metallothionein and Leukemia--Promyelocytic--Acute

We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

Topics: Antigens, CD; Cell Differentiation; Cell Line, Tumor; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Humans; Immunophenotyping; Leukemia, Promyelocytic, Acute; Metallothionein; Proto-Oncogene Proteins; Reproducibility of Results; Trans-Activators; Tretinoin

The PML-RAR alpha hybrid protein generated by the t(15;17) translocation in acute promyelocytic leukemia (APL) is thought to play a central role in the oncogenic process. However, analysis of the oncogenic activity of the fusion protein in tissue culture assays or in mice has been hampered by its apparent toxicity in multiple murine cells. To circumvent this problem, we generated an inducible line of transgenic mice, MT135, in which the expression of PML-RAR alpha is driven by the metallothionein promoter. After 5 days zinc stimulation, 27 out of 54 mice developed hepatic preneoplasia and neoplasia including foci of basophilic hepatocytes, dysplasia and carcinoma with a significantly higher incidence of lesions in females than in males. The rapid onset of liver pathologies was dependent on overexpression of the transgene since it was not detected in noninduced transgenic animals of the same age. The PML-RAR alpha protein was always present in altered tissues at much higher levels than in the surrounding normal liver tissues. In addition, overexpression of PML-RAR alpha resulted in a strong proliferative response in the hepatocytes. We conclude that overexpression of PML-RAR alpha deregulates cell proliferation and can induce tumorigenic changes in vivo.

Topics: Animals; Cell Division; Female; Gene Expression; Leukemia, Promyelocytic, Acute; Liver; Liver Neoplasms; Male; Metallothionein; Mice; Mice, Transgenic; Neoplasm Proteins; Oncogene Proteins, Fusion; Precancerous Conditions; Promoter Regions, Genetic; Zinc Sulfate

We have recently shown, using antisense strategy, that the RII beta regulatory subunit of cAMP-dependent protein kinase is essential for cAMP-induced growth inhibition and differentiation of HL-60 human leukemia cells. We constructed a retroviral vector for RII beta (MT-RII beta) by inserting human RII beta complementary DNA into the OT1521 retroviral vector plasmid that contains an internal mouse metallothionein-1 promoter and a neomycin resistance gene. The PA317 packaging cell line was then transfected with MT-RII beta plasmid to produce the amphotrophic stock of MT-RII beta retroviral vector. The infection with MT-RII beta and treatment with CdCl2 brought about growth arrest in HL-60 human leukemia and Ki-ras-transformed NIH 3T3 clone DT cells in monolayer culture with no sign of toxicity. The growth inhibition correlated with the expression of RII beta and accompanied changes in cell morphology; cells became flat, exhibiting enlarged cytoplasm. The growth of these cells in semisolid medium (anchorage-independent growth) was almost completely suppressed. In contrast, overexpression of the RI alpha subunit of protein kinase enhanced the cell proliferation in DT cells. The MT-RII beta-infected cells exhibited an increased sensitivity toward treatment with cAMP analogues, such as 8-Cl-cAMP and N6-benzyl-cAMP, as compared with the parental noninfected cells. In MT-RII beta HL-60 cells, N6-benzyl-cAMP treatment greatly enhanced the expression of monocytic surface markers. These results suggest that the RII beta cAMP receptor, by binding to its ligand, cAMP, acts as a tumor suppressor protein exerting growth inhibition, differentiation, and reverse transformation.

Topics: 3T3 Cells; 8-Bromo Cyclic Adenosine Monophosphate; Allosteric Site; Animals; Cadmium; Cell Differentiation; Cell Division; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Isoenzymes; Leukemia, Promyelocytic, Acute; Metallothionein; Mice; Phenotype; Promoter Regions, Genetic; Recombinant Fusion Proteins; Retroviridae; Signal Transduction; Tumor Cells, Cultured

Hemopexin-mediated heme transport into mouse hepatoma (Hepa) cells and human promyelocytic (HL-60) cells stimulates the expression of heme oxygenase via transcriptional activation (Alam, J., and Smith, A. (1989) J. Biol. Chem. 264, 17637-17640). Incubation of both these cell types in serum-free medium containing heme-hemopexin is shown here also to increase the steady-state level of metallothionein (MT) mRNA in a time- and dose-dependent manner. Heme-hemopexin is a far more effective inducer (12-fold) of the MT isozyme 1 (MT-1) in Hepa cells than nonprotein-bound heme (4-fold). Apohemopexin has no effect on MT-1 expression, and incubation with heme-hemopexin of mouse L fibroblasts that lack hemopexin receptors does not affect MT-1 expression. Thus, an interaction between the heme-hemopexin complex and its receptor is necessary for increased accumulation of MT-1 transcripts. In vitro nuclear "run-on" analysis indicates that the heme-hemopexin-mediated accumulation of MT-1 mRNA is regulated primarily at the level of initiation of transcription. A highly labile protein is required for constitutive MT-1 gene expression and acts to repress transcription. Transcriptional activation by heme or metals may require decreased concentrations or inactivation of the repressor as well as an additional inducer-specific trans-acting factor. Inhibition of protein synthesis augments the heme-hemopexin-mediated accumulation of MT-1 mRNA. Activation of heme oxygenase (HO) gene transcription by heme requires the synthesis of one (or more) heme-inducible proteins that are labile or become labile upon cycloheximide-sensitive processing or activation. Our comparison of MT and HO points to significant differences in the mechanisms of gene regulation by heme. The concomitant regulation of gene expression of MT-1 and HO in response to heme-hemopexin appears to be a concerted adaptive response of the cells, mediated at the level of the plasma membrane hemopexin receptor, and may relate to the proposed role of MT as an intracellular antioxidant or to a need to sequester zinc which otherwise would compete with iron and occupy sites on regulatory proteins such as the iron-responsive elements.

Topics: Animals; Base Sequence; Cell Line; Cell Nucleus; DNA Probes; Gene Expression Regulation, Neoplastic; Heme; Hemopexin; Humans; Leukemia, Promyelocytic, Acute; Liver Neoplasms, Experimental; Metallothionein; Mice; Molecular Sequence Data; Oligonucleotide Probes; RNA, Messenger; Transcription, Genetic

Metallothionein (MT) gene transcription is regulated in a developmental and tissue-specific manner by metal ions and other agents. We examined MT expression in the human promyelocytic leukemia cell line HL-60 during differentiation along macrophage and neutrophil lineages. All HL-60 phenotypes showed similar basal levels of MT RNA with significant induction following exposure to Cd2+ but not activators of PKC. MT RNA did not correlate with growth state or with known levels of PKC activity, thus our data do not support a role for MT in HL-60 differentiation or for PKC in MT expression.

Topics: Amino Acid Sequence; Base Sequence; Cadmium; Calcimycin; Calcitriol; Cell Differentiation; Cell Line; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Metallothionein; Molecular Sequence Data; Oligonucleotide Probes; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate