Limits...
Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex.

Imai R, Komeda S, Shimura M, Tamura S, Matsuyama S, Nishimura K, Rogge R, Matsunaga A, Hiratani I, Takata H, Uemura M, Iida Y, Yoshikawa Y, Hansen JC, Yamauchi K, Kanemaki MT, Maeshima K - Sci Rep (2016)

Bottom Line: Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy.The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity.Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Biological Macromolecules Laboratory, Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.

ABSTRACT
Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers.

No MeSH data available.


Related in: MedlinePlus

5-H-Y binds to chromatin DNA tightly and folds chromatin in vitro and in vivo. (A) Experimental scheme of the two chromatin folding assays: ultracentrifuge assay (left) and nuclear volume assay (right). (B) 5-H-Y induces chromatin folding. Samples of reconstituted nucleosome fibers were exposed to the indicated concentrations of 5-H-Y (left) or cisplatin (center) and analyzed by sedimentation velocity analytical ultracentrifugation. The integral distribution of diffusion-corrected sedimentation coefficients obtained after analysis of the data by the method of Demeler and van Holde are shown6061. (Right) Summary of analytical ultracentrifuge-SV results. Values at the 50% boundary are displayed as a function of drug concentration added 5-H-Y (red) or cisplatin (blue). (C) Nuclear volume was decreased by 5-H-Y in a dose-dependent manner. Nuclei treated with 50 μM 5-H-Y showed a 12-fold decrease in the volume. This indicates that 5-H-Y induces chromatin folding. The nuclei treated with 5 mM Mg2+ were prepared as a control for the nuclei with highly folded chromatin. The error bars represent the standard deviation. For each point, n = ~100. (D) Volumes between Mg2+-pretreated and 5-H-Y-pretreated nuclei after buffer washing. When the volume was normalized by Mg2+-pretreated nuclei, although Mg2+-pretreated nuclei became large after the washing (relative nuclear volume = 1), 5-H-Y-pretreated nuclei did not change (~0.1). 5-H-Y seems to bind tightly to chromatin DNA, in contrast to Mg2+. The error bars represent the standard deviation. (E) 5-H-Y induces chromatin condensation in vivo. HeLa cells were treated with TSA to decondense chromatin and then with 5-H-Y. 5-H-Y induced enrichment of chromatin at nuclear periphery and nucleoli (left) although we cannot exclude the possibility that condensation by 5-H-Y only occur around nucleoli and nuclear periphery. Right plot shows the intensity quantification of nuclear periphery chromatin. **p < 0.01, Chi-square test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4837362&req=5

f5: 5-H-Y binds to chromatin DNA tightly and folds chromatin in vitro and in vivo. (A) Experimental scheme of the two chromatin folding assays: ultracentrifuge assay (left) and nuclear volume assay (right). (B) 5-H-Y induces chromatin folding. Samples of reconstituted nucleosome fibers were exposed to the indicated concentrations of 5-H-Y (left) or cisplatin (center) and analyzed by sedimentation velocity analytical ultracentrifugation. The integral distribution of diffusion-corrected sedimentation coefficients obtained after analysis of the data by the method of Demeler and van Holde are shown6061. (Right) Summary of analytical ultracentrifuge-SV results. Values at the 50% boundary are displayed as a function of drug concentration added 5-H-Y (red) or cisplatin (blue). (C) Nuclear volume was decreased by 5-H-Y in a dose-dependent manner. Nuclei treated with 50 μM 5-H-Y showed a 12-fold decrease in the volume. This indicates that 5-H-Y induces chromatin folding. The nuclei treated with 5 mM Mg2+ were prepared as a control for the nuclei with highly folded chromatin. The error bars represent the standard deviation. For each point, n = ~100. (D) Volumes between Mg2+-pretreated and 5-H-Y-pretreated nuclei after buffer washing. When the volume was normalized by Mg2+-pretreated nuclei, although Mg2+-pretreated nuclei became large after the washing (relative nuclear volume = 1), 5-H-Y-pretreated nuclei did not change (~0.1). 5-H-Y seems to bind tightly to chromatin DNA, in contrast to Mg2+. The error bars represent the standard deviation. (E) 5-H-Y induces chromatin condensation in vivo. HeLa cells were treated with TSA to decondense chromatin and then with 5-H-Y. 5-H-Y induced enrichment of chromatin at nuclear periphery and nucleoli (left) although we cannot exclude the possibility that condensation by 5-H-Y only occur around nucleoli and nuclear periphery. Right plot shows the intensity quantification of nuclear periphery chromatin. **p < 0.01, Chi-square test.

Mentions: How does 5-H-Y inhibit DNA replication and RNA transcription? Because 5-H-Y is positively charged and induces compaction of naked DNA26, we examined the effects of 5-H-Y on higher-order chromatin structure. To quantitate chromatin structure in solution in vitro, arrays of 12 positioned nucleosomes were reconstituted from pure histones and DNA as a model chromatin (Fig. 5A, top left), followed by sedimentation velocity experiments in an analytical ultracentrifuge (Fig. 5A, bottom left). The degree of folding of the 12-mer nucleosomal arrays was described quantitatively by the sedimentation coefficient (S)35. The extended beads-on-a-string conformation sediments at ~29 S, whereas folding causes the nucleosomal arrays to become compact and increases the sedimentation coefficient to ~40–55 S35. When the nucleosomal arrays were exposed to 5-H-Y, the sedimentation coefficient increased, from 27 S to 40–55 S, in a dose-dependent manner. In contrast, cisplatin did not affect the sedimentation of the nucleosomal arrays (Fig. 5B). These results indicate that 5-H-Y, but not cisplatin, induced folding of nucleosomal arrays in vitro.


Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex.

Imai R, Komeda S, Shimura M, Tamura S, Matsuyama S, Nishimura K, Rogge R, Matsunaga A, Hiratani I, Takata H, Uemura M, Iida Y, Yoshikawa Y, Hansen JC, Yamauchi K, Kanemaki MT, Maeshima K - Sci Rep (2016)

5-H-Y binds to chromatin DNA tightly and folds chromatin in vitro and in vivo. (A) Experimental scheme of the two chromatin folding assays: ultracentrifuge assay (left) and nuclear volume assay (right). (B) 5-H-Y induces chromatin folding. Samples of reconstituted nucleosome fibers were exposed to the indicated concentrations of 5-H-Y (left) or cisplatin (center) and analyzed by sedimentation velocity analytical ultracentrifugation. The integral distribution of diffusion-corrected sedimentation coefficients obtained after analysis of the data by the method of Demeler and van Holde are shown6061. (Right) Summary of analytical ultracentrifuge-SV results. Values at the 50% boundary are displayed as a function of drug concentration added 5-H-Y (red) or cisplatin (blue). (C) Nuclear volume was decreased by 5-H-Y in a dose-dependent manner. Nuclei treated with 50 μM 5-H-Y showed a 12-fold decrease in the volume. This indicates that 5-H-Y induces chromatin folding. The nuclei treated with 5 mM Mg2+ were prepared as a control for the nuclei with highly folded chromatin. The error bars represent the standard deviation. For each point, n = ~100. (D) Volumes between Mg2+-pretreated and 5-H-Y-pretreated nuclei after buffer washing. When the volume was normalized by Mg2+-pretreated nuclei, although Mg2+-pretreated nuclei became large after the washing (relative nuclear volume = 1), 5-H-Y-pretreated nuclei did not change (~0.1). 5-H-Y seems to bind tightly to chromatin DNA, in contrast to Mg2+. The error bars represent the standard deviation. (E) 5-H-Y induces chromatin condensation in vivo. HeLa cells were treated with TSA to decondense chromatin and then with 5-H-Y. 5-H-Y induced enrichment of chromatin at nuclear periphery and nucleoli (left) although we cannot exclude the possibility that condensation by 5-H-Y only occur around nucleoli and nuclear periphery. Right plot shows the intensity quantification of nuclear periphery chromatin. **p < 0.01, Chi-square test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837362&req=5

f5: 5-H-Y binds to chromatin DNA tightly and folds chromatin in vitro and in vivo. (A) Experimental scheme of the two chromatin folding assays: ultracentrifuge assay (left) and nuclear volume assay (right). (B) 5-H-Y induces chromatin folding. Samples of reconstituted nucleosome fibers were exposed to the indicated concentrations of 5-H-Y (left) or cisplatin (center) and analyzed by sedimentation velocity analytical ultracentrifugation. The integral distribution of diffusion-corrected sedimentation coefficients obtained after analysis of the data by the method of Demeler and van Holde are shown6061. (Right) Summary of analytical ultracentrifuge-SV results. Values at the 50% boundary are displayed as a function of drug concentration added 5-H-Y (red) or cisplatin (blue). (C) Nuclear volume was decreased by 5-H-Y in a dose-dependent manner. Nuclei treated with 50 μM 5-H-Y showed a 12-fold decrease in the volume. This indicates that 5-H-Y induces chromatin folding. The nuclei treated with 5 mM Mg2+ were prepared as a control for the nuclei with highly folded chromatin. The error bars represent the standard deviation. For each point, n = ~100. (D) Volumes between Mg2+-pretreated and 5-H-Y-pretreated nuclei after buffer washing. When the volume was normalized by Mg2+-pretreated nuclei, although Mg2+-pretreated nuclei became large after the washing (relative nuclear volume = 1), 5-H-Y-pretreated nuclei did not change (~0.1). 5-H-Y seems to bind tightly to chromatin DNA, in contrast to Mg2+. The error bars represent the standard deviation. (E) 5-H-Y induces chromatin condensation in vivo. HeLa cells were treated with TSA to decondense chromatin and then with 5-H-Y. 5-H-Y induced enrichment of chromatin at nuclear periphery and nucleoli (left) although we cannot exclude the possibility that condensation by 5-H-Y only occur around nucleoli and nuclear periphery. Right plot shows the intensity quantification of nuclear periphery chromatin. **p < 0.01, Chi-square test.
Mentions: How does 5-H-Y inhibit DNA replication and RNA transcription? Because 5-H-Y is positively charged and induces compaction of naked DNA26, we examined the effects of 5-H-Y on higher-order chromatin structure. To quantitate chromatin structure in solution in vitro, arrays of 12 positioned nucleosomes were reconstituted from pure histones and DNA as a model chromatin (Fig. 5A, top left), followed by sedimentation velocity experiments in an analytical ultracentrifuge (Fig. 5A, bottom left). The degree of folding of the 12-mer nucleosomal arrays was described quantitatively by the sedimentation coefficient (S)35. The extended beads-on-a-string conformation sediments at ~29 S, whereas folding causes the nucleosomal arrays to become compact and increases the sedimentation coefficient to ~40–55 S35. When the nucleosomal arrays were exposed to 5-H-Y, the sedimentation coefficient increased, from 27 S to 40–55 S, in a dose-dependent manner. In contrast, cisplatin did not affect the sedimentation of the nucleosomal arrays (Fig. 5B). These results indicate that 5-H-Y, but not cisplatin, induced folding of nucleosomal arrays in vitro.

Bottom Line: Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy.The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity.Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Biological Macromolecules Laboratory, Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.

ABSTRACT
Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers.

No MeSH data available.


Related in: MedlinePlus