Cisplatin Induction of ERCC-1 mRNA Expression in A2780/CP70 Human Ovarian Cancer Cells
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23419
ISSN1083-351X
AutoresQingdi Li, Kevin Gardner, Lijuan Zhang, Byron Tsang, Benjamín C. Bostick, Eddie Reed,
Tópico(s)DNA Repair Mechanisms
ResumoERCC-1 is a critical gene within the nucleotide excision repair pathway, and cells without a functionalERCC-1 do not perform cisplatin-DNA adduct repair. We therefore investigated the cisplatin effect on ERCC-1mRNA expression in vitro. In response to a 1-h cisplatin exposure, A2780/CP70 human ovarian cancer cells showed a 6-fold increase in steady-state level of ERCC-1 mRNA. This rise was attributable to increased transcription as measured by nuclear run-on assays and a 60% increase in ERCC-1mRNA half-life. The increase in ERCC-1 mRNA was preceded by a 4–5-fold rise in mRNA expressions of c-fos and c-jun, a 14-fold increase in c-Jun protein phosphorylation, and an increase in in vitronuclear extract binding activity to the AP-1-like site of ERCC-1. These data suggest that the induction of ERCC-1 expression in A2780/CP70 cells exposed to cisplatin results from two major factors: (a) an increase in the expression of transactivating factors that bind the AP-1-like site in the 5′-flanking region of ERCC-1 and (b) an increase in the level of c-Jun phosphorylation that enhances its transactivation property. ERCC-1 is a critical gene within the nucleotide excision repair pathway, and cells without a functionalERCC-1 do not perform cisplatin-DNA adduct repair. We therefore investigated the cisplatin effect on ERCC-1mRNA expression in vitro. In response to a 1-h cisplatin exposure, A2780/CP70 human ovarian cancer cells showed a 6-fold increase in steady-state level of ERCC-1 mRNA. This rise was attributable to increased transcription as measured by nuclear run-on assays and a 60% increase in ERCC-1mRNA half-life. The increase in ERCC-1 mRNA was preceded by a 4–5-fold rise in mRNA expressions of c-fos and c-jun, a 14-fold increase in c-Jun protein phosphorylation, and an increase in in vitronuclear extract binding activity to the AP-1-like site of ERCC-1. These data suggest that the induction of ERCC-1 expression in A2780/CP70 cells exposed to cisplatin results from two major factors: (a) an increase in the expression of transactivating factors that bind the AP-1-like site in the 5′-flanking region of ERCC-1 and (b) an increase in the level of c-Jun phosphorylation that enhances its transactivation property. cis-diamminedichloroplatinum (II) nucleotide excision repair excision repair cross-complementation group 1 activator protein 1 glyceraldehyde-3-phosphate dehydrogenase c-Jun NH2-terminal kinase/stress-activated protein kinase microculture tetrazolium electrophoretic mobility shift assay cAMP response element-binding protein base pair(s). cis-Diamminedichloroplatinum (II) (cisplatin)1 is one of the most widely used chemotherapeutic agents for the treatment of human ovarian cancer and other tumors (1Reed E. PPO Update. 1996; 10: 1-12Google Scholar, 2Reed E. Dabholkar M. Chabner B.A. Chabner B.A. Longo D.L. 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EMBO J. 1993; 12: 479-487Crossref PubMed Scopus (342) Google Scholar). Therefore, it is possible that the effect of cisplatin on ERCC-1 could be through AP-1 induction or c-Jun phosphorylation. Because methodological problems would limit investigations of mechanism in ovarian cancer tissues taken from patients, we have now conducted studies in the human ovarian cancer cell line, A2780/CP70, to investigate these possibilities. The human ovarian cancer cell line A2780/CP70 has been described previously (42Behrens B.C. Hamilton T.C. Masuda H. Cancer Res. 1987; 47: 414-418PubMed Google Scholar) and were used in all experiments. Cells were cultured in monolayer using RPMI 1640 media supplemented with 10% (v/v) fetal calf serum, 2 mml-glutamine, 0.2 units/ml human insulin, 50 units/ml penicillin, 50 μg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD). Cells were grown in logarithmic growth at 37 °C in a humidified atmosphere consisting of 5% CO2, 95% air. Cells were routinely tested for mycoplasmal infection using a commercial assay system (MycoTect; Life Technologies, Inc.), and new cultures were established monthly from frozen stocks. All media and reagents contained <0.1 ng/ml endotoxin as determined by Limulus polyphemus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, MD). Cell viability was determined in triplicate by trypan blue dye exclusion. Before starting the experiments, the cells were grown to ∼90% confluence after subculturing. Cisplatin (Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD) was initially dissolved in phosphate-buffered saline without Ca2+ or Mg2+at 1.0 mg/ml (3.33 mm cisplatin), and dilutions from this solution were made in media to obtain the desired drug treatment concentrations. The cisplatin dose for A2780/CP70 cells was 40 to 80 μm unless otherwise indicated. Cisplatin treatments were for 1 h. After drug treatments, cells were washed twice with phosphate-buffered saline without Ca2+ or Mg2+, given fresh drug-free media, and incubated for 24 to 48 h or the time indicated. Thereafter, the cells were harvested for use in the RNA and protein isolation assays. α-Amanitin (Calbiochem, San Diego, CA) and cycloheximide (Calbiochem) were dissolved in water. Cisplatin cytotoxicity was determined using the microculture tetrazolium assay, as described previously (43Lee K.B. Parker R.J. Reed E. Cancer Lett. 1995; 88: 57-66Crossref PubMed Scopus (14) Google Scholar). The concentrations of other drugs used in the studies were not toxic to the cells as confirmed by cell recoveries, trypan blue dye exclusion, and cytotoxicity assay. Total RNA was isolated from cells by acid guanidinium thiocyanate-phenol-chloroform extraction (44Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63169) Google Scholar), or using a commercial total RNA isolation reagent kit (Life Technologies, Inc.) according to the manufacturer's instructions. Thirty micrograms of denatured RNA per lane were separated by electrophoresis (Life Technologies, Inc.) through 1% agarose-formaldehyde and transferred to nylon membrane (Zeta-Probe GT; Bio-Rad) by electrophoretic transfer (Trans-Blot Cell; Bio-Rad). Membranes were prehybridized in Quik-Hyb (Stratagene, Menasha, WI) for 15–30 min at 68 °C and then hybridized for 1 to 2 h at 68 °C in Quik-Hyb containing 0.67 μg/ml denatured salmon testes DNA (Stratagene) and 32P-labeled cDNA probe. After washings of increasing stringency, the membranes were air dried, exposed to Kodak XAR-5 x-ray film with intensifying screens at −80 °C, and then analyzed by Collage Analysis (Fotodyne Inc., New Berlin, WI) and quantitated by densitometrical scanning. Before hybridization with a second labeled cDNA probe, the first probe was removed by washing for 2 h at 75 °C in 1 mmTris-HCl (pH 8.0) containing 1 mm EDTA and 0.1 × Denhardt's solution (45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The entire sequence of experiments (including growth of A2780/CP70 cells, drug treatment, and Northern blotting and hybridization) was performed and the results reproduced in two or more separate experiments. Equal RNA loading was determined by visualization of 18 S and 28 S ribosomal RNA bands in ethidium bromide-stained gels and quantification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene transcript on Northern blots. A 1.05-kilobase cDNA probe for human ERCC-1 was obtained from Dr. Aziz Sancar (University of North Carolina, Chapel Hill, NC). A 0.8-kilobase cDNA for human GAPDH was obtained from Dr. Mitchell Olman (University of California, San Diego, CA). The c-fos and a c-jun probes were obtained commercially from Oncogene Research Products (Cambridge, MA). cDNA inserts were excised using appropriate restriction enzymes, isolated by electrophoresis through 1% agarose onto DEAE-membrane (NA-45; Schleicher & Scheull) (45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and purified by using the GeneClean II Kit (BIO 101 Inc., La Jolla, CA). cDNA was labeled with 32P using a commercial random primer kit (Life Technologies, Inc.) according to the manufacturer's instructions. ERCC-1transcription rate was measured using a modification of previously described nuclear run-on analysis (46Raj N.B.K. Pitha P.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3923-3927Crossref PubMed Scopus (85) Google Scholar). A2780/CP70 cells grown to ∼90% confluence were lysed in 10 ml of lysis buffer, containing 10 mm Tris (pH 8.0), 2.5 mm MgCl2, 0.25% Triton X-100, 0.3 m sucrose, and 1 mmdithiothreitol, and nuclei were collected by centrifugation for 5 min at 500 × g. Isolated nuclei were incubated with 250 μCi of [α-32P]UTP (NEN Life Science Products, Wilmington, DE) for 30 min at 37 °C. cDNA for ERCC-1and GAPDH, or vector DNA (the plasmid without ERCC-1cDNA insert) (5 μg of DNA per blot) used in the run-on assay was heat denatured and transferred to supported nitrocellulose (Life Technologies, Inc.) by vacuum filtration using a 24-well manifold (Hybri-Dot; Life Technologies, Inc.). The membrane was rinsed with 6 × sodium chloride-sodium citrate (SSC), air dried, and baked at 80 °C for 2 h in a vacuum oven. Membranes were prehybridized for 1 h at 42 °C in 50% formamide, 6 × SSC, 5 × Denhardt's solution, 0.5% sodium dodecyl sulfate, and 100 μg/ml denatured salmon testes DNA and then hybridized at 42 °C for 3 days in prehybridization buffer containing run-on reaction mixtures adjusted to equalize radioactivity added in all reactions. Membranes were extensively washed with increasing stringency and then treated with 1 μg/ml ribonuclease for 30 min at 37 °C. Membranes were air dried, exposed to XAR-5 film, and quantitation of the results was achieved by densitometric scanning normalized to the signal for GAPDH. A standard technique for measuring stability of labile transcripts was used (47Belasco J.G. Brawerman G. Belasco J.G. Brawerman G. Control of Messenger RNA Stability. Academic Press, San Diego, CA1993: 475-493Crossref Google Scholar). Cells were incubated with fresh drug-free medium for 36 h following treatment with 40 μm cisplatin for 1 h or left without treatment as control, after which α-amanitin (5 μg/ml) or actinomycin D (5 μg/ml) was added to the treated and control flasks. Total RNA was isolated at the time of α-amanitin or actinomycin D addition or at different times thereafter. Northern blot analysis was performed to determine mRNA levels. To prepare whole cell lysates, 2 × 107cells were washed 3 times in ice-cold phosphate-buffered saline, and resuspended in 500 μl of buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mm EDTA, 1 μg/ml leupeptin, 1 μg/ml pepstain, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 1 mm sodium fluoride at 4 °C. Lysates were sheared through a 21-gauge needle and clarified at 4 °C by microcentrifugation. Protein content in the supernatants was determined by means of the BCA protein assay (Pierce, Rockford, IL) using bovine serum albumin as the standard. The cell extract proteins (25 μg) were loaded on a 12% Tris glycine gel (Novex, San Diego, CA), electrophoresed, and transblotted to a Protran (pure nitrocellulose) membrane (Schleicher & Schuell) by the procedure described by Towbin et al. (48Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44915) Google Scholar). The blot was then rinsed with TBS, and incubated with a blocking buffer (1 × TBS, 0.1% Tween 20 with 5% (w/v) nonfat dry milk) overnight at 4 °C. After rinsing with TBS/Tween (0.1%) three times, the blot was incubated with phosho-c-Jun (Ser63 and Ser73) antibodies (1:1,000 in TBS/Tween with milk) overnight at 4 °C on a shaker. The blot was washed three times with TBS/Tween, and incubated with a 1:2,000 dilution of secondary antibody coupled with horseradish peroxidase for 2 h at room temperature on a shaker. The blot was washed three times with TBS/Tween and then incubated with ECL Western blotting detection reagents (Amersham) for 1 min at room temperature. The blot was exposed to x-ray film to visualize the results. Nuclear extracts were prepared from resting or cisplatin-treated A2780/CP70 cells by a modification of the procedure described by Dignam et al.(49Dignam J.P. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9155) Google Scholar). Cells were harvested by scrapping and washed once with ice-cold phosphate-buffered saline. The cells were then resuspended in 1.5 volumes of lysis buffer (70 mm KCl, 1.5 mmMgCl2, 0.5 mm sodium orthovanadate, 0.4 mm sodium fluoride, 0.5 mm phenylmethylsulfonyl fluoride, 1.0 mm dithiothreitol, 25 mm HEPES, pH 7.5). The mixture was incubated on ice for 20 min and then extracted by adding 1.6 volumes of extraction buffer (0.5 mm EDTA, 20% glycerol, 1.66 m KCl, 0.4 mm sodium fluoride, 0.4 mm sodium orthovanadate, 0.1 mmphenylmethylsulfonyl fluoride, 1.0 mm1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, 25 mm HEPES, pH 7.5) with constant shaking at 4 °C for 4 h. Samples were centrifuged at 55,000 × g for 1 h at 4 °C, and the supernatant was dialyzed at 4 °C for 4 h in a buffer containing 20 mm HEPES (pH 7.5), 50 mm KCl, 0.1 mm EDTA, 10% glycerol, 0.4 mm sodium fluoride, 0.4 mm sodium orthovanadate, 0.1 mm phenylmethylsulfonyl fluoride, 1.0 mm dithiothreitol. Samples were stored at −80 °C. Protein content was determined by the BCA protein assay (Pierce). The oligonucleotide sequence used in the following electrophoretic mobility shift assay was based on sequence analysis of the 5′-flanking region of ERCC-1 gene as described previously (28van Duin M. Koken M.H.M. van den Tol J. Odijk H. Bootsma D. Hoeijmakers J.H.J. Nucleic Acids Res. 1987; 15: 9195-9213Crossref PubMed Scopus (71) Google Scholar). Two duplex 21-bp oligonucleotides which encompassed a ERCC-1 AP-1-like site (5′-TCACTGCTGTGTCACCAGCAC-3−, within −355 to −375 from the transcriptional start site at +1 in theERCC-1 promoter region) (see Fig. 1) and an alteredERCC-1 AP-1-like site (5′-TCACTGCTGAGTCACCAGCAC-3−, −355 to −375) were synthesized by Lofstrand Labs Limited (Gaithersburg, MD) and purified by reverse-phase cartridge chromatography. The alteredERCC-1 AP-1-like site contains a concensus AP-1 site produced by a 1-bp substitution from thymidine to adenosine as indicated by the underline. The double-stranded oligonucleotides were labeled with [α-32P]ATP by phosphorylation with bacteriophage T4 polynucleotide kinase and unincorporated precursors were removed using G-25 Sephadex columns (Boehringer Mannheim). 21-bp oligonucleotides that contained the accepted consensus sequence for AP-1, AP-2, CREB, TFIID, and NFκB were obtained from Promega Corp. (Madison, WI) and used in binding or competition studies described below. The nuclear extracts were analyzed for transcription factor binding activity by gel mobility shift assays. Briefly, nuclear extracts were incubated in a 20-μl volume with 1 × binding buffer (1 mmMgCl2, 0.5 mm EDTA, 0.5 mm1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, 50 mm NaCl, 4% glycerol, 10 mm Tris-HCl, pH 7.5) and 2 μg of poly(dI-dC)·poly(dI-dC) (Pharmacia, Piscataway, NJ) at room temperature for 10 min. The 32P-labeled target DNA was then added and the mixture was incubated for 20–30 min at room temperature. In some experiments, a 50-fold concentration of unlabeled competitor DNA was included in the sample prior to the addition of the radiolabeled probe. After the completion of the binding reaction, 2 μl of 10 × gel loading buffer (250 mm Tris-HCl, pH 7.5, 0.2% bromphenol blue, 0.2% xylene cyanol, and 40% glycerol) was added and samples were electrophoresed at room temperature through a 4% nondenaturing polyacrylamide gel in 0.5 × TBE running buffer (0.045 m Tris borate, 0.001 m EDTA, pH 8.0) for 4 h at 100 V which had been pre-run at 100 V for 30 min prior the sample loading. The gels were dried under vacuum and visualized by autoradiography. The nuclear extracts were preincubated with antiserum at room temperature for 20–30 min before analysis by EMSA as described above. The human anti-Fos and anti-AP-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); the affinity purified human anti-Jun antibody has been previously described (50Powers C. Krutzsch H. Gardner K. J. Biol. Chem. 1996; 271: 30089-30095Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These sera specifically detect the presence of the corresponding transcription factor and do not interfere with nuclear factor binding. The A2780/CP70 cell line was treated with 40 μm cisplatin for 1 h, and the expression of ERCC-1 mRNA was measured at various time points following drug exposure. Cisplatin caused time and dose-dependent increases in ERCC-1 mRNA levels (51Li Q. Tsang B. Dabholkar M. Bostick-Bruton F. Reed E. Proc. Am. Assoc. Cancer Res. 1998; 39: 241Google Scholar). Northern analysis showed that ERCC-1 mRNA accumulation was increased by more than 2-fold as early as 6 h after incubation with 40 μm cisplatin and eventually attained a peak level of 5.5-fold increase at 24–48 h after cisplatin administration. Dose-response experiments showed that the effect of cisplatin was maximal at 40–80 μm with about a 6-fold increase in the ERCC-1 mRNA level. The ERCC-1increase was
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