Biomedical subjects

F Mollinedo

Publications and source records attributed to F Mollinedo.

At least 37 records · Page 2Linked to original sources

PubMed1997

Selective induction of apoptosis in cancer cells by the ether lipid ET-18-OCH3 (Edelfosine): molecular structure requirements, cellular uptake, and protection by Bcl-2 and Bcl-X(L).

The ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; Edelfosine) has been shown to be a rapid inducer of apoptosis in human leukemic cells and has been considered as a promising drug in cancer treatment. Here we have found that ET-18-OCH3 induced apoptosis not only in human tumor cell lines but also in primary tumor cell cultures from cancer patients. Human leukemic cells were highly sensitive to ET-18-OCH3, whereas normal cells remained unaffected. Among the distinct modifications of the ET-18-OCH3 molecule assayed, we found that substitutions in positions sn-2 and sn-3 of the glycerol backbone resulted in a complete loss of its capacity to induce apoptosis, highlighting the importance of the molecular structure of ET-18-OCH3 in its apoptotic effect. Induction of apoptosis by ET-18-OCH3 was very well correlated with the uptake of this ether lipid. ET-18-OCH3-resistant 3T3 fibroblasts became sensitive and incorporated significant amounts of the ether lipid following transformation with the SV40 virus. ET-18-OCH3-induced apoptosis as well as ET-18-OCH3 uptake were not mediated through binding of the ether lipid to the platelet-activating factor receptor. Overexpression of bcl-2 or bcl-xL by gene transfer in the human erythroleukemic HEL cells abrogated apoptosis induced by ET-18-OCH3. ET-18-OCH3 did not affect the expression of bcl-2, bcl-xL, or bax in HEL and HL-60 human leukemic cells but induced expression of c-myc, an important effector of apoptosis in several systems. Thus, ET-18-OCH3 behaves as a potent and highly selective antitumor drug able to induce an apoptotic pathway of cell death in tumor cells but not in nonmalignant cells.

Albumins↗

PubMed1997

Identification of two isoforms of the vesicle-membrane fusion protein SNAP-23 in human neutrophils and HL-60 cells.

We have identified and cloned two isoforms of SNAP-23 in human neutrophils and human promyelocytic HL-60 cells. SNAP-23A which is similar to the recently cloned SNAP-23, and a novel isoform, termed SNAP-23B. SNAP-23B results from a deletion of 159 bp of the SNAP-23A cDNA, encoding a protein of approximately 17.8 kDa. SNAP-23B is identical to SNAP-23A, but lacks fifty-three amino acid residues, from 90 to 142. This region of SNAP-23A has a target sequence for posttranslational fatty acid acylation, suggesting that the two isoforms can differ in their capacity to interact with membranes. The level of SNAP-23 mRNA is increased during HL-60 neutrophil differentiation. These findings demonstrate the existence of two SNAP-23 proteins in human neutrophils; and therefore, these cells have all the proteins needed to carry out the exocytic process.

Amino Acid Sequence↗

PubMed1997

Prevention of cytokine-induced changes in leukocyte adhesion receptors by nonsteroidal antiinflammatory drugs from the oxicam family.

OBJECTIVE: To explore the effect of the nonsteroidal antiinflammatory drugs (NSAIDs) piroxicam and meloxicam on quantitative and qualitative changes in leukocyte adhesion receptors induced by cytokines and other activation stimuli. METHODS: The expression of CD11b and L-selectin during neutrophil activation with tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), FMLP, phorbol myristate acetate (PMA), and calcium ionophore A23187 was assessed by flow cytometry. Enzyme-linked immunosorbent assays were used to quantitate soluble L-selectin shed after neutrophil stimulation. Enzyme release was measured to determine neutrophil degranulation by proinflammatory stimuli. Changes in affinity state of beta 1 and beta 2 integrins after neutrophil and T lymphocyte stimulation were assessed, by flow cytometry, using the monoclonal antibodies (MAb) HUTS-21 (anti-beta 1) and CBRM1/5 (anti-CD11b), which recognize activation-dependent epitopes on these two integrins. RESULTS: Pretreatment of neutrophils with either NSAID prevented the changes in L-selectin and CD11b expression induced by TNF alpha, GM-CSF, and FMLP, but not those induced by PMA or A23187. Furthermore, piroxicam significantly decreased the amount of L-selectin shed by cytokine-treated neutrophils, whereas it did not exert this effect on PMA- or A23187-treated neutrophils. Piroxicam also decreased the release of gelatinase and lysozyme induced by TNF alpha, but not by PMA. Interestingly, piroxicam prevented the conformational changes that beta 2 integrins underwent upon activation of neutrophils: the appearance of the activation epitope of CD11b, detected by the CBRM1/5 MAb, was blocked by piroxicam in TNF alpha-treated neutrophils. Moreover, in chemokine-treated T lymphocytes, the expression of activation epitopes on beta 1 integrins was also diminished by piroxicam. In contrast, this NSAID did not affect the beta 1 integrin conformational changes induced by PMA or Mn++. CONCLUSION: Our results indicate that members of the oxicam family are able to interfere with events of neutrophil function, such as their degranulation and cytokine-mediated activation changes in adhesion molecules, both in neutrophils and in lymphocytes. Such effects may significantly contribute to the antiinflammatory activity of these drugs.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1997

Dissociation of the effects of the antitumour ether lipid ET-18-OCH3 on cytosolic calcium and on apoptosis.

1. We have compared the effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) on the cytosolic free calcium concentration ([Ca2+]i) and on apoptosis in several normal and leukaemia cells, including human polymorphonuclear neutrophils (PMNs), U937 cells, and undifferentiated as well as dimethylsulphoxide-differentiated HL60 cells (uHL60 and dHL60, respectively). 2. ET-18-OCH3 produced apoptosis, as evidenced by DNA degradation into oligonucleosome-size fragments, in U937 and uHL60 cells, but not in dHL60 cells or PMNs. 3. ET-18-OCH3 induced an increase in [Ca2+]i mediated through the platelet-activating factor (PAF) receptor in U937, dHL60 cells and PMNs, as shown by cross-desensitization experiments and by prevention of the [Ca2+]i changes by the PAF antagonist WEB-2170. The EC50 values for the increase in [Ca2+]i induced by PAF and ET-18-OCH3 were 5 x 10(-11) and 2.5 x 10(-7) M, respectively. In uHL60 cells the effect of ET-18-OCH3 on [Ca2+]i was very small and was not affected by WEB-2170. 4. PAF did not produce apoptosis in any of the cell types tested. WEB-2170 did not prevent the apoptosis induced by ET-18-OCH3. 5. The uptake of [3H]-ET-18-OCH3 was much larger in U937 and uHL60 cells than in dHL60 cells and PMNs. 6. Our results indicate that the apoptotic effect of ET-18-OCH3 is not related to the changes in [Ca2+]i, effected by interaction with plasma membrane PAF receptors, but to other actions which are associated with the uptake of this drug into the cells.

Antineoplastic Agents↗

PubMed1997

Recurrent infectious diseases in human CD53 deficiency.

We report a familiar syndrome of recurrent heterogeneous infectious diseases, caused by bacteria, fungi, and viruses, which has as its only detectable defect the lack of CD53 antigen expression in neutrophils. All other assays ruled out known causes of recurrent infectious diseases due to either leukocyte adhesion or phagocytosis defects. CD53 belongs to the transmembrane-4 superfamily of proteins, which are a novel group of membrane proteins implicated in growth regulation and cell motility and possibly cell adhesion. We postulate that defects in these membrane proteins can be clinically manifested as complex recurrent infections.

Adolescent↗

PubMed1996

C-Fos is not essential for apoptosis.

The transcription factor AP-1, made up of dimers of Fos and Jun proto-oncogene products, is involved in distinct cellular processes, including cell proliferation, differentiation and apoptosis. In this study, we have used mice in which both copies of the c-fos gene were disrupted by targeted mutagenesis in order to analyze how the apoptotic response was affected in these mice. We prepared primary cultures from the lymphoid organs, spleen and thymus, obtained from both wild-type and c-fos -/- mice and analyzed the induction of apoptosis in these cultures in the absence and presence of etoposide, an inducer of apoptosis in distinct cell types. Primary cultures from both organs, spleen and thymus, isolated from wild-type mice underwent apoptosis after 3 and 6 h of culture, respectively. Addition of etoposide enhanced the apoptotic response and c-fos mRNA levels in both spleen and thymic cells. Nevertheless, we found that induction of apoptosis in primary cultures of cells obtained from spleen and thymus of c-Fos-deficient mice was practically identical to that observed in wild-type mice. These results demonstrate that c-Fos is not essential for apoptosis and that cells lacking c-Fos may undergo normal apoptosis.

Animals↗

PubMed1996

Transient inhibition of capacitative calcium entry in human neutrophils by a monoclonal antibody directed against a 19-kDa antigen.

P1C3 is a monoclonal antibody that binds p19, a novel neutrophil activation antigen that translocates to the cell surface upon neutrophil activation. We find that P1C3 inhibits capacitative Ca2+ entry, induced by emptying the intracellular Ca2+ stores with thapsigargin. The effect is transient, reaching its maximum at 30-60 s, but becomes permanent upon pretreatment of the cells with the protein phosphatase inhibitor calyculin A, suggesting the involvement of protein phosphorylation. The inhibitory action is similar to the one reported previously for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), although the transduction mechanism may be different. Inhibition of Ca2+ entry by fMLP was prevented by pretreatment with pertussis toxin, whereas inhibition by P1C3 was not. Pretreatment with cholera toxin had no effect. This suggests that the effect of P1C3 may not be mediated by a heterotrimeric G protein. Tyrosine kinase inhibitors did not prevent inhibition by either fMLP or P1C3. Phospholipase C activation seems not to be involved as P1C3, contrarily to fMLP, was unable to induce Ca2+ release from the intracellular Ca2+ stores.

Antibodies, Monoclonal↗

PubMed1996

Aceclofenac, a new nonsteroidal antiinflammatory drug, decreases the expression and function of some adhesion molecules on human neutrophils.

OBJECTIVE: To study the effect of aceclofenac, a new nonsteroidal antiinflammatory drug (NSAID), on the expression and function of adhesion molecules in human neutrophils. METHODS: We used flow cytometry analysis to determine peripheral blood neutrophil expression of L-selectin, CD11a, CD11b, CD31, CD43, CD44, and intercellular adhesion molecule 2 (ICAM-3) surface adhesion molecules after treatment with aceclofenac, diclofenac, or dexamethasone. Granular enzyme activity was quantitated in extracellular medium of neutrophils treated with different NSAID: In vitro adhesion assays were developed to examine the effects of aceclofenac on both neutrophil adhesion to tumor necrosis factor alpha stimulated human umbilical vein endothelial cells under nonstatic conditions, and homotypic neutrophil aggregation induced by anti-ICAM-3 and anti-CD18 monoclonal antibodies (Mab). RESULTS: Aceclofenac induced a dramatic decrease of L-selectin expression, whereas a moderate and slight decrement of CD43 and ICAM-3 expression was also observed. In contrast, the expression of other adhesion molecules by neutrophils was unaffected (CD11a, CD31, CD44) or slightly increased (CD11b). Cell adhesion assays, performed under nonstatic conditions, revealed that aceclofenac significantly diminished the L-selectin dependent neutrophil adhesion to endothelial cells. Neutrophil aggregation induced with anti-CD43 Mab was also significantly inhibited by aceclofenac. CONCLUSION: Aceclofenac had a faster and more potent effect than the other NSAID studied, mainly on the expression of cell adhesion molecules. This new NSAID efficiently interferes with neutrophil adhesion to endothelium and this effect may represent an additional relevant mechanism in its antiinflammatory activity.

Anti-Inflammatory Agents↗

PubMed1995

Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease.

Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na+/H+ antiporter induces a raise in pHi which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pHi which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pHi was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOV-induced intracellular acidification was not due to a complete inhibition of the Na+/H+ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na+/H+ antiporter with a marked increase in pHi. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na+/H+ antiporter and the suppression of apoptosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells.

Inhibition of protein N-glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL-60 cells. Internucleosomal DNA fragmentation could be detected after short-time incubation (between 6 and 9 h) of HL-60 cells with low doses of tunicamycin (0.05 micrograms/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin-induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL-60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)-induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL-60 cells is the induction of apoptosis.

Apoptosis↗

PubMed1995

Prevention of in vitro neutrophil-endothelial attachment through shedding of L-selectin by nonsteroidal antiinflammatory drugs.

The activation of the endothelial cells by extravascular stimuli is the key event in the extravasation of circulating leukocytes to target tissues. L-selectin, a member of the selectin family, is constitutively expressed by white cells, and is the molecule involved in the initial binding of leukocytes to activated endothelium. After activation, leukocytes rapidly release L-selectin from the cell surface, suggesting that the functional activity of this molecule is controlled in large part by its appearance and disappearance from cell surface. We have studied in a neutrophil-activated endothelial cell binding assay, the effect of different antiinflammatory drugs (steroidal and nonsteroidal) in the L-selectin-mediated interaction of neutrophils with activated endothelial cells. Some nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin, diclofenac, ketoprofen, and aspirin, but not steroids, strongly inhibited the neutrophil-endothelial cell attachment. Furthermore, we also investigated the underlying mechanism of this functional effect. The expression of L-selectin on the neutrophil surface rapidly decreased in the presence of different NSAIDs, in a dose- and time-dependent manner, whereas no changes in the expression of other adhesion molecules such as CD11a, CD11b, CD31, or ICAM-3 (CD50) were observed. Interestingly, studies in vivo on healthy volunteers treated with physiological doses of indomethacin showed a significant decrease of L-selectin neutrophil expression. Only diclofenac induced an upregulation of CD11b expression, suggesting an activating effect on neutrophils. No enzyme release was observed upon treatment of neutrophils with different NSAIDs, indicating a lack of degranulatory activity of NSAIDs, with the exception of diclofenac. The downregulation of L-selectin expression was due to the rapid cleavage and shedding of the membrane L-selectin, as determined by both immunoprecipitation from 125I-labeled neutrophils, and quantitative estimation in cell-free supernatants. These results suggest that NSAIDs exert a specific action on adhesion receptor expression in neutrophils, which might account, at least in part, for the antiinflammatory activities of NSAIDs.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1994

Involvement of phospholipase D in the activation of transcription factor AP-1 in human T lymphoid Jurkat cells.

The induction of the AP-1 transcription factor has been ascribed to the early events leading to T lymphocyte activation. We have examined the possibility that stimulation of phospholipase D (PLD) may regulate activation of transcription factor AP-1 in human T cells by transfecting human T lymphocyte Jurkat cells with a plasmid containing an AP-1 enhancer element and a chloramphenicol acetyltransferase reporter gene. We have detected activatable PLD in Jurkat cells, and we have found that addition of phosphatidic acid (PA), the physiologic product of PLD action on phospholipids, is rapidly incorporated into Jurkat cells and leads to activation of transcription factor AP-1. Treatment of Jurkat cells with anti-CD3 mAb activated both PLD and transcription factor AP-1. Wortmannin, an inhibitor of receptor-coupled PLD activation, blocked the anti-CD3-induced increases in both PLD activity and AP-1 enhancer activity. We found a good correlation in the transfected cells between PLD activation and induction of AP-1 enhancer activity under different experimental conditions. Furthermore, ethanol, an inhibitor of the PLD pathway, blocked the anti-CD3-stimulated AP-1 enhancer activity. However, this anti-CD3-mediated response was not inhibited by neomycin, an inhibitor of phosphoinositide hydrolysis. The increases in AP-1 enhancer activity induced by PA or anti-CD3 mAb were efficiently abrogated by the presence of propranolol, an inhibitor of PA phosphohydrolase and protein kinase C (PKC). Furthermore, the PA- and the anti-CD3-induced increases in AP-1 enhancer activity were blocked by the presence of PKC inhibitors or by PKC down-regulation. These data indicate that PLD stimulation can activate the transcription factor AP-1 in T lymphocytes, and suggest that the induction of AP-1 enhancer factor activity by PA is mediated via PKC stimulation, either through a direct activating effect of PA or through PA-derived diacylglycerol formation. These data also provide evidence for a role of PLD-derived lipids in the induction of AP-1 enhancer activity resulting from stimulation of the TCR/CD3 complex, suggesting that increased PLD activity can play an important role in T lymphocyte activation.

Arachidonic Acid↗

PubMed1994

The ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine induces expression of fos and jun proto-oncogenes and activates AP-1 transcription factor in human leukaemic cells.

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) has been recently shown to induce apoptosis in the human leukaemic HL-60 and U937 myeloid cell lines [Mollinedo, Martinez-Dalmau and Modolell (1993) Biochem. Biophys. Res. Commun. 192, 603-609]. We have found that ET-18-OCH3 is also able to promote apoptosis in the human leukaemic Jurkat T lymphoid cell line. This lymphoid cell line as well as the two myeloid HL-60 and U937 cell lines incorporated significant amounts of exogenously added radiolabelled ET-18-OCH3. Addition of ET-18-OCH3 to these human leukaemic cells induced an increase in the steady-state mRNA levels of fos and jun proto-oncogenes, components of the transcription factor AP-1. These increases in fos and jun mRNA levels were associated with the activation of the AP-1 transcription factor after addition of ET-18-OCH3 to human leukaemic cells, as assessed by an enhanced binding activity of transcription factor AP-1 to its cognate DNA sequence as well as by stimulation of transcription from an AP-1 enhancer element. These data demonstrate that the ether lipid ET-18-OCH3 can affect gene expression by inducing expression of fos and jun proto-oncogenes and by modulating the activity of transcription factor AP-1.

Apoptosis↗

PubMed1994

Dexamethasone modifies the functional responses of the granulocytic differentiating HL-60 cells.

We have studied the effect of dexamethasone on the granulocytic differentiation of the human promyelocytic cell line HL-60 induced by treatment with retinoic acid (RA) or dimethyl sulphoxide (DMSO). Dexamethasone potentiated the immunophenotypic and functional parameters associated with the granulocytic differentiation induced by RA, including changes in CD11b and CD71 expression, inhibition of cell proliferation, enhancement of secretory and oxidative responses and increase of the phospholipase C (PLC), phospholipase A2 (PLA2) and phospholipase D (PLD) activities. However, dexamethasone had selective effects on several parameters of DMSO-induced cell differentiation. Dexamethasone inhibited the DMSO-induced increase of CD11b cell surface expression as well as the oxidative response and PLD activation triggered by 4 beta-phorbol 12-myristate 13-acetate. Nevertheless, dexamethasone potentiated the receptor-mediated PLC activation and the receptor-mediated secretory and oxidative responses in DMSO-treated cells. Unlike RA-treated HL-60 cells, the DMSO-treated cells contained high values of activatable PLA2 activity which were not affected by dexamethasone. Thus dexamethasone affected differently functional parameters and effector systems of granulocytic HL-60 cells, depending on the differentiation agent used. Dexamethasone by itself did not induce HL-60 cell differentiation, but enhanced the receptor- and non-receptor-mediated secretory responses and induced the appearance of stimulated PLA2 activity in undifferentiated HL-60 cells. These data provide evidence for the selective modulation of functional responses by dexamethasone through alterations in signalling processes.

Antigens, CD↗

PubMed1994

Inhibition of isoprenoid biosynthesis induces apoptosis in human promyelocytic HL-60 cells.

Protein isoprenylation is a posttranslational modification that facilitates membrane association and biological activity of a number of proteins. Mevalonate is the precursor of cellular sterols as well as of isoprenoid lipids involved in protein modification. In this study we show that HL-60 cells treated with lovastatin, an inhibitor of mevalonate synthesis, exhibit alterations in growth and morphology, as well as changes in the subcellular distribution of isoprenylated proteins like nuclear lamin A and p21Ras. Moreover, they are induced to die via apoptosis, as evidenced by the appearance of a typical DNA fragmentation pattern. Lovastatin-induced DNA fragmentation can be specifically prevented by mevalonate. The failure of several products of the mevalonate pathway, including cholesterol, to overcome lovastatin effect, points to the involvement of isoprenylated proteins in the mechanisms suppressing cell death.

Apoptosis↗

PubMed1994

Diacylglycerol and alkylacylglycerol stimulate ram sperm phospholipase A2.

1. We have investigated the susceptibility of ram sperm phospholipase A2 (PLA2) to stimulation by diacyl- and alkylacylglycerols and by monoacyl- and monoalkylglycerols. 2. PLA2 activity in sonicates from ram spermatozoa was enhanced when 1-stearoyl-2-arachidonoyl-sn-glycerol, the diacylglycerol usually generated by polyphosphoinositide breakdown, was added to a radioactive phosphatidylcholine substrate; the effect was time- and Ca(2+)-dependent. 3. Both diacyl- and alkylacylglycerol considerably enhanced PLA2 activity; 1-O-hexadecyl-2-O-methyl-rac-glycerol, however, only showed slight stimulatory ability. 4. The monoradylglycerols 1-monohexadecanoyl-rac-glycerol, 2-monohexadecanoylglycerol, and 1-O-hexadecyl-sn-glycerol had very little effect on the enzyme's activity. 5. Exposure of spermatozoa to 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 1-O-hexadecyl-2-acetyl-rac-glycerol (1-O-C16/2-C2), when cells were stimulated with the ionophore A23187 and Ca2+, resulted in higher PLA2 activity in sperm sonicates. Furthermore, parallel experiments showed that exocytosis was enhanced if spermatozoa were treated with A23187/Ca2+ and either OAG or 1-O-C16/2-C2. Since both diacyl- and alkylacylglycerols increased PLA2 activity and exocytosis, stimulation of PLA2 activity by these diglycerides may take place independently from protein kinase C activation.

Animals↗

PubMed1993

Differences in expression of transcription factor AP-1 in human promyelocytic HL-60 cells during differentiation towards macrophages versus granulocytes.

Commitment of HL-60 cells to macrophage or granulocytic differentiation was achieved by incubation with 4 beta-phorbol 12-myristate 13-acetate (PMA) for 30-60 min or with dimethyl sulphoxide (DMSO) for 24 h respectively. The commitment stage towards PMA-induced macrophage differentiation was associated with increases in jun B and c-fos mRNA levels, as well as with an increase in the binding activity of transcription factor AP-1. Nevertheless, gel retardation analysis indicated that the AP-1 activity detected in untreated cells was drastically reduced during the commitment stage of DMSO-induced HL-60 differentiation towards granulocytes. When HL-60 cells were treated with sodium butyrate, which induced monocytic differentiation, a remarkable increase in AP-1 binding activity was detected. Treatment of HL-60 cells with 1 alpha,25-dihydroxyvitamin D3, another monocytic differentiation agent, induced a weak, but appreciable, increase in AP-1 activity. Furthermore, addition of sodium butyrate or 1 alpha,25-dihydroxyvitamin D3 to HL-60 cells induced the expression of c-fos, c-jun, jun B and jun D proto-oncogenes. In contrast, when HL-60 cells were treated with retinoic acid, a granulocytic differentiation inducer, no enhanced AP-1 binding activity was observed, and only a weak increase in jun D mRNA level was detected. These data indicate that formation of AP-1 is not required for the induction of HL-60 differentiation towards granulocytes, whereas induction of monocytic differentiation is correlated with an increase in AP-1 activity. The differential expression of AP-1 activity may be critical in the differentiation of HL-60 cells towards monocytic or granulocytic lineages.

Base Sequence↗

PubMed1993

Localization of rap1 and rap2 proteins in the gelatinase-containing granules of human neutrophils.

The subcellular localization of rap proteins in resting human neutrophils was investigated by immunoblot analysis with specific anti-rap2 and anti-rap1 antibodies of the membrane proteins obtained from distinct subcellular fractions. Rap2 protein was mainly located in gelatinase-containing granules, whereas rap1 protein was detected both in gelatinase-containing granules and in fractions enriched in plasma membrane. Neither rap1 nor rap2 proteins were found in the cytosol or in azurophilic granules. Rap2B, not rap2A, appeared to be the major rap2 protein in human neutrophils. The identification and subcellular localization of rap1 and rap2 proteins at the membranes of gelatinase-rich granules suggest that these proteins could play a role in the regulation of the rapid and selective mobilization of gelatinase-containing granules in human neutrophils.

Amino Acid Sequence↗