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RA/arsenic activate a PML/P53 axis in NPM-1c AML
AML3pml-/-, which were treated or not with RA for 1 week followed by either doxorubicin or cytarabine as single agents, for an additional 1 week. RA induced PML-depen- dent NPM-1c downregulation and human P53 stabilization in vivo (Figure 4A-D), while all three drugs ultimately induced human P53 phosphorylation (Figure 4A, B).
Importantly, RA synergized with doxorubicin or cytarabine to decrease abundance of human cells in treated mice, only in cells bearing intact PML (Figure 4E-H). These observa- tions suggest that, at least in this model, RA cooperates with chemotherapy to decrease AML burden, resembling the clinical observations made in AML.
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Figure 3. NPM-1c-expressing cells exhibit high Pin1 level and activity. (A) Western blot analysis of Pin1 in OCI-AML2, OCI-AML2-NPM-1wt, OCI-AML2-NPM-1c, OCI- AML3, OCI-AML3pml-/- and OCI-AML3P53-/-. (B) Western blot analysis of NPM-1c and Pin1 in primary blasts derived from seven patients with acute myeloid leukemia (AML) with NPM-1c (p4, p2, p5, p8, p7, p1 and p6) and six AML patients with wild-type NPM-1 (NPM-1 wt) (p11, p13, p14, p10, p15 and p16). Densitometry histograms represent an average of Pin1 expression level in the seven tested NPM-1c AML patients and the six tested NPM-1 wt AML patients. Densitometry was performed using ImageJ software. Statistical analysis was done using a Student t-test. (C) Pin1 relative activity in OCI-AML2, OCI-AML3 and in primary blasts derived from three NPM-1c AML patients and two NPM-1 wt AML patients after treatment with 1 mM of RA, as indicated. Statistical analysis was done using a Student t-test. (D) Cell growth (percent of control) was assessed using the trypan blue exclusion dye assay in OCI-AML2 and OCI-AML3 cells following treatment with RA alone, AG-17724 alone or their combination with arsenic trioxide (ATO), cytarabine (Ara-C) and doxorubicin for 72 h as indicated (n=3). Statistical analysis was done using a Student t-test, *P<0.05; **P<0.01; ***P<0.001.
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