Cycloheximide reduces PGD2 or delta 12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD2 or delta 12-PGJ2 (a biologically active metabolite of PGD2), we examined the effect of various compounds on PGD2 or delta 12-PGJ2 cytotoxicity, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD2 cytotoxicity on NCG cells. When delta 12-PGJ2 was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and alpha-amanitin did not affect PGD2 or delta 12-PGJ2 cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD2 or delta 12-PGJ2 exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD2 or delta 12-PGJ2.     

Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.     

Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: correlation with prostaglandin E2-induced growth inhibition

When L-1210 murine leukemia cells were incubated with 60 microM PGE2 in culture medium containing fetal calf serum for various time, cell proliferation was inhibited dependent on incubation time. However, when the medium containing PGE2 was changed every 6 h during 24 h exposure to cells, growth inhibition became much weaker. Moreover, when the medium containing PGE2 was aged by preincubating without cells at 37 degrees C, growth inhibitory effect of the medium was enhanced with preincubation time, suggesting that active growth inhibitory compound(s) accumulated during preincubation. In culture medium containing fetal calf serum, PGE2 degraded time-dependently and the major product was identified as PGA2 by HPLC. Furthermore, when cells were incubated with the medium containing 60 microM[3H]PGE2 or the same medium aged by preincubation, we observed that the radioactivity was taken up by the cells time-dependently, and identified the incorporated radioactivity as PGA2. This uptake was closely correlated with decrease in viable cell number during incubation. These results suggested that growth inhibitory effect of PGE2 was due to the metabolic dehydration of PGE2 to PGA2, and PGA2, after taken up by cells, exerted cell growth inhibition.     

Delta 12-prostaglandin J2, a plasma metabolite of prostaglandin D2, causes eosinophil mobilization from the bone marrow and primes eosinophils for chemotaxis

PGD(2), a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD(2) is rapidly transformed into its major metabolite delta(12)-PGJ(2), the effect of which on eosinophil migration has not yet been characterized. In this study we found that delta(12)-PGJ(2) was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD(2), PGJ(2), or 15-deoxy-delta(12,14)-PGJ(2). Moreover, pretreatment of eosinophils with delta(12)-PGJ(2) markedly enhanced the chemotactic response to eotaxin, and in this respect delta(12)-PGJ(2) was more effective than PGD(2). delta(12)-PGJ(2)-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that delta(12)-PGJ(2) signaled through the same receptor, CRTH2, as PGD(2). Finally, delta(12)-PGJ(2) was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that delta(12)-PGJ(2) is present in the systemic circulation at relevant levels, a role for this PGD(2) metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E2 (PGE2) on fibroblast proliferation was examined. The presence of PGE2 for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [3H]thymidine occurred at concentrations greater than 10(-7) M. The inhibitory effect of PGE2 was less potent in exponentially growing cells and was not the result of conversion of PGE2 to PGA2 during incubation in growth medium. The G1 phase was determined to be 12-14 h in untreated cultures. The extent of growth inhibition by PGE2 was similar with addition of PGE2 at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE2-induced growth inhibition. Short-term exposure to PGE2 (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE2 affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE2 for 24 h, indicated that release from PGE2 exposure is associated with prolongation of the G1 phase of the cell cycle.     

Delta 12-prostaglandin J2 mimics heat shock in inducing cell cycle arrest at G1 phase

Using a human neuroblastoma cell line GOTO, the effects of delta 12-prostaglandin (PG) J2 on the modulation of cell cycle progression and protein synthesis were examined in comparison with those caused by heat shock (HS). delta 12-PGJ2 induced G1 arrest, the peak of which was obtained at 24 h and continued for 72 h. HS was found to induce G1 arrest earlier than delta 12-PGJ2. Furthermore, sequential HS could maintain G1 arrest. delta 12-PGJ2 induced the synthesis of several heat shock proteins (HSPs) in a manner similar to HS. Using immunoblot analysis, HSP72 was detected prior to inducing G1 arrest and accumulated during the subsequent 72h. The content of HSP72 induced by HS also correlated well with the induction, release, and maintenance of G1 arrest. In addition, both delta 12-PGJ2 and HS induced HSP72 mRNA and simultaneously suppressed N-myc mRNA expression. These results suggest that delta 12-PGJ2 and HS regulate cell cycle progression of GOTO cells via similar mechanisms.     

Selective synthesis and retention of 66k protein in a human neuroblastoma cell line (NCG) treated with a cytotoxic dosage of delta 12-prostaglandin J2

delta 12-prostaglandin(PG)J2 (7.5 micrograms/ml) significantly inhibited protein synthesis and cell growth in a human neuroblastoma cell line (NCG), decreasing these factors by 31.5% and 78.2% of the control values, respectively. Two protein synthesis inhibitors, cycloheximide (CHM) and emetine, exhibited a dose-dependent protective effect for neuroblastoma cells against delta 12-PGJ2 cytotoxicity. At a concentration of 15 micrograms/ml CHM, the number of viable cells increased from 21.8% to 36.7% of the control value (p less than 0.01). The sodium dodecyl sulfate-polyacrylamide gel analysis of [35S]methionine-incorporated proteins revealed an increased synthesis of 86k, 70k and 66k proteins in the delta 12-PGJ2-treated NCG cells under the condition that delta 12-PGJ2 exerts cytotoxicity. Of these proteins, the amount of 66k protein was particularly increased in cell cytosol; however, its synthesis did not occur when CHM prohibited the delta 12-PGJ2 cytotoxic effect. When emetine was used instead of CHM, similar results were obtained. These results strongly suggest that the 66k protein plays a critical role in the delta 12-PGJ2 cytotoxicity.     

Prostaglandin J2 and its metabolites promote neurite outgrowth induced by nerve growth factor in PC12 cells

Although A- and J-type prostaglandins (PG's) arrest the cell cycle at the G1 phase in vitro and suppress tumor growth in vivo, their effects on neuronal cells have not so far been clarified. Here, we found promotion of neurite outgrowth as a novel biological function of PGJ's. In PC12h cells, PGJ's (PGJ2, Delta12-PGJ2 and 15-deoxy-Delta12,14-PGJ2) promoted neurite outgrowth in the presence of nerve growth factor (NGF), whereas they themselves did not show such a promotion. The potency of promoting neurite outgrowth was PGJ2 < Delta12-PGJ2 < 15-deoxy-Delta12,14-PGJ2. However, troglitazone, an activator of peroxisome proliferator-activated receptorgamma (PPARgamma), and other PG's including PGA1, PGA2 and PGD2 did not promote neurite outgrowth. These results suggest that PGJ's promote neurite outgrowth independently of PPARgamma activation.     

Synergistic antiproliferative effect of delta 12-prostaglandin J2 (delta 12-PGJ2) and hyperthermia on human esophageal cancer cell lines

delta 12-PGJ2, one of the cyclopentenone prostaglandins and the ultimate metabolite of prostaglandin D2, has been reported to have potent antiproliferative activity on various tumor cells in vitro and in vivo. In this study, the combined effect of delta 12-PGJ2 and hyperthermia on six established cell lines of human esophageal carcinoma (SGF series) was analyzed by an in vitro assay, and the degree of apoptosis induced by this combination was examined to clarify the mechanism of supra-additive effects. In five SGF cell lines, except SGF-7 cells, combination therapy with delta 12-PGJ2 and hyperthermia showed synergistic antiproliferative effects. The supra-additive combined effect of delta 12-PGJ2 and hyperthermia on esophageal cancer cells is attributed to the synergistic induction of apoptosis. delta 12-PGJ2 induced G1 accumulation and apoptosis was induced by delta 12-PGJ2 from G1 phase. Hyperthermia induced G1 accumulation and apoptosis was induced by hyperthermia during all cell phases. Both augmented G1 arrest followed by G1 phase-selective induction of apoptosis and increased apoptotic induction without cell-cycle specificity are responsible for the synergism of combined treatment with delta 12-PGJ2 and hyperthermia.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression.

The effects of prostaglandins (PGs) A and J, which are anti-tumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated. Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and delta 12-PGJ2 in similar dose-dependent fashions. The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and delta 12-PGJ2. PGs were effective even if they were added immediately before entry into S phase. Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G1/S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea. Levels of c-myc mRNA formed two peaks during the G1 phase, at 1-2 h and at 8-12 h. The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18-24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase. The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells. We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G1/S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated with PGs.     

Induction of heme oxygenase by delta 12-prostaglandin J2 in porcine aortic endothelial cells.

delta 12-Prostaglandin (PG)J2 stimulated the synthesis of a 31,000-dalton protein (termed p31) and the induction of cellular heme oxygenase activity in porcine aortic endothelial cells. A good correlation was observed between the time courses and dose dependencies of the induction of p31 synthesis and that of heme oxygenase activity by delta 12-PGJ2. Hemin, a known inducer of heme oxygenase, also induced p31 synthesis as well as heme oxygenase activity in the cells. On two-dimensional gel electrophoresis, p31 induced by delta 12-PGJ2 exhibited an isoelectric point of 5.4, which coincided exactly with that induced by hemin. These results indicate that the p31 induced by delta 12-PGJ2 in porcine aortic endothelial cells is heme oxygenase.     

Induction of 68,000-dalton heat shock proteins by cyclopentenone prostaglandins. Its association with prostaglandin-induced G1 block in cell cycle progression

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.     

Stimulation of NGF expression and secretion in 3T3-L1 adipocytes by prostaglandins PGD2, PGJ2, and Delta12-PGJ2

Nerve growth factor (NGF) has recently been shown to be secreted from white adipocytes, its production being strongly stimulated by the proinflammatory cytokine tumor necrosis factor-alpha. In this study, we have examined whether a series of prostaglandins and other inflammation-related factors also stimulate NGF expression and secretion by adipocytes, using 3T3-L1 cells. Although interleukin (IL)-1beta, IL-10, and IL-18 each induced a small decrease in NGF mRNA level in 3T3-L1 adipocytes, there was no significant effect of these cytokines on NGF secretion. A small reduction in NGF expression and/or secretion was also observed with adiponectin and prostaglandins PGE(2), PGF(2alpha), and PGI(2). In marked contrast, prostaglandin PGD(2) induced a major, dose-dependent increase (up to 20- to 40-fold) in NGF expression and secretion. The PGD(2) metabolites, PGJ(2) and Delta(12)-PGJ(2), also induced major increases (up to 30-fold) in NGF production. A further metabolite of PGJ(2), 15-deoxy-Delta(12,14)-PGJ(2), a peroxisome proliferator-activated receptor-gamma agonist, led paradoxically to a small increase in NGF mRNA level but a fall in NGF secretion. Both PGD(2) and PGJ(2) induced significant increases in NGF gene expression by 4 h after their addition. It is concluded that PGD(2) and the J series prostaglandins, PGJ(2) and Delta(12)-PGJ(2), can play a significant role in the regulation of NGF production by white adipocytes. These results provide support for the view that NGF is an important inflammatory response protein, as well as a target-derived neurotrophin, in white adipose tissue.     

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.     

Mutagenic effects of brief exposure to bromodeoxyuridine on mouse FM3A cells

Abstract. The time- and dose-dependency of the mutagenic effects of bromodeoxyuridine (BrdU), a thymidine analogue used for cell kinetics studies in vivo and in vitro , were investigated in FM3A cells. Cells incubated with 50–1000 fin BrdU for 72 h showed some inhibition of growth. Cells cultured in BrdU-free medium for 3 d after a 30 min or 2 h exposure to BrdU showed no growth inhibition, while those previously exposed for 24 h to BrdU showed retarded growth. After a 30 min exposure, 60% of cells were labelled with BrdU; after 2 h 70%; and after 24 h almost 100%. After incubation in BrdU-free medium for 3 d (the time required for this cell line to express mutation), cells previously treated for 30 min or 2 h showed reduced BrdU positivity, whereas almost 100% of those treated for 24 h remained BrdU positive. The mutation rate, determined by the number of colonies resistant to ouabain (2 mM) and 6-thioguanine (10 μ) 3 d after exposure to BrdU, was not affected by a 30 min treatment with up to 1000 μ BrdU. Cells treated for 1 or 2 h showed increased resistance to ouabain after exposure to BrdU at concentrations above 100 μM; cells treated for 12 or 24 h showed an increased mutation rate at BrdU concentrations above 50 μM… The number of colonies resistant to 6-thioguanine did not increase in cells treated with BrdU at concentrations up to 1000 μM for 1, 12 or 24 h. We cannot conclude with certainty that brief exposure to BrdU does not modulate DNA to the point of mutation. This study may serve as a guideline for limiting the dose and time of exposure to BrdU for cell kinetics studies in vivo and in vitro.     

Differential effects of serum constituents on apoptosis induced by the cyclopentenone prostaglandin 15-deoxy-delta12,14-prostaglandin J2 in WISH epithelial cells

Cyclopentenone prostaglandins, delta12-PGJ2 and 15d-PGJ2, have potent anti-tumour and anti-inflammatory activities, and have been shown to induce apoptosis in amnion-derived WISH cells. In this study, we have investigated the protective effects of serum and its constituents (growth factors and albumin) on delta12-PGJ2 and 15d-PGJ2-induced apoptosis in WISH cells. Serum (0.5% w/v) was protective against both delta12-PGJ2 and 15d-PGJ2-induced apoptosis. This was not due to the presence of serum-derived growth factors (EGF, IGF-1 and IGF-2), since they had no significant effect on 15d-PGJ2-induced cell death. In contrast, IGF-1 partially inhibited etoposide-induced apoptosis, confirming the presence of a functional IGF-1 receptor signalling system. Albumin was identified as the key survival factor in serum, since albumin and delipidated albumin exhibited the same level of protection from 15d-PGJ2-induced apoptosis as serum itself. The potential for serum albumin to regulate the bioactivity of cyclopentenone PGs may be of considerable importance in pathological conditions where roles for cyclopentenone PGs have been identified.