Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.     

Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3).

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1alpha,25-dihydroxyvitamin D(3). We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50-55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.     

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.     

Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca(2+) dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45-74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.     

Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented HL-60 cells

The objective of this research was to develop a method for measuring Cu/Zn-superoxide dismutase (Cu/Zn-SOD) (E.C. 1.15.1.1) in HL-60 cells and subsequently examine the relationship between cellular copper levels and the activity of this copper-requiring enzyme. In cells such as the neutrophil or HL-60 promyelocyte cell line, the activity of Cu/Zn-SOD cannot be measured because of an increase in the oxidation rate of the substrate by some unknown compound in the cells. Others have utilized heat treatment to inactivate the responsible compounds, however, we found that heat treatment of HL-60 cells resulted in a loss of over half of the activity of the enzyme. The method described here utilizes sodium azide to inhibit the substance(s) that are responsible for the enhanced rate of pyrogallol's oxidation. Gel filtration data confirmed that the compound responsible for the enhanced rate of pyrogallol oxidation was sensitive to azide and did not affect Cu/Zn-SOD activity. When HL-60 cells were incubated with various levels of copper, Cu/Zn-SOD activity did not reflect the cellular copper levels.     

Ouabain- and Ca2(+)-sensitive ATPase activity of chimeric Na- and Ca-pump molecules.

HL-60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET-18-OCH₃ on the activity of protein kinase C. HL-60 cells were incubated with ET-18-OCH₃ at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL-60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser²⁵]-protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane-bound protein kinase C was increased in HL-60 cells treated with ET-18-OCH₃ compared to untreated HL-60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12-myristate 13-acetate. In contrast, solubilized protein kinase C from HL-60 cells could be inhibited or stimulated in vitro by ET-18-OCH₃, dependent on the mode of addition of ET-18-OCH₃ and phospholipids.     

Kinetics of human peptidylarginine deiminase 2 (hPAD2)--reduction of Ca2+ dependence by phospholipids and assessment of proposed inhibition by paclitaxel side chains

Multiple sclerosis is a complex human neurodegenerative disease, characterized by the active destruction of the insulating myelin sheath around the axons in the central nervous system. The physical deterioration of myelin is mediated by hyperdeimination of myelin basic and other proteins, catalysed by the Ca2+ -dependent enzyme peptidylarginine deiminase 2 (PAD2). Thus, inhibition of PAD2 may be of value in treatment of this disease. Here, we have first characterized the in vitro kinetic properties of the human peptidylarginine deiminase isoform 2 (hPAD2). Phosphatidylserine and phosphatidylcholine reduced its Ca2+ dependence by almost twofold. Second, we have explored the putative inhibitory action of the methyl ester side chain of paclitaxel (TSME), which shares structural features with a synthetic PAD substrate, viz., the benzoyl-L-arginine ethyl ester (BAEE). Using the known crystallographic structure of the homologous enzyme hPAD4 and in silico molecular docking, we have shown that TSME interacted strongly with the catalytic site, albeit with a 100-fold lower affinity than BAEE. Despite paclitaxel having previously been shown to inhibit hPAD2 in vitro, the side chain of paclitaxel alone did not inhibit this enzyme's activity.     

The structure-function relationship in the clostripain family of peptidases.

In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1). Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE). Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto-activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.     

Alkyllysophospholipid ET-18-OCH3 acts as an activator of protein kinase C in HL-60 cells

HL-60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET-18-OCH₃ on the activity of protein kinase C. HL-60 cells were incubated with ET-18-OCH₃ at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL-60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser²⁵]-protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane-bound protein kinase C was increased in HL-60 cells treated with ET-18-OCH₃ compared to untreated HL-60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12-myristate 13-acetate. In contrast, solubilized protein kinase C from HL-60 cells could be inhibited or stimulated in vitro by ET-18-OCH₃, dependent on the mode of addition of ET-18-OCH₃ and phospholipids.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

Stimulation of sialidase activity during cell differentiation of human promyelocytic leukemia cell line HL-60

Sialidase activity of human promyelocytic leukemia cell line HL-60 was assayed by a modification of the fluorometric method using 4MU-NANA as a substrate. The pH optimum was 4.1 and the apparent Km value was 0.10 mM. When the cells were induced to differentiate into granulocytes by either retinoic acid or DMSO, sialidase activity increased markedly. After incubation of HL-60 cells with 1 μM retinoic acid for 6 days and with 1.3% DMSO for 8 days, 91% and 75% of total cells, respectively, differentiated into morphologically mature myeloid cells and the sialidase activity increased to 2.5–2.7 times as much as that of the corresponding controls. In other human myeloid leukemia cell lines, K562 and KG-1, the sialidase activity was found to be 1.5- and 3.8-fold that of HL-60, respectively.     

Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells

The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar Km and Vmax (approximately 0.2 mM and approximately 3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37 degrees C; (2) the aSMase is much less sensitive to Triton X-100 ( > 5 mM), compared with < or = 0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFalpha, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect Km or Vmax of the nSMase in HL-60 sonicates. This suggests that TNFalpha may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed.     

Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes.

Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrulline residues Ca(2+)-dependently. PAD V was recently found in granulocyte-differentiated HL-60 cells. To find a target of PAD V, we incubated HL-60 granulocytes with the calcium ionophore A23187 and studied deiminated proteins by immunocytochemistry and immunoblotting using a monospecific antibody to modified citrulline residues. Immunocytochemical signals were found in the nucleus upon incubation with A23187. Immunoblotting indicated that 40-, 18-, 17-, and 14-kDa proteins were preferentially deiminated. The 40-kDa protein, which was focused to pI 5.0 on two-dimensional gel electrophoresis, was identified as nucleophosmin/B23 by mass spectrometry. The 18-, 17-, and 14-kDa proteins extracted with 0.4 N H(2)SO(4) comigrated with histones H3, H2A, and H4, respectively, on two-dimensional gel electrophoresis specialized for histones. The citrulline content of histones amounted to about 10% of the histone molecules. We discuss the implications of deimination of these proteins for their nuclear functions.     

Peptidylarginine deiminase expression and activity in PAD2 knock-out and PAD4-low mice

Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2−/− mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2−/− mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.     

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. Specific esterolytic activities of the kallikrein-like enzyme on N-tosyl-L-arginine methylester (TAME) and N-benzoyl-L-arginine ethylester (BAEE) are 109.5 and 23.6 mumol/min/mg, respectively. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. The enzyme is not lethal at 15 micrograms/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.     

Effects of dimaprit on growth and differentiation of human promyelocytic cell line, HL-60

The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyryl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE2, and cholera toxin). HL-60 is also known to be rich in H2 -histamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H2-stimulation on growth and differentiation of HL-60 using the potent H2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30-60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10(-7)M to 10(-4)M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxide (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10(-7)M to 10(-4)M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyelocyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, ii) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H2 antagonist. Dimaprit-induced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation.     

Development of a sensitive in vitro method for identifying tumor promoters

The identification and characterization of nongenotoxic carcinogens represents a significant challenge to toxicologists. In vitro methods for identifying tumor promoters with suitable sensitivity and specificity have been particularly elusive. Experiments are described which suggest that the human promyelocytic leukemia cell line HL-60 provides a sensitive indicator of promoter-induced changes to gene regulation and expression. As a result of differentiation these cells undergo a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture which exhibits high phagocytic activity. Fluorescent latex particles were used as sensors to highlight the phagocytic phenotype and permitted the use of flow cytometry to automatically quantitate particle internalization. To evaluate specificity, HL-60 cells were treated with a series of phorbol esters covering a range of in vivo tumor promoting activity. Results indicate that this family of compounds induces HL-60 cells to differentiate in proportion to their in vivo promoting activity. To closely assess the sensitivity of the phagocytic endpoint, HL-60 cells were treated with picogram levels of 12-O-tetradecanoyl phorbol-13-acetate (TPA), whereupon increments as low as 50 pg of TPA per ml caused statistically significant increases in phagocytic activity. The experiments described herein suggest that in vitro differentiation of HL-60 cells may reflect the promoter-dependent modifications to gene expression that are observed in vivo during the promotion phase of carcinogenesis. The described method may represent a sensitive promoter screening assay which is both rapid and economical.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

5－氮－2＇－脱氧胞苷（5－aza－CdR）对HL－60细胞分化和DNA甲基化酶的影响

以５－氮－２＇－脱氧胞苷（５－ａｚａ－ＣｄＲ）为诱导物,在０．５μｍｏｌ／Ｌ的最佳浓度下,可诱导ＨＬ－６０细胞分化达１５％左右。同时,用［￣３Ｈ］－ｍｅｔｈｙｌ－ｓ－ａｄｅｎｏｓｙｌｍｅｔｈｉｏｎｉｎｅ（￣３Ｈ－ＳＡＭ）为底物,通过同位素参入法,测定了不同浓度诱导物对ＨＬ－６０细胞ＤＮＡ甲基化酶活力的影响,发现在最佳诱导物浓度下,可使ＨＬ－６０细胞ＤＮＡ甲基化酶活力明显下降,此外,也比较了不同分化水平的ＨＬ－６０细胞中具有不同甲基化水平的ＤＮＡ在体外接受甲基的能力,从而证明５－ａｚａ－ＣｄＲ诱导ＨＬ－６０细胞分化与其ＤＮＡ甲基化状态密切相关。     

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.