Abstract

Ahi-1 (Abelson helper integration site-1) is a novel gene that is commonly activated by proviral insertional mutagenesis in v-abl or myc-induced murine leukemias and lymphomas. Ahi-1 encodes a unique protein with known signaling features including SH3 and WD40-repeat domains but its normal function is unknown. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemic cell lines, as well as in the primary lin−CD34+CD38− leukemic stem cell-enriched population in patients with chronic phase CML. To further investigate the role of Ahi-1 as a potential co-operating oncogene relevant to BCR-ABL-mediated leukemogenesis, we compared the biological behavior of primitive murine hematopoietic cells from the bone marrow of 5-FU-treated adult C57BL/6 mice after their transduction with MSCV-Ahi-1-IRES-YFP, MSCV-BCR-ABL-IRES-GFP retroviruses, either alone or in combination. Quantitative real-time RT-PCR analysis of RNA from FACS-purified lin−YFP+ (Ahi-1+), lin−GFP+ (BCR-ABL+) and lin−YFP+GFP+ (Ahi-1+ + BCR-ABL+)-transduced bone marrow cells showed that Ahi-1 transcripts were present at 40-fold higher levels in the Ahi-1-transduced cells by comparison to the control cells transduced with the empty MIY vector. Immediately post-transduction, the Ahi-1-transduced cells produced a similar number of colonies as the MIY-transduced control cells in semi-solid cultures containing Steel factor (SF) + IL-3 + IL-6 + EPO, although a small proportion of the Ahi-1+ CFCs (~10%) were already growth factor-independent. In addition, the proliferative activity of the FACS-purified lin−YFP+ (Ahi-1+) cells (as indicated by the rate of expansion of viable cells in a week in liquid cultures containing SF, IL-3, and IL-6) was ~3-fold higher than in cultures initiated with control (lin−YFP+) cells. Moreover, after 4 weeks in longterm culture-initiating cell (LTC-IC) assays, the Ahi-1+ cells produced 2x more CFCs than the control cells. All of these endpoints (proliferative activity, growth factor-dependence and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. Interestingly, in cells that were co-transduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced as compared to cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold). Thus, overexpression of Ahi-1 alone deregulates the proliferation control of primitive murine hematopoietic cells and this is additive with the effects of BCR-ABL, suggesting that Ahi-1 and BCR-ABL can cooperate to promote the progression of BCR-ABL-associated diseases like CML.

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