Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Cells and cell culture

We used two human bladder cancer cell lines, T24 (Bubenik et al., 1973) and RT4 (Rigby and Franks, 1970). We also used A431 cells (human epidermal cancer) as a positive control for STAT activation after EGF treatment. Chin et al. (1996) reported that EGF activated STAT1 and STAT3 in A431 cells, and induced the expression of p21waf1 mRNA, then EGF inhibited the growth of A431 cells. All three cancer cells were grown in DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS) (GIBCO), 100 mg/ml streptomycin, and 100 U/ml penicillin (GIBCO), and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37¡C.

1T-1, 1T-2, and 1T-3 cells were immortalized human urothelial cells which were established in our laboratory from normal ureter after infection with a retroviral vector carrying human papilloma virus onco-protein E6 and E7 (Hattori and Oyasu, manuscript in preparation). Cells were grown in Keratinocyte-SFM (GIBCO) supplemented with 100 mg/ml streptomycin, and 100 U/ml penicillin (GIBCO), and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37¡C.

We examined the in vitro effect of EGF on the cell growth by MTT [3-(3, 4-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (Sigma, St. Louis, MO)] assay (Carmichael et al., 1987). Cells were seeded on 96-well plate (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) at 2 x 103 cells per well for A431, and 5 x 103 cells per well for T24 and RT4 cells in DMEM containing 10% FCS, and Keratinocyte-SFM for 1T-1, 1T-2 and 1T-3 cells. After 24 hr, cells were washed with DMEM without FCS or Keratinocyte-SFM twice and placed in DMEM without FCS or Keratinocyte-SFM in the presence or absence of EGF (GIBCO; 0, 10, 100 ng/ml). After 2 and 4 days, the number of cells was quantitated by MTT assay.

Cells were cultured to sub-confluence. After treatment with EGF (100 ng/ml) for 15 min, the cells were washed with 100 mM phosphate buffered saline on ice three times, and lysed with 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 0.5% Nonidet P-40, 20 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 100 mM p-nitrophenyl phosphate, 5 U/ml aprotinin, 1 mM PMSF. The protein concentration of the samples were determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). Twenty microgram protein samples were electrophoresed on SDS-polyacrylamide gel. Proteins from gels were transferred to nitrocellulose (Bio-Rad), and EGFR, phosphorylated tyrosine, MAPKs (ERK1 and ERK2), and activated MAPKs were detected on nitrocellulose with rabbit anti-human EGFR antibody (Upstate Biotechnology, Lake Placid, NY), rabbit anti-phospho-tyrosine antibody (Oncogene Science, Uniondale, NY), with rabbit anti-human MAPK antibody (New England Biolabs, Beverly, MA), with rabbit anti-human phospho-MAPK antibody (New England Biolabs) and an Amersham ECL kit (Amersham, Arlington Heights, IL).

Cell lysates were prepared according to the method of Chin et al. (1996). In brief, cells were lysed with 50 mM HEPES-KOH buffer (pH 7.9) containing 400 mM NaCl, 0.2% NP-40, 10% Glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 0.5 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstain. Double stranded oligonucleotides, p21-SIE1 (5'-CTTCCCGGAAG-3') which was located at -640 nucleotides from the TATA box of p21 promoter (Chin et al., 1996; El-Deiry et al., 1993), and M67-SIE (5'-GTCGACATTTCCCGTAAATC-3') which was a STAT-binding site of the c-fos promoter, were labeled with [g-32P]ATP using T4 polynucleotide kinase, and purified by a spin column system (Pharmacia). Binding reactions were containing 5 mg of cell lysate in a binding buffer of 20 mM HEPES-KOH (pH 7.9), 0.1 mM EDTA, 0.01% NP-40, 100 mg/ml of poly(dI-dC) (Pharmacia), and 5% glycerol. The reactions were incubated for 20 min at room temperature before loading on 6% polyacrylamide gel in low ionic strength 0.5 x TBE buffer. The gels were run at 150 V on ice for approximately 1 hr, and dried. The dried gels were exposed to an X-ray film with an intensifying screen at -70 ¡C. For supershift experiments, polyclonal antiserum against STAT1 (Santa Cruz Biotechnology, Santa Cruz, CA) and STAT3 (Santa Cruz Biotechnology) were added to the binding reaction and were incubated for 20 min at room temperature before addition of the radio-labeled oligonucleotide.

Cells grown in monolayers were harvested at early confluency. RNA was prepared by lysing of cells in a hypotonic buffer containing NP-40 (Sigma), followed by removal of nuclei. Cytoplasmic RNA (20 mg) was electrophoresed onto a formaldehyde/1.0% agarose gel and blotted onto a nylon filter (Amersham). The nylon filter was hybridized with 32P-labeled cDNA probes in 50% formamide, 5 x saline-sodium phosphate-EDTA, 0.1% sodium dodecyl sulfate (SDS), 5 x Denhardt's solution, and 100 mg/ml salmon sperm DNA at 42¡C for 15-20 h. Extensive washing was done, twice with 0.1 x standard saline citrate-0.5% SDS at room temperature and once at 65¡C for 40 min with the same washing buffer. Subsequently, the filter was exposed to an X-ray film with an intensifying screen at -70¡C. The probes used were a an 875 bp fragment of human p21waf1 cDNA isolated in our laboratory (Sato et al., 1997), and a 2.1 kb Xho1-Xho1 fragment of pHFbA-1 for human b-actin (American Type Culture Collection).

For semi-quantitative RT-PCR, cytoplasmic RNA from the cells was reverse-transcribed by Moloney-Murine Leukemia virus reverse-transcriptase (Gibco) at 42 ¡C for 60 min using random primer (5 mM; Gibco) in 20 ml of the reaction mixture. Subsequently one microliter of the products was subjected to the PCR amplification. PCR was performed as follows; the final concentration of dNTPs and primers in the reaction mixture is 200 mM and 1 mM respectively. Taq DNA polymerase (Takara, Otsu, Japan) was added to the mixture at a final concentration of 0.05 U/ml and the reaction was carried out in Takara Thermal Cycler MP (Takara) under following conditions; 94 ¡C for 3 min and then 94 ¡C for 1 min, 55 ¡C for 1.5 min, 72 ¡C for 2.5 min for desired cycles and an extension of 72 ¡C for 4 min.

To determine the appropriate condition for semi-quantitative RT-PCR, we reverse-transcribed 0.05, 0.5, 5, 10 mg of cytoplasmic RNA and amplified the fragment by the same PCR condition with different cycle number (16, 20, 24, 28, 32 cycles). Then we decided to use 5 mg of cytoplasmic RNA as a template for reverse-transcriptation and to use 28 cycles of PCR amplification for EGFR, and 20 cycles for GAPDH. The primers used here were; EGFR-UP, 5'-ATCATCCAATTCTCCAAAATGGC-3', EGFR-DN, 5'-GGGTTCAGAGGCTGATTGTGATAG-3', GAPDH-UP, 5'-GAAATCCCATCACCATCTTCCAGG-3', and GAPDH-DN, 5'-CATGTGGGCCATGAGGTCCACCAC-3'.

The human wild-type p21waf1 promoter luciferase fusion plasmid, WWP-Luc (El-Deiry et al., 1993), was kindly gifted from Dr. B. Vogelstein (Johns Hopkins Oncology Center). The 2.4 kb genomic fragment containing the p21-SIE1 at -640 from the TATA box of p21waf1 promoter was excised from WWP-Luc by Hind III, and the fragment was subcloned into the Hind III site of the luciferase reporter plasmid, pGL3-Basic (Promega, Madison, WI) to generate pGL3-WWP.

T24 cells and RT4 cells (2.5 x 105 cells/dish) were seeded in 60 mm culture dishes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) in DMEM supplemented with 10% FCS. Twenty-four h later, the cells were transfected with 5 mg of pGL3-WWP using Superfect regent (Qiagen, Valencia, CA). Fifteen hr after transfection, EGF (100 ng/ml) treatment was started. After 10 hour EGF treatment, cell lysates were collected for a luciferase assay. The luciferase activities of the cell lysates were measured by Promega luciferase assay kit according to the manufactures' instruction. Luciferase activities were normalized by the amount of the protein in cell lysates. As a preliminary experiment, we examined the transfection efficiency of Superfect regent on our cells. The transfection efficiency of Superfect regent was much higher than those of other cationic regents in our cells, and the efficiency was very stable beyond the experiments (data not shown). Therefore, we normalized the luciferase activities by the amount of the protein content rather than by the expression of the other transfection control vector. Each experiment was repeated at least three times.

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