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Isolation of Diallyl Trisulfide Inducible Differentially Expressed Genes in Human Gastric Cancer Cells by Modified Cdna Representational Difference Analysis

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Isolation of Diallyl Trisulfide Inducible Differentially Expressed Genes in Human Gastric Cancer Cells by Modified Cdna Representational Difference Analysis http://www.paper.edu.cn DNA AND CELL BIOLOGY Volume 21, Number 11, 2002 © Mary Ann Liebert, Inc. Pp. 771–780 Isolation of Diallyl Trisulfide Inducible Differentially Expressed Genes in Human Gastric Cancer Cells by Modified cDNA Representational Difference Analysis YONG LI and YOU-YONG LU ABSTRACT Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gas- tric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to de- velop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirec- tional subtractive hybridization between the two resultant first-round difference products. Bidirectional sub- tractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible dif- ferentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downreg- ulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sam- ple cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer. INTRODUCTION fects by modulating intracellular Ca21 concentration (Sakamoto et al., 1997), cytochrome P4501 (Chun et al., 2001), NAD ARLIC HAS BEEN USED for thousands of years as a tradi- (P)H:quinone oxidoreductase (Singh et al., 1998), quinone re- Gtional Chinese medicine and is used as a prophylactic drug ductase, and glutathione transferase (Munday and Munday, throughout the world. Epidemiologic studies in China and Italy 2001). However, thorough investigations into the molecular indicated protective effects of garlic against human gastric can- mechanisms of action of DATS are rare, and a definitive mech- cer (HGC) (Buiatti et al., 1989; You et al., 1989, 1998; Fleis- anism of anticarcinogenic activities of garlic has not been es- chauer et al., 2000). Allyl sulfides such as diallyl sulfide (DAS), tablished. diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical cDNA representational difference analysis (cDNA RDA) flavor components of garlic, were shown to inhibit prolifera- (Hubank and Schatz, 1994) and mRNA differential display tion of tumor cells in culture and chemically induced tumors in (mRNA DD) (Liang and Pardee, 1992) are both PCR-based experimental animals, such as colon cancer (Wargovich, 1987), techniques for cloning differentially expressed genes. To effi- breast cancer (Sundaram and Milner, 1993), and lung cancer ciently isolate difference mRNA species with very small dif- (Sakamoto et al., 1997). DATS, as a critical organic allyl sul- ferences between two related sample cDNAs, we combined the fur component, was reported to exert its chemopreventive ef- high specific subtractive hybridization of cDNA RDA with the Peking University, School of Oncology & Beijing Institute for Cancer Research, Beijing Molecular Oncology Laboratory, Beijing, China. 转载 771 中国科技论文在线 http://www.paper.edu.cn 772 LI AND LU sensitive bidirectional radioactive detection of mRNA DD and anol. Approximately 106 cells were incubated with 25 mg/ml developed a subtractive hybridization differential display propidium iodide (Sigma, St. Louis, MO), and DNA content (SHDD) method. Compared with cDNA RDA, the major char- was determined with a FACSCalibur (Becton Dickinson, Fuller- acteristics of this modified method include: first, performing a ton, CA). Data were plotted by using CellQuest software. first round of bidirectional subtractive hybridization between two sample cDNAs and getting two first-round difference prod- Total RNA isolation and complementary ucts (DP1s); next, performing a second round of bidirectional DNA (cDNA) synthesis subtractive hybridization between two resultant DP1s and ob- Approximately 400 mg of total RNA from BGC823 cells af- taining two second-round difference products (DP2s); finally, ter exposure to DATS treatment for 48 h was isolated using labeling resultant DP1s and DP2s with a-35S-dATP and sepa- TRIzol reagent and 2 mg of poly(A)1RNA was purified using rating these difference products on DNA sequencing gel. Bidi- rectional subtractive hybridizations, especially the second round Poly(A)1Quick mRNA Isolation Kit (Stratagene, La Jolla, of hybridization, magnified the differences between the two CA). Double-stranded cDNA (ds-cDNA) was synthesized us- sample cDNAs and thus favored isolating mRNA species with ing cDNA Synthesis Kit (Gibco BRL). RNA samples derived from paternal BGC823 cells were processed in parallel. only tiny differences in expression. In our study, we carried out flow cytometric analysis and Subtractive hybridization differential display (SHDD) DNA fragmentation analysis, and showed that DATS treatment induces G1/S arrest and apoptosis in HGC cell line BGC823 Generation of representations. Double-stranded cDNAs cells. Based on these findings, we further employed the SHDD prepared from DAT823 and BGC823 cells were digested with method to isolate DATS inducible differentially expressed Xho I in a mixture containing 2 mg of cDNAs, 20 units of genes between two sample cDNAs derived from DATS-treated Xho I (Promega, Madison, WI), and 1 3 buffer E in a final vol- BGC823 (DAT823) cells and paternal BGC823 cells. ume of 40 ml at 37°C for 4 h. After phenol extraction, ethanol precipitation and resuspension in 20 ml of 1 3 TE, digested cDNAs were then ligated to Xho I Linker-15 (59-TCGAG- MATERIALS AND METHODS GATCCATTCA-39) and Xho I Linker-13 (59-ACTGAATG- Cell culture and treatment of DATS GATCC-39) in a total volume of 98 ml containing 1.5 mM Xho I Linker-15, 1.5 mM Xho I Linker-13 and 1 3 T4 ligase buffer. The HGC cell line BGC823 was constructed by The First Oligonucleotides were annealed to digested cDNAs by cooling People’s Hospital of Peking University, China (Deng et al., the mixture gradually from 50 to 10°C within 1 h, and ligation 1987). BGC823 cells were grown in DMEM medium supple- carried out by adding 800 units of T4 DNA ligase (New En- mented with 5% fetal calf serum and 5% CO2 concentration. gland Biolabs, Beverly, MA) and incubating at 16°C for 16 h. Diallyl trisulfide (DATS) used in this study (97.98% purity) Ligations were digested with BamH I and then ligated to 1R- was ordered from Shang-Hai-He-Feng Pharmacy Company Bam-12/24 adapters in the same manner described above. Fol- (Shanghai, China). BGC823 cells were incubated in medium lowing the procedure provided by Lisitsyn et al. (1993), mul- containing 25 mg/ml DATS for 24, 48, 72, or 96 h. Morpho- tiple PCR reactions were set up to generate amplicons logic changes and growth ability were monitored under the light (representations) using 1R-Bam-24 as the primer. Subse- microscopy, then the cells were harvested and total RNA was quently, the R-adapters were removed from the representa- isolated using TRIzol reagent (Gibco BRL, Grand Island, NY). tions of DAT823 and BGC823 (DAT representation and BGC representation) with BamH I to form the drivers (D-DAT rep- DNA fragmentation analysis resentation and D-BGC representation, about 20 mg each). Two micrograms of D-DAT representation and 2 mg of D- Paternal BGC823 cells and DATS-treated (DAT823) cells BGC representation were ligated to 2J-Bam-12/24 adapters to (5 3 106 each) were collected and washed with cold phosphate- generate the testers (T-DAT representation and T-BGC rep- buffered saline (PBS). The cell pellets were suspended in 100 resentation). Sequences of the oligonucleotides used here were ml of 10 mM Tris-HCl (pH7.6), 100 mM NaCl, 10 mM EDTA, as follows: 1R-Bam-24 59-AGCACTCTCCAGCCTCTCAC- and 0.5% SDS, and incubated at 37°C for 15 min. The sus- CGAG-39; 1R-Bam-12 59-GATCCTCGGTGA-39; 2J Bam-24 pensions were transferred to 1.5-ml tubes and treated with 59-ACCGACGTCGACTATCCATGAACG-3 9; 2J-Bam-12 59- RNase A (1 mg/ml) at 37°C for 30 min. Proteininase K (1 GATCCGTTCATG-39; 3N-Bam-24 59-AGGCAACTGTGC- mg/ml) was added and the mixture then was incubated at 37°C TATCCGAGGGAG-39; 3N-Bam-12 59-GATCCTCCCTCG-39. overnight. After phenol extraction and cold ethanol precipita- The 59 end of 12-mer adapters is dephosphorylated. tion, the DNA pellets were suspended in 40 ml of 1 3 TE (10 mM Tris-HCl (pH7.6), 1 mM EDTA [pH8.0]). Aliquots (10 ml) The first round of bidirectional subtractive hybridization be- were loaded on a 1.5% agarose gel and electrophoresis was car- tween two representations derived from two sample cDNAs. ried out at 5 V/cm for 50 min in 1 3 TBE buffer (89 mM Tris- One microgram of T-DAT representation (tester) was mixed borate, 2 mM EDTA [pH 8.0]). The DNA was stained with with 20 mg of D-BGC representation (driver) and 1 mg of T- ethidium bromide and visualized under an UV illuminator. BGC representation (tester) mixed with 20 mg of D-DT repre- sentation (driver). The hybridization, PCR amplification of hy- Flow cytometric analysis bridized DNAs, and Mung Bean Nuclease (New England Paternal BGC823 cells and DAT823 cells after exposure to Biolabs) digestion of single-stranded DNAs (ss-DNAs) was DATS for 48 or 96 h were harvested and fixed with 70% eth- performed following the protocol of typical cDNA RDA 中国科技论文在线 http://www.paper.edu.cn ISOLATION OF DIALLYL TRISULFIDE INDUCIBLE EXPRESSED GENES BY MODIFIED cDNA RDA 773 FIG.
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