cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells.

Abstract

Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic Xgtll expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by rinding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid selection of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of :3.6 and r2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.Myeloperoxidase (MPO), a critical microbicidal protein of mature polymorphonuclear neutrophils, is a hemoprotein composing 3-5% of the protein weight of these cells (1, 2). It appears in the primary azurophilic granules during the differentiation of the neutrophil (3, 4) and has been used as a marker for these granules (5). Cytochemical staining of MPO activity is used clinically to distinguish acute myeloid leukemia from acute lymphoid leukemia (6). Furthermore, approximately 1 in every 2000 individuals has been shown to be deficient in MPO (7), an apparently benign but poorly understood hereditary disorder of polymorphonuclear neutrophils.Although the function of MPO in the oxygen-dependent respiratory burst of polymorphonuclear neutrophils has been firmly established, relatively little is known about MPO biosynthesis, posttranslational modification, and genetic organization and expression. The exact structure of MPO remains a source of some controversy, and its amino acid sequence is not known.The appearance of MPO in the primary granules marks the transition from the myeloblast to the promyelocyte in the course of normal neutrophilic differentiation (3). Promyelocytes, however, are not found in the peripheral blood of normal individuals and are therefore difficult to study biochemically. The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, contains virtually 100%o promyelocytes with azurophilic granules containing MPO (8).When HL-60 cells are cultured in the presence of compounds such as dimethyl sulfoxide (Me2SO) (8) or retinoic acid (9), these cells mature into metamyelocytes, neutrophili...

Functional activity of three distinct myeloperoxidase (MPO) promoters in human myeloid cells

“…This finding agrees with previous studies by ourselves 4,13 and others 22,23 showing that MPO mRNA synthesis is downregulated almost completely by 24 h after TPA treatment, and it shows that MPO transcription initiated at this site, presumably by the P1 promoter, is physiologically regulated. In addition, Figure 3 shows that using RNA from either untreated or TPA-treated K-562 cells (lanes 3 and 4) or equivalently treated HeLa cells (lanes 5 and 6), no detectable cDNA product is obtained using primer I.…”

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“…MPO forms are isolated by immunoprecipitation and changes in size secondary to processing are identified by SDS-PAGE. The results obtained (48-55) are consistent with results deduced from cDNA clones for MPO (56)(57)(58)(59)(60)(61). The first 41 amino acids at the aminoterminus of the cDNA represent a signal peptide, followed by a propeptide, a light subunit and a heavy subunit.…”

miR-638 Regulates Differentiation and Proliferation in Leukemic Cells by Targeting Cyclin-dependent Kinase 2

“…When treated with ATRA, the similar results were also observed for the two markers of granulocyte differentiation, ITGAM and G-CSF receptor (CSF3R), in miR-638-transfected HL-60 cells. On the contrary, MPO, a marker for myeloblasts and promyelocytes (32), was much lower in miR-638-overexpressed cells than in control cells (Fig. 2H).…”