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Mutational analysis of DBD*--a unique antileukemic gene sequence.
Yan-shan Ji1, Betty H Johnson, M Scott Webb
1Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77555-0645, USA.
Neoplasia (New York, N.Y.)
|August 23, 2002
Summary
A novel gene, DBD*, exhibits potent antileukemic activity by inducing cell death in leukemic cells. Mutations reveal its lethality mechanism is distinct from the native DNA binding domain, localizing optimal activity to its C-terminal sequence.
Area of Science:
- Molecular biology
- Cancer research
- Genetics
Background:
- DBD* is a novel 89 amino acid peptide derived from the DNA binding domain of the human glucocorticoid receptor.
- A frameshift mutation in DBD* replaces the C-terminal amino acids, suggesting altered function compared to the native domain.
Purpose of the Study:
- To investigate the functional significance of specific amino acid positions within DBD*.
- To confirm and extend previous findings on DBD*'s mechanism of action and DNA binding domain (DBD) function.
- To identify the specific DBD* sequence responsible for its antileukemic activity.
Main Methods:
- Site-directed mutagenesis of DBD* at 29 critical positions.
- Transient transfection of steroid-resistant leukemic ICR-27-4 cells via electroporation.
- Evaluation of cell viability using trypan blue exclusion, WST-1 assay, propidium iodide exclusion, and Hoechst 33258 staining.
Main Results:
- Eleven of 29 point mutants showed enhanced antileukemic activity, while four exhibited decreased activity.
- The observed effects of mutations on DBD* activity did not consistently correlate with native DBD function.
- Optimal antileukemic activity was localized to the C-terminal 32 amino acids of DBD*.
Conclusions:
- The cell-lethal mechanism of DBD* is distinct from that of the native glucocorticoid receptor DNA binding domain.
- Specific mutations can modulate the antileukemic potency of DBD*.
- The C-terminal region of DBD* is crucial for its potent antileukemic effects.