In this report we describe a quantitative in vitro assay for the most primitive type of leukemic precursors yet defined in patients with chronic myeloid leukemia (CML). This assay is based on the recently described "long-term culture-initiating cell" (LTC-IC) assay for primitive normal human hematopoietic cells. Such cells, when cocultured with competent fibroblast feeder layers, give rise after a minimum of 5 weeks to multiple single and multilineage clonogenic progenitors detectable in secondary semisolid assay cultures. Similar cultures initiated by seeding a highly enriched source of leukemic cells from patients onto normal feeders showed the clonogenic cell output after 5 weeks to be linearly related to the input innoculum over a wide range down to limiting numbers of input cells, thus allowing absolute frequencies of leukemic LTC-ICs to be determined using standard limiting dilution analysis techniques. Leukemic LTC-IC concentrations in CML marrow were found to be decreased, on average to <10% ofthe normal LTC-IC concentration in normal marrow, but were greatly increased (up to > 10 times) in CML blood. Assessment of the number of clonogenic cells produced per leukemic LTC-IC by comparison to normal blood or marrow LTC-IC values showed this function to be unchanged in leukemic LTC-ICs [i.e., 3.1 ± 0.4 clonogenic cells per CML LTC-IC (mean ± SEM, n = 6) versus 3.7 ± 1.2 (n = 3) and 4.3 ± 0.4 (n = 5), respectively, for normal blood and marrow LTC-ICs].In contrast, leukemic LTC-IC maintenance in LTC proved to be highly defective by comparison to normal LTC-IC of either blood or marrow origin. Thus, when cells from primary LTC were subcultured into secondary LTC-IC assays, leukemic LTC-IC rapidly declined (>30-fold) within the first 10 days of culture, whereas normal LTC-IC numbers remained unchanged during this period. These findings illustrate how self-maintenance and differentiation events in primitive human hematopoietic cells can be differentially modulated by an oncogenic process and provide a framework for further studies of their manipulation, analysis, and therapeutic exploitation. (Ph') (1). The initial cell transformed, and hence the origin of the leukemic clone, is believed to be a totipotent hematopoietic cell with lymphoid as well as myeloid differentiation potential since Ph'-positive cells in these lineages are frequently demonstrable (2). This has suggested that production of the BCR-ABL kinase in a totipotent hematopoietic cell gives it a selective growth advantage. Recent experiments involving retroviral infection of murine bone marrow (BM) cells with BCR-ABL constructs are consistent with this (3, 4), although an underlying molecular mechanism has not been determined. In particular, the biological consequences of BCR-ABL kinase expression in very primitive human hematopoietic cells have been difficult to investigate because methods for their selective isolation have not been available.CML patients with elevated leukocyte (WBC) counts show dramatic increases in the number of Ph'-posi...
Chronic myeloid leukemia (CML) has long served as a prototype malignancy for basic as well as clinical studies aimed at developing curative cancer treatment protocols. Well established features of chronic phase CML are its origin in a pluripotent stem cell, a now well defined molecular genetic basis involving the creation of a BCR-ABL fusion gene and evidence of resultant abnormalities in the mechanisms that normally control primitive hemopoietic cell proliferation. We have recently shown how the long-term marrow culture system can be adapted to quantitate and characterize a very primitive cell type in normal blood and marrow samples, as well as their normal and leukemic counterparts in patients with CML. This system has also been used to dissect mechanisms of normal progenitor regulation and to identify specific anomalies affecting leukemic (CML) progenitors. Our studies show that cells detected by their ability to initiate long-term cultures (LTC) of leukemic cells (i.e., CML LTC-initiating cells or LTC-IC) are differently distributed between marrow and blood by comparison to LTC-IC in normal individuals and, although functionally similar in terms of the number and differentiation types of clonogenic cells they produce, CML LTC-IC exhibit defective self-maintenance. Phenotypically these primitive leukemic cells are heterogeneous; the majority display features of activated/proliferating cells but a significant proportion do not. We have also documented heterogeneity in primitive CML cell responses to two factors that specifically and reversibly arrest the cycling of primitive normal hemopoietic cells; i.e., TGF-beta and MIP-1 alpha, to which CML cells are normally responsive and abnormally unresponsive, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell- depleted fraction of normal blood are maintained on irradiated, marrow- derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4- hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell- depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)
The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.
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