Normal Myeloid Progenitor Cells Research Articles

Binase induces apoptosis of transformed myeloid cells and does not induce T-cell immune response

Microbial RNases along with such animal RNases as onconase and BS-RNase are a promising basis for developing new antitumor drugs. We have shown that the Bacillus intermedius RNase (binase) induces selective apoptosis of transformed myeloid cells. It attacks artificially expressing activated c-Kit myeloid progenitor FDC cells and chronic myelogenous leukemia cells K562. Binase did not induce apoptosis in leukocytes of healthy donors and in normal myeloid progenitor cells. The inability of binase to initiate expression of activation markers CD69 and IFN-γ in CD4+ and CD8+ T-lymphocytes testifies that enzyme is devoid of superantigenic properties. Altogether, these results demonstrate that binase possesses therapeutic opportunities for treatment of genotyped human neoplasms expressing activated kit.

Acute Promyelocytic Leukemia Stem Cell Derived from Leukemic Progenitor.

Efforts to identify the leukemic stem cell (or leukemia-repopulating cell, LRC) of acute myeloid leukemia (AML) have led to considerable insights, but also to a number of unresolved questions. While the LRC is believed to arise primarily from the hematopoietic stem cell, data from chronic myelogenous leukemia blast crisis and acute promyelocytic leukemia (APL) suggest that they may arise from more committed cells. In order to clarify the potential of committed progenitors to become LRCs in some human AMLs, we utilized the MRP8-PML/RARA transgenic murine model of APL to perform initial studies directed at the identification of the LRC in APL. Unsorted spleen cells of MRP8-PML/RARA leukemic mice injected into cohorts of sublethally irradiated mice using limiting dilutions showed that the LRC of APL is present in 1:100 to 1:300 cells. FACS analysis of six primary and serially transplanted leukemic specimens revealed that normal hematopoetic stem cells and myeloid progenitors were rare, comprising .007% and .078% of total splenic cells, respectively. These data raised the possibility that the LRC is present at higher frequency than these normal progenitors and does not belong to any of these populations. Given the scarcity of normal progenitors, we surmised that the LRC may be part of the leukemic population. We performed FACS analysis of seven primary leukemias to elucidate potential heterogeneity within the leukemic cell population. Myeloblasts had the immunophenotype lymph−Gr1+ckit+CD16/32+CD34mod/+. A smaller population that made up 1.4–12.7% of all cells was lymph−Gr1−ckitlo/mod and had variable expression of CD16/32 and CD34. Such cells have been identified as myelomonocytic cells and represent a stage in myelopoiesis beyond the GMP. Our findings suggest a hierarchy in which Gr1−ckitlo/mod leukemia repopulating cells proliferate and differentiate to maintain the population of abnormal promyelocytes. We infer from the dilution analysis data that the LRC in this MRP8-PML/RARA mouse model of acute promyelocytic leukemia cannot be a hematopoietic stem cell or a normal myeloid progenitor cell. Also, it is apparent that the entire blast population is not capable of transferring disease; otherwise, all the mice injected would have developed leukemia. While our data does indicate that while leukemogenesis in APL is a hierarchy, it originates from a newly identified leukemia progenitor cell that is Gr1−ckitlo/mod that may be more mature than a GMP. It is quite likely that the LRC lies within this population of cells. Future work will further delineate the immunophenotype of the LRC in this model and in human leukemia, as well as molecular abnormalities that are important in differentiating from the LRC into the myeloblast phenotype.

Identification of Genes Abnormally Expressed in Both Human and Murine MLL-ELL and/or MLL-ENL Leukemia.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. Their abnormally expressed genes identify specific markers for their clinical diagnosis. Important biological properties are often conserved across species. However, although genetically engineered mouse leukemia models are well-established, few systematic studies have validated the genes that exhibit similar abnormal expression patterns in both human and mouse leukemia models. MLL-ELL and MLL-ENL fusion genes resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3), respectively, are frequently involved in human acute leukemia, and in retrovirus-mediated mouse leukemia models. We used the SAGE technique to compare gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both human and mouse. We analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL or MLL-ENL fusions, and a leukemia cell line with an MLL-ELL fusion. 484,303 SAGE tags were identified from the nine samples, yielding 103,899 unique tags in human and 60,993 in mouse samples. We identified 40 candidate genes that appear to be abnormally expressed in both human and murine MLL-ELL leukemias (2 up- and 38 down-regulated), and 72 in both human and murine MLL-ENL leukemias (23 up and 49 down). 25 candidate genes are down-regulated in both types of leukemias, and many of them can bind with and/or regulate other candidate genes in the candidate list. For example, LCN2 can bind directly with and positively regulate MMP9; MMP9 and TMSB4X may positively regulate FOS; FOS and JUNB can bind directly and positively regulate each other. JUNB may inhibit proliferation and promote apoptosis, and it was reported that inactivation of JunB in LT-HSC leads to MPD while its inactivation in committed myeloid progenitors also predisposes to leukemia evolution. LCN2 may also positively regulate apoptosis. Meanwhile, some important candidate genes are observed only in one type of leukemia. For example, both PXN and ARHGEF1 are down-regulated only in MLL-ELL leukemias. PXN can bind directly with ARHGEF1, and the latter may inhibit proliferation. Similarly, MYB is significantly upregulated only in MLL-ENL leukemias, which was reported to play a role in MLL-ENL-mediated transformation. Taken together, some common pathways may exist in the development of both types of leukemias, whereas each may also have their own pathway. The deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes, and are studying the functions and pathways of the validated candidate genes. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute myeloid leukemia, which may lead to more effective therapy for these leukemias.

Gene expression profiles in acute myeloid leukemia with common translocations using SAGE

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15(+) myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15(+) cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes/pathways for more specifically targeted therapy.

Genes Similarly Abnormally Expressed in Both Human and Murine MLL-Associated Leukemia.

Although more than 50 different loci translocate to the MLL gene at chromosome band 11q23, resulting in either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), no unifying property is shared by all partner genes. The translocations result in a functional fusion of the N-terminal part of MLL gene and the C-terminal part of each partner gene, presumably leading to changes in the expression of the normal target genes, most of which have not been identified. Although genetically engineered mouse leukemia models have been widely used, few systematic studies have evaluated whether such models are valid equivalents of human leukemia. We used serial analysis of gene expression (SAGE) to obtain genome-wide gene expression profiles in normal myeloid progenitor cells from human CD15+ and mouse Gr-1+ cells. We also analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL [t(11;19)(q23;p13.1)] or MLL-ENL [t(11;19)(q23;p13.3)] fusions, and a cell line from a leukemia mouse transduced with an MLL-ELL fusion. MLL-ELL and MLL-ENL fusions are frequently involved in human AML, while MLL-ENL is also seen in human ALL. 484,303 SAGE tags were identified from the nine samples (40,000 to 100,000 tags per sample), yielding 103,899 unique SAGE tags in the human and 60,993 in the mouse samples. Analysis of the SAGE data identified 43 candidate genes that appear to be abnormally expressed in both human and mouse myeloid leukemia progenitor cells with either MLL-ELL or MLL-ENL fusions (9 up-regulated and 34 down-regulated; Table 1). Increasing evidence suggests that endogenous antisense RNAs may play critical roles in gene regulation and cancer. Natural antisense RNAs include cis-encoded antisense RNAs transcribed from the opposite strand of the same genomic locus as the sense target genes, and trans-encoded antisense RNAs such as microRNAs (miRNAs) transcribed from a genomic locus different from the sense target genes. 26 of the 43 candidate genes have antisense partners (with a total of 7 cis-encoded antisense RNAs and 36 trans-encoded miRNAs) and thereby might be regulated by endogenous antisense RNAs. We are currently validating the expression pattern of the 43 candidate genes in at least 30 different human and mouse leukemia and normal control samples with quantitative RT-PCR, and measuring the level of expression of all known miRNAs via microarray in these samples. Our studies on the abnormally expressed genes and their potential antisense partners will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute leukemia, which may lead to more effective therapy for these leukemias.Table 1Genes deregulated in both human and mouse leukemiasaTotal numberUp-regulated genesDown-regulated genesGenes with antisense partner(s)In MLL-ELL fusions2112012In MLL-ENL fusions3382521In both types of fusions110117Total unique genes4393426aThe genes have at least 3 fold difference in expression with a significance P < 0.05 between each leukemia sample and the normal control sample.

Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism.

We previously reported that N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) treatment caused large increases of ceramide levels in neuroblastoma cell lines and induced cell death by a combination of apoptosis and necrosis through p53 (also known as TP53)-independent and caspase-independent pathways. Our goal was to determine if several molecules that inhibit enzymes involved in ceramide metabolism-L-threo-dihydrosphingosine (safingol), d, l-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), and tamoxifen-enhanced 4-HPR-mediated cytotoxicity and/or affected ceramide levels. Cellular lipids were quantified by radiolabeling and thin-layer chromatography. Cytotoxicity and cytotoxic synergy (expressed as combination index, where combination index <1 indicates synergy and >1 indicates antagonism) were measured in cultured cancer cell lines with the use of a fluorescence-based assay of cell viability employing digital imaging microscopy. Statistical tests were two-sided. 4-HPR increased ceramide levels by de novo synthesis. Safingol (1-4 microM) was incorporated into a stereochemical variant of ceramide and synergized with a 3:1 molar ratio of 4-HPR (3-12 microM), to produce a 100-fold to 10 000-fold (2 to 4 logs) increase in cytotoxicity relative to 4-HPR alone in neuroblastoma (combination index <0.1), lung (combination index <0.1-0.2), melanoma (combination index <0.1-0.2), prostate (combination index <0.1-1.0), colon (combination index 0.1-0.3), breast (combination index = 0.1-0.5), and pancreas (combination index = 0.2) cell lines, including p53 mutant and alkylator-resistant cell lines. The 4-HPR and safingol combination was cytotoxic in low-oxygen conditions and was minimally toxic to normal fibroblasts and bone marrow myeloid progenitor cells. Addition of agents that retard ceramide glucosylation and/or acylation, such as PPMP or tamoxifen, to 4-HPR or to the combination of 4-HPR and safingol further increased cytotoxicity to tumor cells. Combinations of 4-HPR and modulators of ceramide metabolism may form the basis for a novel chemotherapy that is functional under hypoxic conditions (e.g., such as those within tumors) and is p53 independent and caspase independent.

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway.

The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CbetaII (PKCbetaII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCbetaII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCbetaII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.

Estrone potentiates myeloid cell differentiation: A role for 17β-hydroxysteroid dehydrogenase in modulating hemopoiesis

Hormones such as 1α,25-dihydroxy vitamin D 3 (D 3), all-trans retinoic acid, and 9-cis retinoic acid stimulate differentiation of myeloid progenitor cells via their interaction with specific hormone receptors. However, the sensitivity of cells to these agents is not merely governed by the expression of their receptors and the availability of ligand to bind them. Recent studies from our group suggested that the actions of D 3 and retinoids on myelopoiesis also are influenced by endogenous mechanisms involving other steroid hormones. In this study we examined the influence of local estrogen metabolism on the differentiation of HL60 cells and normal primitive myeloid progenitor cells. Quantitative thin-layer chromatography (TLC) analyses showed that HL60 and normal cells are able to generate estrone (E 1) from estradiol (E 2). Neither cell population generated significant amounts of E 2 from E 1. Reverse transcriptase polymerase chain reaction and Northern analyses confirmed that normal and leukemic myeloid progenitor cells expressed mRNA for the type I and IV isoforms of 17β-hydroxysteroid dehydrogenase. Conversion of E 2 to E 1 was upregulated within 24 hours when HL60 cells were treated with either all-trans retinoic acid or D 3 at doses that induce their differentiation toward neutrophils or monocytes, respectively. Similarly, D 3-induced monocyte differentiation of normal myeloid progenitor cells was associated with increased capacity to generate E 1 from E 2. When HL60 cells or normal myeloid progenitor cells were exposed to exogenous E 1 they became more sensitive to the differentiation-inducing effects of D 3. Data presented provide further evidence for the local modulation of myelopoiesis by intracrine mechanisms. In particular, our findings suggest that local metabolism of steroids by normal as well as leukemic myeloid cells influences their responsiveness to D 3 and retinoids.

Differentiation of Myeloid Cells and 1, 25-Dihydroxyvitamin D3

It is well established that1, 25(OH)2D3 induces monocyte/macrophage (Mo/Mø) colonies when added to culture of granulocyte/macrophage progenitors. Recently, we demonstrated that one of the target cells of1, 25(OH)2D3 in Mo/Mø differentiation is the neutrophilic promyelocyte that is believed to belong to the neutrophilic lineage. This fact overthrows the established theory that normal hematopoietic precursors are committed to respective cell lineages and do not deviate from their own lineage. The lineage switching from the promyelocyte to Mo/Mø was suggested to be operating in vivo because1, 25(OH)2D3 is a physiological substance produced by Mø. More recently, we have shown that transient exposure (24 h) of promyelocytes to1, 25(OH)2D3 causes Mo/Mø differentiation. This strategy could be useful for examining the effects of1, 25(OH)*D3 on the growth and differentiation of normal myeloblasts and myeloid progenitor cells. Recent advances in molecular biology have enabled investigators to identify a number of genes involved in Mo/Mø differentiation induced by1, 25(OH)2D3. Some of these may be the determinant genes for Mo/Mø differentiation; however, further studies are required to determine the underlying mechanisms of Mo/Mø differentiation.

Differential Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on the Radiation Sensitivity of Normal Versus Leukemic Bone Marrow Progenitor Cell Populations

We examined the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), interleukin 3 (rhIL-3) and interleukin 6 (rhIL-6) on the radiation sensitivity of normal and leukemic bone marrow progenitor cell populations. Conditioning of leukemic progenitor cells (LPC) from acute lymphoblastic leukemia (ALL) patients with rhG-CSF enhanced their radiation sensitivity, whereas conditioning with rhIL-3 or rhIL-6 had the opposite effect. In contrast to its effects on LPC derived from ALL patients, rhG-CSF reduced the radiation sensitivity of normal myeloid progenitor cells as well as LPC from acute myeloblastic leukemia (AML) patients. Differential modulation of the radiation sensitivity of LPC by rhG-CSF may provide the basis for better total body irradiation (TBI) regimens for ALL patients undergoing autologous bone marrow transplantation (BMT).

Involvement of p21cip-1 and p27kip-1 in the Molecular Mechanisms of Steel Factor-Induced Proliferative Synergy In Vitro and of p21cip-1 in the Maintenance of Stem/Progenitor Cells In Vivo

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip-1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip-1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.

Expression of rhombotin 2 in normal and leukaemic haemopoietic cells.

The rhombotin 2 gene was identified by its association with a recurrent chromosome translocation (11;14)(p13;q11), occurring in T-cell acute lymphoblastic leukaemias (ALLs). High levels of RBTN2 have been found in T-cell leukaemias carrying the translocation and in some T-cell ALLs that lack, by cytogenetic and molecular techniques, translocations involving 11p13. In normal murine tissues RBTN2 has been found to be widely expressed, with high levels present in brain and early B cells. Studies carried out in mice lacking RBTN2 have demonstrated the importance of this gene in erythropoiesis. We have investigated the expression of RBTN2 in human leukaemic cells, and in human and murine normal myeloid progenitor cells. RBTN2 is expressed in the leukaemic cells of patients with pre-B ALL, T-ALL and acute myeloblastic leukaemia (AML). By cytogenetic and molecular techniques no abnormalities were noted in 11p13. Using clonogenic assays and single cell PCR we found that RBTN2 is expressed strongly in the precursors of mixed erythrocyte/macrophage/mast, erythrocyte, megakaryocyte, neutrophil and macrophage colonies. In contrast, RBTN2 message was low to undetectable in the mature progeny. These findings indicate that RBTN2 is expressed in leukaemias of both the myeloid and lymphoid lineages. Further, these studies show that in normal myeloid and lymphoid cells the expression of RBTN2 is present in progenitor cells and is lost as the cells terminally differentiate. This latter finding further illustrates that the detection of a RNA in a population of cells should not be interpreted to mean that all of the cells in that population express the RNA.

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells

Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL-3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13-7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G-CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

The RAR-RXR as Well as the RXR-RXR Pathway Is Involved in Signaling Growth Inhibition of Human CD34+ Erythroid Progenitor Cells

Abstract Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL- 3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13– 7410) and RXR (Ro 25–6603 and Ro 25–7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13–7410 potently inhibited G- CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25–6603 and Ro 25–7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

Preferential inhibitory effect of soluble factor(s) in human bone marrow stromal cells on proliferation of K562 leukemia cells versus normal myeloid progenitor cells.

We examined the effect of diffusible factors generated during the culture of the KM102 stromal cell line as well as in long-term bone marrow culture (LTBMC) on K562 leukemia cells, with respect to proliferation of clonogenic cells as well as total cells, and compared it with the effect on normal myeloid progenitors (CFU-GM). Proliferation of K562 cells plated in diffusion chambers was inhibited by coculture for 3-5 days in the fluid phase of stromal cell cultures or stromal cell-conditioned medium (CM), while CFU-GM proliferation was not inhibited under the same culture conditions. The inhibitory action was not attributed to the exhaustion of nutrients or growth promoting factors such as stem cell factor. These findings suggest that bone marrow stromal cells secrete diffusible molecule(s) which exert a preferential inhibitory effect on K562 leukemic cells vs. normal CFU-GM. Neutralization with antibodies against hematopoiesis-inhibiting cytokines such as TGF-beta 1, IFN-gamma, MIP-1 alpha and IL-4 which were detected in stromal cell-CM, failed to abrogate the inhibitory effect of KM102-CM on K562 cells. IL-1, TNF-alpha, IFN-alpha and lipopolysaccharides, known as stimulators of various cytokines from stromal cells, could not enhance the inhibitory activity. Further characterization of the factors may have implications for the treatment of leukemias.

Effects of Human FLT3 Ligand on Myeloid Leukemia Cell Growth: Heterogeneity in Response and Synergy With Other Hematopoietic Growth Factors

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL-responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.

Myeloid cell proliferation stimulated by steel factor is pertussis toxin sensitive and enhanced by cholera toxin

Effects of G-protein toxins on Steel factor (SLF) and granulocyte-macrophage colony stimulating factor (GM-CSF) stimulated proliferation of human factor-dependent cell line, M07e, were evaluated. Pertussis toxin pretreatment suppressed GM-CSF- or Steel factor-induced proliferation by 54±8%; however, proliferation induced by the combination of GM-CSF plus Steel factor was suppressed to a much lesser extent (14±8%). Pretreatment of M07e cells with cholera toxin, suppressed GM-CSF- and GM-CSF plus Steel factor-stimulated proliferation by 57 ± 6% and 79%, respectively, but increased the proliferative response to Steel factor alone by twofold. Similar effects of pertussis toxin and cholera toxin were observed on proliferation of normal myeloid progenitor cells from human umbilical cord blood. Pertussis toxin treatment of M07e cells for 4 h resulted in the ADP-ribosylation of 40–42 kDa protein band but did not significantly increase cyclic AMP levels. Cholera toxin pretreatment was associated with a 10-fold increase in intracellular cyclic AMP levels. These results implicate pertussis toxin sensitive pathways for both GM-CSF and Steel factor, but suggest that these pathways may not be required for synergistic proliferation stimulated by the combination. In addition, proliferation stimulated by GM-CSF, ± Steel factor, is sensitive to cholera toxin pretreatment; whereas cholera toxin pretreatment enhanced proliferation stimulated by Steel factor, possibly via increased cyclic AMP. This suggests divergent signal transduction pathways for the two cytokines.

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.

Guanine arabinoside as a bone marrow-purging agent.

Arabinosylguanine (araG) is a nucleoside analogue that is rapidly converted by cells of the T lymphoid lineage to its corresponding arabinosylguanine nucleotide triphosphate (araGTP), resulting in inhibition of DNA synthesis and selective in vitro toxicity to T lymphoblastoid cell lines as well as to freshly isolated leukemia cells from patients with T cell acute lymphoblastic leukemia (ALL). We have previously demonstrated that araG is an effective agent to use for chemoseparation of malignant T lymphoblasts from human bone marrow. When freshly isolated human T leukemia cells or T lymphoblastoid cells were treated with 100 microM araG for 18 hours, up to 6 logs of clonogenic T cells are eliminated without appreciable toxicity to the normal myeloid, erythroid, and megakaryocytoid clonal progenitor cells. We subsequently described studies in a murine model of T cell acute lymphoblastic leukemia (ALL) in which we tested whether bone marrow contaminated with malignant T cells and purged ex vivo with araG, could reconstitute both the lymphoid and myeloerythroid lineages in the absence of leukemic relapse. The model utilized 6C3HED tumor cells, derived from a Thy 1.2+ malignant murine T cell line, which were shown to cause lethal leukemia in C3H/HeN mice. Intravenous injection of 10(6) 6C3HED cells resulted in 100 percent mortality within 18 days, with autopsy revealing tumor infiltration of multiple organs. Evidence of araG's ability to purge bone marrow of malignant tumor cells without causing significant toxicity to normal marrow-derived hematopoietic progenitor cells was documented in experiments in which 75 percent of lethally irradiated mice receiving transplants of syngeneic bone marrow contaminated with 6C3HED tumor cells and treated ex vivo with 100 mM araG for 18 hours survived for 250 to > 400 days. Reconstitution of the lymphoid, myeloid, and erythroid lineages with donor cells in surviving mice was documented. The data presented indicate that araG may effectively purge bone marrow of malignant T cells without irreversible toxicity to hematopoietic stem cells. This purging regimen is recommended for consideration for clinical trials in patients with T cell malignancies undergoing autologous bone marrow transplantation and may also be a viable option for T cell depletion as a strategy to prevent graft versus host disease.