The interference effects of hexadecylphosphocholine on proliferation and membrane phospholipid metabolism in human myeloid leukemia cell lines

Membrane phospholipids are important regulators of cellular function. The phospholipid activities, such as lipid composition and transportation, contribute to cellular homeostasis in the lifespan of cells. Alterations in phospholipids result in the movement of bilayer lipids and the initiation of coagulation, recognition and internalization. Hexadecylphosphocholine (HePC) exerts antitumor potencies and represents a new class of antitumor agents targeted to the cellular membrane. Human myeloid leukemia cell lines HL-60 and K562 employed in this study were inhibited by HePC in vitro. The results indicate that the HL-60 cell line was sensitive, while K562 was resistant to HePC. Synthetic HePC is an alkyllysophospholipid analog which interacted with the cell membrane, thereby altering lipid composition and metabolism of membrane phospholipids and modulating intracellular calcium in human myeloid leukemia HL-60 and K562 cell lines. The contents of membrane phospholipids, including phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), were determined quantitatively with high performance liquid chromatography. The sensitivity of myeloid leukemia HL-60 and K562 cell lines to HePC probably depends on the different distribution of these four phospholipids in the cellular membrane, or on the response of these phospholipids to HePC. The cytosolic free calcium ([Ca++]i) concentration increased by HePC confirmed that [Ca++]i was released from the intracellular calcium pool and is associated with cell differentiation and apoptosis. We investigated the hypothesis that the antiproliferative effect of HePC was mediated through the interference with cellular membrane phospholipids, including choline-containing phospholipids (PC), aminophospholipids (PE and PS) and PI, in eukaryotic cells.     

Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog, hexadecylphosphocholine

The prototype of a new class of antiproliferative phospholipid analogs, hexadecylphosphocholine (HePC), has been shown to inhibit tumor growth and is currently used for the treatment of cutaneous metastases of mammary carcinomas. Although several cellular targets of HePC, e.g. protein kinase C and CTP:phosphocholine cytidylyltransferase, have been proposed, the mechanisms of HePC-induced anticancer activity are still unclear. Considering that the antiproliferative effect of HePC correlates with inhibition of phosphatidylcholine biosynthesis, which is tightly coupled to sphingomyelin biosynthesis, we tested the hypothesis that treatment of cells with the anticancer drug leads to increased cellular ceramide and subsequently to apoptotic cell death. In the present study, we showed that 25 micromol/liter HePC induced apoptosis. In further experiments, we demonstrated that HePC inhibited the incorporation of radiolabeled choline into phosphatidylcholine and at a later time point into sphingomyelin. This was confirmed by metabolic labeling of the lipid backbone using radiolabeled serine, and it was shown that HePC decreased the incorporation of serine into sphingomyelin by 35% and simultaneously increased the incorporation of serine into ceramide by 70%. Determination of the amount of ceramide revealed an increase of 53% in HePC-treated cells compared with controls. In accordance with the hypothesis that elevated ceramide levels may be the missing link between the metabolic effects of HePC and its proapoptotic properties, HePC-induced apoptosis was blocked by fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, we found that membrane-permeable ceramides additively increased the apoptotic effect of HePC.     

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT

In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.     

Correlation between expression of mdr-1 gene and oncogenes in human promyelocytic leukemic HL60 cell line and sublines

Non-ischaemic high flow priapism resulting from trauma of the genito-perineal area is an uncommon occurrence. Resolution by selective embolization of the internal pudendal artery is currently the choice treatment. This paper presents the case of a 9 year-old patient where embolization with autologous coagulation achieved healing.     

Induction of apoptosis by benzene metabolites in HL60 and CD34+ human bone marrow progenitor cells

Two cell types, HL60 human promyelocytic leukemia cells and CD34+ human bone marrow progenitor cells, were used as model systems to explore a possible role for apoptosis in the myelotoxicity of the phenolic metabolites of benzene. HL60 cells were treated with either phenol, catechol, hydroquinone, or 1,2,4-benzenetriol and then stained with Hoechst 33342 and propidium iodide and subjected to fluorescent microscopy. Cells with nuclear condensation and fragmentation were scored as apoptotic, and etoposide (40 microM) was used as a positive control. Catechol, 1,2,4-benzenetriol, and hydroquinone induced marked time- (0-24 hr) and concentration- (25-100 microM) dependent apoptosis, whereas phenol (750 microM) did not. Under these conditions, no significant necrosis was observed. The induction of apoptosis was confirmed by internucleosomal cleavage of DNA, assessed by agarose gel electrophoresis. CD34+ cells treated with etoposide (40 microM) or hydroquinone (50 microM) for 18 hr were stained and subjected to fluorescent microscopy as above. The percentage of cells exhibiting nuclear condensation and/or fragmentation as well as high intensity staining significantly increased in both cases. The induction of apoptosis was confirmed using a terminal deoxynucleotidyl transferase assay. These data show that apoptosis can be induced in both HL60 and CD34+ human bone marrow progenitor cells by benzene metabolites. The ability of phenolic metabolites of benzene to induce apoptosis in human bone marrow progenitor cells may contribute to benzene myelotoxicity.     

Isolation and characterization of a variant HL60 cell line defective in the activation of the NADPH oxidase by phorbol myristate acetate

Promyelocytic human leukemia HL60 cells can be differentiated into neutrophil-like cells that exhibit an NADPH oxidase activity through direct stimulation of protein kinase C (PKC) with PMA or through formyl peptide receptor activation. We have isolated a variant HL60 clone that exhibited a conditional PMA-induced oxidative response depending on the agent used for the differentiation. While cells differentiated with DMSO responded to either PMA or N-formyl peptide (N-formyl-Met-Leu-Phe-Lys or fMLFK), cells differentiated with dibutyryl-cAMP (Bt2cAMP) responded to fMLFK but very poorly to PMA. However, in Bt2cAMP-differentiated cells, the expression of the different PKC isoforms was similar to that observed in DMSO-differentiated cells. Moreover, PMA was able to induce a normal phosphorylation of the cytosolic factor p47phox and to fully activate extracellular signal-regulated kinases (Erk1/2). Interestingly, Bt2cAMP-differentiated cells exhibited a strong and sustained O2- production when costimulated with PMA and suboptimal concentrations of fMLFK which were, per se, ineffective. This sustained response was only slightly reduced by the conjunction of the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor. Variant HL60 cells that were stably transfected with a constitutively active form of Rac1 were able, when differentiated with Bt2cAMP, to secrete oxidant following PMA stimulation. Altogether, the results suggest that, in addition to the phosphorylation of p47phox, the activation of NADPH oxidase requires the activation of a Rac protein through a pathway that diverges at a point upstream of MEK and that is independent of the activation of wortmannin sensitive PI3K.     

环氧合酶-2抑制剂对白血病细胞HL-60的化疗增敏作用及机制

目的 观察环氧合酶-2(COX-2)抑制剂塞来昔布对白血病细胞株HL-60的化疗增敏作用,并对其机制进行初步探讨.方法 MTT法评估塞来昔布、多柔比星及二者联合对HL-60细胞的生长抑制效应;流式细胞术(FCM)检测细胞的凋亡;反转录聚合酶链反应(RT-PCR)检测Survivin基因的表达;Western blotting检测Survivin蛋白的表达.结果 多柔比星联合塞来昔布5和10μmol/L对HL-60细胞的半数抑制浓度(IC50)分别为0.25及0.16μg/ml,明显低于多柔比星单用的IC50(0.48μg/ml);多柔比星0.10μg/ml联合10 μmol/L塞来昔布下调Survivin基因mRNA及蛋白的表达;联合塞来昔布5和10 μmol/L的凋亡率[分别为(13.07±1.66)%及(22.36±1.84)%]较多柔比星0.10μg/ml单用[(5.72±1.25)%]明显增加(P<0.01).结论 COX-2抑制剂塞来昔布对白血病细胞株HL-60具有明显的化疗增敏作用,其初步机制涉及下调Survivin的表达,增加细胞凋亡.     

Effects of phospholipid base analogues on the subcellular membrane ether composition of suspension cultured LM cells

Morbidity and mortality from pulmonary complications following urinary tract operations can be reduced by preoperative identification of the high risk patient. Pulmonary function tests and arterial blood gases are necessary to identify these patients and to delineate the severity of their pulmonary disease. Respiratory complications can be prevented in many patients with the proper use of pre- and postoperative chest physical therapy and oxygen therapy. Despite the most careful pulmonary management, some patients develop acute respiratory failure following urologic operations. Respiratory failure results from a combination of physiologic abnormalities which impair alveolar ventilation and oxygenation. Utilizing controlled ventilation, supplemental oxygen, and a physiologic approach to treating the underlying cause of respiratory failure, three fourths of urologic patients in respiratory failure may be expected to survive.     

Comparative assessment of the activity of beta-carotene, retinoyl-beta-D-glucuronide and retinoic acid on growth and differentiation of a human promyelocytic leukemia cell line HL-60

We compared the ability of all-transretinoic acid (RA), all-trans-retinoyl-beta-D-glucuronide (RAGL), and all-trans-beta-carotene (BC) to inhibit growth and to induce differentiation of the human promyelocytic leukemia cell line HL-60 into morphologically mature granulocytes. BC was made water-soluble by the solutol-solvent-system. RA (1 microM) could induce differentiation of 85% of the HL-60 cells after a total incubation time of 180 hours, RAGL (5 microM) induced 64% of the cells, whereas 33% of the HL-60 cells were differentiated after incubation with BC (10 microM), which was determined by assessing cell functional capacity to reduce nitroblue tetrazolium dye in response to phorbolesters. The absence of RA in RAG and BC treated cells gives strong evidence that RAG and BC exert intrinsic biological effects.     

Induction of apoptotic cell death in human leukemic cell line, HL-60, by extremely low frequency electric magnetic fields: analysis of the possible mechanisms in vitro

An anthropomorphic Rando phantom was used to compare radiation doses sustained during helical and conventional axial CT of the pelvis. The values obtained with the Rando phantom were validated against cadaveric phantoms, and show good agreement. For the authors' particular CT unit, helical scanning was found to deliver a lower radiation dose than conventional axial scanning. This was most prominent at 1.0-s tube rotation times (average dose ratio 1.24). For realistic scanning parameters and exposure factors, the ratio of radiation dose to pelvic organs can be expected to lie in the range of 40-100 mGy. The whole-body effective dose (ED) depends on selection of scanning parameters and patients anatomy. In a favourable case scenario, the ED for CT scanning of the pelvis in a male can be expected to be between 10 and 20 mSv if the scrotum is not included in the radiation field, while the ED in a female will be approximately 20 mSv. An examination of scatter radiation fall-off curves from a single slice shows that the spread of scatter radiation is only marginally affected by slice thickness. A total of 10-12 cm of human soft tissue acts as a good barrier against internal scattered radiation. The use of such scatter fall-off curves, together with manufacturers' dosimetry specifications, allows a fast estimate of absorbed dose.     

Photodynamic effects of meso-tetra (4-sulfonatophenyl)porphine on human leukemia cells HEL and HL60, human lymphocytes and bone marrow progenitor cells

A new measure is presented, suitable for documenting severity and response to treatment in chronic low-back pain syndromes. It is self-administered and combines two pain and seven mobility items. These were selected from pre-existing validated instruments on the basis of their sensitivity to change after treatment. Their Italian validated translation was adopted. The measure was administered to 32 chronic low-back pain Italian patients, all refractory to previous conservative treatments. In most cases they presented with herniation or protrusion of 1-3 lumbar discs. Patients were treated with 3-6 sessions of autotraction over a 5- to 15-day period. Scores on the scale were recorded at admission, discharge and follow-up, 1-3 months after treatment. At discharge and at follow-up, patients were asked if, overall, they felt improved, the same or worse. At follow-up, 20 patients out of 32 reported overall improvement. Scalometric properties of the measure were tested using Rasch analysis. For admission and follow-up, items followed a consistent hierarchical relationship along a unidimensional pain/disability variable, which is being called back illness. The items were not redundant, in that they spread well along a wide range of difficulty/severity. The hierarchy matched well with the expected expression of the conditions of the patients. A study was conducted on 34 chronic back pain patients, showing satisfactory test-retest reliability. Depending on the various items, Cohen's unweighted K ranged from 0.27 to 0.78, with ten of the 11 items above the 0.45 level of acceptability, while intraclass correlation coefficients ranged from 0.42 to 0.89. At follow-up, changes in BACKILL of plus 15% or more, with respect to admission, were consistent with patients' reports of improvement in 19 out of 20 cases. Changes in BACKILL of less than 15% were consistent with patients reports of being the same or worse in 11 out of 12 cases.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

Induction of apoptosis in HL-60 human promyelocytic leukemia cells by adenosine A(3) receptor agonists

The effects of adenosine (ADO) analogs on cells of the human promyelocytic HL-60 line were examined. ADO A(3) receptor agonists, N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA, 30-60 microM) and 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA, 10-30 microM) induced apoptotic cell death. In contrast, neither an A(1)/A(2) antagonist (XAC) nor other selective ADO receptor agonists (CPA, NECA and CGS21680) induced apoptosis at concentrations of <30 microM. Both IB-MECA and CI-IB-MECA significantly induced Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx, suggesting the presence of phospholipase C-coupled ADO A(3) receptors on HL-60 cells. This was further supported by the presence of mRNA of ADO A3 receptor in the cells. These results suggest that activation of ADO A(3) receptors is responsible for the ADO-induced apoptosis in HL-60 cells and could be of potential therapeutic value in the treatment of leukemia.     

Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning

Camptothecins are a new class of anticancer drugs that target DNA topoisomerase I; current efforts are directed toward elucidating optimal combinations of these drugs with other antineoplastic agents. A rationale for the use of sequential therapy involving the combination of camptothecins with topoisomerase II-targeting drugs, such as etoposide, has arisen from observations of increased topoisomerase II protein levels in cell lines resistant to camptothecin. In an effort to understand potential mechanisms of resistance to this strategy, we developed a U-937 cell subline, denoted RERC, that is capable of surviving exposure to sequential topoisomerase poisoning. The RERC cells are 200-fold resistant to camptothecin, 8-fold resistant to etoposide, and 10-fold hypersensitive to cisplatin compared to the parental U-937 cells. Biochemical analyses indicate that the resistant phenotype involves alterations in both topoisomerase I and topoisomerase IIalpha. Topoisomerase I catalytic activity in the resistant cells is similar to that of the parental line but is resistant to camptothecin. Moreover, the resistant cells express a single mRNA species of topoisomerase I that codes for a mutation in codon 533. In addition, topoisomerase IIalpha protein levels are decreased 10-fold in the resistant line, coincident with a two-fold decrease in the expression of topoisomerase IIalpha mRNA. Collectively, these results indicate that resistance to sequential topoisomerase poisoning may involve a reduction in total cellular topoisomerase activity.     

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.     

Induction of magnetic resonance-visible lipid in a transformed human breast cell line by tetraphenylphosphonium chloride

Proton magnetic resonance spectroscopy (1H MRS) and DNA flow cytometry were used to monitor the effects of the cationic lipophilic phosphonium salt and potential antineoplastic agent tetraphenylphosphonium chloride (TPP) on the transformed human breast cell line HBL-100. TPP treatment for 48 hr was cytostatic at low concentrations and cytotoxic at higher concentrations with an IC50 of 55 microM as measured by Trypan blue exclusion. At micromolar concentrations, TPP caused a significant increase in the methylene MR signal arising from mobile lipid as measured by the ratio of the lipid CH2 peak height to either the CH3 peak height (internal referencing) or the peak height for p-aminobenzoic acid (PABA) as an external reference in a co-axial capillary within the sample. Over the same concentration range, TPP caused a slowing of passage through S phase as demonstrated by a significant depletion of cells in G2/M phase with a concurrent but non-significant increase in cells in S. Time-dependent increases in MR-visible lipid were observed with 2 microM TPP treatment, and the removal of TPP from the culture medium caused no significant reduction in mobile lipid. Two-dimensional 1H-1H COSY spectra of TPP-treated HBL-100 cells revealed concentration-dependent increases in cross-peak volume ratios arising from lipid acyl chains relative to both internal (lysine, polyamines) and external (PABA) standards. Increases in choline and glycerophosphocholine cross-peak volume ratios were observed, indicating that the catabolism or rearrangement of phospholipids may be responsible for the observed MR-visible lipid increases.