Histiocytic Lymphoma Cell Line U937 Research Articles

Preventive Effect of Trimidox on Oxidative Stress in U937 Cell Line

Trimidox (3,4,5-trihydroxybenzamidoxime) is one of the most potent ribonucleotide reductase inhibitors, revealing an antitumor effect in several experimental studies. We have examined the effect of trimidox on the induction of cytotoxicity and apoptosis via oxidative stress by typical free radical inducers, hydrogen peroxide (H(2)O(2)), tert-butylhydroperoxide (tBuOOH) or ultraviolet (UV) irradiation in a human diffuse histiocytic lymphoma U937 cell line. Trimidox showed strong radical scavenging activity by the DPPH reduction assay. The 50% rate inhibited the DPPH reduction concentration of trimidox, and its derivates didox, or gallic acid were 8.8 microM, 117.5 microM, or 41.8 microM, respectively. Induction of cytotoxicity by H(2)O(2) (500 microM) or tBuOOH (100 microM) was concentration-dependently attenuated by incubation with Trimidox (10-150 microM). Trimidox also prevented the effect of UV-induced apoptosis estimated by both nuclear morphological change and DNA fragmentation. This effect was due to inhibition of the production of reactive oxygen species. Moreover, the activity and mRNA expression of catalase, an antioxidant enzyme, was significantly increased by trimidox. These results indicate that trimidox has radical scavenging activity and prevents exogenous oxidative stress and increase in catalase; therefore, trimidox is suggested as an anticancer agent exhibiting potent antioxidant properties in this study.

Topoisomerase II beta levels are a determinant of melphalan-induced DNA crosslinks and sensitivity to cell death

The role of topoisomerase (topo) II in DNA repair has yet to be fully elucidated. Current evidence suggesting a role for topo II in the repair of DNA damage has been obtained by using in vitro model systems or inferred from correlative data in drug resistant cell lines. In this study we directly examined the role of topo IIα and β in mediating the repair of melphalan-induced crosslinks in cellular DNA. To accomplish this, we used siRNA technology to knock down either topo IIα or β in human chronic myelogenous leukemia K562 and histiocytic lymphoma U937 cell line. Our data demonstrate that topo IIβ levels, (but not α), are a determinant of melphalan-induced crosslinks and sensitivity to melphalan. Furthermore, we show that knocking down topo IIβ inhibits the repair of melphalan-induced crosslinks in K562 cells. These studies represent the first direct evidence that topo IIβ participates in the repair of DNA damage induced by an alkylating agent in cellular DNA. Finally, these results suggest non-redundant roles for these two isoforms in mediating repair of DNA crosslinks.

Chromosomal Rearrangements Induced by DNA Double-Strand Breaks.

It has been suggested that introduction of double-strand DNA breaks into human chromosomes can lead to gross chromosomal rearrangements (GCRs) through improper repair mechanisms, and that these GCRs may be leukemogenic through the loss of tumor suppressor genes or the creation of fusion oncogenes. Using the human histiocytic lymphoma cell line U937, we developed a model system in which we were able to introduce a double-strand DNA break (DSB) in vivo at a predetermined location, and subsequently examine the ensuing chromosomal changes flanking the breakage site. In this model, U937 cells are stably transfected with a construct that contains the 18 bp recognition site for a rare cutting endonuclease, I-SceI, whose recognition sequence is not normally present in the human genome. The I-SceI site is placed between a strong constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker by conferring ganciclovir (GCV) sensitivity to the cells. Following transfection of an I-SceI expression vector, double-strand DNA breakage occurs and abrogatesHSV-tk expression, leading to a dramatic increase in GCV resistant cells. We showed that, even in the abscence of GCV selection, the IsceI enzyme was able to cleave its recognition sequence in at least half of the transfected cells. The predominant type of rearrangement detected following the appearance of a double-strand break was an interstitial deletion. These deletions typically displayed uneven recession of free DNA ends flanking the cleavage site, generally resulting in either very short (<10 bp) or very long (>1000) bp deletions extending into the EF1alpha promoter and homogeneous deletions of 100–1700 bp into the HSV-tk gene. Moreover, in many clones, nucleotide microhomologies of 1–6 basepairs, palindromes, or both were detected at the breakpoint junctions, suggesting repair via a non-homologous end joining (NHEJ) mechanism.. We also showed that in 25 % of the recovered clones the DSB was repaired by formation of direct or inverted repeats derived from the breakpoint junction. Several clones showed similarly complex rearrangements, including short nucleotide insertions, inversions and duplications. Finally, approximately 4.5% (7/150) of the clones demonstrated insertion events that involved integration of nucleotide sequences derived from distant genomic regions, including LINE elements, coding regions of endogenous genes and sequences exhibiting known genomic instability. These insertions may have been derived via reverse transcription of mRNA sequences; alternatively the distant genomic regions may have served as templates for the inserted sequences, or genomic DNA fragments may have been directly incorporated into the I-SceI-induced break. A step-by-step modification of this loss of function selection scheme will provide a platform for studying DNA rearrangements induced by faulty dsDNA repair.

A combination of proteomics, principal component analysis and transcriptomics is a powerful tool for the identification of biomarkers for macrophage maturation in the U937 cell line.

The monocyte-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. We have used two-dimensional gel electrophoresis (2-DE), oligonucleotide microarrays and principal component analysis (PCA) to characterize the U937 cell line as a model system for the differentiation of monocytes into macrophages. A total of 226 differentially expressed proteins were found, of which 41 were selected by PCA for identification using matrix-assisted laser desorption/ionization tandem mass spectrometry. Based on the PCA results, three marker proteins were selected for confirmation of differential expression using Western blot and quantitative real time-PCR. The selected marker proteins were: gamma interferon inducible lysosomal thiol reductase, cathepsin D and adipocyte-fatty acid binding protein. All three proved to be good differentiation markers for macrophage maturation of U937 cells as well as peripheral blood-derived macrophages. The transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. These include osteospontin, matrix metalloproteinase 9, and HC-gp39. Our results show that the characteristics of U937 macrophages resemble those of inflammatory (exudate) macrophages, exemplified by the down-regulation of 5' nucleotidase and the up-regulation of leucine aminopeptidase mRNAs. In conclusion, using the powerful combination of transcriptomics, 2-DE and PCA, our results show that U937 cells differentiated by PMA treatment are an excellent model system for monocyte derived macrophage generation from blood.

Monocytes stimulate COX-2 expression in pancreatic cancer cells

COX-2 is an enzyme that modulates human pancreatic cancer (PaCa) cell survival, proliferation, and tumor metastasis. 60–80% of human PaCa express COX-2. However, the regulation of COX-2 expression in PaCa cells is not clear. We hypothesize that inflammatory cell infiltrate—namely monocytes/macrophages found commonly in PaCa—regulates the expression and activity of COX-2. The human histiocytic lymphoma cell line U937 was terminally differentiated with phorbol ester (PMA) and served as a human monocyte/macrophage model. IL-1β levels in the culture medium were measured by ELISA. PaCa cell lines (BxPC-3, Capan-2, HPAF-II) were stimulated with U937 conditioned culture medium (CM). COX-2 protein expression was determined by immunoblotting and prostaglandin E2 (PGE2) production by ELISA. To determine, whether the effect of U937 CM was mediated by IL-1β, we used an IL-1 receptor antagonist (IL-1RA). The effect of recombinant IL-1β on COX-2 expression, PGE2 production, and ERK1/ERK2 phosphorylation in human PaCa cells was analyzed. Since PMA-differentiated U937 cells secreted low levels of IL-1β (15 ± 2 pg/ml), we stimulated differentiated U937 cells with LPS markedly increasing IL-1β secretion (1670 ± 145 pg/ml). CM of U937 cells increased COX-2 protein expression after 24 hours 5.6-fold, 7.7-fold, and 5.3-fold in the BxPC-3, Capan-2, and HPAF-II cells, respectively. Compared to control, U937 CM increased PGE2 production in BxPC-3 (202 ± 8 vs. 516 ± 24 pg/ml, p < 0.01), Capan-2 (19 ± 1 vs. 39 ± 1 pg/ml, p < 0.01), and HPAF-II cells (173 ± 10 vs. 429±33 pg/ml, p<0.01). The effect of U937 CM on COX-2 expression and PGE2 production was only partially inhibited by IL-1RA. Recombinant IL-1β dose- and time dependently increased COX-2 expression, PGE2 production, and ERK1/ERK2 activation in BxPC-3, Capan-2, and HPAF-II cells, an effect which was inhibited by IL-1RA. IL-1β secreted by monocytes stimulates expression of COX-2 in human pancreatic cancer cells. Other cytokines secreted from U937 cells may also activate COX-2 expression. Studies combining immunomodulation with COX-2 inhibition for PaCa treatment may yield significant results.

Overexpression of manganese superoxide dismutase promotes survival in cell lines after doxorubicin treatment.

Overexpression of manganese superoxide dismutase (MnSOD) has been postulated as one possible mechanism of protection from oxidative damage and free radicals. Doxorubicin treatment induces oxygen free radicals, leading to cytotoxicity and myelosuppression. The present study was performed to determine whether over-expression of MnSOD may play a role in resistance to doxorubicin. Retroviral constructs having the human MNSOD gene in the sense orientation and the neomycin phosphotransferase gene (NEOR) as a selectable marker were transduced into the human melanoma cell line A375 and the human histiocytic lymphoma cell line U937. Stably transduced A375 and U937 cells were subjected to 10-100 ng/ml doxorubicin for 24 h and compared with doxorubicin-treated A375 and U937 cells transduced with vector only. A colony forming assay was used to determine cell viability in semi-solid medium. Results demonstrated that wild-type A375 and U937 cells display low levels of endogenous MnSOD mRNA and protein, and are sensitive to doxorubicin treatment. In contrast, A375 and U937 cells transduced with the MNSOD gene consistently demonstrate increased colony formation in the presence of increasing concentrations of doxorubicin. MnSOD-transduced A375 and U937 cells also demonstrate increased MnSOD mRNA and protein levels when compared with wild type or those cells transduced with vector only. These results indicate that overexpression of MnSOD can enhance resistance to doxorubicin treatment.

Apoptosis-inducing effect of a new bisphosphonate, YM529, on various hematopoietic tumor cell lines.

In recent years, it has been reported that bisphosphonates inhibited the cell cycle of myeloma cells to inhibit cell proliferation directly, and it was also reported that bisphosphonates induced apoptosis of myeloma cells in vitro. Recently, YM529 was developed as a new third-generation bisphosphonate. In our experiment, we investigated whether YM529 showed an antitumor effect on hematopoietic tumor cell lines other than myeloma, and we compared YM529 with YM175, which had a relatively more potent antitumor effect than that of existing bisphosphonates. We found that YM529 inhibited cell proliferation in various hematopoietic tumor cell lines (acute promyelocytic leukemia cell line HL-60, chronic myeloid leukemia cell line K562, histiocytic lymphoma cell line U937, lymphoblastic leukemia T cell line Jurkat, acute lymphoblastic leukemia T cell line MOLT-4, lymphoblastic leukemia B cell line CCRF-SB) including myeloma (myeloma cell line HS-Sultan) dose-dependently and time-dependently to a degree equivalent or superior to that in myeloma, and induced apoptosis at a lower concentration as compared with YM175. We confirmed many dead cells as well as apoptosis based on the detection of the nuclei with separate globular structure, the activation of caspase-3, and the decrease in mitochondrial transmembrane potential. Therefore, it is concluded that further utilization of YM529 can be expected against hematopoietic tumor cells in the future.

DNA Microarray Analyses of Genes Elicited by Ultrasound in Human U937 Cells

The gene expression of human histiocytic lymphoma cell line U937 at 6 h after 1 MHz ultrasound treatment in the presence of Ar or N2O gas was examined by DNA microarrays. Of the 9,182 genes analyzed, only the keratin gene was identified as down-regulated in the cells exposed to ultrasound in the presence of N2O where no internal cavitation was observed. In contrast, five up-regulated and two down-regulated genes were identified in the cells exposed to ultrasound in the presence of Ar where internal cavitation was apparently observed. Six changes of the gene expression were confirmed by the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Gene expression of heme oxygenase was augmented by a factor of 6.6 in microarray and by 4.0 by RT-PCR. These results indicate that internal cavitation increased the expression of genes responsive to oxidative stress in sonicated cells but non-inertial cavitation had minimal effects on gene expression.

Constitutive expression of MCP-1 and RANTES in the human histiocytic lymphoma cell line U-937

Constitutive expression of MCP-1 and RANTES in the human histiocytic lymphoma cell line U-937

Apoptosis Induced by Cadmium in Human Lymphoma U937 Cells through Ca2+-calpain and Caspase-Mitochondria- dependent Pathways

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Deltapsi(m)) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-x(L) and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 microm), a voltage-dependent Ca(2+) channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 microm and decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) but not type 2 or type 3 IP(3)R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP(3)R or of calpain inhibitors on the loss of Deltapsi(m) were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca(2+)-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

Apoptosis induced by calcein acetoxymethyl ester in the human histiocytic lymphoma cell line U-937 GTB

Effects of calcein acetoxymethyl ester (calcein/AM) on macromolecular synthesis, mitochondrial membrane potential, and mode of death were studied in U-937 GTB lymphoma cells. This was accomplished by measurements of 14C-labeled thymidine and leucine incorporation, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) and caspase-3 activity measurements, TdT-mediated dUTP nick end labeling (TUNEL) staining, morphology, and a newly developed assay of apoptosis detection, the microculture kinetic assay (MiCK). This assay, based on absorbance measurements of cells, has been reported to reflect morphological changes in apoptosis. At 2.5 μg/mL, rapid inhibition of DNA and protein synthesis resembling that of the known inhibitors, aphidicholin and cycloheximide, was observed. Decreased mitochondrial membrane potential was evident after 1 hr of exposure and was followed by an increase in caspase-3 activity, while at 6 hr 30% of cells appeared positive with TUNEL staining. After 12 hr of exposure, viability was less than 5% as judged by morphological examination. In the MiCK assay, calcein (2.5 μg/mL) gave a rapid rise in absorbance after 3.5 hr of exposure with a peak at 5 hr, indicating maximum extent of apoptosis at that time. This was similar to the pattern generated for etoposide and doxorubicin. The results indicate that calcein, similar to cytotoxic drugs, induces a strong apoptotic response within hours of exposure.

Alginates from wound dressings activate human macrophages to secrete tumour necrosis factor- α

Alginates are used to manufacture a number of wound dressings. Clinical observations indicate that they may initiate or accelerate healing of chronic wounds after treatment of underlying pathology. Wound granulation tissue contains large numbers of macrophages and they are thought to regulate the healing process. As purified alginates have been demonstrated to activate macrophages this study was initiated to determine whether alginates present within wound dressings may interact with wound macrophages. Alginate fibres taken from four commercially available dressings were co-cultured with the human histiocytic lymphoma cell line U937 following its differentiation with PMA. Activation was assessed by measurement of TNF α production. Two of the dressings, Seasorb and Tegagen, had a minimal effect whilst Sorbsan at 1 mg/ml induced 302+19 pg/ml TNF α. This effect was inhibited by polymyxin B indicating that activation was due to endotoxin contamination. Kaltostat induced production of 839+36 pg/ml TNF α. This effect was induced both by polymyxin inhibitable endotoxin and a direct interaction with the alginate fibres. These data indicate that some alginate containing dressings have the potential to activate macrophages within the chronic wound bed and generate a pro-inflammatory signal which may initiate a resolving inflammation characteristic of healing wounds.

Mass spectrometric analysis of arachidonyl-containing phospholipids in human U937 cells.

The human histiocytic lymphoma U937 cell line contains a rich source of the 85 kDa cytosolic phospholipase A2 (cPLA2). DMSO-differentiated U937 cells were used as a model to investigate the free arachidonic acid release, the arachidonate distribution and the phospholipid source of arachidonate upon Ca2+ ionophore stimulation. A combination of several chromatographic and mass spectrometric techniques was employed in this study. The amount of free arachidonic acid (AA) released upon stimulation, the arachidonate content in total lipids and in each of the phospholipid classes were determined by gas chromatography/mass spectrometry (GC/MS). Glycerophosphoethanolamine (GPE) was found to be the major pool of arachidonate in differentiated human U937 cells (55%) and glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) contributed 22 and 8%, respectively. Upon Ca2+ ionophore stimulation, GPE class lost the largest amount of arachidonate, followed by GPC class. GPI class, however, gained a substantial amount of arachidonate. Most of the arachidonate depleted from GPE and GPC was recovered as free AA, some of which was rapidly esterified into GPI species. GC/MS with electron capture negative chemical ionization provided excellent sensitivity for the measurement of arachidonic acid which was derivatized to its pentafluorobenzyl ester. Intact phospholipid molecular species including the arachidonyl-containing phospholipid species were identified using capillary high-performance liquid chromatography/continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS). No specificity was found for releasing free AA among the arachidonyl-containing GPE and GPC species upon Ca2+ ionophore stimulation. CF-LSIMS provided a sensitive and effective means of detecting intact phospholipid species.

Relationships of apoptotic signaling mediated by ceramide and TNF-alpha in U937 cells.

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis. We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the histiocytic lymphoma cell line U937. We found considerable differences between cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-alpha are committed early and irreversibly to the apoptotic pathway and start to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment. The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceramide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the first 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics and the commitment to the apoptotic pathway suggest that, (a) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide elevations, in TNF-alpha treated cells, are a consequence rather than a cause of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.

Tetracycline derivatives induce apoptosis selectively in cultured monocytes and macrophages but not in mesenchymal cells.

Evidence for a non-antibiotic activity displayed by certain tetracycline derivatives is presented. This activity is a selective cytotoxicity toward cells of the monocytic lineage (the human histiocytic lymphoma U937 cell line and the mouse macrophage line RAW264) but not toward various cells of a mesenchymal lineage (including primary ovine articular chondrocytes and meniscal cells, murine calvarial osteoblasts and MG-63 osteosarcoma cells, and primary human neonatal foreskin fibroblasts). Cells were incubated with various chemically modified tetracycline derivatives (CMTs) or doxycycline for 24 hrs at a range of concentrations between zero and 50 micrograms/mL in both serum-containing and serum-free culture conditions. Assessment of cell viability by means of the MTT assay demonstrated a potent dose-dependent cytotoxic effect induced by compound CMT-3 and a less potent effect induced by doxycycline, but no apparent cytotoxic effect in the presence of either CMT-2 or CMT-5. Cytospin preparations analyzed by the labeling of DNA fragments indicated the same trends and suggested that cell death was via an apoptotic mechanism. The cytotoxic potency of these tetracyclines toward cells of the monocytic lineage could be diminished but not abolished by either the presence of 10% fetal calf serum within the culture medium, or pre-treatment with phorbol esters to promote a more macrophage-like phenotype. These data provide evidence that, in addition to well-characterized antibiotic and MMP-inhibitory characteristics, tetracyclines may function by a novel mechanism to induce selective apoptosis.

A Glass Fiber/Diethylaminoethyl Double Filter Binding Assay That Measures Apoptotic Internucleosomal DNA Fragmentation

A filter binding assay that measures internucleosomal DNA fragmentation associated with apoptosis is described. The assay is based on a novel principle that consists of using simultaneously two kinds of glass fiber filters to harvest [3H]thymidine-prelabeled cells following their incubation with inducers of apoptosis. One filter, which is neutral, traps intact chromatin and high-molecular-weight DNA. The other filter, which is positively charged with DEAE active groups, traps low-molecular-weight DNA fragments. DNA fragmentation is quantified by measuring the radioactivity retained by each of the filters. The assay was evaluated with the histiocytic lymphoma cell line U937 and the topoisomerase inhibitors camptothecin, etoposide, and doxorubicin. These agents caused a dose-dependent decrease of radioactivity in the neutral filter and a parallel increase of radioactivity in the DEAE filter. Irradiation-induced single strand breaks and topoisomerase-mediated primary DNA damage were not detected by this method. Consistent with the detection of internucleosomal DNA fragmentation, the effects measured by this assay were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using this assay were validated by observation of DNA ladders on agarose gels and by morphologic examination of apoptotic features. Evaluation of the assay in a mock screen demonstrated that the introduction of the DEAE filter increases the assay sensitivity and eliminates false positives. Thus, this assay may be used in high-throughput screening approaches to discover novel modulators of apoptosis.

H1 and H2 histamine receptors in histiocytic lymphoma cell line U-937

H1 and H2 histamine receptors in histiocytic lymphoma cell line U-937

Inhibitors of Dipeptidyl Peptidase IV (DP IV, CD26) Specifically Suppress Proliferation and Modulate Cytokine Production of Strongly CD26 Expressing U937 Cells

Various studies from different laboratories have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) expressed in T and NK cells is involved in the regulation of DNA synthesis and cytokine production. In this paper, we performed a biochemical and functional characterization of dipeptidyl peptidase IV on the human histiocytic lymphoma cell line U937. Using U937 clones expressing low to high levels of membrane localized CD26, we found that the synthetic reversible inhibitors of DP IV, Lys-[Z(NO 2)]-thiazolidide and Lys-[Z(NO 2)]-piperidide, have different effects on all functions. In U937-H cells that strongly express high levels of CD26, DP IV inhibitors were shown to suppress DNA synthesis and production of IL-1β, but stimulate the secretion of the IL-1 receptor antagonist (IL-1RA) and of TNF-α. In contrast, both inhibitors did not influence the cytokine production and DNA synthesis in U937-L cells exhibiting low level CD26 expression. These data support the hypothesis that CD26 plays a crucial role in proliferation and cytokine production, not only in T cells, but also in other cell systems, and that enzymatic activity is essential for its function.

Effects of DMSO-induced differentiation on arachidonate mobilization in the human histiocytic lymphoma cell line U937: Responsiveness to sub-micromolar calcium ionophore A23187 and phorbol esters

The human histiocytic lymphoma cell line, U937, is a rich source for isolation and purification of the 85 kDa cytosolic phospholipase A 2 (cPLA 2). Recent studies suggest that this enzyme catalyzes the agonist-stimulated release of arachidonate from membrane phospholipids, thereby initiating eicosanoid synthesis. We therefore investigated in situ regulation of phospholipase A 2 activity in intact U937 cells. The results indicate that calcium ionophore A23187 stimulatable release in intact undifferentiated U937 is low and only weakly dose dependent. Dimethyl sulfoxide (DMSO) differentiation of U937 cells results in a dramatic increase of A23187-stimulated arachidonate mobilization. Consistent with the characteristics of cPLA 2 in vitro, A23187-stimulated arachidonate release in differentiated U937 cells is highly specific for arachidonate and is activated by submicromolar A23187 concentrations. Phorbol myristate acetate (PMA) further potentiates arachidonate release in differentiated U937 cells by 4–6-fold over A23187 alone. However, treatment of differentiated U937 cells with PMA alone is an ineffective stimulus for arachidonate release, suggesting that a calcium transient is necessary for in situ arachidonate mobilization. A23187-stimulated arachidonate release increases during distinct temporal phases of differentiation (0–36 h, 84–96 h). By contrast PMA enhancement of the response to A23187 develops early in differentiation, and is complete by 36 h. These results suggest that differentiation-induced alterations in cPLA 2 regulatory elements, such as intracellular free calcium and/or phosphorylation, may regulate mobilization of arachidonate in U937 cells.

Quantitative PCR for detection of femtogram quantities of interleukin-8 mRNA expression.

A quantitative polymerase chain reaction (PCR) assay for mRNA expression of interleukin-8 (IL-8), a neutrophil chemotactant and activator, was developed to examine the expression of this cytokine by colonic mucosa. A synthetic IL-8 RNA deleted in size of native IL-8 mRNA was used as an external control. The synthetic IL-8 RNA was mixed with total RNA from cells and converted to cDNA and amplified by PCR simultaneously. The lower limit of sensitivity for the assay was found to be more than 1 femtogram of IL-8 mRNA. The assay determined IL-8 mRNA expression when the RNA was isolated from either human histiocytic lymphoma cell line U937 cells or human colonic mucosa obtained from colitis patients and healthy controls. The development of the rapid and sensitive assay should provide a means to more fully evaluate the role of this cytokine in diverse disease states with small scale.