Related Experiment Videos
Involvement of a DNA binding protein, MDR-NF1/YB-1, in human MDR1 gene expression by actinomycin D
Abstract:
The human multidrug resistance 1 (MDR1) gene is an SOS gene that responds to environmental stress including various anticancer agents. The chloramphenicol acetyltransferase (CAT) gene was linked to various lengths of MDR1 promoter, and these constructs were integrated into the genome of human cancer KB cells. Using these cell lines, we previously demonstrated that various environmental stimuli lead to an increased abundance of both CAT enzymatic activity and CAT mRNA in a sequence dependent manner. We examined the molecular mechanism of this stress response using actinomycin D, a potent RNA synthesis inhibitor. We found that CAT activity was significantly increased more than 10 fold by actinomycin D itself without comparable elevation of CAT mRNA. CAT induction was, however, lost in the presence of a deletion from position -136 to -76. Gel mobility shift assays showed that the specific DNA binding activity of the transacting protein, MDR-NF1/YB-1, which binds to the inverted CCAAT box, was augmented in nuclear extracts from the cells treated with actinomycin D. We also found that actinomycin D increased the steady state levels of MDR-NF1/YB-1 mRNA, which encodes the inverted CCAAT box binding protein. These results indicate that MDR-NF1/YB-1 mediates the response of the MDR1 gene to environmental stress.
Insights
Environmental stress activates the human multidrug resistance 1 (MDR1) gene via the MDR-NF1/YB-1 protein. This protein mediates increased chloramphenicol acetyltransferase (CAT) activity and mRNA levels in response to stress.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The human multidrug resistance 1 (MDR1) gene is an SOS gene.
- MDR1 responds to environmental stress, including anticancer agents.
- Previous studies linked chloramphenicol acetyltransferase (CAT) gene constructs to MDR1 promoters in human cancer cells, showing stress-induced increases in CAT activity and mRNA.
Purpose of the Study:
- To investigate the molecular mechanism of the MDR1 gene's stress response.
- To identify the specific DNA elements and proteins involved in stress-induced gene expression.
Main Methods:
- Utilized actinomycin D, an RNA synthesis inhibitor, to study stress response.
- Created cell lines with varying lengths of the MDR1 promoter linked to the CAT gene.
- Performed gel mobility shift assays to assess protein-DNA interactions.
- Quantified CAT enzymatic activity and CAT mRNA levels.
- Analyzed the effect of a specific promoter deletion (-136 to -76) on CAT induction.
- Measured steady-state levels of MDR-NF1/YB-1 mRNA.
Main Results:
- Actinomycin D significantly increased CAT activity (over 10-fold) without a comparable rise in CAT mRNA.
- This CAT induction was abolished by a deletion in the MDR1 promoter region (-136 to -76).
- Gel mobility shift assays revealed augmented DNA binding activity of MDR-NF1/YB-1 to the inverted CCAAT box in nuclear extracts from actinomycin D-treated cells.
- Actinomycin D also increased the steady-state levels of MDR-NF1/YB-1 mRNA.
Conclusions:
- MDR-NF1/YB-1 mediates the stress response of the MDR1 gene.
- The inverted CCAAT box in the MDR1 promoter is crucial for this stress-induced activation.
- The mechanism involves enhanced DNA binding and increased expression of the MDR-NF1/YB-1 protein.