Figure 4.
Figure 4. B220+ Gr1– c-Kit+ leukemic populations differ in phenotypic characteristics as a consequence of Gata2 haploinsufficiency. (A-B) Correlation between the percentage of B220+Gr1– cell populations in the bone marrows and expression levels of mouse endogenous Gata2 (A) and total (human + mouse) EVI1 (B) mRNAs in the B220+Gr1–c-Kit+ cells of 3q21q26 (n = 10) or 3q21q26::Gata2+/− (n = 6) mice. The abundance of Gata2 and total (human + mouse) EVI1 mRNAs was normalized to Gapdh abundance. Average values for the 3q21q26 Type III leukemic mice were set to 1. Linear approximations and R2 values of the 3q21q26 leukemic mice are shown. (C) Representative flow cytometric profiles of CD150, CD48, IL7Rα, Flt3, CD19, FcγR II/III, Mac1, and myeloperoxidase (MPO) in the B220+Gr1–c-Kit+ bone marrow cells from leukemic 3q21q26 Type I (upper panels) and III (middle panels) and 3q21q26::Gata2+/− Type I (lower panels) mice (red lines). Negative (unstained) control samples are shown in gray. (D) D-J and V-D-J rearrangements in B220+Gr1–c-Kit+ cells of the 3q21q26 Type I and III leukemia and the 3q21q26::Gata2+/− Type I leukemia. Arrows indicate the position of amplified fragments of ∼1033, ∼716, or ∼333 bp (D-J rearrangements) or ∼1058, ∼741, or ∼358 bp (V-D-J rearrangements) using primer pairs J3-VH558, J3-VH7183, and J3-VHQ52 (see supplemental Data). M and P indicate lanes loaded with a DNA marker or a positive control (polymerase chain reaction products from genomic DNA of WT mouse CD19+ cells). (E) Expression levels of Ebf1 (left panel), Pax5 (center panel) and Cebpa mRNA (right panel) in the B220+Gr1–c-Kit+ cells from 3q21q26 Type I (n = 3), Type III (n = 4), or the 3q21q26::Gata2+/− Type I (n = 6) leukemic mice. CD19+ B cells and granulocyte-macrophage progenitor (GMP) cells in WT mice are used for positive controls of Ebf1 and Pax5 and as a positive control for Cebpa expression, respectively. The abundance of each mRNA was normalized to Gapdh. Average values for the 3q21q26 Type I leukemic mice were set to 1. Arrows indicate undetectable or slight expression levels. (F) The percentages of BrdU+ cells in the B220+Gr1–c-Kit+ population of the 3q21q26 Type III (n = 3) or 3q21q26::Gata2+/− Type I (n = 6) leukemic bone marrows. B220+Gr1–c-Kit+ cells were analyzed 2 hours after intraperitoneal injection of BrdU. Bar graphs represent mean ± SD. *P < .05; **P < .01.

B220+Gr1–c-Kit+leukemic populations differ in phenotypic characteristics as a consequence of Gata2 haploinsufficiency. (A-B) Correlation between the percentage of B220+Gr1– cell populations in the bone marrows and expression levels of mouse endogenous Gata2 (A) and total (human + mouse) EVI1 (B) mRNAs in the B220+Gr1–c-Kit+ cells of 3q21q26 (n = 10) or 3q21q26::Gata2+/− (n = 6) mice. The abundance of Gata2 and total (human + mouse) EVI1 mRNAs was normalized to Gapdh abundance. Average values for the 3q21q26 Type III leukemic mice were set to 1. Linear approximations and R2 values of the 3q21q26 leukemic mice are shown. (C) Representative flow cytometric profiles of CD150, CD48, IL7Rα, Flt3, CD19, FcγR II/III, Mac1, and myeloperoxidase (MPO) in the B220+Gr1–c-Kit+ bone marrow cells from leukemic 3q21q26 Type I (upper panels) and III (middle panels) and 3q21q26::Gata2+/− Type I (lower panels) mice (red lines). Negative (unstained) control samples are shown in gray. (D) D-J and V-D-J rearrangements in B220+Gr1–c-Kit+ cells of the 3q21q26 Type I and III leukemia and the 3q21q26::Gata2+/− Type I leukemia. Arrows indicate the position of amplified fragments of ∼1033, ∼716, or ∼333 bp (D-J rearrangements) or ∼1058, ∼741, or ∼358 bp (V-D-J rearrangements) using primer pairs J3-VH558, J3-VH7183, and J3-VHQ52 (see supplemental Data). M and P indicate lanes loaded with a DNA marker or a positive control (polymerase chain reaction products from genomic DNA of WT mouse CD19+ cells). (E) Expression levels of Ebf1 (left panel), Pax5 (center panel) and Cebpa mRNA (right panel) in the B220+Gr1–c-Kit+ cells from 3q21q26 Type I (n = 3), Type III (n = 4), or the 3q21q26::Gata2+/− Type I (n = 6) leukemic mice. CD19+ B cells and granulocyte-macrophage progenitor (GMP) cells in WT mice are used for positive controls of Ebf1 and Pax5 and as a positive control for Cebpa expression, respectively. The abundance of each mRNA was normalized to Gapdh. Average values for the 3q21q26 Type I leukemic mice were set to 1. Arrows indicate undetectable or slight expression levels. (F) The percentages of BrdU+ cells in the B220+Gr1–c-Kit+ population of the 3q21q26 Type III (n = 3) or 3q21q26::Gata2+/− Type I (n = 6) leukemic bone marrows. B220+Gr1–c-Kit+ cells were analyzed 2 hours after intraperitoneal injection of BrdU. Bar graphs represent mean ± SD. *P < .05; **P < .01.

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