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Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs.

Stathopoulou A, Roukos V, Petropoulou C, Kotsantis P, Karantzelis N, Nishitani H, Lygerou Z, Taraviras S - PLoS ONE (2012)

Bottom Line: RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA.Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis.Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Medical School, University of Patras, Patras, Greece.

ABSTRACT

Background: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis.

Methodology/principal finding: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA.

Conclusion/significance: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

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Related in: MedlinePlus

5-Fluoruracil (5-FU) does alter Cdt1 protein expression levels in HepG2 but not in HeLa cells.HeLa and HepG2 cells were incubated for 6 h with 5-FU (0.1, 10 and 100 µg/ml) in the absence (lanes 1–4 and 9–10) or in the presence (lanes 5–8 and 12–14) of MG-132 (20 µM). Protein extracts were analyzed by Western blotting using antibodies against Cdt1, PARP, Geminin and Tubulin.
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pone-0034621-g004: 5-Fluoruracil (5-FU) does alter Cdt1 protein expression levels in HepG2 but not in HeLa cells.HeLa and HepG2 cells were incubated for 6 h with 5-FU (0.1, 10 and 100 µg/ml) in the absence (lanes 1–4 and 9–10) or in the presence (lanes 5–8 and 12–14) of MG-132 (20 µM). Protein extracts were analyzed by Western blotting using antibodies against Cdt1, PARP, Geminin and Tubulin.

Mentions: To address a possible effect of the chemotherapeutic agent 5-FU on Cdt1 targeting upon DNA damage, HeLa cells were treated with the pyrimidine analogue for 6 h and Cdt1 protein levels were asssesed by western blotting. As shown in Figure 4, (lanes 2–4) no alteration of Cdt1 protein levels upon 5-FU treatment was observed. On the contrary, incubation of 5-FU in HepG2 cells resulted in a mild downregulation of Cdt1 expression (Figure 4, lanes 10–11), which was proteolysis-dependent as revealed by stabilization of Cdt1 protein levels in MG-132 treated cells (Figure 4, lanes 13–14). In addition, in accordance with previous results, Geminin protein levels remained unaffected.


Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs.

Stathopoulou A, Roukos V, Petropoulou C, Kotsantis P, Karantzelis N, Nishitani H, Lygerou Z, Taraviras S - PLoS ONE (2012)

5-Fluoruracil (5-FU) does alter Cdt1 protein expression levels in HepG2 but not in HeLa cells.HeLa and HepG2 cells were incubated for 6 h with 5-FU (0.1, 10 and 100 µg/ml) in the absence (lanes 1–4 and 9–10) or in the presence (lanes 5–8 and 12–14) of MG-132 (20 µM). Protein extracts were analyzed by Western blotting using antibodies against Cdt1, PARP, Geminin and Tubulin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316709&req=5

pone-0034621-g004: 5-Fluoruracil (5-FU) does alter Cdt1 protein expression levels in HepG2 but not in HeLa cells.HeLa and HepG2 cells were incubated for 6 h with 5-FU (0.1, 10 and 100 µg/ml) in the absence (lanes 1–4 and 9–10) or in the presence (lanes 5–8 and 12–14) of MG-132 (20 µM). Protein extracts were analyzed by Western blotting using antibodies against Cdt1, PARP, Geminin and Tubulin.
Mentions: To address a possible effect of the chemotherapeutic agent 5-FU on Cdt1 targeting upon DNA damage, HeLa cells were treated with the pyrimidine analogue for 6 h and Cdt1 protein levels were asssesed by western blotting. As shown in Figure 4, (lanes 2–4) no alteration of Cdt1 protein levels upon 5-FU treatment was observed. On the contrary, incubation of 5-FU in HepG2 cells resulted in a mild downregulation of Cdt1 expression (Figure 4, lanes 10–11), which was proteolysis-dependent as revealed by stabilization of Cdt1 protein levels in MG-132 treated cells (Figure 4, lanes 13–14). In addition, in accordance with previous results, Geminin protein levels remained unaffected.

Bottom Line: RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA.Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis.Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Medical School, University of Patras, Patras, Greece.

ABSTRACT

Background: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis.

Methodology/principal finding: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA.

Conclusion/significance: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

Show MeSH
Related in: MedlinePlus