3 protein transport conforms to the known internal route of plasma membrane proteins. Importantly, we have not found an overlap with the signal from the mitochondria-specific probe mitotracker Red CMXRos. Since another tumour-specific apoptosis inducer, the viral apoptin gene is differentially localized in normal versus tumour cells (3), we determined the localization of the ORCTL3-ECFP fusion protein in WT and ras-transformed NRK cells but found no difference in its cellular locale between these cell types (not shown). As ORCTL3 could cause cell death at intracellular sites during transit, we applied the pan-caspase inhibitor zVAD to block apoptosis and expected that those cells that accrue contain ORCTL3 at the relevant locale for apoptosis induction. Figure 6A shows that upon cell death inhibition the percentage of cells with a prominent intracellular accumulation of the ORCTL3-ECFP protein increased while the percentage of apoptotic cells decreased proportionally (see also supplementary figure 3). Hence, we speculated that ORCTL3 exerts its apoptosis activity at the ER or the Golgi. A fusion construct with an ER retention signal at the C-terminus of ORCTL3 led to higher apoptosis induction than wild type ORCTL3, while a fusion construct with a Golgi retention signal caused less apoptosis (Fig. 6B). Consequently, we speculated that ORCTL3 is inducing the signal for apoptosis at the ER, an organelle with a well-recognized role in apoptosis. In support of this hypothesis we found that co-transfection of the ER stress inhibitor BiP/GRP78 caused a reduction in apoptosis (Fig 6C). ER stress is a complex cellular response that leads to the upregulation of both pro- and anti-apoptotic genes. We determined the protein levels of the ER stress factors ATF4/CREB2, as a pro-, and BiP as an anti-apoptotic protein in ORCTL3 transfected cells. In these experiments we used the ER stressor tunicamycin or ionomycin as controls whose apoptotic effects are influenced by the expression level of BiP (23, 24). Our results show that in cells that undergo apoptosis upon ORCTL3 expression (Hela and HEK293T cells), ATF4 was upregulated to an extent comparable with control-treated cells. BiP, on the other hand, did not increase when compared to tunicamycin-or ionomycin-treated cells (Fig. 6D,E). In contrast, HUVEC cells that do not succumb to apoptosis upon ORCTL3 transfection failed to show any signs of ER stress, with neither BiP nor ATF4 being upregulated (Fig. 6F). Transfection efficiency in these experiments, as determined by GFP transfected cells, was 40% in Hela cells, 60% in HEK293T and 65% in HUVEC cells (not shown). Treatment with the ER stress inducer tunicamycin could also increase BiP in HUVEC cells (Fig. 6F) and did not indicate that Hela cells are more susceptible to ER stress than HUVEC cells (supplementary fig. 4).

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