Analysis of genomic breakpoints in p190 and p210 BCR-ABL indicate distinct mechanisms of formation.

We sought to understand the genesis of the t(9;22) by characterizing genomic breakpoints in chronic myeloid leukemia (CML) and BCR-ABL-positive acute lymphoblastic leukemia (ALL). BCR-ABL breakpoints were identified in p190 ALL (n=25), p210 ALL (n=25) and p210 CML (n=32); reciprocal breakpoints were identified in 54 cases. No evidence for significant clustering and no association with sequence motifs was found except for a breakpoint deficit in repeat regions within BCR for p210 cases. Comparison of reciprocal breakpoints, however, showed differences in the patterns of deletion/insertions between p190 and p210. To explore the possibility that recombinase-activating gene (RAG) activity might be involved in ALL, we performed extra-chromosomal recombination assays for cases with breakpoints close to potential cryptic recombination signal sequence (cRSS) sites. Of 13 ALL cases tested, 1/10 with p190 and 1/3 with p210 precisely recapitulated the forward BCR-ABL breakpoint and 1/10 with p190 precisely recapitulated the reciprocal breakpoint. In contrast, neither of the p210 CMLs tested showed functional cRSSs. Thus, although the t(9;22) does not arise from aberrant variable (V), joining (J) and diversity (D) (V(D)J) recombination, our data suggest that in a subset of ALL cases RAG might create one of the initiating double-strand breaks.

Effects of Flt3 ligand and interleukin-7 on in vitro growth of acute lymphoblastic leukemia cells.

We investigated the effects of Flt3/Flk-2 ligand (FL) and interleukin-7 (IL-7) on DNA synthesis and proliferation of blast cells from patients with acute lymphoblastic leukemia (ALL). After 7 days of serum-free suspension culture of 19 samples, FL induced maximal DNA synthesis in two cases, whereas the combination of FL and IL-7 did so in another eight samples with a stimulation index (SI) >2. However, the number of viable cells after 7 days of liquid culture decreased in all but one sample. In this case of a pre-pre-B-ALL with a translocation t(4;11), FL induced dose-dependent proliferation (maximal 100 ng/mL) and cells stimulated with FL could be cultured for up to 4 weeks. A homogeneous population with 98% CD19-positive cells was detected before and after culture, and there was no evidence of nonleukemic cell proliferation as determined by immunophenotyping. The flt3 gene was transcribed in all seven cases studied by reverse-transcriptase polymerase chain reaction (RT-PCR). In the ALL cells responsive to FL, the expression of functional Flt3 receptors was confirmed by demonstrating FL-dependent tyrosine phosphorylation of Flt3. Furthermore, FL-dependent tyrosine phosphorylation of cellular proteins of estimated molecular weights of 70, 115, and 140 kD was detectable in these cells. These data demonstrate the functional heterogeneity of ALL samples and show that functional Flt3 receptors capable of mediating FL-dependent mitogenic signaling are expressed in a subset of ALL.

The role of marrow accessory cell populations in the augmentation of human erythroid progenitor cell (BFU-E) proliferation by prostaglandin E.

The requirement for CD8+ T lymphocytes in the stimulation of erythroid progenitor cells by prostaglandin E (PGE) was examined. When low density bone marrow (LD-BM) or non-adherent bone marrow (NA-BM) cells were depleted of CD8+ cells the enhancing effect of PGE on BFU-E proliferation was abrogated. However, further enrichment of marrow progenitor cells by depletion of accessory cells using a cocktail of specific monoclonal antibodies, immunoadherence and fluorescence activated cell sorting with the MY10 monoclonal antibody resulted in a population of erythroid progenitor cells which were responsive to the enhancing effect of PGE despite the absence of CD8+ cells. Stepwise individual cell lineage depletion of marrow cell populations indicated that prostaglandin E enhanced erythroid burst formation in the absence of CD8+ cells provided that glycophorin-A+ cells were removed from LD-BM or NA-BM cells. These results suggest that nucleated erythroid cell populations may modulate the enhancement of BFU-E by PGE. The ability of GP-A+ cells to block the enhancement of erythroid burst formation by PGE following removal of CD8+ T cells was confirmed by readdition of conditioned medium prepared from positively selected GP-A+ marrow cells. These results expand the role of CD8+ T cells in the PGE enhancement of BFU-E proliferation and suggest another mechanism by which accessory cells regulate the proliferation of BFU-E in bone marrow.