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Liquid crystalline phases of DNA and nucleosome core particles can be formed in vitro, indicating the crucial roles of these phases in the maintenance and compaction of genomes in vivo. In the present study, sequential levels of liquid crystalline decompaction were identified in highly purified nuclei of Karenia papilionacea in response to the gradual chelation of divalent counterions by ethylenediaminetetraacetic acid (EDTA); the decompaction was observed using polarizing light microscopy, confocal laser scanning microscopy, and transmission electron microscopy and further confirmed utilizing microcalorimetry. Nested fibrous coils in 150nm arc-like bands of chromatin were observed in the early stages of chromosomal decompaction. The microcalorimetry spectra of isolated nuclei revealed that the dynamic processes of nuclear decompaction occurred in a nonlinear manner; in addition, an EDTA-sensitive thermal transition between 60 degrees C-70 degrees C, corresponding to a liquid-crystalline-phase transition of chromosomes, was found. The results suggested that nested coils of fibrous chromatin filaments are responsible for the establishment and stabilization of the liquid crystalline and bire-fringence features of the chromosomes of dinoflagellates. The results also indicated that positively charged divalent counterions play significant roles in modulating liquid crystalline phases to compact the chromosomes of dinoflagellates.

From prokaryotes to eukaryotes, the DNA condensation process by basic proteins is essential to store the genome in vivo. The surface charge inversion with a DNA/HCcp3 charge ratio of 0.7/1 indicates a role for HCcp3-mediated counter ion-driven mechanism in DNA condensation. The formation of the cholesteric liquid-crystalline phase of DNA HCcp3 complexes occurred when DNA charges were neutralized. The results suggest that DNA condensation processes by HCcp3 can be divided into two distinctive binding stages: the first state, prior to condensation, involves an entropy-driven binding process of HCcp3 to the DNA, while the second state is an electrostatically driven condensing and assembling process.

From prokaryotes to eukaryotes, the DNA condensation process by basic proteins is essential to store the genome in vivo. The surface charge inversion with a DNA/HCcp3 charge ratio of 0.7/1 indicates a role for HCcp3-mediated counter ion-driven mechanism in DNA condensation. The formation of the cholesteric liquid-crystalline phase of DNA HCcp3 complexes occurred when DNA charges were neutralized. The results suggest that DNA condensation processes by HCcp3 can be divided into two distinctive binding stages: the first state, prior to condensation, involves an entropy-driven binding process of HCcp3 to the DNA, while the second state is an electrostatically driven condensing and assembling process.

Liquid crystalline phases of DNA and nucleosome core particles can be formed in vitro, indicating the crucial roles of these phases in the maintenance and compaction of genomes in vivo. In the present study, sequential levels of liquid crystalline decompaction were identified in highly purified nuclei of Karenia papilionacea in response to the gradual chelation of divalent counterions by ethylenediaminetetraacetic acid (EDTA); the decompaction was observed using polarizing light microscopy, confocal laser scanning microscopy, and transmission electron microscopy and further confirmed utilizing microcalorimetry. Nested fibrous coils in 150nm arc-like bands of chromatin were observed in the early stages of chromosomal decompaction. The microcalorimetry spectra of isolated nuclei revealed that the dynamic processes of nuclear decompaction occurred in a nonlinear manner; in addition, an EDTA-sensitive thermal transition between 60 degrees C-70 degrees C, corresponding to a liquid-crystalline-phase transition of chromosomes, was found. The results suggested that nested coils of fibrous chromatin filaments are responsible for the establishment and stabilization of the liquid crystalline and bire-fringence features of the chromosomes of dinoflagellates. The results also indicated that positively charged divalent counterions play significant roles in modulating liquid crystalline phases to compact the chromosomes of dinoflagellates.

From prokaryotes to eukaryotes, the DNA condensation process by basic proteins is essential to store the genome in vivo. The surface charge inversion with a DNA/HCcp3 charge ratio of 0.7/1 indicates a role for HCcp3-mediated counter ion-driven mechanism in DNA condensation. The formation of the cholesteric liquid-crystalline phase of DNA HCcp3 complexes occurred when DNA charges were neutralized. The results suggest that DNA condensation processes by HCcp3 can be divided into two distinctive binding stages: the first state, prior to condensation, involves an entropy-driven binding process of HCcp3 to the DNA, while the second state is an electrostatically driven condensing and assembling process.

Liquid crystalline phases of DNA and nucleosome core particles can be formed in vitro, indicating the crucial roles of these phases in the maintenance and compaction of genomes in vivo. In the present study, sequential levels of liquid crystalline decompaction were identified in highly purified nuclei of Karenia papilionacea in response to the gradual chelation of divalent counterions by ethylenediaminetetraacetic acid (EDTA); the decompaction was observed using polarizing light microscopy, confocal laser scanning microscopy, and transmission electron microscopy and further confirmed utilizing microcalorimetry. Nested fibrous coils in 150nm arc-like bands of chromatin were observed in the early stages of chromosomal decompaction. The microcalorimetry spectra of isolated nuclei revealed that the dynamic processes of nuclear decompaction occurred in a nonlinear manner; in addition, an EDTA-sensitive thermal transition between 60 degrees C-70 degrees C, corresponding to a liquid-crystalline-phase transition of chromosomes, was found. The results suggested that nested coils of fibrous chromatin filaments are responsible for the establishment and stabilization of the liquid crystalline and bire-fringence features of the chromosomes of dinoflagellates. The results also indicated that positively charged divalent counterions play significant roles in modulating liquid crystalline phases to compact the chromosomes of dinoflagellates.

Purpose: Rhabdomyosarcoma (RMS) is a common pediatric soft-tissue tumor. In this study, we evaluated the efficacy and selectivity of drozitumab, a death receptor DR5-targeted therapeutic antibody, in RMS preclinical models. Experimental Design: A panel of 11 RMS cell lines was used for in vitro studies. The molecular marker predictive of response to drozitumab was interrogated. Selected RMS cell lines were injected into the gastrocnemius muscle of mice for in vivo assessment of the potency and selectivity of drozitumab. Results: We report that DR5, but not DR4, persisted at high levels and on the surface of all RMS cell lines. DR5 antibody drozitumab was effective in vitro against the majority of RMS cell lines. There was a strong correlation between caspase-8 expression and the sensitivity to drozitumab, which induced the rapid assembly of the death-induced signaling complex and the cleavage of caspase-8 only in sensitive cells. More importantly, caspase-8 catalytic activity was both necessary and sufficient for mediating the sensitivity to drozitumab. Furthermore, drozitumab had potent antitumor activity against established RMS xenografts with a specificity predicted from the in vitro analysis and with tumor-free status in half of the treated mice. Conclusion: Our study provides the first preclinical evaluation of the potency and selectivity of a death receptor antibody in RMS. Drozitumab is effective, in vitro, against the majority of RMS cell lines that express caspase-8 and, in vivo, may provide long-term control of RMS. ©2011 AACR.

Purpose: Rhabdomyosarcoma (RMS) is a common pediatric soft-tissue tumor. In this study, we evaluated the efficacy and selectivity of drozitumab, a death receptor DR5-targeted therapeutic antibody, in RMS preclinical models. Experimental Design: A panel of 11 RMS cell lines was used for in vitro studies. The molecular marker predictive of response to drozitumab was interrogated. Selected RMS cell lines were injected into the gastrocnemius muscle of mice for in vivo assessment of the potency and selectivity of drozitumab. Results: We report that DR5, but not DR4, persisted at high levels and on the surface of all RMS cell lines. DR5 antibody drozitumab was effective in vitro against the majority of RMS cell lines. There was a strong correlation between caspase-8 expression and the sensitivity to drozitumab, which induced the rapid assembly of the death-induced signaling complex and the cleavage of caspase-8 only in sensitive cells. More importantly, caspase-8 catalytic activity was both necessary and sufficient for mediating the sensitivity to drozitumab. Furthermore, drozitumab had potent antitumor activity against established RMS xenografts with a specificity predicted from the in vitro analysis and with tumor-free status in half of the treated mice. Conclusion: Our study provides the first preclinical evaluation of the potency and selectivity of a death receptor antibody in RMS. Drozitumab is effective, in vitro, against the majority of RMS cell lines that express caspase-8 and, in vivo, may provide long-term control of RMS. ©2011 AACR.

Purpose: Rhabdomyosarcoma (RMS) is a common pediatric soft-tissue tumor. In this study, we evaluated the efficacy and selectivity of drozitumab, a death receptor DR5-targeted therapeutic antibody, in RMS preclinical models. Experimental Design: A panel of 11 RMS cell lines was used for in vitro studies. The molecular marker predictive of response to drozitumab was interrogated. Selected RMS cell lines were injected into the gastrocnemius muscle of mice for in vivo assessment of the potency and selectivity of drozitumab. Results: We report that DR5, but not DR4, persisted at high levels and on the surface of all RMS cell lines. DR5 antibody drozitumab was effective in vitro against the majority of RMS cell lines. There was a strong correlation between caspase-8 expression and the sensitivity to drozitumab, which induced the rapid assembly of the death-induced signaling complex and the cleavage of caspase-8 only in sensitive cells. More importantly, caspase-8 catalytic activity was both necessary and sufficient for mediating the sensitivity to drozitumab. Furthermore, drozitumab had potent antitumor activity against established RMS xenografts with a specificity predicted from the in vitro analysis and with tumor-free status in half of the treated mice. Conclusion: Our study provides the first preclinical evaluation of the potency and selectivity of a death receptor antibody in RMS. Drozitumab is effective, in vitro, against the majority of RMS cell lines that express caspase-8 and, in vivo, may provide long-term control of RMS. ©2011 AACR.

Background: Perifosine, an alkylphospholipid tested in phase II clinical trials, modulates the extrinsic apoptotic pathway and cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to augment apoptosis. The current study focuses on revealing the mechanisms by which perifosine enhances TRAIL-induced apoptosis.Results: The combination of perifosine and TRAIL was more active than each single agent alone in inducing apoptosis of head and neck squamous cell carcinoma cells and inhibiting the growth of xenografts. Interestingly, perifosine primarily increased cell surface levels of DR5 although it elevated the expression of both DR4 and DR5. Blockade of DR5, but not DR4 upregulation, via small interfering RNA (siRNA) inhibited perifosine/TRAIL-induced apoptosis. Perifosine increased phosphorylated c-Jun NH2-terminal kinase (JNK) and c-Jun levels, which were paralleled with DR4 and DR5 induction. However, only DR5 upregulaiton induced by perifosine could be abrogated by both the JNK inhibitor SP600125 and JNK siRNA. The antioxidants, N-acetylcysteine and glutathione, but not vitamin C or tiron, inhibited perifosine-induced elevation of p-c-Jun, DR4 and DR5. Moreover, no increased production of reactive oxygen species was detected in perifosine-treated cells although reduced levels of intracellular GSH were measured.Conclusions: DR5 induction plays a critical role in mediating perifosine/TRAIL-induced apoptosis. Perifosine induces DR5 expression through a JNK-dependent mechanism independent of reactive oxygen species. © 2010 Fu et al; licensee BioMed Central Ltd.

The largest known genomes are encoded by the histoneless liquid crystallinechromosomes (LCCs) of dinoflagellates. LCC represents a good model for studying higher-order structure of chromosome and the interactions of chromosomal DNA condensation. In the present study, the nuclear decondensation was investigated in response to extranuclear EDTA chelation. Optical microscopy identified several decondensation states of the LCCs with various EDTA concentrations. The dynamic decondensation processes were found to be in a nonlinear manner. The thermal stability assessment of condensed and EDTA-treated isolated nuclei revealed a thermal transition at 64 ℃ corresponding to liquid crystalline phase transition of LCCs. AFM and electron microscopy showed the presence of Large Nucleosome-Like Domains in LCCs with the diameter about 80 nm. This size of nucleosome-like domains is close to the minimum theoretical organelle size, as predicted by the ‘A-O’ theory, that will generate sufficient entropy-driven depletion forces to condensation. We propose that entropy-driven depletion forces and counterionic attractions of divalent cation cooperatively modulate nucleosome-like fundamental units for stabilization and assembly to higher-order structure of liquid crystalline chromosomes of dinoflagellates. The study of DNA condensation by dinoflagellate histone-like protein HCc3p demonstrated that the cholesteric liquid crystalline DNA can be formed by the interaction of DNA and HCc3p in vitro. The dynamic DNA-HCc3p interactions were studied by CD spectroscopy and ITC. The results suggest that the homogenously formation of cholesteric liquid crystalline DNA-HCc3p condensate involved two stage process, first by entropy-driven counterionic attraction, and followed by intermolecular electrostatic-driven assemble of bundle-like DNA-HCc3p complexes assembly when DNA charges were neutralized. The DNA charge inversion is also observed in electrophoretic mobility assays, which indirectly support t

Background: Perifosine, an alkylphospholipid tested in phase II clinical trials, modulates the extrinsic apoptotic pathway and cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to augment apoptosis. The current study focuses on revealing the mechanisms by which perifosine enhances TRAIL-induced apoptosis.Results: The combination of perifosine and TRAIL was more active than each single agent alone in inducing apoptosis of head and neck squamous cell carcinoma cells and inhibiting the growth of xenografts. Interestingly, perifosine primarily increased cell surface levels of DR5 although it elevated the expression of both DR4 and DR5. Blockade of DR5, but not DR4 upregulation, via small interfering RNA (siRNA) inhibited perifosine/TRAIL-induced apoptosis. Perifosine increased phosphorylated c-Jun NH2-terminal kinase (JNK) and c-Jun levels, which were paralleled with DR4 and DR5 induction. However, only DR5 upregulaiton induced by perifosine could be abrogated by both the JNK inhibitor SP600125 and JNK siRNA. The antioxidants, N-acetylcysteine and glutathione, but not vitamin C or tiron, inhibited perifosine-induced elevation of p-c-Jun, DR4 and DR5. Moreover, no increased production of reactive oxygen species was detected in perifosine-treated cells although reduced levels of intracellular GSH were measured.Conclusions: DR5 induction plays a critical role in mediating perifosine/TRAIL-induced apoptosis. Perifosine induces DR5 expression through a JNK-dependent mechanism independent of reactive oxygen species. © 2010 Fu et al; licensee BioMed Central Ltd.

The largest known genomes are encoded by the histoneless liquid crystallinechromosomes (LCCs) of dinoflagellates. LCC represents a good model for studying higher-order structure of chromosome and the interactions of chromosomal DNA condensation. In the present study, the nuclear decondensation was investigated in response to extranuclear EDTA chelation. Optical microscopy identified several decondensation states of the LCCs with various EDTA concentrations. The dynamic decondensation processes were found to be in a nonlinear manner. The thermal stability assessment of condensed and EDTA-treated isolated nuclei revealed a thermal transition at 64 ℃ corresponding to liquid crystalline phase transition of LCCs. AFM and electron microscopy showed the presence of Large Nucleosome-Like Domains in LCCs with the diameter about 80 nm. This size of nucleosome-like domains is close to the minimum theoretical organelle size, as predicted by the ‘A-O’ theory, that will generate sufficient entropy-driven depletion forces to condensation. We propose that entropy-driven depletion forces and counterionic attractions of divalent cation cooperatively modulate nucleosome-like fundamental units for stabilization and assembly to higher-order structure of liquid crystalline chromosomes of dinoflagellates. The study of DNA condensation by dinoflagellate histone-like protein HCc3p demonstrated that the cholesteric liquid crystalline DNA can be formed by the interaction of DNA and HCc3p in vitro. The dynamic DNA-HCc3p interactions were studied by CD spectroscopy and ITC. The results suggest that the homogenously formation of cholesteric liquid crystalline DNA-HCc3p condensate involved two stage process, first by entropy-driven counterionic attraction, and followed by intermolecular electrostatic-driven assemble of bundle-like DNA-HCc3p complexes assembly when DNA charges were neutralized. The DNA charge inversion is also observed in electrophoretic mobility assays, which indirectly support t

The largest known genomes are encoded by the histoneless liquid crystallinechromosomes (LCCs) of dinoflagellates. LCC represents a good model for studying higher-order structure of chromosome and the interactions of chromosomal DNA condensation. In the present study, the nuclear decondensation was investigated in response to extranuclear EDTA chelation. Optical microscopy identified several decondensation states of the LCCs with various EDTA concentrations. The dynamic decondensation processes were found to be in a nonlinear manner. The thermal stability assessment of condensed and EDTA-treated isolated nuclei revealed a thermal transition at 64 ℃ corresponding to liquid crystalline phase transition of LCCs. AFM and electron microscopy showed the presence of Large Nucleosome-Like Domains in LCCs with the diameter about 80 nm. This size of nucleosome-like domains is close to the minimum theoretical organelle size, as predicted by the ‘A-O’ theory, that will generate sufficient entropy-driven depletion forces to condensation. We propose that entropy-driven depletion forces and counterionic attractions of divalent cation cooperatively modulate nucleosome-like fundamental units for stabilization and assembly to higher-order structure of liquid crystalline chromosomes of dinoflagellates. The study of DNA condensation by dinoflagellate histone-like protein HCc3p demonstrated that the cholesteric liquid crystalline DNA can be formed by the interaction of DNA and HCc3p in vitro. The dynamic DNA-HCc3p interactions were studied by CD spectroscopy and ITC. The results suggest that the homogenously formation of cholesteric liquid crystalline DNA-HCc3p condensate involved two stage process, first by entropy-driven counterionic attraction, and followed by intermolecular electrostatic-driven assemble of bundle-like DNA-HCc3p complexes assembly when DNA charges were neutralized. The DNA charge inversion is also observed in electrophoretic mobility assays, which indirectly support t

Background: Perifosine, an alkylphospholipid tested in phase II clinical trials, modulates the extrinsic apoptotic pathway and cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to augment apoptosis. The current study focuses on revealing the mechanisms by which perifosine enhances TRAIL-induced apoptosis.Results: The combination of perifosine and TRAIL was more active than each single agent alone in inducing apoptosis of head and neck squamous cell carcinoma cells and inhibiting the growth of xenografts. Interestingly, perifosine primarily increased cell surface levels of DR5 although it elevated the expression of both DR4 and DR5. Blockade of DR5, but not DR4 upregulation, via small interfering RNA (siRNA) inhibited perifosine/TRAIL-induced apoptosis. Perifosine increased phosphorylated c-Jun NH2-terminal kinase (JNK) and c-Jun levels, which were paralleled with DR4 and DR5 induction. However, only DR5 upregulaiton induced by perifosine could be abrogated by both the JNK inhibitor SP600125 and JNK siRNA. The antioxidants, N-acetylcysteine and glutathione, but not vitamin C or tiron, inhibited perifosine-induced elevation of p-c-Jun, DR4 and DR5. Moreover, no increased production of reactive oxygen species was detected in perifosine-treated cells although reduced levels of intracellular GSH were measured.Conclusions: DR5 induction plays a critical role in mediating perifosine/TRAIL-induced apoptosis. Perifosine induces DR5 expression through a JNK-dependent mechanism independent of reactive oxygen species. © 2010 Fu et al; licensee BioMed Central Ltd.