| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
The E2A-HLF fusion transcription factor, which is generated by the t(17;19)(q22;p13) translocation, is found in a small population of pro-B cell ALL. Patients associated with this chimera share distinct clinical features such as hypercalcemia, coagulopathy, and very poor prognosis due to resistance to intensive chemotherapy including aggressive conditioning for BMT, all of which are unusual for this type of ALL. We previously demonstrated that inhibition of the trans-activation potential of E2A-HLF chimera by its dominant negative mutant results in apoptosis in t(17;19)^+ leukemia cells but does not affect cell cycle. Moreover, E2A-HLF blocks apoptosis induced by cytokine deprivation in IL-3-dependent cells, suggesting that this fusion protein contributes to leukemogenesis by substituting for the antiapoptotic function of cytokines. Here we show that survivin is a downstream target molecule of E2A-HLF. Four t(17;19)^+ ALL cell lines expressed survivin at high levels and downregulation
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of E2A-HLF function by its dominant negative mutant suppressed survivin expression. In addition, forced expression of E2A-HLF in Nalm-6, a t(17;19)^- ALL cell line, upregulated survivin expression. Survivin is known to be expressed in G2/M phase predominantly. Indeed, separation of the fractions enriched for in each phase of the cell cycle using a counterflow centrifugal elutriator revealed G2M phase-dominant survivin expression in t(17;19)^- ALL cells including Nalm-6. In t(17;19)^+ leukemia cells, however, survivin was expressed throughout the cell cycle. Moreover, Nalm-6 cells enforcedly expressing E2A-HLF showed cell cycle-independent survivin expression. Reporter assay revealed that E2A-HLF induced luciferase activity by transfecting with each reporter construct that contains survivin promoter in a different length from the initial ATG, suggesting that E2A-HLF induces survivin expression at the transcriptional level, but this effect is not by direct binding of E2A-HLF to survivin promoter. To test whether survivin plays antiapoptotic roles in t(17:19)^+ cells, we used a survivin mutant that lacks a phosphorylation site (T34A-survivin) and is considered to inhibit survivin function in a dominant negative manner. T34A-survivin induced massive apoptosis throughout cell cycle in t(17;19)^+ cells. By contrast, in t(17;19)^- cells, T34A-survivin induced cell death in only a small population in, G2/M phase. In addition to caspase-dependent pathways, T34A-survivin induced apoptosis in t(17;19)^+ ALL cells through caspase-independent pathways, in which apoptosis inducing factor (AIF) translocated from cytoplasm to nucleus. These results indicate that cell cycle-independent upregulation of survivin by the E2A-HLF chimera is indispensable for the survival of t(17;19)^+ ALL cells, and that inhibition of survivin may be an effective therapeutic strategy for this refractory ALL. Less
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