Myeloperoxidase Gene Expression Research Articles

Changes of DNA Methylation and Chromatin Structure in the HumanMyeloperoxidase Gene During Myeloid Differentiation

Expression of the myeloperoxidase (MPO) gene is tightly regulated in a tissue- and development-specific manner. Accumulation of MPO messenger RNA (mRNA) occurs only at the late myeloblastic and promyelocytic stages of myeloid differentiation and is negligible at other stages of myeloid development and in other tissues. The goal of our studies was to begin to understand the events that occur to control MPO gene expression during normal granulocytopoiesis. Chromatin structure of the MPO gene was evaluated by DNase I treatment of isolated nuclei and Southern blot analysis. No detectable DNase I hypersensitive sites were found in the region of the MPO gene in non-myeloid cells. One site was present in the 5' upstream region in myeloid cells that are developmentally too immature to transcribe MPO. Three sites of hypersensitivity in the regions of the putative MPO promoter and upstream region occurred in MPO-expressing promyelocytes. These sites were markedly reduced in terminally differentiated, non-expressing myeloid cells. Analysis of DNA methylation of the MPO gene using methylation-sensitive restriction enzymes showed that the gene was highly methylated in non-myeloid cells. Stepwise demethylation occurred in myeloid cells developmentally too immature to transcribe MPO. Maximal demethylation in the 5' gene region occurred in MPO-expressing promyelocytes. This methylation pattern did not change in terminally differentiated, MPO non-expressing myeloid cells. A somatic hybrid cell formed by fusion of HL-60 (MPO-expressing cells) and PUT (MPO non-expressing lymphoid cells) extinguished expression of MPO and showed a chimeric pattern of MPO gene methylation, suggesting that demethylation is necessary but not sufficient for expression of the MPO gene. Our studies show that demethylation and DNase I hypersensitivity of the MPO gene were associated with a tissue-dependent potential for MPO gene expression that preceded the developmental ability to express MPO mRNA.

Regulation of human bone marrow lactoferrin and myeloperoxidase gene expression by tumor necrosis factor-alpha.

Lactoferrin (LF) and myeloperoxidase (MPO) are glycoproteins synthesized in early myeloid cells (promyelocytes, myelocytes) and stored in granules of polymorphonuclear neutrophilic granulocytes. Both proteins are involved in the host inflammatory response, and LF has been found to have myelosuppressive activity in vivo and in vitro. Little is known, however, about the regulation of their production. We investigated the stability of LF and MPO mRNA and the effects of purified recombinant human TNF-alpha on LF and MPO levels in normal human bone marrow. Low density human bone marrow cells were cultured in the presence or absence of actinomycin D (10 micrograms/ml) or TNF-alpha (200 U/ml). LF and MPO RNA levels were analyzed by Northern blots using, respectively, a 650-bp insert from the plasmid pHL41, and a 2.3-kb insert from the plasmid pMPO2 as probes. It was found that: 1) LF mRNA is a fairly stable molecule, with a half-life of between 8 and 9 h, whereas MPO is less stable, with a half-life of between 4 and 5 h; 2) TNF-alpha decreases both LF and MPO mRNA levels, an effect seen by 24 h with MPO mRNA and 48 h with LF mRNA; 3) nuclear run-on assays revealed that TNF decreases transcription of the LF gene by 70% and the MPO gene by 50%; and 4) the suppressive effect of TNF-alpha on LF and MPO mRNA levels is not due to cell killing or selective differentiation and is reversible.

Human Myeloperoxidase Gene Expression in Acute Leukemia

Abstract To evaluate the relationship between myeloperoxidase (MPO) gene expression and specific lineages of hematopoietic differentiation, we analyzed expression of the MPO gene in a variety of normal and leukemic human cells. As a first step, we synthesized several oligonucleotide probes and isolated cDNA clones for the MPO gene. MPO expression was detected in most acute myeloid leukemias, and the level of expression correlated well with cytochemical enzymatic activity. No MPO message was detected in pure lymphoid leukemias, whereas very low basal levels of MPO transcripts were found in normal bone marrows. In four cases of acute undifferentiated leukemias cytochemically negative for MPO, significant levels of MPO transcripts were detected, suggesting a myeloid origin for these cases. These results indicate that MPO gene expression may serve as an additional marker for subclassification of acute leukemias and may be used to identify leukemic cells arrested at an early stage of the myeloid differentiation pathway.

Human myeloperoxidase gene expression in acute leukemia

To evaluate the relationship between myeloperoxidase (MPO) gene expression and specific lineages of hematopoietic differentiation, we analyzed expression of the MPO gene in a variety of normal and leukemic human cells. As a first step, we synthesized several oligonucleotide probes and isolated cDNA clones for the MPO gene. MPO expression was detected in most acute myeloid leukemias, and the level of expression correlated well with cytochemical enzymatic activity. No MPO message was detected in pure lymphoid leukemias, whereas very low basal levels of MPO transcripts were found in normal bone marrows. In four cases of acute undifferentiated leukemias cytochemically negative for MPO, significant levels of MPO transcripts were detected, suggesting a myeloid origin for these cases. These results indicate that MPO gene expression may serve as an additional marker for subclassification of acute leukemias and may be used to identify leukemic cells arrested at an early stage of the myeloid differentiation pathway.

Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia

By using a cDNA clone of the myeloperoxidase (MPO) gene, we have studied, by Northern blot analysis, the level of MPO mRNA in eight cases of acute lymphoblastic leukemia (ALL). The blast cell populations studied were characterized by morphologic, cytochemical, immunochemical, and molecular criteria. With all the methods used the populations were found to be highly homogeneous and showed a typical lymphoid phenotype. In particular, the Ig heavy-chain gene rearrangement was largely prevalent, and the germ line configuration was almost absent. However, in three of eight cases, high levels of MPO mRNA were detected. The remarkable homogeneity of the cell populations examined suggests that the MPO mRNA observed was present in cellular elements certainly identified as lymphoid. The absence of contamination by myeloid cells was confirmed by the results of Western blot analysis of the proteins of the cell population studied: no MPO protein was detectable. The levels of mRNA observed were high enough to be comparable to those observed in a promyelocytic cell population.

Myeloperoxidase Gene Expression in Blast Cells With a Lymphoid Phenotype in Cases of Acute Lymphoblastic Leukemia

By using a cDNA clone of the myeloperoxidase (MPO) gene, we have studied, by Northern blot analysis, the level of MPO mRNA in eight cases of acute lymphoblastic leukemia (ALL). The blast cell populations studied were characterized by morphologic, cytochemical, immunochemical, and molecular criteria. With all the methods used the populations were found to be highly homogeneous and showed a typical lymphoid phenotype. In particular, the Ig heavy-chain gene rearrangement was largely prevalent, and the germ line configuration was almost absent. However, in three of eight cases, high levels of MPO mRNA were detected. The remarkable homogeneity of the cell populations examined suggests that the MPO mRNA observed was present in cellular elements certainly identified as lymphoid. The absence of contamination by myeloid cells was confirmed by the results of Western blot analysis of the proteins of the cell population studied: no MPO protein was detectable. The levels of mRNA observed were high enough to be comparable to those observed in a promyelocytic cell population.

Regulation of gene expression of myeloperoxidase during myeloid differentiation.

Myeloperoxidase (MPO) is a major heme enzyme involved in inflammatory responses of polymorphonuclear leukocytes. Using cDNA and intron specific probes for MPO we studied the regulation of MPO expression during myeloid differentiation of the promyelocytic HL-60 leukemia cell line. Mature MPO mRNA species of 3.3, 2.8 and 1.6 kb and heterogeneous nuclear (hn) RNA of greater than 8 and approximately 4 kb were observed in wildtype HL-60 cells. Induction of differentiation of the cells towards either granulocytes or macrophages resulted in a profound decrease (greater than 95%) in the concentration of MPO mRNA levels, showing that gene expression of MPO mRNA is closely linked to the stage of development of myeloid cells. Studies using normal and leukemic hematopoietic cells confirmed these findings and showed that myeloblasts and promyelocytes contain MPO mRNA. Rate of transcription of MPO was measured by a nuclear run-on assay in wild-type and day 3- and day -4 differentiated HL-60 cells and was nearly the same in all three. In contrast, rate of transcription of c-myc in the same nuclei became almost undetectable with induction of differentiation. Overall transcription decreased by 60% and 80% on day 3 and 4 of differentiation, respectively, compared to wild-type cells. Stability of mature MPO mRNA was also measured and found to be the same in wild-type and differentiated HL-60. Half-life of MPO hnRNA was less than or equal to 30 min in wild-type HL-60; nevertheless, this hnRNA was easily detectable 3 days after induction of differentiation of these cells. Taken together, the results show that decreased expression of MPO mRNA with differentiation occurs in part post-transcriptionally, possibly due to a failure in RNA processing. In addition, as overall transcription decreases during differentiation, MPO transcription is concomitantly reduced. This indicates that transcriptional and post-transcriptional mechanisms cooperate in the control of MPO gene expression.

Transcriptional regulation of myeloperoxidase gene expression in myeloid leukemia HL-60 cells during differentiation into granulocytes and macrophages

Myeloperoxidase gene transcription in isolated nuclei from HL-60 cells induced to differentiate into granulocytes by dimethyl sulfoxide or into macrophages by 12-O-tetradecanoylphorbol-13-acetate was studied by dot-blot hybridization of a myeloperoxidase cDNA to the 32P-labeled nuclear transcripts. Myeloperoxidase gene transcription, like that of c- myc gene transcription, was reduced to a low level within 12 h after the inductions of these differentiations. In contrast, transcription of the actin gene remained constant. These results indicate that decrease in myeloperoxidase synthesis in HL-60 cells during differentiation is regulated at a transcriptional level.

Human Myeloperoxidase Gene: Molecular Cloning and Expression in Leukemic Cells

We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity-purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.

Human myeloperoxidase gene: molecular cloning and expression in leukemic cells

We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity- purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.

Regulation of myeloperoxidase gene expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in human leukemia HL-60 cells.

Myeloperoxidase synthesis during induction of differentiation of human promyelocytic leukemia HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. Differentiation was characterized by morphological changes, arrest of cell proliferation, development of cell adherence, and increased secretion of lysozyme. The cellular myeloperoxidase activity decreased early during induction of differentiation by TPA. Pulse-labeling experiments indicated that the rate of myeloperoxidase synthesis decreased to an undetectable level in cells exposed to TPA for 22 h. The relative amounts of myeloperoxidase mRNA in TPA-treated and untreated cells were determined by measuring translatable mRNA activity in a reticulocyte lysate system. Reduction in the myeloperoxidase mRNA level was observed as early as after 3 h treatment with TPA, and no myeloperoxidase mRNA was detected after 24 h. Time course experiments indicated that the time required for 50% reduction of myeloperoxidase mRNA in TPA-treated cells was approximately 5 h. These results suggest that TPA induces decrease of myeloperoxidase activity in HL-60 cells at a pretranslational level.

Regulation of myeloperoxidase gene expression during differentiation of human myeloid leukemia HL-60 cells.

When human myeloid leukemia HL-60 cells were induced to differentiate into mature cells by dimethyl sulfoxide or retinoic acid, the amount of myeloperoxidase activity per cell decreased to 20 to 30% of that of uninduced cells, and the rate of myeloperoxidase biosynthesis decreased to an undetectable level in 19 h after induction of differentiation. After 19-h exposure to an inducer, the cells could not resume myeloperoxidase synthesis on further incubation in inducer-free medium. When polysomes and mRNAs prepared from untreated and treated cells were translated in rabbit reticulocyte lysates, the former showed myeloperoxidase polypeptide synthesis, and the latter did not. These results indicate that the inability of induced cells to synthesize myeloperoxidase is due to the absence of myeloperoxidase mRNA.