tetracycline and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Chronic myeloid leukemia (CML) is a clonal disorder originating in the pluripotent hematopoietic stem cell (HSC), the hallmark of which is the constitutively activated p210-type of Bcr-Abl tyrosine kinase protein. Studies in recent years have helped us to understand the molecular processes involved in the initiation and progression of CML. Although a great amount of knowledge has been accumulated, the effect of Bcr-Abl on the HSC is still unclear. We have developed an in vitro system that mirrors the chronic phase of CML with a combination of in vitro embryonic stem cell differentiation and tetracycline-inducible Bcr-Abl expression. Enforced Bcr-Abl expression was sufficient to increase the number of both multilineage progenitors and myeloid progenitors. The current system is powerful for analyzing the genetic changes in hematopoietic development. This review focuses on how Bcr-Abl affects HSCs and how Bcr-Abl expression alters the properties of HSCs.

Topics: Animals; Cell Differentiation; Cell Division; Fusion Proteins, bcr-abl; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Tetracycline

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.

Topics: Acute-Phase Proteins; Animals; Apoptosis; Blotting, Western; Bone Marrow Cells; Cell Death; Coculture Techniques; Dose-Response Relationship, Drug; Fusion Proteins, bcr-abl; Hematopoiesis; Hematopoietic Stem Cells; Humans; Interleukin-3; Lentivirus; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lipocalin-2; Lipocalins; Mice; Mice, Inbred C3H; Mice, Inbred NOD; Mice, SCID; Oligonucleotides, Antisense; Oncogene Proteins; Plasmids; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Spleen; Tetracycline; Time Factors

P210 Bcr-Abl is an activated tyrosine kinase oncogene encoded by the Philadelphia chromosome associated with human chronic myelogenous leukemia (CML). The disease represents a clonal disorder arising in the pluripotent hematopoietic stem cell. During the chronic phase, patients present with a dramatic expansion of myeloid cells and a mild anemia. Retroviral gene transfer and transgenic expression in rodents have demonstrated the ability of Bcr-Abl to induce various types of leukemia. However, study of human CML or rodent models has not determined the direct and immediate effects of Bcr-Abl on hematopoietic cells from those requiring secondary genetic or epigenetic changes selected during the pathogenic process. We utilized tetracycline-regulated expression of Bcr-Abl from a promoter engineered for robust expression in primitive stem cells through multilineage blood cell development in combination with the in vitro differentiation of embryonal stem cells into hematopoietic elements. Our results demonstrate that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. These effects are reversible upon extinguishing Bcr-Abl expression. These findings are consistent with Bcr-Abl being the sole genetic change needed for the establishment of the chronic phase of CML and provide a powerful system for the analysis of any genetic change that alters cell growth and lineage choices of the hematopoietic stem cell.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Lineage; Coculture Techniques; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Promoter Regions, Genetic; Tetracycline; Transfection

The BCR/ABL gene product of the Philadelphia (Ph) chromosome induces chronic myelogenous leukemia (CML). We generated a hematopoietic cell line, TonB210.1, with tetracycline-dependent BCR/ABL expression to investigate the pathways by which BCR/ABL transforms cells. TonB210.1 demonstrates conditional growth factor independence in tissue culture and rapidly forms tumors in mice fed the tetracycline analog doxycycline. The tumors regress completely upon doxycycline withdrawal, but ultimately reform in all animals. After a long latency, tumors also develop in animals never exposed to doxycycline. Subclones of TonB210.1 established from doxycycline-independent tumors demonstrate distinct mechanisms of transformation. Most subclones manifest increased basal levels of BCR/ABL expression; some have lost the capacity to augment expression upon induction, whereas others remain inducible. More interestingly, some subclones maintain tight conditional expression of BCR/ABL and are therefore transformed by secondary mechanisms that no longer require BCR/ABL expression. These subclones show constitutive phosphorylation of the STAT5 protein, suggesting that activating mutations have occurred upstream in the signaling pathway to STAT5. The tight conditional expression of BCR/ABL in the TonB210.1 cell line affords the opportunity to study several interesting aspects of the biology of BCR/ABL, including activation of critical signaling pathways and transcriptional programs, and its potential role in genomic instability.

Topics: Animals; Cell Transformation, Neoplastic; DNA Transposable Elements; Doxycycline; Escherichia coli; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Genes, Synthetic; Hematopoietic Stem Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Neoplastic Stem Cells; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Recombinant Fusion Proteins; Signal Transduction; Tetracycline; Tetracycline Resistance; Trans-Activators; Transfection; Tumor Cells, Cultured