Disruption of the estrogen receptor beta gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis

Abstract

Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor beta knockout (ERbeta-/-) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15-30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERbeta-/- mice than in WT littermates. Some of the ERbeta-/- mice also had a severe lymphoproliferative phenotype. Thus the absence of ERbeta results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERbeta in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERbeta-/- mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERbeta agonists might have clinical value in the treatment of leukemia.

Fig. 1.

Aged ERβ^–/– mice develop myeloproliferative disease. Massive splenomegaly (A) and enlargement of Peyer's patches (B) and mesenteric lymph nodes (C) from 18-month-old ERβ^–/– mice (Left) compared with an age-matched WT littermate (Right) are shown. (D–G) Spleen histology of WT (D) and ERβ^–/– (E) mice and bone marrow histology of WT (F) and ERβ^–/– (G) mice. The femurs were surgically removed and fixed in 4% paraformaldehyde-buffered PBS and decalcified in 5.5% EDTA/10% formalin for 7 days. Nonhematopoietic organs such as liver (H) and lung (I) show a massive infiltration of myeloid cells.

Fig. 2.

Mice lacking ERβ have a striking increase in cellularity in bone marrow (A) and spleen (B). ERβ^–/– (black column) and age-matched WT male and female (white column) 1.5-year-old mice were killed, and from each mouse one femur and one tibia were flushed with PBS. The cells were washed and resuspended in 10 ml of PBS. The total cells per leg were calculated based on hematocytometer counts [data are shown as mean (n = 5 per group)].

Fig. 3.

Blood smears of WT and ERβ^–/– mice. Blood was withdrawn from the mice by cardiac puncture and smeared on microscope slides. The slides were stained with May–Grünewald Giemsa solution.

Fig. 4.

(A) Single-cell suspensions from bone marrow of ERβ^–/– mice and their WT littermates were stained with indicated combinations of antibodies for flow-cytometric studies. The numbers in the boxes indicate the percentage of myeloid cells. The data show one analysis from a single mouse, which is representative of the data obtained with six pairs of mice including males and females. In mice used in this analysis, total cellularity of ERβ^–/– bone marrow was increased >2-fold over control, and there was splenomegaly with granulocytosis in the blood. (B) The estimated total number of myeloid and B lymphoid cells in bone marrow of ERβ^–/– (black column) and WT (white column) mice is shown. The analysis shown is one of six and is from the mice shown in A.

Fig. 5.

Histological examination of spleen (A) and nonhematopoietic organs lung (B) and liver (C and D) indicates a massive myeloid and blast cell infiltration. The tissues of WT mice do not show any infiltration. Analysis by myeloperoxidase immunohistochemistry (A–C) and chloroacetate esterase staining (D) shows focal areas of blastic cells. (E–H) In the spleens of 2-year-old ERβ^–/– mice various histological phenotypes are evident.

Fig. 6.

Myeloid cells in the bone marrow of WT mice express ERβ protein. Bone marrow cells from 3-month-old female WT mice were separated by flow cytometry into Gr-1^–/Mac-1^– (nonmyeloid) and Gr-1^hi/Mac-1^hi (myeloid) cells and analyzed for the expression of ERβ by Western blotting.