Short and long term effects of cytoskeleton-disrupting drugs on cytochrome P450 Cyp1a-1 induction in murine hepatoma 1c1c7 cells: suppression by the microtubule inhibitor nocodazole.
Cultured murine hepatoma 1c1c7 cells were treated with either the actin filament-disrupting drug cytochalasin D or the microtubule inhibitors colchicine and nocadazole (NOC) to assess the role of the cytoskeleton in the process of cytochrome P450 Cyp1a-1 induction. Indirect fluorescence analyses demonstrated that microtubule or actin networks were disrupted within 1 hr of treatment and remained altered as long as cultures were maintained in the presence of the drugs. Treatment of cultures with cytochalasin D, colchicine, or NOC for 1 hr before the addition of dibenz[a,c]anthracene had no effect of Cyp1a-1 induction, as monitored by measurements of CYP1A1 mRNA. Pretreatment with NOC for > or = 18 hr produced populations of cells that had either a flat or rounded morphology. Both populations, when isolated 20-24 hr after NOC treatment, were arrested in the G2/M phase of the cell cycle (83-98% in G2/M versus approximately 7-10% in nontreated or solvent-treated cultures). Cyp1a-1 induction was suppressed in both of these populations, as monitored by measurement of CYP1A1 mRNA content (reductions of > 68%), 7-ethoxyresorufin O-deethylase activity (reductions of > 80%), or microsomal CYP1A1 protein content (reductions of > 80%). In contrast, overall [3H]leucine incorporation into protein was not affected. Cytosol prepared from these NOC-treated cultures bound approximately 39% of the radiolabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin bound by cytosol isolated from solvent-treated cultures. Nuclear extracts prepared from cultures treated with NOC for 20-24 hr before in vivo exposure to inducer and cytoplasmic extracts isolated from similarly NOC-treated cultures that were exposed to inducer in vitro demonstrated reductions of > or = 54% and > or = 55%, respectively, in their abilities to bind to DNA, when analyzed by gel retardation analyses using an oligonucleotide corresponding to dioxin-responsive element D of the Cyp1a-1 gene. These studies suggest that ligand-dependent induction of Cyp1a-1 transcription is unaffected by short term disruption of the microfilament or microtubule network. However, long term exposure to microtubule inhibitors causes cells to pause in the G2/M stage of the cell cycle and modulates processes involved in the induction of Cyp1a-1 in these cells.
Schöller
A
A
Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201.
Hong
N J
NJ
Bischer
P
P
Reiners
J J
JJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000199:Actins; D065607:Cytochrome P-450 Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D015739:Nocodazole