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A homogeneous cell-based assay to identify N-linked carbohydrate processing inhibitors
A Datti1, R S Donovan, B Korczak
1GlycoDesign Inc., Toronto, Ontario, Canada. adatti@glycodesign.com
Analytical Biochemistry
|May 11, 2000
Summary
Researchers developed a cell-based assay to find anticancer drugs that block sugar molecule (beta1-6GlcNAc-branched glycan) production. This assay identifies compounds that protect tumor cells from a toxic lectin, aiding cancer research.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Malignant transformation involves changes in cell surface glycosylation.
- N-linked oligosaccharides with beta1-6GlcNAc branches correlate with tumor invasion and metastasis.
Purpose of the Study:
- To develop a cell-based assay for identifying compounds that inhibit beta1-6GlcNAc-branched glycan biosynthesis.
- To find potential anticancer agents that block specific glycan structures without causing significant cell death.
Main Methods:
- A homogeneous cell-based assay using MDAY-D2 tumor cells was developed.
- The assay detects compounds that block beta1-6GlcNAc-branched glycan expression by assessing protection against the cytotoxic plant lectin leukoagglutinin.
- Alkaline phosphatase activity, which is proportional to cell number, was measured spectrophotometrically to quantify cell viability.
Main Results:
- The assay successfully identified agents that inhibit beta1-6GlcNAc-branched glycan expression.
- The alkaline phosphatase assay could detect as few as 1,500 cells.
- The assay demonstrated adaptability for high-throughput screening with a coefficient of variation of 4.2% for controls.
Conclusions:
- The developed cell-based assay is effective for screening compounds that inhibit beta1-6GlcNAc-branched glycan biosynthesis.
- This assay format shows potential for broader applications in high-throughput screening for compounds targeting other cellular growth inhibitors.