died. CFC colonies formed by CML cells with suppression of AHI 1 were found to be smaller than those resulting from cells transduced with a control vector. It was interesting that more signifi cant reduction in CFC numbers was observed in transduced primary PD184352 MEK inhibitor CML cells from IM nonresponders and blast crisis compared with IM responders. Coexpression of Ahi 1 in BCR ABL inducible cells regulates transforming activities of BCR ABL cells To further investigate potential regulatory roles of Ahi 1 in mediating BCR ABL transforming activities, we evaluated cooperative  transduced BaF3 cell line in which the level of expression of p210 BCR ABL can be variably down regulated by exposure to doxycycline.
Reduction in BCR ABL protein expression in the presence of Dox results in a corresponding BAY 73-4506 decrease in GF independence of BaF3 cells both in liquid suspension cultures and in semisolid cultures in vitro. As shown in Fig. 6, cell growth and survival was dramatically reduced when Dox was added to culture at 48 h, while the same cells showed a marked increase in cell expansion in the absence of both IL 3 and Dox. Similarly, down regulation of BCR ABL expression completely inhibited CFC generation in semisolid cultures in the absence of IL 3. Interestingly, introduction of Ahi 1 into cells with inhibited BCRABL expression under these stringent conditions enabled them to grow continuously in liquid suspension culture, to have less Annexin V apoptotic cells, and to produce more factorindependent CFCs than cells transduced with BCR ABL alone.
These results were consistently observed in two individual clonal cell lines. In the presence of IL 3 and Dox, BCR ABL transduced BaF3 cells showed a signifi cant reduction in CFC production, addition of IL 3 cannot rescue inhibitory effects caused by suppression of BCR ABL expression. This can be enhanced signifi cantly by cotransduction of Ahi 1. Collectively, these results demonstrate the ability of Ahi 1 to immediately reverse in vitro growth defi ciencies resulting from down regulation of BCRABL in vitro, and provide direct evidence of the regulatory role of Ahi 1 in BCR ABL mediated transformation.
Coexpression of Ahi 1 in BCR ABL inducible cells sustains tyrosine phosphorylation of BCR ABL and enhances activation of JAK2 and STAT5 To determine whether coexpression of Ahi 1 in BCR ABL inducible BaF3 cells infl uences protein expression and tyrosine kinase activity of BCR ABL, and candidate downstream signaling that refl ects enhanced transforming phenotypes observed, we performed Q RT PCR and Western blot analyses in these cells in the presence and absence of Dox. As expected, BCR ABL transcripts were highly expressed without Dox and down regulated in the presence of Dox in the BCR ABL inducible cells after 24 48 h in culture. Elevated BCR ABL transcript levels were again observed in BCR ABL and Ahi 1 cotransduced cells generated from two individual cells lines. As expected, Ahi 1 transcripts were highly elevated in cotransduced cells when compared with control BaF3 cells. Strikingly, tyrosine phosphorylation of p210 BCR ABL could not sufficiently be suppressed in Ahi 1 and BCR ABL cotransduced cells, as compared with BCR ABL transduced cells alone in the presence of the same amount of D