3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL): cloning and characterization of a mouse liver HL cDNA and subchromosomal mapping of the human and mouse HL genes

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) is a homodimeric mitochondrial matrix enzyme that catalyzes the last step of ketogenesis. Using a human HL cDNA as a probe, we isolated a 1.4-kb mouse HL cDNA (HLM) from a mouse liver library and extended the sequence in the 5 direction, using RACE PCR to include the complete coding sequence. The nucleotide sequence of the mouse HL coding region is 85.7% identical to human HL, and 52.6% to Ps. mevalonii HL. Peptide identities of 87.4% and 54.3% respectively were observed. Southern analysis of 29 strains of laboratory mice and of Mus spretus revealed a total of about 25 kb of hybridizing fragments and three polymorphic fragments in both EcoRI and HindIII digestions. The mouse HL locus (Hmgcl) was localized on Chromosome (Chr) 4: Pmv-19-12.6±3.6 cM-Hmgcl-7.3±2.3 cM-Xmv-8-1.5±1.0 cM-Gpd1. The human HL locus (HMGCL) was mapped to distal Chr 1p by analysis of a human-hamster hybrid cell panel and by in situ hybridization.     

Preparation, physicochemical properties and biological activity of copper(II) complexes with 6-(2-chlorobenzylamino)purine (HL1) or 6-(3-chlorobenzylamino)purine (HL2). The single-crystal X-ray structure of

Copper(II) complexes of 6-(2-chlorobenzylamino)purine (HL1) and 6-(3-chlorobenzylamino)purine (HL2), respectively, were prepared. Depending on the pH of the medium and the molar ratio of reactants the following mononuclear (trigonal-bipyramidal) and dinuclear (octahedral, trigonal-bipyramidal or tetrahedral) complexes were isolated: [Cu2(mu-HL1)2(mu-Cl2)2(HL1)2Cl2] (1a,b), [Cu2(mu-Cl)2(mu-L1)2(H2O)2] (2a), [Cu2(mu-Cl)2(mu-L2)2(H2O)2] (2b), [Cu(H+L2)2Cl3]Cl.H2O (3a,b), [Cu2(mu-Cl)2(HL1)2Cl2] (4a), and [Cu2(mu-Cl)2(HL2)2Cl2] (4b). The compounds were characterized by elemental analyses, electronic, infrared and mass (FAB+, ES+) spectral data, magnetic susceptibility temperature dependence measurements and molar conductivity data. An X-ray single-crystal structural analysis of [Cu(H+L2)2Cl3]Cl.2H2O (3b) showed that the Cu2+ ion is penta-coordinated by three chloride ions and by two H+L2 ligands. Thus, the Cu2+ ion adopts a distorted trigonal bipyramidal coordination geometry with the protonated H+L2 ligands coordinated in trans apical positions, while the three chloride ions are situated in an equatorial plane. The cytotoxic activity of the complexes was determined by a calcein AM assay. Mouse melanoma cell line B16-FO, human malignant melanoma cell line G361, human osteogenic sarcoma cell line HOS and human breast adenocarcinoma cell line MCF7 were used. IC50 values, the drug concentrations lethal to 50% of the tumor cells, were estimated. One of the important mechanisms responsible for the cytotoxicity of cytokinin-derived compounds, the inhibition of cyclin-dependent kinases by the studied complexes, was also determined.     

Cyclic AMP-dependent protein kinase isoenzymes in human myeloid leukemia (HL60) and breast tumor (MCF-7) cells

Combinations of retinoic acid (RA) and cAMP mediate many biological responses in a large variety of cell types. While the basis for the apparent synergistic effects of RA and cAMP are not clearly defined, it is likely that activation of PKA by cAMP is involved. However, literature reports concerning the identity of PKA isoforms in HL60 and MCF-7 cells are conflicting. The purpose of the present investigation is to identify PKA isoforms in HL60 and MCF-7 cells. Utilization of high-performance anion-exchange liquid chromatography, immunoblotting, and 8-azido-cAMP photoaffinity binding resulted in the finding that HL60 cells contain PKA types I alpha and II alpha, while MCF-7 cells contain PKA types I alpha, II alpha, and II beta. PKA type I alpha in both HL60 and MCF-7 cells eluted from columns as two well-separated peaks. One peak eluted at a low salt concentration in agreement with previous reports. The second HL60 PKA type I alpha peak eluted at a salt concentration intermediate between that eluting the first peak and that eluting PKA type II alpha and contained approximately 62% of the total RI alpha protein. However, the second MCF-7 PKA type I alpha peak contained approximately 66% of the total RI alpha protein and co-eluted with PKA types II alpha and II beta. This "contamination" of PKA type II fractions with PKA type I has led, in some cases, to interpretations that may need reevaluation.     

MAPK-dependent expression of p21(WAF) and p27(kip1) in PMA-induced differentiation of HL60 cells

Treatment of HL60 cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest and differentiation towards the macrophage lineage. PMA-induced changes are easily monitored by morphological changes while cells in suspension start adhering onto substrate. PMA induces rapid activation of the extracellular signal-regulated kinases (ERKs). Activation of the ERK pathway is essential to PMA-induced differentiation of HL60 cells. PMA also induces the expression of the cyclin-dependent kinase inhibitors p21(WAF) and p27(kip1), which is modulated by the use of an inhibitor of the ERK cascade. This implies that a link exists between ERK activation and p21(WAF) and p27(kip1) induction in the process of terminal differentiation.     

Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells.

Inhibitors of protein phosphatases 1/2A (okadaic acid and calyculin A) exhibited differential cytotoxicity toward three human leukemia cell lines, in an increasing order of resistance, HL60 less than HL60/ADR less than K562 cells. Cytotoxicity of the toxins was associated with marked mitotic arrest of the cells, characterized by chromatid scattering/overcondensation and abnormal mitotic spindles. In all cases, calyculin A was more potent than okadaic acid. Protein phosphorylation experiments in intact cells revealed that HL60/ADR, the adriamycin-resistant variant, showed a higher overall phosphorylation of nuclear proteins than the drug-sensitive parental HL60, and that phorbol ester (protein kinase C activator) and calyculin A appeared to more specifically stimulate phosphorylation of p66 and p60, respectively. It was suggested that the toxins might be useful in delineating mechanisms underlying certain properties of cancer cells (such as multidrug resistance, mitosis and differentiation) related to protein phosphorylation/dephosphorylation reactions.     

PARP degradation in apoptotic Syrian hamster embryo (SHE) cells compared to HL60 cell line

In this study, we attempted to identify apoptotic Syrian hamster embryo (SHE) cells by detecting the specific cleavage of poly(ADP-ribose)polymerase (PARP). Apoptosis was unequivocally identified in serum-deprived SHE cells. After protein electrophoresis and transfer, the anti-PARP antibody (C-2-10) was applied in order to visualize PARP degradation and the anti-polymer antibody (LP96-10) was used to identify PARP and its expected 89-kDa fragment on the membrane after renaturation and NAD+ addition. Results showed that PARP rapidly disappeared during apoptosis in SHE cells, but the resulting fragment remained undetectable with the anti-PARP antibody and no stable polymerase activity of this fragment was measured using anti-polymer antibody. Serum-starved SHE cells were compared to the etoposide-treated HL60 cell line as a control for typical apoptosis-related PARP cleavage. These results underline the fact that while PARP degradation is a criterion for apoptosis, the diagnosis of apoptosis can not rely exclusively on the appearance of its 89-kDa fragment as this signal may fail to appear in some cell systems.     

Skipping of exon 2 and exons 2 plus 3 of HMG-CoA lyase (HL) gene produces the loss of beta sheets 1 and 2 in the recently proposed (beta-alpha)8 TIM barrel model of HL

HMG-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine catabolism. We report a new Spanish patient who bears the frequent nonsense mutation G109T (Mediterranean mutation). This mutation can produce aberrant splicing with three mRNA variants: one of the expected size, the second with deletion of exon 2, and the third with deletion of exons 2 and 3. Recently our group proposed a 3D model for human HL containing a (beta-alpha)(8) (TIM) barrel structure. We have studied the effect of the deletions of exon 2 and exons 2 plus 3 on the proposed HL model. Exon 2 skipping led to the loss of beta-sheet 1, and the skipping of exons 2 and 3 caused the disappearance of alpha helix 1 and beta-sheets 1 and 2.-     

Changes in cell kinetics associated with differentiation of a human promyelocytic cell line (HL60)

Abstract. Terminal cell differentiation results in an irreversible arrest in the G₁ phase of the cell cycle and loss of the capacity for cell renewal. In the murine erythroleukaemia cell line (MELC), commitment to erythroid differentiation was found also to be preceded by an early, transient, phase of inhibition of growth due to prolongation of the G₁ phase. We determined the effect of differentiation-inducing agents on the growth kinetics of a human promyelocytic cell line (HL60) which undergoes differentiation into mature granulocyte. At concentrations of inducers optimal for cell differentiation, an early, transient stimulation of cell multiplication was found. DNA synthesis was enhanced in HL60 cells as early as 5 hr after exposure to inducer. Nevertheless, HL60 cell maturation eventually also resulted in a loss of the multiplication ability. The duration of exposure to inducer required for irreversible loss of the potential for self-renewal was determined by the fall in the cloning efficiency of induced cells; the results indicate that it preceded the switch-off of the replication mechanism; the majority of the cells lost their ability to form large colonies at the time of peak DNA synthesis and were able to complete an additional two to three cell cycles at a rate similar to uninduced cells. These changes occurred before HL60 cells became committed and might play a pivotal role in the process of cell differentiation.     

Catalase regulates cell growth in HL60 human promyelocytic cells: evidence for growth regulation by H(2)O(2)

Reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)) are generated constitutively in mammalian cells. Because of its relatively long life and high permeability across membranes, H(2)O(2) is thought to be an important second messenger. Generation of H(2)O(2) is increased in response to external insults, including radiation. Catalase is located at the peroxisome and scavenges H(2)O(2). In this study, we investigated the role of catalase in cell growth using the H(2)O(2)-resistant variant HP100-1 of human promyelocytic HL60 cells. HP100-1 cells had an almost 10-fold higher activity of catalase than HL60 cells without differences in levels of glutathione peroxidase, manganese superoxide dismutase (MnSOD), and copper-zinc SOD (CuZnSOD). HP100-1 cells had higher proliferative activity than HL60 cells. Treatment with catalase or the introduction of catalase cDNA into HL60 cells stimulated cell growth. Exposure of HP100-1 cells to a catalase inhibitor resulted in suppression of cell growth with concomitant increased levels of intracellular H(2)O(2). Moreover, exogenously added H(2)O(2) or depletion of glutathione suppressed cell growth in HL60 cells. Extracellular signal regulated kinase 1/2 (ERK1/2) was constitutively phosphorylated in HP100-1 cells but not in HL60 cells. Inhibition of the ERK1/2 pathway suppressed the growth of HP100-1 cells, but inhibition of p38 mitogen-activated protein kinase (p38MAPK) did not affect growth. Moreover, inhibition of catalase blocked the phosphorylation of ERK1/2 but not of p38MAPK in HP100-1 cells. Thus our results suggest that catalase activates the growth of HL60 cells through dismutation of H(2)O(2), leading to activation of the ERK1/2 pathway; H(2)O(2) is an important regulator of growth in HL60 cells.     

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.     

Molecular and clinical analysis of Japanese patients with 3-hydroxy-3-methylglutaryl CoA lyase (HL) deficiency

Deficiency of mitochondrial 3-hydroxy-3-methylglutaryl CoA lyase (HL, EC4.1.3.4.) is an autosomal recessive genetic disorder characterized by acute episodes of vomiting, hypotonia, and lethargy in the neonatal period or in infancy. Except in Saudi Arabia, where HL deficiency is the most common organic acidemia, the disorder is quite rare with only 41 cases being reported in the English literature, and only five known cases among Japanese. In this study, we present the results of a molecular analysis of all five Japanese patients together with their clinical phenotypes. Five different mutations in the HL gene were identified: one large deletion, one nonsense mutation, one missense mutation, and two splice mutations. Except for G835A (E279K) with its relatively common occurrence among Japanese, these mutations were unique to each family. The results of expression studies with mutated HL cDNAs confirmed the pathogenicity of these mutations and supported the importance of previously identified functional domains of the HL molecule, i.e., the putative catalytic site or dimerization site. In addition, we identified an alternative splicing event that resulted in the skipping of exons 5 and 6. This alternatively spliced product did not show HL activity and was present in various tissues of normal subjects. Clinically, all patients presented with similar symptoms, except that the timing of the initial presentation varied considerably, from 1 day to 1 year 3 months. In general, patients with null-activity mutations presented earlier in life, whereas those with residual activities presented later.     

Functional Characterization of Recombinant Endo-Levanase (LevBk) from Bacillus koreensis HL12 on Short-Chain Levan-Type Fructooligosaccharides Production

The Protein Journal - Levan-type fructooligosaccharides (L-FOSs) are a prominent class of non-digestible oligosaccharides with potential as nutritional prebiotics. Endo-levanase, which randomly...     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectin-like molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.