Journal of Biomolecular Structure and Dynamics, 1992
The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary amm... more The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary ammonium heterocycles has been investigated by means of CD spectroscopy and melting measurements. From the spectroscopic results and binding data it is concluded that the agents SN-6132, SN-6131 and SN-6113 have relatively high affinity to AT base pair sequences whereas the binding to GC pairs is very low. The binding selectivity to AT base pair sequences decreases in the order netropsin > SN-6132 > SN-6113 > SN-6131. Poly(dA).poly(dT) has the highest binding preference for SN-6132 relative to that of SN-6131. The different binding behavior of the ligands is related to their distinct changes in the chemical structure and to the DNA minor groove properties which determines the adaptability of the ligands in the groove.
The Jerusalem Symposia on Quantum Chemistry and Biochemistry, 1990
The cytotoxicity of a series of DNA-binding drugs was measured in a number of both mouse and huma... more The cytotoxicity of a series of DNA-binding drugs was measured in a number of both mouse and human cell lines and some multidrug resistant sublines. The intercalating drugs used were derivatives of 9-anilinoacridine, including amsacrine. The nonintercalating drugs were phenylbisbenzimidazole derivatives, including the Hoechst dyes H33258 and H33342. Comparison of the ability of the intercalators to inhibit the growth of cultured L1210 cells showed that the presence of certain substituents on the anilino moiety, such as a methoxy group or a benzenesulphonamide group in the correct place, greatly enhances the degree of growth inhibition for a given amount of DNA binding. Divergence between activity and DNA binding was also found with two multidrug resistant Jurkat sublines but in this case the anilino substituents for optimal activity were quite different. The Hoechst dyes at low concentrations did not inhibit cell growth, but some did strongly potentiate the cytotoxicity of the amsacrine analogue CI-921. In this case, potentiation was strongly dependent on the substituents on the terminal phenyl ring. The results can be explained if it is assumed that both the anilino substituents of the intercalators and the phenyl substituents of the Hoechst dyes interact with an enzyme receptor while the remainder of the molecule binds to DNA. The most likely enzyme receptors are isozymes of the topoisomerase II isozymes.
PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thou... more PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thought to control MITF expression during normal melanocyte differentiation. However, it is not clear whether this is still true in melanoma and whether the effects of knockdown of PAX3 on the inhibition of melanoma growth or survival are by its regulation of MITF. By western blot and quantitative real-time reverse transcription-PCR, we investigated the relationship between PAX3 and MITF expression in 27 metastatic melanoma and one immortalized melanocyte cell lines. All lines were found to express both PAX3 and MITF proteins but levels varied by 15 fold and more than 100 fold, respectively. The expression of PAX3 protein was correlated with that of MITF (r=0.75; P<0.001) but the expression of PAX3 protein and MITF mRNA was not. Immunofluorescence microscopy showed that individual cells expressed widely differing relative amounts of PAX3 and MITF protein. By MTT cell proliferation and flow cytometry assays, both MITF and PAX3 proteins seemed to be functional, as knockdown with siRNA led to reduced proliferation and induction of apoptosis. However, knockdown of PAX3 with small interfering RNA did not decrease MITF expression and vice versa. In one cell line (NZM15), silencing of PAX3 induced terminal differentiation whereas silencing of MITF induced expression of FOXD3, a repressor of melanogenesis. The results suggest that the melanoma lines used in this study show considerable phenotypic variation of expression of these two transcriptional activators and reflect a deregulation of the developmental process operating in the genesis of the melanocyte lineage, and that they probably function independently to enhance the survival of melanoma cells.
Journal of Biomolecular Structure and Dynamics, 1987
Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied b... more Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.
The production of nitric oxide in endotoxin-resistant C3H/HeJ mice in response to flavone-8-aceti... more The production of nitric oxide in endotoxin-resistant C3H/HeJ mice in response to flavone-8-acetic acid (FAA), derivatives of xanthenone-4-acetic (XAA), endotoxin and recombinant human tumour necrosis factor-alpha (TNF-alpha) was investigated and compared with the induction of haemorrhagic necrosis in subcutaneous M16/C tumours. FAA and XAA analogues stimulated nitric oxide production both in vitro (activated macrophages) and in vivo (plasma nitrate elevation) in both C3H/HeJ and C3H/HeN mice (5,6-dimethyl-XAA greater than 5-methyl-XAA greater than FAA greater than XAA greater than 8-methyl-XAA). Recombinant human TNF-alpha stimulated nitric oxide production equally from both murine strains while endotoxin stimulated nitric oxide production only by C3H/HeN mice. The extent of induction of haemorrhagic necrosis in tumour-bearing mice treated with FAA, 5,6-dimethyl XAA or endotoxin paralleled the effects on nitric oxide production, showing a differential between the two strains of mice only in the case of endotoxin.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1992
... of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrin... more ... of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrine Yoshihisa Iwamoto, Lynnette R. Ferguson, Amira Pearson and Bruce C ... Acri-dine derivatives were synthesized in this labora-tory (Baguley et al., 1981; Atwell et al., 1987; Robbie et al ...
The interaction of a series of bisquaternary ammonium heterocycles (BQA) with the triple helix of... more The interaction of a series of bisquaternary ammonium heterocycles (BQA) with the triple helix of poly(dA).2poly(dT) was investigated using thermal denaturation and circular dichroism spectroscopy. The BQA-bound triplexes undergo two distinct transitions during thermal melting: a first melting step from the triplex to the duplex state with the BQA ligand remaining bound and a second step from the duplex to single strands. The ionic strength dependence of the triplex stability at increasing ligand concentration was analyzed by phase diagrams. The results demonstrate that some BQA ligands thermally stabilize the triplex at Na+ concentrations of < or = 150 mM and destabilize this structure above the range 150-220 mM Na+, indicating promotion of triplex formation under low ionic strength and diminution of the affinity of the major groove-bound third strand in the triplex at high ionic strength. SN-6999 most strongly destabilizes the triplex structure but stabilizes the DNA duplex, wh...
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a low-molecular-weight biological response modifier... more 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a low-molecular-weight biological response modifier scheduled for clinical evaluation, induced synthesis of tumour necrosis factor-alpha (TNF-alpha) in serum of mice, with maximal activity being observed at 2-3 h after administration. At a dose of 27.5 mg/kg, DMXAA induced similar TNF-alpha concentrations as did flavone-8-acetic acid given at its maximum tolerated dose (MTD; 330 mg/kg), whereas 8-methylxanthenone-4-acetic acid, which has no antitumour activity, did not induce serum TNF-alpha at its MTD (440 mg/kg). The dependence of schedule on TNF-alpha induction was studied by giving DMXAA to mice in two doses of 27.5 mg/kg each separated by different intervals. An interval of 0 (i.e. 55 mg/kg given in a single dose) produced a TNF-alpha concentration 9-fold that produced by a single dose of 27.5 mg/kg. This dose, although higher than the MTD of 30 mg/kg, did not affect the health of mice at the time of assay (3 h). An interval of 1 da...
Journal of Biomolecular Structure and Dynamics, 1992
The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary amm... more The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary ammonium heterocycles has been investigated by means of CD spectroscopy and melting measurements. From the spectroscopic results and binding data it is concluded that the agents SN-6132, SN-6131 and SN-6113 have relatively high affinity to AT base pair sequences whereas the binding to GC pairs is very low. The binding selectivity to AT base pair sequences decreases in the order netropsin &gt; SN-6132 &gt; SN-6113 &gt; SN-6131. Poly(dA).poly(dT) has the highest binding preference for SN-6132 relative to that of SN-6131. The different binding behavior of the ligands is related to their distinct changes in the chemical structure and to the DNA minor groove properties which determines the adaptability of the ligands in the groove.
The Jerusalem Symposia on Quantum Chemistry and Biochemistry, 1990
The cytotoxicity of a series of DNA-binding drugs was measured in a number of both mouse and huma... more The cytotoxicity of a series of DNA-binding drugs was measured in a number of both mouse and human cell lines and some multidrug resistant sublines. The intercalating drugs used were derivatives of 9-anilinoacridine, including amsacrine. The nonintercalating drugs were phenylbisbenzimidazole derivatives, including the Hoechst dyes H33258 and H33342. Comparison of the ability of the intercalators to inhibit the growth of cultured L1210 cells showed that the presence of certain substituents on the anilino moiety, such as a methoxy group or a benzenesulphonamide group in the correct place, greatly enhances the degree of growth inhibition for a given amount of DNA binding. Divergence between activity and DNA binding was also found with two multidrug resistant Jurkat sublines but in this case the anilino substituents for optimal activity were quite different. The Hoechst dyes at low concentrations did not inhibit cell growth, but some did strongly potentiate the cytotoxicity of the amsacrine analogue CI-921. In this case, potentiation was strongly dependent on the substituents on the terminal phenyl ring. The results can be explained if it is assumed that both the anilino substituents of the intercalators and the phenyl substituents of the Hoechst dyes interact with an enzyme receptor while the remainder of the molecule binds to DNA. The most likely enzyme receptors are isozymes of the topoisomerase II isozymes.
PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thou... more PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thought to control MITF expression during normal melanocyte differentiation. However, it is not clear whether this is still true in melanoma and whether the effects of knockdown of PAX3 on the inhibition of melanoma growth or survival are by its regulation of MITF. By western blot and quantitative real-time reverse transcription-PCR, we investigated the relationship between PAX3 and MITF expression in 27 metastatic melanoma and one immortalized melanocyte cell lines. All lines were found to express both PAX3 and MITF proteins but levels varied by 15 fold and more than 100 fold, respectively. The expression of PAX3 protein was correlated with that of MITF (r=0.75; P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001) but the expression of PAX3 protein and MITF mRNA was not. Immunofluorescence microscopy showed that individual cells expressed widely differing relative amounts of PAX3 and MITF protein. By MTT cell proliferation and flow cytometry assays, both MITF and PAX3 proteins seemed to be functional, as knockdown with siRNA led to reduced proliferation and induction of apoptosis. However, knockdown of PAX3 with small interfering RNA did not decrease MITF expression and vice versa. In one cell line (NZM15), silencing of PAX3 induced terminal differentiation whereas silencing of MITF induced expression of FOXD3, a repressor of melanogenesis. The results suggest that the melanoma lines used in this study show considerable phenotypic variation of expression of these two transcriptional activators and reflect a deregulation of the developmental process operating in the genesis of the melanocyte lineage, and that they probably function independently to enhance the survival of melanoma cells.
Journal of Biomolecular Structure and Dynamics, 1987
Binding to DNA&#39;s of the non-intercalative ligands SN-6999 and SN-18071 has been studied b... more Binding to DNA&#39;s of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA&#39;s, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.
The production of nitric oxide in endotoxin-resistant C3H/HeJ mice in response to flavone-8-aceti... more The production of nitric oxide in endotoxin-resistant C3H/HeJ mice in response to flavone-8-acetic acid (FAA), derivatives of xanthenone-4-acetic (XAA), endotoxin and recombinant human tumour necrosis factor-alpha (TNF-alpha) was investigated and compared with the induction of haemorrhagic necrosis in subcutaneous M16/C tumours. FAA and XAA analogues stimulated nitric oxide production both in vitro (activated macrophages) and in vivo (plasma nitrate elevation) in both C3H/HeJ and C3H/HeN mice (5,6-dimethyl-XAA greater than 5-methyl-XAA greater than FAA greater than XAA greater than 8-methyl-XAA). Recombinant human TNF-alpha stimulated nitric oxide production equally from both murine strains while endotoxin stimulated nitric oxide production only by C3H/HeN mice. The extent of induction of haemorrhagic necrosis in tumour-bearing mice treated with FAA, 5,6-dimethyl XAA or endotoxin paralleled the effects on nitric oxide production, showing a differential between the two strains of mice only in the case of endotoxin.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1992
... of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrin... more ... of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrine Yoshihisa Iwamoto, Lynnette R. Ferguson, Amira Pearson and Bruce C ... Acri-dine derivatives were synthesized in this labora-tory (Baguley et al., 1981; Atwell et al., 1987; Robbie et al ...
The interaction of a series of bisquaternary ammonium heterocycles (BQA) with the triple helix of... more The interaction of a series of bisquaternary ammonium heterocycles (BQA) with the triple helix of poly(dA).2poly(dT) was investigated using thermal denaturation and circular dichroism spectroscopy. The BQA-bound triplexes undergo two distinct transitions during thermal melting: a first melting step from the triplex to the duplex state with the BQA ligand remaining bound and a second step from the duplex to single strands. The ionic strength dependence of the triplex stability at increasing ligand concentration was analyzed by phase diagrams. The results demonstrate that some BQA ligands thermally stabilize the triplex at Na+ concentrations of < or = 150 mM and destabilize this structure above the range 150-220 mM Na+, indicating promotion of triplex formation under low ionic strength and diminution of the affinity of the major groove-bound third strand in the triplex at high ionic strength. SN-6999 most strongly destabilizes the triplex structure but stabilizes the DNA duplex, wh...
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a low-molecular-weight biological response modifier... more 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a low-molecular-weight biological response modifier scheduled for clinical evaluation, induced synthesis of tumour necrosis factor-alpha (TNF-alpha) in serum of mice, with maximal activity being observed at 2-3 h after administration. At a dose of 27.5 mg/kg, DMXAA induced similar TNF-alpha concentrations as did flavone-8-acetic acid given at its maximum tolerated dose (MTD; 330 mg/kg), whereas 8-methylxanthenone-4-acetic acid, which has no antitumour activity, did not induce serum TNF-alpha at its MTD (440 mg/kg). The dependence of schedule on TNF-alpha induction was studied by giving DMXAA to mice in two doses of 27.5 mg/kg each separated by different intervals. An interval of 0 (i.e. 55 mg/kg given in a single dose) produced a TNF-alpha concentration 9-fold that produced by a single dose of 27.5 mg/kg. This dose, although higher than the MTD of 30 mg/kg, did not affect the health of mice at the time of assay (3 h). An interval of 1 da...
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