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Xinbo Zhang - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial Membrane Potential Change Induced by Hoechst 33342 in Myelogenous Leukemia Cell Line HL-60
2016Co-Authors: Address Timothy, Xinbo Zhang, P Singleton, Jenn C. Chen, Timothy P. Singleton, Fl KiechleAbstract:Abstract. Hoechst 33342’s effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3’-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70 % by 1 hr and 70-90 % at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40 % and then partially normalized, and cell viability was>92 % at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis. (received 3 May 2004; accepted 24 May 2004
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Abstract 1921: The effect of Hoechst 33342 and Hoechst 33258 on side population (SP) cells or stem cell-like population
Tumor Biology, 2014Co-Authors: Naimei Tang, Crystal Zhang, Cyndi Noraian, Anil Wali, Harvey I. Pass, Michael Harbut, Xinbo ZhangAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Hoechst 33342 side population (SP) analysis is widely used for identifying and sorting side population (SP) or stem cell-like population from a variety of tissues and species including cancer cell lines. However, Hoechst 33342, but not its derivative Hoechst 33258, is an apoptotic inducer. Our previous results showed that Hoechst 33342 induced apoptosis in seven mesothelioma cell lines through cytochrome C release, caspase activation and degradation of Poly (ADP-ribose) polymerase. We hypothesize Hoechst 33342 may cause SP cell death during Hoechst 33342 staining and Hoechst 33258 may be a safe indicator for identification and isolation of SP cells. The aims of this study are 1) to detect the effect of H 33342 on SP cells, and 2) to identify if H33258 can be an indicator for stem cell isolation. Procedure: The effect of Hoechst dyes on the tumorigenicity of H2373 mesothelioma cells was analyzed by soft agar clonogenic colony formation. MTT assay have been employed to estimate the effect of Hoechst dyes on cell growth in H2373 mesothelioma cells, and Hoechst 33342 and Hoechst 33258 SP analysis was performed on a FACSDiVa cell sorter. Results: Our result demonstrated that Hoechst 33342 staining can causes SP cell damage and also decreases cell colony formation and cell proliferation after using the routine staining doses and intervals for SP cell analysis. In contrast, Hoechst 33258 has little effect on the cell colony formation and cell proliferation. We have found that verapamil, ABC transporter inhibitor, significantly inhibits the efflux of intracellular Hoechst 33258, which means Hoechst 33258 may be used as a marker in isolating ABC transporter positive cells, as Hoechst 33342 does. Conclusion: 1) Hoechst 33342 can induce apoptosis of SP cells of mesothelioma cancer cells 2) Hoechst 33258 may be a safe indicator and replace Hoechst 33342 for identification and isolation of side population. Future Studies: 1) More in vitro methods such as clonogenic assay, drug resistant assay, and invasion assay are required for further determination of stem cell-like properties of side population isolated by Hoechst 33258 when compared to that isolated by Hoechst 33342. 2) Xenograft (or animal) study is ultimately required to determine the capacity of in vivo tumor formation of the side population cells isolated by Hoechst 33258 after the completion of in vitro study Note: This abstract was not presented at the meeting. Citation Format: Naimei Tang, Crystal Zhang, Cyndi Noraian, Anil Wali, Harvey Pass, Michael Harbut, Xinbo Zhang. The effect of Hoechst 33342 and Hoechst 33258 on side population (SP) cells or stem cell-like population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2014-1921
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Fatty Acid Synthase and its mRNA Concentrations Are Decreased at Different Times Following Hoechst 33342-induced Apoptosis in BC3H-1 Myocytes
Annals of clinical and laboratory science, 2006Co-Authors: Xinbo Zhang, Fl KiechleAbstract:Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 microg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not. Hoechst 33342 at 20 microg/ml reduced the concentration of FAS protein, which was followed more than 6 hr later by a reduction in FAS mRNA. In conclusion, the inhibition of fatty acid synthesis induced by 20 microg/ml of Hoechst 33342 is attributed to the degradation of FAS protein by activated caspases rather than by inhibition of FAS enzyme activity or FAS mRNA synthesis.
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Mitochondrial membrane potential change induced by Hoechst 33342 in myelogenous leukemia cell line HL-60.
Annals of clinical and laboratory science, 2004Co-Authors: Jenn C. Chen, Xinbo Zhang, Timothy P. Singleton, Fl KiechleAbstract:Abstract. Hoechst 33342's effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70% by 1 hr and 70-90% at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40% and then partially normalized, and cell viability was > 92% at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis.
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Hoechst 33342 alters luciferase gene expression in transfected BC3H-1 myocytes
Archives of pathology & laboratory medicine, 2003Co-Authors: Xinbo Zhang, Fl KiechleAbstract:○ Background.-Hoechst 33342 and Hoechst 33258 bind to the minor groove of DNA. Hoechst 33342 induces apoptosis in a variety of cell types by a mechanism that is associated with disruption of the formation of the TATA box-binding protein/DNA complex. Objective.-To further investigate the role of Hoechst 33342 in gene regulation using BC3H-1 myocytes transfected with 4 different pGL3 luciferase reporter vectors constructed with or without the SV40 promoter and/or enhancer regions or with 2 synthetic Renilla luciferase vectors (phRL-null and phRL-TK). Methods.-Luciferase messenger RNA content was measured by reverse transcriptase-polymerase chain reaction, and luciferase activity was measured by luminometry. The ability of transcription factors in nuclei prepared from BC3H-1 myocytes to bind to a [ 32 P]-labeled 24-base pair oligonucleotide containing the TATA box-binding element was determined by a gel mobility shift assay. Results.-In vivo, 4.4 and 8.9 μM of Hoechst 33342 (sublethal doses) increased luciferase enzyme activity in cells transfected with each of the 4 pGL3 luciferase reporter vectors and both of the Renilla luciferase vectors. Hoechst 33258 had no effect on luciferase enzyme activity. In vitro, Hoechst 33342 increased transcription factor binding to the 24-mer oligonucleotide containing the TATA box-binding element, which would be favorable to increased RNA polymerase II efficiency. Conclusion.-Hoechst 33342 stimulates luciferase activity by a pathway that is independent of the integrity of the promoters in the luciferase gene expression vectors used (pGL3 basic, pGL3 control, pGL3 enhancer, and pGL3 promoter vectors, phRL-null, or phRL-TK).
Fl Kiechle - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial Membrane Potential Change Induced by Hoechst 33342 in Myelogenous Leukemia Cell Line HL-60
2016Co-Authors: Address Timothy, Xinbo Zhang, P Singleton, Jenn C. Chen, Timothy P. Singleton, Fl KiechleAbstract:Abstract. Hoechst 33342’s effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3’-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70 % by 1 hr and 70-90 % at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40 % and then partially normalized, and cell viability was>92 % at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis. (received 3 May 2004; accepted 24 May 2004
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Fatty Acid Synthase and its mRNA Concentrations Are Decreased at Different Times Following Hoechst 33342-induced Apoptosis in BC3H-1 Myocytes
Annals of clinical and laboratory science, 2006Co-Authors: Xinbo Zhang, Fl KiechleAbstract:Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 microg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not. Hoechst 33342 at 20 microg/ml reduced the concentration of FAS protein, which was followed more than 6 hr later by a reduction in FAS mRNA. In conclusion, the inhibition of fatty acid synthesis induced by 20 microg/ml of Hoechst 33342 is attributed to the degradation of FAS protein by activated caspases rather than by inhibition of FAS enzyme activity or FAS mRNA synthesis.
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Mitochondrial membrane potential change induced by Hoechst 33342 in myelogenous leukemia cell line HL-60.
Annals of clinical and laboratory science, 2004Co-Authors: Jenn C. Chen, Xinbo Zhang, Timothy P. Singleton, Fl KiechleAbstract:Abstract. Hoechst 33342's effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70% by 1 hr and 70-90% at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40% and then partially normalized, and cell viability was > 92% at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis.
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Hoechst 33342 alters luciferase gene expression in transfected BC3H-1 myocytes
Archives of pathology & laboratory medicine, 2003Co-Authors: Xinbo Zhang, Fl KiechleAbstract:○ Background.-Hoechst 33342 and Hoechst 33258 bind to the minor groove of DNA. Hoechst 33342 induces apoptosis in a variety of cell types by a mechanism that is associated with disruption of the formation of the TATA box-binding protein/DNA complex. Objective.-To further investigate the role of Hoechst 33342 in gene regulation using BC3H-1 myocytes transfected with 4 different pGL3 luciferase reporter vectors constructed with or without the SV40 promoter and/or enhancer regions or with 2 synthetic Renilla luciferase vectors (phRL-null and phRL-TK). Methods.-Luciferase messenger RNA content was measured by reverse transcriptase-polymerase chain reaction, and luciferase activity was measured by luminometry. The ability of transcription factors in nuclei prepared from BC3H-1 myocytes to bind to a [ 32 P]-labeled 24-base pair oligonucleotide containing the TATA box-binding element was determined by a gel mobility shift assay. Results.-In vivo, 4.4 and 8.9 μM of Hoechst 33342 (sublethal doses) increased luciferase enzyme activity in cells transfected with each of the 4 pGL3 luciferase reporter vectors and both of the Renilla luciferase vectors. Hoechst 33258 had no effect on luciferase enzyme activity. In vitro, Hoechst 33342 increased transcription factor binding to the 24-mer oligonucleotide containing the TATA box-binding element, which would be favorable to increased RNA polymerase II efficiency. Conclusion.-Hoechst 33342 stimulates luciferase activity by a pathway that is independent of the integrity of the promoters in the luciferase gene expression vectors used (pGL3 basic, pGL3 control, pGL3 enhancer, and pGL3 promoter vectors, phRL-null, or phRL-TK).
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The Effect of Hoechst 33342 on Luciferase Gene Transcription and Translation.
TheScientificWorldJournal, 2001Co-Authors: Xinbo Zhang, Fl KiechleAbstract:INTRODUCTION. Hoechst 33342 (H342) and Hoechst 33258 (H258) bind to AT regions of the DNA minor groove. H342, but not H258, induces apoptosis in various cells (1). Sequence-selective DNA-binding drugs inhibit transcription factors from binding to their target sites on gene promoters in vitro (2). In vivo studies demonstrate that H342-induced apoptosis is associated with degradation of TATA box binding protein (TBP) (3) and accumulation of intracellular E2F-1 (4). Here, we determine the effect of H342 and H258 on in vivo luciferase gene expression in BC3H-1 myocytes using 4 pGL-3 vectors: Basic, Promoter, Enhancer, and Control.
Kenneth Gable - One of the best experts on this subject based on the ideXlab platform.
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Activation and inhibition of the sarcoplasmic reticulum Ca^2+ channel by the polycationic dyes Hoechst 33342 and Hoechst 33258
The Journal of Membrane Biology, 1993Co-Authors: Troy J. Beeler, Kenneth GableAbstract:The polycationic dyes, Hoechst 33342 (Bisbenzimide,2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl) 2,5′-bi 1H benzimidazole) and Hoechst 33258 (Bisbenzimide,2′-(4-hydroxyphenyl) 5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole) alter the activity of the sarcoplasmic reticulum Ca^2+ channel. Although they act competitively, Hoechst 33342 decreases, while Hoechst 33258 increases, the rate of channel-mediated Ca^2+ efflux from junctional sarcoplasmic reticulum vesicles. Unlike other cationic sarcoplasmic reticulum Ca^2+ channel antagonists, Hoechst 33342 blocks the ryanodine-activated Ca^2+ channel. Both Hoechst 33342 and Hoechst 33258 inhibit the channel incorporated into the planar lipid bilayer. Since the only structural difference between the two dyes is that the agonist Hoechst 33258 has a hydroxy group where the antagonist Hoechst 33342 has an ethoxy group, it is possible that the more hydrophobic, bulky ethoxy group blocks Ca^2+ movement through the channel, whereas the hydroxy group only reduces the rate of Ca^2+ movement. The opinions or assertions contained herein are private ones of the author ad are not to beconstrued as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
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Activation and inhibition of the sarcoplasmic reticulum Ca2+ channel by the polycationic dyes Hoechst 33342 and Hoechst 33258.
The Journal of membrane biology, 1993Co-Authors: Troy Beeler, Kenneth GableAbstract:The polycationic dyes, Hoechst 33342 (Bisbenzimide,2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl) 2,5′-bi 1H benzimidazole) and Hoechst 33258 (Bisbenzimide,2′-(4-hydroxyphenyl) 5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole) alter the activity of the sarcoplasmic reticulum Ca2+ channel. Although they act competitively, Hoechst 33342 decreases, while Hoechst 33258 increases, the rate of channel-mediated Ca2+ efflux from junctional sarcoplasmic reticulum vesicles. Unlike other cationic sarcoplasmic reticulum Ca2+ channel antagonists, Hoechst 33342 blocks the ryanodine-activated Ca2+ channel. Both Hoechst 33342 and Hoechst 33258 inhibit the channel incorporated into the planar lipid bilayer. Since the only structural difference between the two dyes is that the agonist Hoechst 33258 has a hydroxy group where the antagonist Hoechst 33342 has an ethoxy group, it is possible that the more hydrophobic, bulky ethoxy group blocks Ca2+ movement through the channel, whereas the hydroxy group only reduces the rate of Ca2+ movement.
Paul J. Smith - One of the best experts on this subject based on the ideXlab platform.
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abcg2 associated resistance to Hoechst 33342 and topotecan in a murine cell model with constitutive expression of side population characteristics
Cytometry Part A, 2009Co-Authors: Paul J. Smith, Emeline Furon, Marie Wiltshire, Lee Campbell, Graham P. Feeney, R. D. Snyder, Rachel J. ErringtonAbstract:Drug resistant tumor “side-populations,” enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations. © 2009 International Society for Advancement of Cytometry
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ABCG2‐associated resistance to Hoechst 33342 and topotecan in a murine cell model with constitutive expression of side population characteristics
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2009Co-Authors: Paul J. Smith, Emeline Furon, Marie Wiltshire, Lee Campbell, Graham P. Feeney, R. D. Snyder, Rachel J. ErringtonAbstract:Drug resistant tumor “side-populations,” enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations. © 2009 International Society for Advancement of Cytometry
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Detection of multidrug resistance and quantification of responses of human tumour cells to cytotoxic agents using flow cytometric spectral shift analysis of Hoechst 33342-DNA fluorescence
Cancer chemotherapy and pharmacology, 1991Co-Authors: Paul J. Smith, Sally A. Morgan, James V. WatsonAbstract:We describe the application of a flow cytometric technique for assessing the radiation or drug sensitivity characteristics of human tumour cells. The technique makes use of the phenomenon that a red shift occurs in the fluorescence emission spectrum of a DNA-specific dye (Hoechst 33342) as an increasing number of dye molecules bind to nuclear DNA. Intact, viable cells undergo a time-dependent spectral shift that can be distinguished from the rapid shift observed in cells with damaged membranes by the use of multiparametric flow cytometry. The responses of various human cell lines were compared, namely, those of normal and ataxia-telangiectasia (A-T) lymphoblastoid lines, a small-cell lung carcinoma line and its (in vitro) derived multidrug-resistant variants. A close correlation was found between dye toxicity and the degree of DNA binding of Hoechst 33342 independent of cellular DNA content, with lymphoblastoid and multidrug-resistant small-cell lung cancer cells showing enhanced and restricted dye-binding rates, respectively. VP16-and radiation-induced cell kill was found to result in a quantifiable increase in the fraction of cells undergoing a rapid spectral shift and was capable of detecting the increased radiation sensitivity of A-T-derived cells. Spectral shift analysis provides a rapid method for assessing the responses of tumour cells to cytotoxic agents and for determining the general ability of cells to protect cellular DNA from a model DNA-binding agent (Hoechst 33342) that participates in the multidrug resistance phenotype.
Ronald T. Borchardt - One of the best experts on this subject based on the ideXlab platform.
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Bidirectional transport of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein, across MDCK-MDR1 cell monolayers
Journal of pharmaceutical sciences, 2004Co-Authors: Fuxing Tang, Hui Ouyang, Jerry Z. Yang, Ronald T. BorchardtAbstract:The bidirectional permeation characteristics of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein (P-gp), across monolayers of MDCK cells transfected with the human MDR1 gene (MDCK–MDR1) were investigated. The ratios of the apparent permeability coefficients (Papp) of rhodamine 123 and Hoechst 33342 flux measured in the basolateral (BL) to apical (AP) direction versus the flux in the AP-to-BL direction (Papp BL-to-AP/Papp AP-to-BL) were 115 and 177, respectively. The P-gp inhibitor GF-120918 could significantly reduce the polarized efflux of both rhodamine 123 and Hoechst 33342. Rhodamine 123 appeared to “stimulate” the polarized efflux of Hoechst 33342 across MDCK–MDR1 cell monolayers. In contrast, Hoechst 33342 partially inhibited the polarized efflux of rhodamine 123 across these cell monolayers whereas daunorubicin partially inhibited the polarized efflux of both rhodamine 123 and Hoechst 33342. The uptake characteristics of rhodamine 123 and Hoechst 33342 in MDCK–MDR1 cells were measured in the absence and presence of GF-120918 and known P-gp substrates (Hoechst 33342, rhodamine 123, and daunorubicin). The uptake of rhodamine 123 and Hoechst 33342 in MDCK–MDR1 cells was enhanced more than twofold by inclusion of GF-120918 (2 μM) in the incubation medium. Daunorubicin (160 μM) increased the relative fluorescence unit (RFU) values of cytoplasm-associated rhodamine 123 by up to 30%. However, daunorubicin (40 μM) and rhodamine 123 (5 μM) decreased the RFU values of cell membrane-associated Hoechst 33342 by 70% and 40%, respectively. To further explore what appears to be a “stimulatory” effect of daunorubicin and rhodamine 123 on the uptake of Hoechst 33342 and a stimulatory effect of daunorubicin on Hoechst 33342 transport across cell monolayer, uptake of Hoechst 33342 into liposomes in the presence and absence of GF-120918, daunorubicin, and rhodamine 123 was determined. GF-120918 exhibited no effect on the RFU values of liposome-associated Hoechst 33342. In contrast, rhodamine 123 and daunorubicin decreased the fluorescence of liposome-associated Hoechst 33342 suggesting these molecules were either quenching the fluorescence of this chemical probe or displacing it from the lipid bilayer. In conclusion, these bidirectional transport data indicate that rhodamine 123 and Hoechst 33342 are excellent substrates of P-gp in MDCK–MDR1 cells. The ability of Hoechst 33342 to partially inhibit the polarized efflux of rhodamine 123 is consistent with these substrates binding to the same site on P-gp. In contrast, the ability of rhodamine 123 to apparently “stimulate” the efflux of Hoechst 33342 in both the transport and uptake experiments suggests the substrates might bind to different sites on P-gp. However, experimental results using liposomes suggested that this “stimulation” phenomenon by rhodamine 123 on Hoechst 33342 uptake and efflux might simply be an artifact. Thus, the use of Hoechst 33342 to probe the binding sites on a membrane-bound protein such as P-gp might be problematic. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1185–1194, 2004