Michiyuki Yamada

High production of catalase in hydrogen peroxide-resistant human leukemia HL-60 cell lines

The catalase activities of HP50-2 and HP100-1 cells, which are H2O2-resistant cell lines derived from human leukemia HL-60 cells, were 3 and 18 times higher, respectively, than that of HL-60 cells. These catalase activities of the resistant cells were precipitated with anti-catalase serum. The glutathione peroxidase activity of HP50-2 cells was about twice that of HL-60 or HP100-1 cells. The superoxide dismutase activities of HP50-2 and HP100-1 cells were, respectively, about 4 and 2 times that of HL-60 cells. In addition, both the resistant cell lines were completely devoid of myeloperoxidase activity. Pulse-labeling experiments showed that the syntheses of catalase in HP50-2 and HP100-1 cells were, respectively, 2 and 4 times that in HL-60 cells, and that, unlike the parent cells, neither line synthesized myeloperoxidase. Thus the alteration of catalase, glutathione peroxidase, and superoxide dismutase activity could be linked to the resistance of H2O2 of human leukemia cells.

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.

Similarity of kinetics of three types of myeloperoxidase from human leukocytes and four types from HL-60 cells.

Km values for H2O2 and Vmax values for three types of myeloperoxidase (MPO) from human leukocytes (MPO-I, -II, and -III) and four types from human myeloid leukemia HL-60 cells (MPO-IA, -IB, -II, and -III) were determined. Km values of human leukocyte MPOs decreased with increasing pH from 4.4 to 6.2 and increased with increasing NaCl concentration from 0.025 to 0.14 M. There was no significant difference among Km values of leukocyte MPO-I, -II, and -III. NaBr also showed a tendency similar to that of NaCl with regard to the effects of pH and halide concentration on Km values. However, Km values in the presence of NaBr were lower than those in the presence of NaCl. Effects of pH and NaCl concentration on Vmax values of MPO-I, -II, and -III were also examined. Vmax values of MPO-I, -II, and -III were higher at pH 4.9 and 5.4 and increased with increasing NaCl concentration. In addition, no difference was observed between Km values of leukocyte and those of HL-60 cells. MPO-IB, the half-molecular-weight enzyme of HL-60 cells, also had the same Km values as the others. Furthermore, inhibition of the activities of seven MPOs of leukocytes and HL-60 cells by H2O2 was similarly observed at concentrations above 1 mM at pH 5.4 but not at pH 4.4. These results indicate that there is no difference in the affinity to H2O2 among leukocyte MPO-I, -II, and -III and HL-60 cell MPO-IA, -IB, -II, and -III.

pH dependence of salt activation of human leukocyte elastase

The effects of pH on salt stimulation of the rates of hydrolysis of three substrates by human leukocyte elastase were studied. The enzyme was most active at pH 10.5, 8.0-8.5, and 9.5 for the hydrolyses of fluorescein isothiocyanate-labeled S-carboxymethylated bovine serum albumin (FITC-CM-BSA), succinyl-L-Ala-L-Pro-L-Ala-7-methylcoumaryl-4-amide (Suc-APA-MCA), and succinyl-L-Ala3-p-nitroanilide (Suc-Ala3-pNA), respectively, in the absence of NaCl. The enzyme was activated by 0.5 M NaCl similarly at all pHs tested for the hydrolysis of Suc-Ala3-pNA, but more at neutral and alkaline pH values, respectively, for the hydrolyses of FITC-CM-BSA and Suc-APA-MCA. Thus, in the presence of 0.5 M NaCl, the enzyme was most active at pH 8.0 and 10.0 with FITC-CM-BSA and Suc-APA-MCA, respectively. In contrast, the proteolytic activity of porcine pancreatic elastase was somewhat inhibited by 0.5 M NaCl.

Isolation and characterization of extracellular myeloperoxidase precursor in HL-60 cell cultures☆

An extracellular myeloperoxidase precursor of HL-60 cells was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose chromatography, and monoclonal antibody affinity chromatography. The purified protein was a glycoprotein of approximately 89 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino-terminal amino acid sequence of the protein began at amino acid residue 49 of the 745-amino acid sequence deduced from a myeloperoxidase cDNA, suggesting that the protein consisted of 697 amino acid residues. The implications of the precursor in the processing of myeloperoxidase are discussed.

Adaptation of human leukemia HL-60 cells to hydrogen peroxide as oxidative stress

The growth inhibitory effect on human leukemia HL-60 cells of hydrogen peroxide as an oxidative stress and the possibility of adaptation of the cells to this stress were examined. When HL-60 cells were treated with various concentrations of hydrogen peroxide, concentrations of 5-200 microM were found to be sublethal. When HL-60 cells were repeatedly exposed to 50 and 100 microM hydrogen peroxide, they began to grow stably in the medium with the drug after a few months, indicating that they had become adapted to the drug. Sublines HP50-2 and HP100-1 cloned from these cultures were approximately 40-fold and 340-fold more resistant, respectively, than the parent cells. These resistant sublines tended to form cell aggregates in stationary culture, suggesting an alteration in their surface membrane. The HP50-2 and HP100-1 lines will be useful in the studies of many cellular mechanisms associated with reactive oxygens.

Identification of myeloperoxidase in human colostrum

The properties of a peroxidase in human colostrum were studied using antiserum against human myeloperoxidase. The peroxidase in human colostrum gave a single precipitin line against the antiserum on double immunodiffusion, and this precipitin line fused completely with the precipitin line formed between myeloperoxidase and the antiserum. The peroxidase activity in human colostrum was precipitated completely with anti-myeloperoxidase IgG, like myeloperoxidase activity. The peroxidase of colostral whey was purified to homogeneity. The purified enzyme consisted of two subunits of Mr 59,000 and 15,000, corresponding in size to the two subunits of myeloperoxidase. Immunostaining of a protein blot from a sodium dodecyl sulfate-polyacrylamide electrophoresis gel also showed that the peroxidase in the whey extract consisted of the same two subunits as myeloperoxidase. These results indicate that the peroxidase of human colostrum is identical with myeloperoxidase.

ASSIGNMENT OF THE MYELOPEROXIDASE GENE MPO TO HUMAN CHROMOSOME 17 USING SOMATIC CELL HYBRIDS AND FLOW-SORTED CHROMOSOMES

SummaryA cDNA coding for human myeloperoxidase (MPO) was used as a probe to study MPO gene structure and to determine the chromosomal location of the gene in the human genome. Southern blot hybridization of restriction endonuclease digests of human DNA with the MPO cDNA probe showed that a single gene for human MPO was present in the human genome. Southern blot hybridization experiments with human-mouse cell hybrid DNAs containing various subsets of human chromosomes revealed that the human MPO gene is located on chromosome 17. This conclusion was supported by DNA spot-blot hybridization using flow-sorted human chromosomes.