The Isolation Of Differentially Expressed Genes In The G₂/M Arrest Induced By Diallyl Disulfide In HL-60 Cells Using Suppression Subtractive Hybridization

Objective: Diallyl disulfide(DADS), an oil-soluble allyl sulfur compound found in processed garlic, was found to inhibit the growth of various tumors by decreasing the cell growth rate, inducing apoptosis and cell cycle arrest. The previous studies in our laboratory showed that DADS induced the growth arrest of HL-60 cells in the G₂/M phase, but the detail mechanism by which DADS induced the cell cycle arrest remains to be elucidated. The aim of this study was to isolate and identify G2/M arrest-related genes, which were differentially expressed in HL-60 treated with or without DADS using suppression subtractive hybridization (SSH).Methods: An SSH cDNA library of HL-60 cells was constructed using DADS-treatedHL-60 cell mRNA as tester and untreated-HL-60 cell mRNA as driver, respectively. Positive clones in the library were randomly selected. The patterns of the differentially expressing-gene fragments were analyzed by PCR. Sequences of resulting cDNA fragments were compared with that of genes in GenBank by Blastn.The resulting expressed sequence tags (EST) were confirmed by RT-PCR based on mRNA from HL-60 cells with or without DADS treatment. Both 3'and 5'-terminal sequences of one novel EST were amplified by rapid amplification of cDNA ends (RACE). The primers for RACE were designed in the basis of a highly conserved region in the mentioned EST by Primer5.0. The cDNA from 3'RACE was cloned into pGEM-T Easy Vector, and the positive clones were sequenced. The construction and functions of the resulting gene were ananlyzed by Bioinformatics.Result: 220 positive clones, which contain 120 positive directional clones and 100...