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Identification of 5-Iodotubercidin as a genotoxic drug with anti-cancer potential.

Zhang X, Jia D, Liu H, Zhu N, Zhang W, Feng J, Yin J, Hao B, Cui D, Deng Y, Xie D, He L, Li B - PLoS ONE (2013)

Bottom Line: We found that 5-Iodotubercidin could cause DNA damage, verified by induction of DNA breaks and nuclear foci positive for γH2AX and TopBP1, activation of Atm and Chk2, and S15 phosphorylation and up-regulation of p53.Moreover, 5-Iodotubercidin showed anti-tumor activity as it could reduce the tumor size in carcinoma xenograft mouse models in p53-dependent and -independent manners.These findings reveal 5-Iodotubercidin as a novel genotoxic drug that has chemotherapeutic potential.

View Article: PubMed Central - PubMed

Affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Tumor suppressor p53, which is activated by various stress and oncogene activation, is a target for anti-cancer drug development. In this study, by screening panels of protein kinase inhibitors and protein phosphatase inhibitors, we identified 5-Iodotubercidin as a strong p53 activator. 5-Iodotubercidin is purine derivative and is used as an inhibitor for various kinases including adenosine kinase. We found that 5-Iodotubercidin could cause DNA damage, verified by induction of DNA breaks and nuclear foci positive for γH2AX and TopBP1, activation of Atm and Chk2, and S15 phosphorylation and up-regulation of p53. As such, 5-Iodotubercidin induces G2 cell cycle arrest in a p53-dependent manner. Itu also induces cell death in p53-dependent and -independent manners. DNA breaks were likely generated by incorporation of 5-Iodotubercidin metabolite into DNA. Moreover, 5-Iodotubercidin showed anti-tumor activity as it could reduce the tumor size in carcinoma xenograft mouse models in p53-dependent and -independent manners. These findings reveal 5-Iodotubercidin as a novel genotoxic drug that has chemotherapeutic potential.

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Itu treatment results in an increase in S and G2/M phase cells.A. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 24 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. B. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 2.5 µM of Itu for 24 hrs. C. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 48 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. D. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 1.0 µM of Itu for 48 hrs.
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pone-0062527-g005: Itu treatment results in an increase in S and G2/M phase cells.A. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 24 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. B. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 2.5 µM of Itu for 24 hrs. C. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 48 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. D. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 1.0 µM of Itu for 48 hrs.

Mentions: Many nucleoside analogs cause cell cycle arrest at the S phase with exceptions such as 20-C-cyano-20-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC), which causes cell cycle arrest at the G2 phase [4], [35]. Incorporation of nucleoside analogs terminates the elongation of nascent DNA strand and leads to stalling of replication forks. To test whether Itu activates cell cycle checkpoints, we treated p53+/+ and p53−/− HCT116 cells with different concentrations of Itu for 24 or 48 hrs. It was found that 24 hr-treatment with higher concentrations of Itu led to a modest increase in the percentage of S phase cells, which was accompanied with a decrease in the percentage of G1 phase cells, without significantly altering the percentage of G2/M phase cells (Fig. 5A and 5B). When the cells were treated with various concentrations of Itu for 48 hrs, more cells were in the G2 phase, accompanied by a decrease in G1 phase but no significant change in the percentage of S phase cells (Fig. 5C and 5D). One explanation is that cells were initially stuck in the S phase but managed to pass through S phase and were eventually blocked in the G2/M phase. Thus Itu mainly activates the G2 checkpoint. This is in contrast to most of the nucleoside analogs, which lead to S phase cell cycle arrest [4], and ionizing radiation (IR), which induces cell cycle arrest at G1 and G2 phase and is accompanied by a decrease in S phase in HCT116 cells [21]. In the absence of p53, 24-hr-treatment led to an initial increase in the S phase at higher doses of Itu (Fig. 5A and 5B), yet 48 hrs after treatment, the difference between control and treated cells became minimal (Fig. 5C and 5D), indicating that p53−/− HCT116 cells, but not the p53+/+ HCT116 cells, undergo S phase arrest and that p53−/− cells managed to escape this checkpoint. Moreover, p53−/− cells did not show a G2 phase arrest (Fig. 5A and 5B). These results indicate that Itu-induced G2 phase arrest requires the presence of p53.


Identification of 5-Iodotubercidin as a genotoxic drug with anti-cancer potential.

Zhang X, Jia D, Liu H, Zhu N, Zhang W, Feng J, Yin J, Hao B, Cui D, Deng Y, Xie D, He L, Li B - PLoS ONE (2013)

Itu treatment results in an increase in S and G2/M phase cells.A. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 24 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. B. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 2.5 µM of Itu for 24 hrs. C. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 48 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. D. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 1.0 µM of Itu for 48 hrs.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646850&req=5

pone-0062527-g005: Itu treatment results in an increase in S and G2/M phase cells.A. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 24 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. B. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 2.5 µM of Itu for 24 hrs. C. HCT116 (p53+/+ and p53−/−) were treated with different concentrations of Itu for 48 hrs. The cells were fixed, stained with PI, and analyzed with FACS. Percentage of cells in different phases was shown. D. Representative cell cycle profiles of p53+/+ and p53−/− HCT116 in the presence of 1.0 µM of Itu for 48 hrs.
Mentions: Many nucleoside analogs cause cell cycle arrest at the S phase with exceptions such as 20-C-cyano-20-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC), which causes cell cycle arrest at the G2 phase [4], [35]. Incorporation of nucleoside analogs terminates the elongation of nascent DNA strand and leads to stalling of replication forks. To test whether Itu activates cell cycle checkpoints, we treated p53+/+ and p53−/− HCT116 cells with different concentrations of Itu for 24 or 48 hrs. It was found that 24 hr-treatment with higher concentrations of Itu led to a modest increase in the percentage of S phase cells, which was accompanied with a decrease in the percentage of G1 phase cells, without significantly altering the percentage of G2/M phase cells (Fig. 5A and 5B). When the cells were treated with various concentrations of Itu for 48 hrs, more cells were in the G2 phase, accompanied by a decrease in G1 phase but no significant change in the percentage of S phase cells (Fig. 5C and 5D). One explanation is that cells were initially stuck in the S phase but managed to pass through S phase and were eventually blocked in the G2/M phase. Thus Itu mainly activates the G2 checkpoint. This is in contrast to most of the nucleoside analogs, which lead to S phase cell cycle arrest [4], and ionizing radiation (IR), which induces cell cycle arrest at G1 and G2 phase and is accompanied by a decrease in S phase in HCT116 cells [21]. In the absence of p53, 24-hr-treatment led to an initial increase in the S phase at higher doses of Itu (Fig. 5A and 5B), yet 48 hrs after treatment, the difference between control and treated cells became minimal (Fig. 5C and 5D), indicating that p53−/− HCT116 cells, but not the p53+/+ HCT116 cells, undergo S phase arrest and that p53−/− cells managed to escape this checkpoint. Moreover, p53−/− cells did not show a G2 phase arrest (Fig. 5A and 5B). These results indicate that Itu-induced G2 phase arrest requires the presence of p53.

Bottom Line: We found that 5-Iodotubercidin could cause DNA damage, verified by induction of DNA breaks and nuclear foci positive for γH2AX and TopBP1, activation of Atm and Chk2, and S15 phosphorylation and up-regulation of p53.Moreover, 5-Iodotubercidin showed anti-tumor activity as it could reduce the tumor size in carcinoma xenograft mouse models in p53-dependent and -independent manners.These findings reveal 5-Iodotubercidin as a novel genotoxic drug that has chemotherapeutic potential.

View Article: PubMed Central - PubMed

Affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Tumor suppressor p53, which is activated by various stress and oncogene activation, is a target for anti-cancer drug development. In this study, by screening panels of protein kinase inhibitors and protein phosphatase inhibitors, we identified 5-Iodotubercidin as a strong p53 activator. 5-Iodotubercidin is purine derivative and is used as an inhibitor for various kinases including adenosine kinase. We found that 5-Iodotubercidin could cause DNA damage, verified by induction of DNA breaks and nuclear foci positive for γH2AX and TopBP1, activation of Atm and Chk2, and S15 phosphorylation and up-regulation of p53. As such, 5-Iodotubercidin induces G2 cell cycle arrest in a p53-dependent manner. Itu also induces cell death in p53-dependent and -independent manners. DNA breaks were likely generated by incorporation of 5-Iodotubercidin metabolite into DNA. Moreover, 5-Iodotubercidin showed anti-tumor activity as it could reduce the tumor size in carcinoma xenograft mouse models in p53-dependent and -independent manners. These findings reveal 5-Iodotubercidin as a novel genotoxic drug that has chemotherapeutic potential.

Show MeSH
Related in: MedlinePlus