Figure 5
In vitro lymphoid transformation and in vivo leukemogenesis evoked by BCR-ABL1 are dependent on GAB2. (A) Agarose colony assays. BM from the indicated Balb/c donor (blue, Gab2+/+; red, Gab2−/−) mice was transduced with retrovirus coexpressing p210 BCR-ABL1 and either GFP or the indicated GAB2 variant. Transduced cells were plated directly in agarose as described in Methods, and transformed pre-B-lymphoid colonies were counted at day 10. The differences in colony numbers between p210-GFP–transduced Gab2+/+ and Gab2−/− BM and between p210-GFP–transduced and p210-Gab2WT–transduced Gab2−/− BM, were significant (P < .0001 and P = .001, respectively, unpaired Student t tests), whereas there was no difference between p210-GFP–transduced Gab2+/+ and p210-Gab2WT–transduced Gab2−/− BM (P = .224). The difference in colony numbers between Gab2−/− BM transduced with p210-Gab2WT retrovirus and either p210-Gab2Δp85 or p210-Gab2ΔShp2 retrovirus was significant (P = .0004 and P = .0002, respectively, unpaired Student t tests), whereas the difference between Gab2−/− BM transduced with p210-Gab2Δp85 and p210-Gab2ΔShp2 retrovirus did not reach significance (P = .0647, unpaired Student t test). (B) Whitlock-Witte assays. Freshly harvested Balb/c BM from Gab2+/+ (top) and Gab2−/− (bottom) donors was transduced with retrovirus coexpressing p210 BCR-ABL1 and either GFP or the indicated GAB2 variant, and plated in triplicate at the indicated cell numbers per well. Nontransduced cells were added to provide 106 total cells for stromal support. Wells were scored as positive when cell number reached 106 viable nonadherent cells per well, as described in Methods. (C) Extracts from immortalized BCR-ABL1–transformed B-lymphoid cell lines (from Figure 5B) from Gab2+/+ (WT) or Gab2−/− (knockout) donors were analyzed for GAB2 protein expression and phosphorylation, as described in Methods. (D) K-M curves of recipients of BM from Gab2+/+ littermates transduced with p210MIGFP retrovirus (blue solid line, n = 8), and of Gab2−/− donors (red lines) transduced with p210-IRESwt-Gab2WT retrovirus (dotted line, n = 8) or the indicated Gab2 mutant retrovirus (dashed lines, n = 8 for each cohort). All deaths were due to B-ALL. There was no significant difference in survival between recipients of p210MIGFP-transduced Gab2+/+ BM and p210-IRESwt-Gab2WT–transduced Gab2−/− BM (P = .493) or between recipients of p210MIGFP-transduced Gab2−/− BM and p210-IRESwt-Gab2ΔShp2–transduced Gab2−/− BM (P = .734). By contrast, the survival of recipients of p210MIGFP-transduced Gab2+/+ BM and p210MIGFP-transduced Gab2−/− BM, and of recipients p210-IRESwt-Gab2WT–transduced and p210-IRESwt-Gab2Δp85–transduced Gab2−/− BM, was different (P < .0001 and P = .001, respectively, Mantel-Cox tests).

In vitro lymphoid transformation and in vivo leukemogenesis evoked by BCR-ABL1 are dependent on GAB2. (A) Agarose colony assays. BM from the indicated Balb/c donor (blue, Gab2+/+; red, Gab2−/−) mice was transduced with retrovirus coexpressing p210 BCR-ABL1 and either GFP or the indicated GAB2 variant. Transduced cells were plated directly in agarose as described in Methods, and transformed pre-B-lymphoid colonies were counted at day 10. The differences in colony numbers between p210-GFP–transduced Gab2+/+ and Gab2−/− BM and between p210-GFP–transduced and p210-Gab2WT–transduced Gab2−/− BM, were significant (P < .0001 and P = .001, respectively, unpaired Student t tests), whereas there was no difference between p210-GFP–transduced Gab2+/+ and p210-Gab2WT–transduced Gab2−/− BM (P = .224). The difference in colony numbers between Gab2−/− BM transduced with p210-Gab2WT retrovirus and either p210-Gab2Δp85 or p210-Gab2ΔShp2 retrovirus was significant (P = .0004 and P = .0002, respectively, unpaired Student t tests), whereas the difference between Gab2−/− BM transduced with p210-Gab2Δp85 and p210-Gab2ΔShp2 retrovirus did not reach significance (P = .0647, unpaired Student t test). (B) Whitlock-Witte assays. Freshly harvested Balb/c BM from Gab2+/+ (top) and Gab2−/− (bottom) donors was transduced with retrovirus coexpressing p210 BCR-ABL1 and either GFP or the indicated GAB2 variant, and plated in triplicate at the indicated cell numbers per well. Nontransduced cells were added to provide 106 total cells for stromal support. Wells were scored as positive when cell number reached 106 viable nonadherent cells per well, as described in Methods. (C) Extracts from immortalized BCR-ABL1–transformed B-lymphoid cell lines (from Figure 5B) from Gab2+/+ (WT) or Gab2−/− (knockout) donors were analyzed for GAB2 protein expression and phosphorylation, as described in Methods. (D) K-M curves of recipients of BM from Gab2+/+ littermates transduced with p210MIGFP retrovirus (blue solid line, n = 8), and of Gab2−/− donors (red lines) transduced with p210-IRESwt-Gab2WT retrovirus (dotted line, n = 8) or the indicated Gab2 mutant retrovirus (dashed lines, n = 8 for each cohort). All deaths were due to B-ALL. There was no significant difference in survival between recipients of p210MIGFP-transduced Gab2+/+ BM and p210-IRESwt-Gab2WT–transduced Gab2−/− BM (P = .493) or between recipients of p210MIGFP-transduced Gab2−/− BM and p210-IRESwt-Gab2ΔShp2–transduced Gab2−/− BM (P = .734). By contrast, the survival of recipients of p210MIGFP-transduced Gab2+/+ BM and p210MIGFP-transduced Gab2−/− BM, and of recipients p210-IRESwt-Gab2WT–transduced and p210-IRESwt-Gab2Δp85–transduced Gab2−/− BM, was different (P < .0001 and P = .001, respectively, Mantel-Cox tests).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal