BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells

Abstract

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo(-/-) and EpoR(-/-) mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210(BCR-ABL) and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR(-/-) mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.

Figure 1

Hematopoietic progenitor assays for BCR-ABL and v-SRC transduced EpoR^−/− fetal liver cells. CFU-E (A) and BFU-E (B) colonies were generated in cultures of 10⁵ EpoR^−/− fetal liver cells infected with retroviral constructs encoding EpoR, BCR-ABL, and v-SRC. Total CFU-E and BFU-E were counted after 2 (A) and 8 days (B), respectively. Results are presented as percentage of EpoR rescued colonies in the optimum conditions (cultures containing Epo, IL-3, IL-6, and SF) shown by the hatched bars. Graphs are from duplicate cultures of 3 (v-SRC) to 10 (all other constructs) independent experiments. Under the optimum condition (cultures containing spleen conditioned media and Epo), the average of the absolute number of CFU-E colonies rescued by EpoR was 1,698 ± 426 (n = 4) per 10⁶ cells plated. Similarly, the average of absolute number of BFU-E rescued by EpoR-infected cells under the optimum condition (Epo, IL-3, IL-6, and SF) was 68 ± 12 per 10⁵ cells (n = 10).

Figure 2

BCR-ABL and v-SRC expression reconstitute the erythroid phenotype in EpoR^−/− fetal liver cells. (A) Epo-independent BCR-ABL-generated bursts (BFU-E) cultured either in 15% serum alone (a) or in serum supplemented with IL-3, IL-6, and SF (b); Epo-dependent EpoR-generated colony cultured in Epo and IL3, IL-6, and SF is shown as control (c) (×100). (B) Cytospin preparations and May-Grünwald Giemsa staining of individual BCR-ABL-generated BFU-E-derived colonies generated in the absence (a) or presence of IL-3, IL-6, and SF (b) show erythroid cells at different stages of differentiation (×1,000). (C) Genomic PCR of BCR-ABL on individual BFU-E from EpoR (lanes 1 and 2)- or BCR-ABL (lanes 3–6)-derived colonies plated in the presence of 15% serum only (lanes 5 and 6), serum supplemented with IL-3, IL-6, and SF (lanes 3 and 4), or Epo and IL-3, IL-6, and SF (lanes 1 and 2). The PCR reaction was run on a 1.5% agarose gel and shows a band of 404 bp corresponding to the amplified breakpoint region of BCR-ABL. A positive control using a BCR-ABL containing plasmid MSCV-P210 is shown (lane 7). (D) GFP-positive v-SRC reconstituted BFU-E from EpoR^−/− fetal liver cells (×100). Results are from one representative experiment.

Figure 3

BCR-ABL mutants rescue erythroid phenotype in EpoR^−/− fetal liver cells. CFU-E (A) and BFU-E (B) colonies generated in cultures of 10⁵ EpoR^−/− fetal liver cells infected with retroviral constructs encoding BCR-ABL mutants (P210Y177F and P210 ΔSH2), wild-type P210^BCR-ABL, or EpoR. Total CFU-E and BFU-E were counted after 2 (A) and 8 days (B), respectively. Results are presented as percentage of EpoR-rescued colonies in the optimum conditions shown by hatched bars (see legend of Fig. 1 A and B). Graphs are from duplicate cultures of at least four independent experiments.