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Nucleolar targeting by platinum: p53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction.

Peterson EJ, Menon VR, Gatti L, Kipping R, Dewasinghe D, Perego P, Povirk LF, Farrell NP - Mol. Pharm. (2014)

Bottom Line: In this article, the downstream effects of nucleolar localization are described.This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems.Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure-activity paradigms for antitumor complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and ‡Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23284, United States.

ABSTRACT
TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 arrest immediately, but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure-activity paradigms for antitumor complexes.

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(A) β-Galactosidaseactivity assay; HCT116 cells were assayedfor β-galactosidase activity after treatment with 20 μMcisplatin or TriplatinNC for 96 h. Shown is a representative of twoindependent experiments. (B) Confocal microscopy; HCT116 cells weretreated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPIstained DNA (blue). Right, top panel; cleaved caspase-3 (yellow).Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (whitestar) containing condensed/fragmented DNA and active caspase-3. Cellswith compacted DNA (white arrows) do not contain active caspase-3.(C) Summary; percentage of HCT116 cells undergoing the indicated cellularprocess after treatment with 20 μM cisplatin or TriplatinNCfor 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm andnucleolus of interphase cells. Cells treated while in S-G2 proceed through mitosis. During prophase, the DNA (blue) condensesand the nuclear membrane (red) disintegrates allowing cytoplasmicpools of TriplatinNC to interact with condensed DNA. Cells undergocytokinesis; however, the DNA does not decondense or progress throughG1. (a) Determined by immunofluorescence: β-tubulin,nucleophosmin/B23, and DAPI DNA staining (Figure 5A,B). (b) Determined by β-galactosidase staining andlight microscopy at 200× magnification (panel A). (c) Determinedby immunofluorescence; cleaved caspase-3; and DAPI DNA staining. Thepercentage of cells undergoing apoptosis was determined as the numberof cells positive for cleaved caspase-3 divided by the total numberof cells. n > 500 cells each for two repeat experiments(Figure 5B).
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fig6: (A) β-Galactosidaseactivity assay; HCT116 cells were assayedfor β-galactosidase activity after treatment with 20 μMcisplatin or TriplatinNC for 96 h. Shown is a representative of twoindependent experiments. (B) Confocal microscopy; HCT116 cells weretreated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPIstained DNA (blue). Right, top panel; cleaved caspase-3 (yellow).Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (whitestar) containing condensed/fragmented DNA and active caspase-3. Cellswith compacted DNA (white arrows) do not contain active caspase-3.(C) Summary; percentage of HCT116 cells undergoing the indicated cellularprocess after treatment with 20 μM cisplatin or TriplatinNCfor 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm andnucleolus of interphase cells. Cells treated while in S-G2 proceed through mitosis. During prophase, the DNA (blue) condensesand the nuclear membrane (red) disintegrates allowing cytoplasmicpools of TriplatinNC to interact with condensed DNA. Cells undergocytokinesis; however, the DNA does not decondense or progress throughG1. (a) Determined by immunofluorescence: β-tubulin,nucleophosmin/B23, and DAPI DNA staining (Figure 5A,B). (b) Determined by β-galactosidase staining andlight microscopy at 200× magnification (panel A). (c) Determinedby immunofluorescence; cleaved caspase-3; and DAPI DNA staining. Thepercentage of cells undergoing apoptosis was determined as the numberof cells positive for cleaved caspase-3 divided by the total numberof cells. n > 500 cells each for two repeat experiments(Figure 5B).

Mentions: Aside from mitosis, condensedchromatin also occurs during senescence,as senescence-associated heterochromatin foci (SAHF).46 In this process, cells grow larger, flatten shape, andexpress senescence-associated β-galactosidase. When HCT116 cellswere treated with 20 μM cisplatin for 96 h, the remaining cellswere large, flattened, and showed β-galactosidase activity,i.e., blue precipitates, representing hydrolysis products of X-galby β-galactosidase (Figure 6A, lowerleft). TriplatinNC treated cells were small, with very little cytoplasmiccontent, and did not show evidence of β-galactosidase activity(Figure 6A, lower right). Therefore, we foundthat the DNA compaction events were not consistent with senescence.


Nucleolar targeting by platinum: p53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction.

Peterson EJ, Menon VR, Gatti L, Kipping R, Dewasinghe D, Perego P, Povirk LF, Farrell NP - Mol. Pharm. (2014)

(A) β-Galactosidaseactivity assay; HCT116 cells were assayedfor β-galactosidase activity after treatment with 20 μMcisplatin or TriplatinNC for 96 h. Shown is a representative of twoindependent experiments. (B) Confocal microscopy; HCT116 cells weretreated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPIstained DNA (blue). Right, top panel; cleaved caspase-3 (yellow).Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (whitestar) containing condensed/fragmented DNA and active caspase-3. Cellswith compacted DNA (white arrows) do not contain active caspase-3.(C) Summary; percentage of HCT116 cells undergoing the indicated cellularprocess after treatment with 20 μM cisplatin or TriplatinNCfor 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm andnucleolus of interphase cells. Cells treated while in S-G2 proceed through mitosis. During prophase, the DNA (blue) condensesand the nuclear membrane (red) disintegrates allowing cytoplasmicpools of TriplatinNC to interact with condensed DNA. Cells undergocytokinesis; however, the DNA does not decondense or progress throughG1. (a) Determined by immunofluorescence: β-tubulin,nucleophosmin/B23, and DAPI DNA staining (Figure 5A,B). (b) Determined by β-galactosidase staining andlight microscopy at 200× magnification (panel A). (c) Determinedby immunofluorescence; cleaved caspase-3; and DAPI DNA staining. Thepercentage of cells undergoing apoptosis was determined as the numberof cells positive for cleaved caspase-3 divided by the total numberof cells. n > 500 cells each for two repeat experiments(Figure 5B).
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fig6: (A) β-Galactosidaseactivity assay; HCT116 cells were assayedfor β-galactosidase activity after treatment with 20 μMcisplatin or TriplatinNC for 96 h. Shown is a representative of twoindependent experiments. (B) Confocal microscopy; HCT116 cells weretreated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPIstained DNA (blue). Right, top panel; cleaved caspase-3 (yellow).Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (whitestar) containing condensed/fragmented DNA and active caspase-3. Cellswith compacted DNA (white arrows) do not contain active caspase-3.(C) Summary; percentage of HCT116 cells undergoing the indicated cellularprocess after treatment with 20 μM cisplatin or TriplatinNCfor 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm andnucleolus of interphase cells. Cells treated while in S-G2 proceed through mitosis. During prophase, the DNA (blue) condensesand the nuclear membrane (red) disintegrates allowing cytoplasmicpools of TriplatinNC to interact with condensed DNA. Cells undergocytokinesis; however, the DNA does not decondense or progress throughG1. (a) Determined by immunofluorescence: β-tubulin,nucleophosmin/B23, and DAPI DNA staining (Figure 5A,B). (b) Determined by β-galactosidase staining andlight microscopy at 200× magnification (panel A). (c) Determinedby immunofluorescence; cleaved caspase-3; and DAPI DNA staining. Thepercentage of cells undergoing apoptosis was determined as the numberof cells positive for cleaved caspase-3 divided by the total numberof cells. n > 500 cells each for two repeat experiments(Figure 5B).
Mentions: Aside from mitosis, condensedchromatin also occurs during senescence,as senescence-associated heterochromatin foci (SAHF).46 In this process, cells grow larger, flatten shape, andexpress senescence-associated β-galactosidase. When HCT116 cellswere treated with 20 μM cisplatin for 96 h, the remaining cellswere large, flattened, and showed β-galactosidase activity,i.e., blue precipitates, representing hydrolysis products of X-galby β-galactosidase (Figure 6A, lowerleft). TriplatinNC treated cells were small, with very little cytoplasmiccontent, and did not show evidence of β-galactosidase activity(Figure 6A, lower right). Therefore, we foundthat the DNA compaction events were not consistent with senescence.

Bottom Line: In this article, the downstream effects of nucleolar localization are described.This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems.Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure-activity paradigms for antitumor complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and ‡Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23284, United States.

ABSTRACT
TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 arrest immediately, but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure-activity paradigms for antitumor complexes.

Show MeSH
Related in: MedlinePlus