Candidate Gene Targets of the c-Myb Protooncogene in Human Myloid Leukemia
Presenter: Susan E. Shetzline
Institution: University of Pennsylvania
- The c-Myb proto-oncogene encoded transcription factor, Myb is required for normal and malignant hematopoeisis. Ectopic c-Myb expression can transform hematopoetic cells, thus Myb may play a role in leukemogenesis.
- To identify potential downstream targets of Myb, which may be targets for future leukemia therapy, K562 cells were engineered to express an inducible dominant negative form of c-Myb. Downstream gene induction was assayed using micro-array technique and confirmed using PCR. 10,000 candidate genes were evaluated using this technique.
Materials and Methods
- A chimeric transcription actor composed of c-Myb's DNA binding domain, the engrailed protein transcription repression domain and a mutated estrogen receptor responsive to tamoxifen was created and stably transfected into a K562 cell line.
- The cells were cultured for 3 days with Tamoxifen, repressing transcription of myb regulated genes.
- These cells were processed for micro-array gene expression analysis. Confirmation of gene over or under-expression was performed for several candidate genes using PCR.
- Culture of this modified K562 cell line with tamoxifen led to reduced cell proliferation in-vitro. Suppression of Myb function led to:
- Greater than two-fold over-expression of 34 of 10,000 tested genes. Several of these, including cdc7 and neuromedin U are associated with erythroid or megakaryocytic differentiation. Cdc7 and neuromedin U over-expression was confirmed using PCR.
- Greater than two-fold under-expression of 70 genes was found.
- Previously identified Myb targets including bcl-2, c-kit, CD34 and c-myb showed less than 50% change in expression.
- It is unclear why dominant negative myb expression slows cell proliferation.
- A manageable number of potential myb regulated genes has been identified for further analysis.
- Neuromedin U and cdc7 overexpression has been confirmed using PCR. Further investigation into the role of these genes in hematopoetic cell cycle regulation is ongoing.
This study has identified several new potential molecular targets which may be important in the pathogenesis of leukemia. Further study of these downstream targets of c-Myb proto-oncogene expression may yield novel genetically targeted anti-leukemia therapies in the same mold as STI-571