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

Induction of differentiation sensitizes chronic myeloid leukemia (CML) cells to the BCR-ABL inhibitor imatinib by mechanisms that remain unknown. We previously identified the BCR-ABL downstream effector CD69 which inhibits imatinib-induced CML cell differentiation. Herein, we found that the erythroid differentiation inducers activin A and aclacinomycin A induced expression of erythroid markers (α-globin, ζ-globin, GATA-1, and glycophorin A) and simultaneously reduced CD69 levels in K562 CML cells. Blockade of p38MAPK by SB203580 and shRNA eliminated the inhibitory effect of activin A on the promoter, mRNA, and protein levels and positive cell population of CD69. CD69 overexpression inhibited activin A-induced erythroid marker expression. Pretreatment of K562 cells with activin A to induce differentiation followed by a subtoxic concentration of imatinib caused growth inhibition and apoptosis that was reduced by CD69 overexpression. Activin A also reduced the expression of CD69's potential downstream molecule metallothionein 2A (MT2A) via p38MAPK. MT2A-knockdown reduced CD69 inhibition of activin A-induced erythroid marker expression. Furthermore, MT2A-knockdown reduced CD69 inhibition of activin A-imatinib sequential treatment-mediated growth inhibition and apoptosis in K562 and BCR-ABL-expressing CD34

Topics: Activins; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Apoptosis; Cell Differentiation; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; K562 Cells; Lectins, C-Type; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metallothionein; p38 Mitogen-Activated Protein Kinases

Overexpression of SGTP and/or MT may contribute to various carcinogenic processes and to resistance to anticancer treatment. The importance of these proteins, although clearly established in solid tumours, has not been fully understood in haematopoietic neoplasm. The aim of this study was to determine the expression of MT and SGTP in the bone marrow of patients with MPD. Twenty paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MPD were evaluated -- osteomyelofibrosis (OMF), n = 9 and chronic myelocytic leukaemia (CML), n = 11. We demonstrate increased SGTP and MT expression in the bone marrow of MPD patients. In our study levels of MT in OMF patients were higher than in CML. This suggests that MT expression may correlate with bone marrow fibrosis. These data, although based on a relatively small number of patients, raise the possibility that SGTP and MT may play a role in the pathogenesis of MPD. The clinical significance of this phenomenon needs further investigation.

Topics: Bone Marrow; Bone Marrow Cells; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Immunohistochemistry; Isoenzymes; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metallothionein; Primary Myelofibrosis

The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T-cell leukemia in transgenic mice.

Topics: Animals; Base Sequence; Clone Cells; DNA Primers; Female; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; Genes, abl; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, T-Cell; Male; Metallothionein; Mice; Mice, Transgenic; Molecular Sequence Data; Pedigree; Phosphotyrosine; Promoter Regions, Genetic; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; RNA, Messenger; Tissue Distribution; Tyrosine

DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190 constructs in transgenic mice. mRNA transcripts were detectable early in embryonic development and also in hematopoietic tissue of adult animals. Expression of BCR/ABL in peripheral blood preceded development of overt disease. P210 founder and progeny transgenic animals, when becoming ill, developed leukemia of B, T-lymphoid, or myeloid origin after a relatively long latency period. In contrast, P190-transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. The observed dissimilarities are most likely due to intrinsically different properties of the P190 and P210 oncoproteins and may also involve sequences that control transgene expression. The delayed progression of BCR/ABL P210-associated disease in the transgenic mice is consistent with the apparent indolence of human chronic myeloid leukemia during the chronic phase. We conclude that, in transgenic models, comparable expression of BCR/ABL P210 and BCR/ABL P190 results in clinically distinct conditions.

Topics: Animals; B-Lymphocytes; Base Sequence; Blast Crisis; Disease Models, Animal; Embryonal Carcinoma Stem Cells; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Leukemia, Experimental; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metallothionein; Mice; Mice, Transgenic; Molecular Sequence Data; Molecular Weight; Neoplastic Stem Cells; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; T-Lymphocytes; Transgenes

Type I interferons have potent antiproliferative activity both in vitro and in vivo, and their tumor suppressor activity has been suggested. A series of eukaryotic vectors containing a synthetic human consensus type I interferon gene (IFN-con1) under the control of different promoters (cytomegalovirus early promoter, murine metallothionein promoter and the Rous sarcoma virus LTR) were constructed and stably transfected into type I IFN-deficient myelogenous leukemic K562 cells. Constitutive expression of IFNcon1 reverted the malignant phenotype, as indicated by loss of tumorgenicity in nude mice. When stably transformed cells were mixed with parental tumor cells, there was retardation of tumor growth. Constitutive expression of IFNcon1 reverted the malignant phenotype in vitro, as indicated by growth inhibition in culture, and reduction in colony formation on soft agar. Furthermore, IFNcon1 gene expression resulted in elevated erythroid differentiation, growth arrest in S phase and induced apoptosis. Thus the presence of an active IFNcon1 gene overcomes the oncogenic potential of K562 by coordinated modulation of cell proliferation, differentiation and programmed cell death, and it acts as a tumor suppressor in vivo.

Topics: Animals; Apoptosis; Base Sequence; Cell Cycle; Cell Differentiation; Cell Division; Chromosomes, Human, Pair 9; Gene Deletion; HeLa Cells; Humans; Interferon Type I; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metallothionein; Mice; Molecular Sequence Data; Multigene Family; Promoter Regions, Genetic; Recombinant Fusion Proteins; S Phase; Transfection; Tumor Cells, Cultured

Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. Heme treatment increased both HO activity and HO-1 mRNA in the human erythroleukemic cell line K562. Electrophoretic mobility-shift assays of nuclear protein extracts from heme-treated and control cells with specific oligonucleotide probes containing binding sites for known transcription factors, including AP-1, AP-2, Sp1, NF-kappa B, CTF/NF1, TFIID, OKT1, and CREB, and oligonucleotides containing serum-, metal-, and glucocorticoid-responsive elements demonstrated a specific and marked increase in the NF-kappa B and AP-2 transcription factors and, to a lesser extent, an increase in AP-1. No significant increase in other transcription factors over the control, untreated cells was observed. DNase I footprint assays using purified transcription factors revealed the presence of NF-kappa B and AP-2 binding sites in the proximal part of the promoter region of the human HO-1 gene. Moreover, nucleotide sequence analysis of the HO-1 promoter region showed that the protected regions encompassed NF-kappa B and AP-2 consensus binding sites. The presence of regulatory sequences for the binding of transcription factors such as NF-kappa B and AP-2, whose activation is associated with the immediate response of the cell to an injury, may be an indication of the important role which HO-1 may play in defense mechanisms against tissue injury.

Topics: Animals; Base Sequence; Binding Sites; Cell Line; Deoxyribonuclease I; DNA Primers; DNA-Binding Proteins; Enzyme Induction; Gene Expression; Heme Oxygenase (Decyclizing); Hominidae; Humans; Isoenzymes; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metallothionein; Molecular Sequence Data; NF-kappa B; Polymerase Chain Reaction; Promoter Regions, Genetic; Restriction Mapping; TATA Box; Tetradecanoylphorbol Acetate; Transcription Factor AP-2; Transcription Factors; Tumor Cells, Cultured