transforming-growth-factor-beta and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Extensive bone marrow necrosis and symptomatic hypercalcemia have been described independently as rare complications of chronic myeloid leukemia. Here we report a 66-year-old man who developed B cell blastic transformation 10 years after diagnosis of CML in the chronic phase. Extensive bone marrow necrosis and symptomatic hypercalcemia concurrently developed after transformation, with development of disseminated intravascular coagulation and multifocal osteolysis. Most necrotic cells were readily identifiable as blasts. Mediators related to hypercalcemia, including prostaglandin E2, transforming growth factor-alpha and transforming growth factor-beta, were significantly elevated in the serum. As far as we know, this is the first case report of chronic myeloid leukemia concurrently developing bone marrow necrosis and hypercalcemia; this association was not reported in other types of leukemia or bone marrow malignancies.

Topics: Blast Crisis; Bone Marrow; Dinoprostone; Disseminated Intravascular Coagulation; Fatal Outcome; Humans; Hypercalcemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Necrosis; Osteolysis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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)

Topics: Blood Cells; Bone Marrow; Cell Differentiation; Cell Division; Chemokine CCL4; Clone Cells; Cytokines; Fusion Proteins, bcr-abl; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophage Inflammatory Proteins; Monokines; Neoplastic Stem Cells; Phenotype; Transforming Growth Factor beta; Tumor Cells, Cultured

Loss of TGF-β-mediated growth suppression is a major contributor to the development of cancers, best exemplified by loss-of-function mutations in genes encoding components of the TGF-β signaling pathway in colorectal and pancreatic cancers. Alternatively, gain-of-function oncogene mutations can also disrupt antiproliferative TGF-β signaling. However, the molecular mechanisms underlying oncogene-induced modulation of TGF-β signaling have not been extensively investigated. Here, we show that the oncogenic BCR-ABL1 of chronic myelogenous leukemia (CML) and the cellular ABL1 tyrosine kinases phosphorylate and inactivate Smad4 to block antiproliferative TGF-β signaling. Mechanistically, phosphorylation of Smad4 at Tyr195, Tyr301, and Tyr322 in the linker region interferes with its binding to the transcription co-activator p300/CBP, thereby blocking the ability of Smad4 to activate the expression of cyclin-dependent kinase (CDK) inhibitors and induce cell cycle arrest. In contrast, the inhibition of BCR-ABL1 kinase with Imatinib prevented Smad4 tyrosine phosphorylation and re-sensitized CML cells to TGF-β-induced antiproliferative and pro-apoptotic responses. Furthermore, expression of phosphorylation-site-mutated Y195F/Y301F/Y322F mutant of Smad4 in Smad4-null CML cells enhanced antiproliferative responses to TGF-β, whereas the phosphorylation-mimicking Y195E/Y301E/Y322E mutant interfered with TGF-β signaling and enhanced the in vivo growth of CML cells. These findings demonstrate the direct role of BCR-ABL1 tyrosine kinase in suppressing TGF-β signaling in CML and explain how Imatinib-targeted therapy restored beneficial TGF-β anti-growth responses.

Topics: Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphorylation; Smad4 Protein; Transforming Growth Factor beta; Tyrosine

To investigate the expression and significance of regulatory T cells (Tregs), FoxP3 and transforming growth factor-β (TGF-β) in different phase of chronic myeloid leukemia (CML).. Peripheral blood of 73 CML patients in Department of Hematology, Heze Municipal Hospital from March 2018 to March 2021 were collected. According to patient's period in CML, they were divided into ND CML group (newly diagnosed), CP CML group (chronic period), and BP CML group (blast phase). The percentage of Tregs, expression level of. The abnormal distribution of Treg subset in different phases of CML and the significant increase of the expression level of. Treg、. 分析调节性T细胞（Treg）、叉头状转录因子P3（FoxP3 ）和转化生长因子-β（TGF-β）在慢性髓系白血病（CML）不同时期中的表达及意义。.. 收集2018年3月-2021年3月在菏泽市立医院血液内科就诊的73例CML患者的外周血，根据患者所处的CML不同时期，将患者分为初诊组、慢性期组和急变期组，应用流式细胞术检测患者Treg水平，RT-qPCR检测. Treg细胞亚群在CML不同时期中的异常分布和. 去甲氧柔红霉素联合大剂量阿糖胞苷巩固治疗首次缓解的老年急性髓系白血病的疗效分析.. 评估去甲氧柔红霉素联合大剂量阿糖胞苷作为老年急性髓系白血病（AML）患者缓解后治疗方案的疗效及安全性。.. 对2017年11月至2021年6月纳入的24例年龄≥60岁的初次诱导缓解的AML患者进行去甲氧柔红霉素联合大剂量阿糖胞苷的巩固化疗（去甲氧柔红霉素10 mg/m

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Cytarabine; Female; Forkhead Transcription Factors; Humans; Idarubicin; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Male; Middle Aged; Remission Induction; Retrospective Studies; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Despite decades of research, standard therapies remain ineffective for most leukemias, pushing toward an essential unmet need for targeted drug screens. Moreover, preclinical drug testing is an important consideration for success of clinical trials without affecting non-transformed stem cells. Using the transgenic chronic myeloid leukemia (CML) mouse model, we determine that leukemic stem cells (LSCs) are transcriptionally heterogenous with a preexistent drug-insensitive signature. To test targeting of potentially important pathways, we establish ex vivo expanded LSCs that have long-term engraftment and give rise to multilineage hematopoiesis. Expanded LSCs share transcriptomic signatures with primary LSCs including enrichment in Wnt, JAK-STAT, MAPK, mTOR and transforming growth factor β signaling pathways. Drug testing on expanded LSCs show that transforming growth factor β and Wnt inhibitors had significant effects on the viability of LSCs, but not leukemia-exposed healthy HSCs. This platform allows testing of multiple drugs at the same time to identify vulnerabilities of LSCs.

Topics: Animals; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Neoplastic Stem Cells; Transcriptome; Transforming Growth Factor beta

Macrophages (Mϕ) have been reported to downmodulate the cytotoxicity of natural killer (NK) cell against solid tumor cells. However, the collaborative role between NK cells and Mϕ remains underappreciated, especially in hematological cancers, such as chronic myeloid leukemia (CML). We observed a higher ratio of innate immune cells (Mϕ and NK) to adaptive immune cells (T and B cells) in CML bone marrow aspirates, prompting us to investigate the roles of NK and Mϕ in CML. Using coculture models simulating the tumor inflammatory environment, we observed that Mϕ protects CML from NK attack only when CML was itself mycoplasma-infected and under chronic infection-inflammation condition. We found that the Mϕ-protective effect on CML was associated with the maintenance of CD16 level on the NK cell membrane. Although the NK membrane CD16 (mCD16) was actively shed in Mϕ + NK + CML trioculture, the NK mCD16 level was maintained, and this was independent of the modulation of sheddase by tissue inhibitor of metalloproteinase 1 or inhibitory cytokine transforming growth factor beta. Instead, we found that this process of NK mCD16 maintenance was conferred by Mϕ in a contact-dependent manner. We propose a new perspective on anti-CML strategy through abrogating Mϕ-mediated retention of NK surface CD16.

Topics: Adaptive Immunity; B-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Cell Survival; Coculture Techniques; Cytokines; Cytotoxicity, Immunologic; GPI-Linked Proteins; Humans; Inflammation; Interleukin-8; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophages; Mycoplasma; Receptors, IgG; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Recent strategies for treating CML patients have focused on investigating new combinations of tyrosine kinase inhibitors (TKIs) as well as identifying novel translational research agents that can eradicate CML leukemia-initiating cells (CML-LICs). However, little is known about the therapeutic benefits such CML-LIC targeting therapies might bring to CML patients. In this study, we investigated the therapeutic potential of EW-7197, an orally bioavailable transforming growth factor-β signaling inhibitor which has recently been approved as an Investigational New Drug (NIH, USA), to suppress CML-LICs in vivo. Compared to TKI treatment alone, administration of TKI plus EW-7197 to CML-affected mice significantly delayed disease relapse and prolonged survival. Notably, combined treatment with EW-7197 plus TKI was effective in eliminating CML-LICs even if they expressed the TKI-resistant T315I mutant BCR-ABL1 oncogene. Collectively, these results indicate that EW-7197 may be a promising candidate for a new therapeutic that can greatly benefit CML patients by working in combination with TKIs to eradicate CML-LICs.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Disease Models, Animal; Humans; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Inbred C57BL; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyridazines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Triazoles

Ornithine decarboxylase antizyme (OAZ) has recently emerged as a potential therapeutic target in various malignant tumors because it plays vital roles in cellular functions including proliferation, differentiation, apoptosis and genomic stability. Therefore, there is a significant interest in discovering its function in chronic myeloid leukemia (CML). Firstly, OAZ1 mRNA was measured by qRT-PCR in 43 cases with CML and 23 controls, and we demonstrated that it is significantly down-regulated in CML patients. To further understand its functions in CML pathogenesis, OAZ1 was overexpressed, and the human leukemia PCR array analysis was used to monitor the expression of key genes commonly involved in leukemia development, classification and therapeutic response. We found several favorable up-regulation factors including CXCL10, DAPK1 and IKZF3. In conclusion, OAZ1 may be a useful therapeutic target in CML due to its potential ability to induce erythroid differentiation and cell apoptosis. These functions were proven to be associated with several gene changes that were directly or indirectly caused by OAZ1. The mechanism of how OAZ1 affects other genes remains to be elucidated.

Topics: Apoptosis; Case-Control Studies; Chemokine CXCL10; Down-Regulation; Erythropoiesis; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Up-Regulation

The persistence leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) despite tyrosine kinase inhibition (TKI) may explain relapse after TKI withdrawal. Here we performed genome-wide transcriptome analysis of highly refined CML and normal stem and progenitor cell populations to identify novel targets for the eradication of CML LSCs using exon microarrays. We identified 97 genes that were differentially expressed in CML versus normal stem and progenitor cells. These included cell surface genes significantly upregulated in CML LSCs: DPP4 (CD26), IL2RA (CD25), PTPRD, CACNA1D, IL1RAP, SLC4A4, and KCNK5. Further analyses of the LSCs revealed dysregulation of normal cellular processes, evidenced by alternative splicing of genes in key cancer signaling pathways such as p53 signaling (e.g. PERP, CDKN1A), kinase binding (e.g. DUSP12, MARCKS), and cell proliferation (MYCN, TIMELESS); downregulation of pro-differentiation and TGF-β/BMP signaling pathways; upregulation of oxidative metabolism and DNA repair pathways; and activation of inflammatory cytokines, including CCL2, and multiple oncogenes (e.g., CCND1). These data represent an important resource for understanding the molecular changes in CML LSCs, which may be exploited to develop novel therapies for eradication these cells and achieve cure.

Topics: Bone Morphogenetic Protein Receptors; Cell Differentiation; Cell Proliferation; Chemokine CCL2; Cyclin D1; DNA Repair; Down-Regulation; Gene Expression Profiling; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Signal Transduction; Stem Cells; Transcriptome; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Disease Models, Animal; Enzyme Activation; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Inbred C57BL; Neoplastic Stem Cells; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Signal Transduction; Transforming Growth Factor beta; Tumor Stem Cell Assay

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by uncontrolled growth of progenitor cells expressing the tyrosine kinase fusion gene product, Bcr-Abl. At present, little is known regarding how TGFbeta, and downstream Smad transcription factors, influence CML cell proliferation in the context of Bcr-Abl expression. Here we show that ectopic Bcr-Abl expression dramatically increases TGFbeta/Smad-dependent transcriptional activity in Cosl cells, and that this may be due to enhancement of Smad promoter activity. Bcr-Abl expressing TF-1 myeloid cells are more potently growth arrested by TGFbeta compared to the parental TF-1 cell line. Additionally, growth of Bcr-Abl-expressing CD34+ cells from chronic phase CML patients is inhibited by TGFbeta and, interestingly, treatment of a non-proliferating CD34+ CML cell sub-population with the TGFbeta kinase inhibitor SB431542 enhanced cell death mediated by the Bcr-Abl inhibitor imatinib. Our data suggest that the expression of Bcr-Abl leads to hyper-responsiveness of myeloid cells to TGFbeta, and we hypothesise that this novel cross-regulatory mechanism might play an important role in maintaining the transformed progenitor cell population in CML.

Topics: Antigens, CD34; Benzamides; Fusion Proteins, bcr-abl; Hematopoietic Stem Cells; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Plasmids; Promoter Regions, Genetic; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Angiogenesis is critical for the clinical progression of haematopoietic malignancies and depends on angiogenic factors. Angiogenin is a powerful factor produced by neoplastic cells and host microenvironment. High levels of soluble angiogenin (sAng) correlate with a poor prognosis in patients affected by acute myeloid leukaemia and myelodysplastic syndromes, but no data are available on sAng in chronic myeloproliferative disorders (CMD). Therefore, in this study we investigated the clinical significance of the angiogenin in sera of patients with chronic myeloid leukaemia (CML) (n = 14) or essential thrombocythaemia (ET) (n = 20), and correlated them with those of soluble transforming growth factor-beta(1) (sTGF beta(1)). Enzyme-linked immunosorbent assay detected (P < 0.05) higher levels of sAng in CMD compared with healthy subjects (1026.74 +/- 464.60 pg/mL and 196.00 +/- 39.90 pg/mL, respectively). The highest levels of sAng were detected in CML patients (1349.23 +/- 549.55 pg/mL). Interestingly, CML patients who achieved haematological remission after interferon therapy showed circulating levels of angiogenin significantly (P < 0.05) decreased when compared with those at diagnosis. In ET patients, levels of angiogenin (889.34 +/- 267.66 pg/mL) and sTGF beta(1) (76.69 +/-6.08 pg/mL) were higher (P < 0.05) compared with healthy controls (57.93 +/- 19.39 pg/mL). No correlation was found between levels of sAng and levels of sTGF beta(1) or platelet count among ET patients. Our results show for the first time that elevated blood levels of angiogenin feature chronic myeloid malignancies, suggesting a role of angiogenin in the pathogenesis of these diseases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Chronic Disease; Female; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Myeloproliferative Disorders; Neoplasm Proteins; Prognosis; Remission Induction; Ribonuclease, Pancreatic; Solubility; Thrombocythemia, Essential; Transforming Growth Factor beta

The growth factor-independent erythroleukemic cell line ERY-1 was established from the peripheral blood of a 87-year-old woman with chronic myeloid leukemia (CML) in the acute phase. Immunophenotyping showed that fresh leukemic cells were positive for CD13, CD33, CD36 and CD235a (glycophorin A), a phenotype compatible with that of erythroblastic cells. Cytogenetic and fluorescence in situ hybridization (FISH) analysis demonstrated classical t(9;22)(q34;q11) chromosomic translocation associated with a duplication of the BCR-ABL fusion gene. Other cytogenetic abnormalities were detected in all analyzed mitosis, the most frequent being a trisomy of chromosome 8. The established ERY-1 cell line retains these immunophenotypic and cytogenetic features, and light and electron microscopy confirmed the relatively mature erythroblastic phenotype of the cells. In addition, ERY-1 cell line expressed beta-globin mRNA and a non-phosphorylable form of the erythropoietin receptor, even in presence of erythropoietin. Of note, the proliferation of ERY-1 cells was inhibited by TGFbeta1 or STI-571 (Gleevec), without significant induction of further differentiation. In conclusion, ERY-1 is a new growth factor-independent human erythroleukemic cell line with a relatively mature phenotype that may be useful to study the molecular events involved in erythroblastic differentiation.

Topics: Aged; Aged, 80 and over; Antigens, CD; Benzamides; Cell Line, Tumor; Chromosomes, Human, Pair 8; Female; Fusion Proteins, bcr-abl; Gene Duplication; Globins; Humans; Imatinib Mesylate; Immunophenotyping; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phenotype; Piperazines; Pyrimidines; Receptors, Erythropoietin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Translocation, Genetic; Trisomy

We report a case of chronic myelocytic leukemia (CML) with cyclic oscillation of platelet and leukocyte counts and attempt to elucidate the oscillatory mechanism from the standpoint of cytokine regulation of hematopoiesis. A 57-year-old woman with a diagnosis of CML exhibited platelet and white blood cell (WBC) count fluctuations of a cyclic nature. The average duration of the cycles was about 8 weeks. The patient suffered from headache, fatigue, and malaise at the peak of the cycle. The peak thrombopoietin concentration in peripheral blood coincided with a period of decrease in platelet numbers. The change in transforming growth factor beta (TGF-beta) level paralleled that of the platelet numbers. A progenitor cell assay revealed the suppression of trilineage colony formation in the presence of plasma from the blood cell peak point, and this suppression was completely blocked when the plasma was incubated with an anti-TGF-beta antibody. From these findings, we concluded that the cyclic oscillation of the platelet, WBC, and reticulocyte counts had been induced by excess negative feedback to megakaryopoiesis by TGF-beta.

Topics: Antibodies, Monoclonal; Blood Cells; Feedback, Physiological; Female; Hematopoiesis; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocyte Count; Middle Aged; Periodicity; Platelet Count; Reticulocyte Count; Transforming Growth Factor beta

Topics: Animals; DNA-Binding Proteins; Genes, abl; Genotype; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MDS1 and EVI1 Complex Locus Protein; Mice; Myelodysplastic Syndromes; Proto-Oncogenes; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Transforming growth factor beta3 (TGF-beta3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-beta3 on highly purified CD34+ cells and more immature CD34-DR(-) cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors.. Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late-acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively.. The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-beta3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3-and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-beta3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-beta3 demonstrated a greater inhibitory activity on primitive CD34+DR cells than on more mature CD34+ cells. Again, CML CD34+DR(-) cells were significantly more inhibited by TGF-beta3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-beta induces cell cycle arrest in G(1) phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-beta3 preferentially triggered the programmed cell death of CML CD34-cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion. We demonstrate that TGF-beta3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.

Topics: Antigens, CD34; Apoptosis; Cell Cycle; Cells, Cultured; Clone Cells; Cytokines; fas Receptor; Hematopoiesis; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Transforming Growth Factor beta; Up-Regulation

Chronic myeloid leukemia (CML) is a malignant stem cell disease characterized by an expansion of myeloid progenitor cells expressing the constitutively activated Bcr-Abl kinase. This oncogenic event causes a deregulation of apoptosis and cell cycle progression. Although the molecular mechanisms protecting from apoptosis in CML cells are well characterized, the cell cycle regulatory event is poorly understood. An inhibitor of the cyclin-dependent kinases, p27, plays a central role in the regulation of growth factor dependent proliferation of hematopoietic cells. Therefore, we have analyzed the influence of Bcr-Abl in the regulation of p27 expression in various hematopoietic cell systems. An active Bcr-Abl kinase causes down-regulation of p27 expression in murine Ba/F3 cells and human M07 cells. Bcr-Abl blocks up-regulation of p27 after growth factor withdrawal and serum reduction. In addition, p27 induction by transforming growth factor-beta (TGF-beta) is completely blocked in Bcr-Abl positive M07/p210 cells. This deregulation is directly mediated by the activity of the Bcr-Abl kinase. A Bcr-Abl kinase inhibitor completely abolishes p27 down-regulation by Bcr-Abl in both Ba/F3 cells transfected either with a constitutively active Bcr-Abl or with a temperature sensitive mutant. The down-regulation of p27 by Bcr-Abl depends on proteasomal degradation and can be blocked by lactacystin. Overexpression of wild-type p27 partially antagonizes Bcr-Abl-induced proliferation in Ba/F3 cells. We conclude that Bcr-Abl promotes cell cycle progression and activation of cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27. (Blood. 2000;96:1933-1939)

Topics: Animals; Blotting, Western; Cell Cycle Proteins; Cell Division; Cell Line; Chromones; Culture Media; Culture Media, Serum-Free; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Endopeptidases; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Growth Substances; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Microtubule-Associated Proteins; Morpholines; Multienzyme Complexes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

We established two novel cell lines, designated as IMS-BC1 and IMS-BC2, from two patients with chronic myelogenous leukemia in blast crisis. The two cell lines were positive for CD13 and CD33 and negative for CD34 and HLA-DR by surface marker analysis. IMS-BC1 had four Philadelphia (Ph1) chromosomes and a breakpoint within the 3'-portion of M-bcr, and IMS-BC2 had five Ph1 chromosomes and two breakpoints within the 3'- and 5'-portions of M-bcr. Both cell lines' growth activities were moderately suppressed by IFN-alpha. The proliferation of IMS-BC2 was inhibited by IFN-gamma and apoptosis was induced within 72 h, while IMS-BC1 was resistant to IFN-gamma. Fibronectin inhibited the proliferation of the two cell lines at higher than 10 micrograms/ml, but only IMS-BC2 showed apoptosis. Transforming growth factor-beta inhibited the proliferation of IMS-BC2 resulting in apoptosis, while it inhibited that of IMS-BC1 moderately but failed to induce apoptosis. All-trans retinoic acid (ATRA) inhibited the proliferation of IMS-BC2 at very low concentration (10(-17) mol/l) and induced apoptosis at doses higher than 10(-9) mol/l within 72 h without terminal differentiation, while IMS-BC1 was completely resistant to ATRA. The two cell lines showed different responses to growth inhibitory cytokines and factors. These cell lines should prove useful in the analysis of mechanisms of apoptosis induced by growth inhibitory cytokines and factors.

Topics: Adolescent; Adult; Antigens, CD; Antineoplastic Agents; Apoptosis; Cell Division; Humans; Interferon-gamma; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The long-term culture (LTC) system has been useful for analyzing mechanisms by which stromal cells regulate the proliferative activity of primitive normal, but not chronic myeloid leukemia (CML), hematopoietic progenitor cells. In previous studies, we identified two endogenous inhibitors in this system. One is transforming growth factor-beta (TGF-beta), which is equally active on primitive normal and CML progenitors. The other we now show to be monocyte chemoattractant protein-1 (MCP-1). Thus, MCP-1, when added to LTC, blocked the activation of primitive normal progenitors but did not arrest the cycling of primitive CML progenitors. Moreover, the endogenous inhibitory activity of LTC stromal layers could be overcome by the addition of neutralizing antibodies to MCP-1, but not to macrophage inflammatory protein-1alpha (MIP-1alpha). However, neither of these antibodies antagonized the inhibitory activity of NAc-Ser-Asp-Lys-Pro (AcSDKP) on primitive normal but not CML progenitor cycling in this system. Moreover, none of six other -C-C- or -C-X-C- chemokines, previously shown to inhibit primitive normal human CFC proliferation in semisolid assays, were found to act as negative regulators when added to normal LTC. These results provide further support for the concept that primitive CML progenitor cell proliferation is deregulated when these cells are exposed to limiting concentrations of multiple inhibitors, only some of which have differential actions on normal and Ph+/BCR-ABL+ cells.

Topics: Bone Marrow; Cell Cycle; Cell Division; Cells, Cultured; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Cytokines; Fusion Proteins, bcr-abl; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophage Inflammatory Proteins; Neoplastic Stem Cells; Oligopeptides; Recombinant Proteins; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-alpha-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor beta1 (TGFbeta1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D3, as judged by the capability to reduce nitro blue tetrazolium, by the appearance of monocyte-related cell surface antigens and by morphologic criteria. K562 cells showed decreased differentiation response to TGFbeta1, but not to hemin, when incubated with antisense oligonucleotides. U-937 antisense RB-transfected cells were also suppressed in their ability to upregulate levels of hypophosphorylated pRb when induced to differentiate. Although U-937 cells incubated with antisense oligonucleotides and clones expressing the antisense RB construct were hampered in their ability to differentiate on incubation with ATRA or Vit D3, the induced G0/G1-accumulation was similar to differentiating control cells treated with ATRA or Vit D3. Intriguingly, U-937 clones overexpressing RB were also inhibited in their differentiation response to ATRA or Vit D3 but not inhibited in their ability to respond with G0/G1 accumulation when induced with these substances. The results indicate that pRb plays a role in induced differentiation of U-937 cells as well as K562 cells involving mechanisms that, at least partially, are distinct from those inducing G1 accumulation.

Topics: Animals; Antigens, Differentiation; Antigens, Neoplasm; Calcitriol; Cell Differentiation; G1 Phase; Gene Expression Regulation, Leukemic; Genes, Retinoblastoma; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Large B-Cell, Diffuse; Mice; Monocytes; Neoplasm Proteins; Oligonucleotides, Antisense; Retinoblastoma Protein; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Spontaneous and induced fetal resorptions have been associated with the infiltration and activation of GM1-positive natural killer (NK)-like cells. Predominance of these cells in the decidua and their reduced lytic activity suggest that regulation of their killing activity could be important for the survival of the fetus. It has therefore been hypothesized that the embryo was regulating NK lytic activity. To test this hypothesis, human and rabbit lymphocytes were cultured with various concentrations of interleukin-2. Their ability to kill 51Cr-labelled NK and lymphokine-activated killer (LAK)-sensitive targets was assessed in the presence of rabbit blastocoelic fluid taken at day-12 of pregnancy (BF D-12). BF D-12 dramatically suppressed the killing activity of NK and LAK cells. This effect was observed on K562 (NK-sensitive targets), P815 cells (LAK-sensitive targets), and freshly isolated cells in rabbit trophoblastic cell preparation. Elimination of prostaglandin E2 (PGE2), but not transforming growth factor beta 2 (TGF beta 2) or 6 keto prostaglandin F1 alpha (6KPGF 1 alpha), by affinity chromatography, completely abolished BF biological activity. These findings clearly suggest that PGE2 in BF regulates the killing activity of NK and LAK cells, and that the semiallograft embryo plays an active role in its own protection. To our knowledge, it is the first demonstration that PGE2 from the embryo inhibits NK and LAK cell lytic activity.

Topics: Animals; Blastocyst; Body Fluids; Cytotoxicity, Immunologic; Dinoprostone; Female; Humans; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Mast-Cell Sarcoma; Mice; Pregnancy; Rabbits; Transforming Growth Factor beta; Tumor Cells, Cultured

The erythromegakaryocytic cell line (LAMA-84) and the erythroeosinophilic cell line (LAMA-87) were used to study receptor expression and receptor-mediated response to monocyte/macrophage colony-stimulating factor (M-CSF) and transforming growth factor beta (TGF-beta), two modulators of cell proliferation. As demonstrated by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), c-fms and M-CSF mRNA were expressed in both cell lines. M-CSF was detected in the supernatant of both cell lines and addition of a neutralizing anti-M-CSF antibody inhibited cell growth. The two LAMA cell lines were found to express TGF-beta1, -beta2, and -beta3 mRNAs and to secrete TGF-beta mostly in latent form. Addition of anti-TGF-beta antibodies to the culture medium increased their proliferation, whereas TGF-beta1 inhibited cell proliferation by downregulating the c-myc mRNA. These results show that the proliferation of both LAMA cell lines is positively and negatively regulated by autocrine mechanisms, implying the presence of M-CSF and TGF-beta, respectively. They suggest that similar autocrine loops could be involved in the growth regulation of leukemic cells in vivo.

Topics: Animals; Blast Crisis; Blotting, Northern; Cell Division; Culture Media, Conditioned; Eosinophils; Erythrocytes; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophage Colony-Stimulating Factor; Megakaryocytes; Mice; Mice, Nude; Neoplasm Transplantation; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

We evaluated the effects of transforming growth factor-beta 1 (TGF-beta 1) on the growth of hematopoietic progenitors in normal donors and in patients with hematologic malignancies now designed as clonal disorders of multipotential stem cells. TGF-beta 1 at 80 pM exhibited differential effects on the normal hematopoietic progenitors when cells were stimulated with different growth factors, such as G-CSF, GM-CSF, interleukin-3 (IL-3), or stem cell factor (SCF). The suppressive effect by TGF-beta 1 was increased for growth with GM-CSF, IL-3, and SCF, and growth with G-CSF was unaffected in hematologic malignancies, TGF-beta 1 suppression for growth with G-CSF was increased for essential thrombocythemia (ET) and polycythemia vera; chronic myelogenous leukemia (CML) in chronic phase; CML in accelerated phase; CML in myeloid crisis; myelodysplastic syndrome (MDS) in refractory anemia; MDS in refractory anemia with an excess of blasts; and acute myeloblastic leukemia (AML). In CML-myeloid crisis and AML, TGF-beta 1 almost completely abolished the growth, with some patient-to-patient variation. The mean ED50s for the growth of leukemic blast progenitors were 1.6, 1.2, 0.7, and 0.2 pM in the presence of G-CSF, GM-CSF, IL-3, and SCF, respectively, c-myc and c-myb antisense oligonucleotides significantly suppressed the growth of leukemic blast progenitors, but not that of clonogenic cells from normal donors and patients with ET. We also demonstrated that TGF-beta 1 inhibits mRNA expression by AML blasts for c-myc and/or c-myb. When the data are taken together, growth suppression by TGF-beta 1 appears to increase with the progression of clonal evolution in hematologic malignancies.

Topics: Base Sequence; Blast Crisis; Bone Marrow; Cell Division; Colony-Forming Units Assay; Gene Expression; Genes, myc; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Molecular Sequence Data; Oligodeoxyribonucleotides; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Proto-Oncogene Proteins c-myc; Recombinant Proteins; Reference Values; RNA, Messenger; Transforming Growth Factor beta

To understand the relationship between transforming growth factor beta-1 (TGF-beta 1) and the integrin profile presented by chronic myeloid leukemia cells, we have studied, using Northern analysis, the expression of TGF-beta 1 messenger RNA (TGF-beta mRNA) in myeloid cell lines and in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition we determined the positivity for alpha 4 and alpha 5 integrin molecules in those cells using specific monoclonal antibodies and flow cytometry. CML patients (N = 3) presented mean values of alpha 4 and alpha 5 higher (alpha 4: 60 +/- 20%; alpha 5: 70 +/- 41%) than AML (N = 10) blast cells (alpha 4: 25 +/- 23%; alpha 5: 18 +/- 16%). Northern analysis revealed an almost four-fold higher expression of TGF-beta mRNA in K562 (derived from a patient with chronic myeloid leukemia) compared to the myeloblastic cell line HL60. The highest TGF-beta mRNA levels were seen in the U937 lineage. CML leukemic cells (N = 3) showed high TGF-beta mRNA levels comparable to the levels expressed by K562 which was paralleled by high beta 1 integrin mRNA. AML blast cells presented a variable degree of expression of TGF-beta mRNA when compared to HL60. One patient with acute megakaryoblastic leukemia (FAB subtype M7), usually associated with myelofibrosis, presented the highest TGF-beta mRNA levels. We conclude that studying TGF-beta 1 and its mechanisms of action will help in understanding fibrosis in leukemic patients, and perhaps to design treatments for such conditions.

Topics: Humans; Integrins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Transforming Growth Factor beta; Tumor Cells, Cultured

The present study proved that TGF-beta 1 significantly inhibited the growth of K 562 cells. The drop in cell numbers after 24 h incubation with increasing concentrations of TGF-beta 1 (0.01, 0.1, 1.0, 10.0 ng/ml) was accompanied by significant suppression of the activity of two key enzymes of polyamine biosynthesis: ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). In contrast to ODC and SAMDC activity, TGF-beta 1 did not significantly affect the absolute concentration of spermidine and spermine in K 562 cells. We suppose that the lack of an evident drop in concentration of spermidine and spermine in spite of a significant decrease in ODC and SAMDC activity in K 562 cells exposed to TGF-beta 1 resulted from the uptake of polyamines from the extracellular space.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosylmethionine Decarboxylase; Apoptosis; Cell Division; Cell Line; Dose-Response Relationship, Drug; Eflornithine; Humans; Isoquinolines; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitoguazone; Ornithine Decarboxylase; Piperazines; Polyamines; Protein Kinase Inhibitors; Putrescine; Spermidine; Spermine; Transforming Growth Factor beta; Tumor Cells, Cultured

In chronic myeloid leukemia (CML) an abnormality at the stem cell level results in unregulated expansion of myeloid progenitors. The mechanism underlying this uncontrolled proliferation remains unclear. An in vitro clonogenic assay which detects the human counterpart of the murine colony forming unit (CFU) CFU-A/CFU-S day 12 was described in a report of our recent findings. CML bone marrow samples were found to proliferate in the CFU-A assay, producing colonies morphologically indistinguishable from normal controls. The bcr/abl transcripts were sought in the RNA from individual colonies using the polymerase chain reaction (PCR). For the five CML samples tested to date, the majority of CFU-A colonies at diagnosis or in early chronic phase were found to be bcr/abl positive. For normal controls both macrophage inflammatory protein-1 alpha (MIP-1 alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibited the proliferation of CFU-A colonies when directly added to the assay. In contrast, CML progenitors responded normally to TGF-beta 1, but showed no response to MIP-1 alpha. In suicide assays, for five normal bone marrow samples, CFU-A progenitors induced into S-phase returned to a quiescent state after treatment with MIP-1 alpha. CML progenitors demonstrated inherently high cycle status which showed no definite response to MIP-1 alpha. However, TGF-beta 1 resulted in quiescence of CML progenitor cycling. In conclusion, the primitive progenitors from CML samples were inhibited normally by TGF-beta 1 but showed no response to MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Division; Chemokine CCL4; Cytokines; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophage Inflammatory Proteins; Monokines; Recombinant Proteins; Transforming Growth Factor beta; Tumor Stem Cell Assay

Long-term maintenance of normal hematopoiesis in vitro is possible when very primitive progenitors are cocultivated with certain non-hematopoietic stromal cells that may co-exist in (or be derived from cells that co-exist in) hematopoietic tissues. Such long-term cultures (LTC) have been used to develop quantitative assays for the most primitive populations of hematopoietic cells currently detectable in adult marrow. In addition they provide a unique model for analysis of the complex molecular mechanisms that may regulate primitive hematopoietic cell population dynamics in vivo. Similar studies with LTC of cells from patients with chronic myeloid leukemia (CML) have made it possible to detect and characterize very primitive neoplastic cell populations in this disease. These latter studies have revealed differences in the properties of primitive CML cells that both reflect and explain their increased turnover and are thus presumably part of the mechanism that enables the neoplastic clone to expand in vivo. In addition, the most primitive neoplastic cells in CML patients are abnormally distributed between the marrow and blood and their ability to maintain their numbers in LTC has also been found to be defective. Assessment of the number and behaviour of primitive cells in LTC of CML marrow has been used to identify those patients most likely to benefit from intensive therapy supported by transplantation of cultured autologous marrow. Twenty-two such CML patients have now been treated with this experimental protocol. The results to date have clearly established the feasibility of this novel treatment strategy and, together with more recent laboratory findings, suggest future avenues for significantly improving the management of CML patients.

Topics: Bone Marrow Transplantation; Cells, Cultured; Hematopoiesis; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Transforming Growth Factor beta; Transplantation, Autologous

Basophils and eosinophils share a common differentiation pathway. Factors regulating terminal commitment toward one cell type, however, have so far not been defined. Interleukin-3 (IL-3) is a potent differentiation factor for both human eosinophils and basophils. In the present study, the effects of various recombinant human (rh) growth regulators on IL-3-dependent growth of eosinophils and basophils were studied in a bone marrow (BM) suspension culture system (normal donors, n = 13). We found that type beta transforming growth factors (TGFs) lead to a significant increase in the absolute numbers of basophils in BM cultures grown in the presence of IL-3 (day 14 of culture; IL-3: 133 +/- 20 v IL-3 + TGF-beta 1: 231 +/- 28 x 10(3)/mL [P less than .01]) and to an increase in the total histamine values (IL-3: 72.6 +/- 22.2 v IL-3 + TGF-beta 1: 142.9 +/- 37.3 ng/mL [P less than .015]) compared with rhIL-3 alone. In contrast, type beta TGFs were found to inhibit the IL-3-dependent growth of eosinophils (IL-3: 170.4 +/- 37.2 v IL-3 + TGF-beta 1: 16.7 +/- 5.2 x 10(3)/mL [P less than .01]) and formation of eosinophil cationic protein in the same culture system. The effect of TGF-beta 1 (and TGF-beta 2) on IL-3-dependent differentiation of basophils and eosinophils was dose- and time-dependent (maximum effects observed with 1 to 10 ng/mL of rhTGF-beta 1 or TGF-beta 2) and could be neutralized by an antibody specific for TGF-beta 1. In contrast to the TGFs, interferon-alpha (IFN-alpha) and IFN-gamma were found to downregulate IL-3-dependent formation of both basophils (IL-3: 167 +/- 33 v IL-3 + IFN-alpha: 67 +/- 25 v IL-3 + IFN-gamma: 65 +/- 33 x 10(3)/mL [P less than .01]) and eosinophils (IL-3: 239 +/- 5 v IL-3 + IFN-alpha: 81 +/- 4 v IL-3 + IFN-gamma: 67 +/- 17 x 10(3)/mL [P less than .05]) in our culture system. Type beta TGFs as well as the IFNs failed to directly induce differentiation of human basophils or eosinophils in the absence of other growth factors. Together, these results show that type beta TGFs and IFNs are potent regulators of cytokine-dependent growth and differentiation of human allergic effector cells.

Topics: Antibodies, Monoclonal; Antigens, Surface; Basophils; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Eosinophils; Hematopoietic Stem Cells; Histamine Release; Humans; Interferon Type I; Interferon-gamma; Interleukin-3; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocytes; Neutrophils; Recombinant Proteins; Transforming Growth Factor beta

Experiments were undertaken to investigate the molecular basis of primitive hematopoietic progenitor cell regulation in both the long-term culture system and in methylcellulose, particularly with a view to characterizing factors either able or unable to influence the behaviour of primitive leukemic cells from patients with chronic myeloid leukemia (CML). Long-term cultures of CML cells with or without irradiated normal marrow feeder layers were initiated from peripheral blood cells of CML patients with high white blood cell counts. Three weeks later the effect of exogenously added transforming growth factor-beta 1 (TGF-beta 1) on progenitor cycling status was examined. A single addition of 5 ng/ml TGF-beta 1 was able to reversibly arrest the otherwise uninterrupted turnover of primitive leukemic erythroid and granulopoietic progenitors for a period of up to 7 days both in the presence and absence of a normal adherent cell population. When TGF-beta 1 was incorporated into methylcellulose cultures, its ability to inhibit colony formation by CML progenitors showed the same differential activity on primitive cell types exhibited by normal progenitors. Dose-response curves for analogous populations of normal and leukemic cells were indistinguishable. Increasing the concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose colony assays decreased the sensitivity displayed by normal clonogenic cells to TGF-beta 1 and no differences were detectable when CML cells were used in such regulator competition experiments. These findings support a general model of primitive hematopoietic cell regulation in which entry into S-phase is determined at the intracellular level by multiple convergent pathways that may deliver either positive or negative signals from activated cell surface receptors for distinct extracellular factors. The present study shows for the first time that primitive CML progenitors exposed to TGF-beta 1 in vitro can be transiently blocked in a noncycling state for several days without loss of viability and that the mechanisms responsible for the emergence and maintenance of a clonal population of CML cells in vivo do not appear to involve changes in their sensitivity to TGF-beta 1. It is thus unlikely that the heightened proliferative activity exhibited by primitive CML progenitors both in vivo and in long-term culture can be explained by an abnormality in the intracellular mechanisms normally activated by TGF-beta 1

Topics: Cell Cycle; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Transforming Growth Factor beta; Tumor Cells, Cultured

Recent in vitro studies indicate that bone marrow mesenchymal elements, residing in close proximity to hematopoietic cell populations, elaborate a network of cytokines that are, at least partially, responsible for modulating the growth and maturation of the latter compartment. Leukemia inhibitory factor (LIF), a molecule with both positive and negative regulatory activities, has been implicated in murine embryogenesis and hematopoiesis. We demonstrate that cultured normal human bone marrow stromal cells constitutively express LIF message. Further, exposure of these cells to other hematopoietic modulators including interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) (but not interferon-alpha [IFN alpha]) increases the level of LIF RNA. Interestingly, cultured stromal cells derived from three of four patients with chronic myelogenous leukemia showed enhanced LIF expression. These observations suggest that LIF may participate, either alone or through interaction with other cytokines, in the bone marrow microenvironment-mediated influence on both normal and malignant hematopoietic processes.

Topics: Bone Marrow; Cell Line; Fibroblasts; Gene Expression; Growth Inhibitors; Hematopoiesis; Humans; Interleukin-1; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocytes; Lymphokines; Monocytes; RNA; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We have shown that incubation of bone marrow (BM) with interleukin 2 (IL-2) generates activated bone marrow cells (ABM) with potent tumoricidal activity in vitro and in vivo. The present study was carried out to define the interaction of other cytokines with IL-2 in generation of ABM. Our data show that interleukin 1 (IL-1), interferon (IFN)- both gamma and alpha, and tumor necrosis factor (TNF-alpha) significantly increased the cytolytic potential of ABM. Interleukin 3, interleukin 4, transforming growth factor-beta and adherent cells were reduced, while granulocyte-macrophage colony-stimulating factor had no influence on the generation of cytolytic activity. IL-1 was enhanced while TNF-alpha depressed the BM progenitor cell activity in vitro. The IL-2-induced purging ability of BM contaminated with leukemic cells was increased by IL-1, TNF-alpha and IFN-gamma. This study shows that biomodulation of BM with combination of cytokines in vitro can be useful in purging a large leukemic burden.

Topics: Bone Marrow; Burkitt Lymphoma; Cell Line; Cytokines; Cytotoxicity, Immunologic; Drug Interactions; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Humans; In Vitro Techniques; Interferon-alpha; Interferon-gamma; Interleukin-1; Interleukin-2; Interleukin-3; Interleukin-4; Killer Cells, Natural; Leukemia; Leukemia, Lymphoid; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Activins; Cell Differentiation; Cell Line; Cells, Cultured; Colony-Forming Units Assay; Erythropoiesis; Globins; Hematopoietic Stem Cells; Humans; Inhibins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macromolecular Substances; Models, Biological; Multigene Family; RNA, Messenger; Transforming Growth Factor beta