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Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner.

Pérez-Torras S, Vidal-Pla A, Cano-Soldado P, Huber-Ruano I, Mazo A, Pastor-Anglada M - Cell Death Dis (2013)

Bottom Line: We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration.Importantly, the translocation-defective transporter triggered these same effects on cell physiology.Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona and National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBERehd), Barcelona, Spain. s.perez-torras@ub.edu

ABSTRACT
Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

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hCNT1 overexpression activates Akt and Erk. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at an MOI of 10. (a) The time-course of Akt and Erk phosphorylation was analysed in total cell extracts by western blotting using the indicated antibodies. (b) Infected NP-9 and NP-29 cells were treated with 50 μℳ PD98059 (MEK inhibitor) or 20 nℳ (+) or 100 nℳ (#) wortmannin (PI3K inhibitor) for 48 h. Total-cell lysates were analysed by immunoblotting with the indicated antibodies. (c) After infection with Adctrol (black bars) and AdhCNT1 (gray bars), NP-9 and NP-29 cells were treated with different concentrations of PD98059 and cell viability was assessed by MTT assay 72 h later. Data are expressed as percentage of viable cells relative to Adctrol- or AdhCNT1-infected cells. Results are means±S.E.M. (n=3). Statistical significance was determined with Student's t-test; ***P<0.005
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fig5: hCNT1 overexpression activates Akt and Erk. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at an MOI of 10. (a) The time-course of Akt and Erk phosphorylation was analysed in total cell extracts by western blotting using the indicated antibodies. (b) Infected NP-9 and NP-29 cells were treated with 50 μℳ PD98059 (MEK inhibitor) or 20 nℳ (+) or 100 nℳ (#) wortmannin (PI3K inhibitor) for 48 h. Total-cell lysates were analysed by immunoblotting with the indicated antibodies. (c) After infection with Adctrol (black bars) and AdhCNT1 (gray bars), NP-9 and NP-29 cells were treated with different concentrations of PD98059 and cell viability was assessed by MTT assay 72 h later. Data are expressed as percentage of viable cells relative to Adctrol- or AdhCNT1-infected cells. Results are means±S.E.M. (n=3). Statistical significance was determined with Student's t-test; ***P<0.005

Mentions: An analysis of the time-course of the events following AdhCNT1 infection revealed an increase in Akt phosphorylation at both S473 and T308 sites, starting at 12 h after infection (Figure 5a). Erk phosphorylation was also potentiated at 18 h of AdhCNT1 infection in NP-9 cells, whereas increased Erk phosphorylation in AdhCNT1 NP-29-infected cells was only evident later (24 and 48 h after infection) (Figure 5a). Treatment with the Erk inhibitor PD98059 prevented Erk phosphorylation in both cell lines, although in hCNT1-overexpressing NP-29 cells, this treatment increased Akt S473 phosphorylation (Figure 5b). Moreover, NP-29 cells also appeared to show increased basal phosphorylation of Erk, as inferred from its status in Adctrol versus AdhCNT1-infected cells at the time of adenovirus removal (4 h; first assayed time-point). Interestingly, inhibition of Erk activation with PD98059 induced a slight but significant, reduction in hCNT1-associated cell death in NP-9, but not in NP-29, cells (Figure 5c). Inhibition of PI3K with wortmannin (20 and 100 nℳ) induced a slight decrease in hCNT1-induced phosphorylation of Akt at both S473 and T308 in NP-9 and N-29 cells (Figure 5b), although no effect on cell viability was observed at 100 nℳ (Supplementary Figure S4).


Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner.

Pérez-Torras S, Vidal-Pla A, Cano-Soldado P, Huber-Ruano I, Mazo A, Pastor-Anglada M - Cell Death Dis (2013)

hCNT1 overexpression activates Akt and Erk. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at an MOI of 10. (a) The time-course of Akt and Erk phosphorylation was analysed in total cell extracts by western blotting using the indicated antibodies. (b) Infected NP-9 and NP-29 cells were treated with 50 μℳ PD98059 (MEK inhibitor) or 20 nℳ (+) or 100 nℳ (#) wortmannin (PI3K inhibitor) for 48 h. Total-cell lysates were analysed by immunoblotting with the indicated antibodies. (c) After infection with Adctrol (black bars) and AdhCNT1 (gray bars), NP-9 and NP-29 cells were treated with different concentrations of PD98059 and cell viability was assessed by MTT assay 72 h later. Data are expressed as percentage of viable cells relative to Adctrol- or AdhCNT1-infected cells. Results are means±S.E.M. (n=3). Statistical significance was determined with Student's t-test; ***P<0.005
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fig5: hCNT1 overexpression activates Akt and Erk. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at an MOI of 10. (a) The time-course of Akt and Erk phosphorylation was analysed in total cell extracts by western blotting using the indicated antibodies. (b) Infected NP-9 and NP-29 cells were treated with 50 μℳ PD98059 (MEK inhibitor) or 20 nℳ (+) or 100 nℳ (#) wortmannin (PI3K inhibitor) for 48 h. Total-cell lysates were analysed by immunoblotting with the indicated antibodies. (c) After infection with Adctrol (black bars) and AdhCNT1 (gray bars), NP-9 and NP-29 cells were treated with different concentrations of PD98059 and cell viability was assessed by MTT assay 72 h later. Data are expressed as percentage of viable cells relative to Adctrol- or AdhCNT1-infected cells. Results are means±S.E.M. (n=3). Statistical significance was determined with Student's t-test; ***P<0.005
Mentions: An analysis of the time-course of the events following AdhCNT1 infection revealed an increase in Akt phosphorylation at both S473 and T308 sites, starting at 12 h after infection (Figure 5a). Erk phosphorylation was also potentiated at 18 h of AdhCNT1 infection in NP-9 cells, whereas increased Erk phosphorylation in AdhCNT1 NP-29-infected cells was only evident later (24 and 48 h after infection) (Figure 5a). Treatment with the Erk inhibitor PD98059 prevented Erk phosphorylation in both cell lines, although in hCNT1-overexpressing NP-29 cells, this treatment increased Akt S473 phosphorylation (Figure 5b). Moreover, NP-29 cells also appeared to show increased basal phosphorylation of Erk, as inferred from its status in Adctrol versus AdhCNT1-infected cells at the time of adenovirus removal (4 h; first assayed time-point). Interestingly, inhibition of Erk activation with PD98059 induced a slight but significant, reduction in hCNT1-associated cell death in NP-9, but not in NP-29, cells (Figure 5c). Inhibition of PI3K with wortmannin (20 and 100 nℳ) induced a slight decrease in hCNT1-induced phosphorylation of Akt at both S473 and T308 in NP-9 and N-29 cells (Figure 5b), although no effect on cell viability was observed at 100 nℳ (Supplementary Figure S4).

Bottom Line: We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration.Importantly, the translocation-defective transporter triggered these same effects on cell physiology.Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona and National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBERehd), Barcelona, Spain. s.perez-torras@ub.edu

ABSTRACT
Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

Show MeSH
Related in: MedlinePlus