[Isolation of E1A-related drug-sensitive new genes by suppression subtractive hybridization].

BACKGROUND & OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug-sensitivity of tumor cells to several antitumor agents, and also increase the radio-sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer-related genes,which were differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH).

METHODS

To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed-gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi-quantitative RT-PCR analysis.

RESULTS

The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over-expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over-expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi-quantitative RT-PCR analysis, and the difference of mRNA expression was approximately 3-8 times.

CONCLUSION

Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full- length genes and studying their functions.