Abstract
Wild-type MCF-7 human breast cancer cells were cultured for 3 months in 1 microM benzo[a]pyrene (BaP), and resistant clones were screened for inducibility of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One of the BaP-resistant (BaPR) clones exhibited unique genotypic expression which distinguished it from both wild-type and drug-resistant (AdrR) variant MCF-7 cells. Glutathione levels, glutathione S-transferase activities, estrogen receptor levels, estrogen responsiveness, and expression of the multidrug-resistant MDR1 and MRP mRNA levels were similar in the wild-type and BaPR cells, whereas these parameters were reported to be altered in AdrR cells. In contrast, TCDD induced CYP1A1 gene expression and inhibited selected estrogen-induced responses in wild-type but not BaPR MCF-7 cells. Treatment of wild-type and BaPR cells with [3H]TCDD resulted in formation of the radiolabeled aryl hydrocarbon (Ah) 6 S nuclear receptor complex in both cell lines. The loss of Ah responsiveness in the BaPR variant cells correlated with the failure of the nuclear or transformed cytosolic Ah receptor complex to bind genomic dioxin-responsive elements as determined in gel retardation assays.
Highlights
Glutathione levels, glutathione S - vitro culturing techniques have been used to isolatpehortransferase activities, estrogen receptor levels, estrogbeon1 ester [19] and adriamycin-resistant (Ad*) MCF-7 clones responsiveness, and expression of the multidrug-resistant MDRl and membrane protein (MRP) mRNA levels were similar in the wild-typeand B e R cells,whereastheseparameters were reported to be altered in A* cells
Cells with [SH]TCDD resulted in formationof the radio- receptor [29, 30], and treatmentof these cells with Ah receptor labeled aryl hydrocarbo(nAh)6 S nuclear receptor com- agonist such as 2,3,7,8-tetrachlorodibenzo-p-dioxi(TnCDD) replex in both cell linesT. he loss of Ah responsiveness in sults in the rapid formation of liganded nuclear Ah receptor the BaPRvariant cells correlated with the failuorfethe complex and the inductioonf CYPIAl gene expression.Vickers nuclear or transformed cytosoliAc h receptor complex toet al. [29]have reported a correlation between Ah responsivebind genomic dioxin-responsive elemeanstdsetermined ness and estrogen receptor (ER) content in several human breast caelnlcleinres, in gel retardation assays
Wild-type MCF-7human breast cancer cells were maintained in long-term culturefor 3 monthsin media containingDMEIF12 and 5%fetal calf serum which containe1dp~ BaP
Summary
Chemicals-Ethoxyresorufin, TCDD,[3H]TCDD (32Ci/mmol),and 2,3,7,8-tetrachlorodibenzofurawn ere prepared in this laboratory to DMSO, dimethyl sulfoxide;BSA, bovine serum albumin; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; BaP,benzo[alpyrene. The resistant cell lines were initially screened by Activity by Laser Cytometry-The effects of TCDD on GSH levels and determining thienducibility of etholryresorufin 0-deethylase activitbyy glutathioneS-transferase activity in wild-type and BaPR mutant. MCF-7 and BaPRcells were plated incover glass chambers(100,000 twice in 30 ml of HEGD buffer (25 m HEPES, 1.5 m~ EDTA, and 1IIM celldchamber), and, after incubation for 24 h, the cells were treated dithiothreitol, and 10% glycerol, pH 7.6). To synthesize cDNA, 100 ng of poly(A+)RNA was incubated at 37 "C for 60 min in a 20-111 reaction mixture containing m~ Tris.HC1,pH 8.3at 37 "C,8 m~ MgCI,, 50 mM KCl, 2 m~ concentration of each dNTP, 20 units of RNAguard The gels were dried, and protein-DNA inter- of each amplification primer
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