Related Experiment Video
Updated: Aug 26, 2026
Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe
Published on: September 13, 2022
[Isolation of E1A-related drug-sensitive new genes by suppression subtractive hybridization]
Ye-Wei Ma1, Xin-Lai Qian, Qing-Zheng Zhao
1Cancer Institute,Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, PR China.
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.
Insights
Researchers identified novel anticancer genes by analyzing gene expression changes in tumor cells treated with the E1A gene. This study isolated ten new gene fragments that may play a role in E1A
Area of Science:
- Molecular Oncology
- Gene Expression Analysis
- Cancer Therapeutics
Context:
- The E1A gene is recognized for its tumor suppressive properties, inhibiting cancer growth, metastasis, and reversing malignant phenotypes.
- E1A enhances tumor cell sensitivity to chemotherapy and radiotherapy, but the specific genes mediating these effects are largely unknown.
- Understanding these E1A-associated genes is crucial for developing novel cancer treatment strategies.
Purpose:
- To identify novel anticancer-related genes differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH).
- To isolate and characterize gene fragments that are upregulated in response to E1A protein treatment in human lymph node metastasis tumor cells (LN686).
Summary:
- A suppression subtractive hybridization (SSH) library was constructed using E1A-treated LN686 cells (tester) and parental LN686 cells (driver).
- Analysis of the SSH library yielded 362 clones with inserted fragments, of which 10 were identified as novel expressed sequence tags (ESTs).
- Semi-quantitative RT-PCR confirmed significant overexpression (3-8 times) of seven novel genes in E1A-treated cells compared to controls.
Impact:
- This study successfully isolated ten novel gene fragments associated with E1A's anticancer effects.
- These findings provide a foundation for further research into the full-length genes and their functional roles in cancer suppression.
- The identified genes represent potential targets for future development of targeted cancer therapies.
