Thymosin alpha 1 down-regulates the growth of human non-small cell lung cancer cells in vitro and in vivo.

The effect of thymosin alpha 1 (THN alpha 1) and its NH2-terminal fragment (THN1-14) and COOH-terminal fragment (THN15-28) on non-small cell lung cancer (NSCLC) growth was evaluated. Using an anti-THN alpha 1 antibody, receptors were identified on NSCLC cells that were pretreated with 10(-6) M THN alpha 1. [3H]Arachidonic acid was readily taken up by NSCLC cells and THN alpha 1 significantly increased the rate of arachidonic acid release. THN1-15 slightly stimulated but THN15-28 and THN beta 4 did not alter arachidonic acid release from NCI-H1299 cells. In clonogenic growth assays in vitro, THN alpha 1 (10(-6) M) significantly decreased NSCLC colony number whereas THN1-14, THN15-28, and THN beta 4 were less potent. Using growth assays in vivo, THN alpha 1 (10 micrograms s.c./day) but not THN1-14, THN15-28, or THN beta 4 inhibited significantly NSCLC xenograft formation in nude mice. These data suggest that biologically active THN alpha 1 receptors are present on NSCLC cells and that native THN alpha 1 inhibits the growth of human NSCLC.

Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells.

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.

Neuromedin B stimulates arachidonic acid release, c-fos gene expression, and the growth of C6 glioma cells.

The effects of neuromedin B (NMB) on C6 glioma cells were investigated. NMB bound with high affinity (IC50 = 1 nM) to C6 cells whereas BN and GRP were less potent (IC50 = 40 and 100 nM). NMB (1 nM) elevated cytosolic Ca2+ in individual C6 cells and the increase in cytosolic Ca2+ was reversed by 1 microM [D-Arg1, D-Pro2,D-Trp7.9,Leu11]substance P [APTTL]SP, a broad spectrum antagonist. NMB stimulated [3H]arachidonic acid release from C6 cells and the increase in [3H]arachidonic acid release was reversed [APTTL]SP. NMB increased transiently c-fos gene expression in C6 cells. NMB increased the number of C6 colonies in soft agar and the increase in growth caused by NMB was reversed by [APTTL]SP. These data suggest that NMB receptors may regulate the proliferation of C6 cells.

Corticotropin-releasing factor stimulates cyclic AMP, arachidonic acid release, and growth of lung cancer cells.

The effects of corticotropin-releasing factor (CRF) on human lung cancer cell lines was investigated. Corticotropin-releasing factor increased the cAMP levels in a dose-dependent manner; CRF (100 nM) elevated the cAMP levels approximately eleven-fold using NCI-H345 cells and increased the gastrin-releasing peptide (GRP) secretion rate by approximately 70%. Similarly, sauvagine, a structural analogue of CRF, elevated the cAMP levels with a half-maximal effective dose (ED50) of 20 nM. The increase in cAMP caused by CRF and sauvagine was reversed by alpha-helical CRF(9-41). Corticotropin-releasing factor had no effect on cytosolic calcium but stimulated [3H]arachidonic acid release from NCI-H1299 cells with an ED50 of 30 nM. The increase in [3H]arachidonic acid release caused by 100 nM CRF was significantly reversed by 1 or 10 microM alpha-helical CRF(9-41). Also, CRF stimulated the clonal growth of NCI-H345 and H720 cells and the growth increase caused by CRF was reversed by alpha-helical CRF(9-41). These data suggest that CRF may be a regulatory peptide in lung cancer.

IL-1 and anti-inflammatory drugs modulate A beta cytotoxicity in PC12 cells.

We studied the effect of the inflammatory cytokine interleukin-1 beta (IL-1) and several anti-inflammatory drugs on amyloid beta-peptide (A beta) cytotoxicity. Incubation of PC12 cells with 10(-6) M A beta 1-42 for 24 h reduced viabilty to 36%; coincubation with 10(-7) M IL-1 further reduced viability to 13% of baseline. With preincubation of PC12 cells with the drugs indomethacin, dexamethasone and chloroquine, reduction in viability was limited to 51%, 48% and 44%, respectively, compared to 32% with A beta alone. These experiments support further study of anti-inflammatory therapy in Alzheimer's disease.

Interleukin-1 potentiation of beta-endorphin secretion and the dynamics of interleukin-1 internalization in pituitary cells.

1. This study demonstrates that human recombinant interleukin-1 (IL-1) stimulates beta-endorphin release and potentiates the secretion of beta-endorphin in both a mouse anterior pituitary cell line AtT-20 and rat pituitary cell cultures. 2. In pituitary cell cultures, prolonged treatment with phorbol ester had no effect on IL-1-induced beta-endorphin release, but abolished the potentiating effects of IL-1 on vasopressin-induced beta-endorphin secretion. 3. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in normal pituitary cell cultures following depletion of protein kinase C. 4. The late IL-1-induced secretion of beta-endorphin does not require the continuous presence of the cytokine. 5. Incubation of monolayers with 125I-IL-1 alpha (10(-9) M) at 8 degrees C and then at 37 degrees C for various times revealed that IL-1 alpha was internalized. There was a progressive increase in the ratio of cytoplasmic to cell-surface-associated 125I-IL-1 alpha. 6. These results indicate that the IL-1-induced beta-endorphin release and its potentiation of beta-endorphin secretion involves internalization of this cytokine, perhaps via cell surface IL-1 receptors.

Separation and purification of peptides by high-speed counter-current chromatography.

Preparative separations of peptides have been accomplished using high-speed counter-current chromatography. This has been made possible by the use of a particular solvent system that does not exhibit solvent carryover at high speed flow and centrifugation conditions. The solvent system composed of tert.-butyl methyl ether, n-butanol, acetonitrile and aqueous trifluoroacetic acid which can be adjusted in volume ratios and percent acid is employed in two instruments for counter-current chromatography. The cross-axis coil planet centrifuge was used for separation of various dipeptides. Superior resolution was obtained with the multi-layer coil planet centrifuge in the separation of six dipeptides and in the preparative purification of two synthetic peptides.

A fatal case of trifluoperazine poisoning.

A fatal case of trifluoperazine poisoning is reported. Case history, pathological findings, and the concentrations of trifluoperazine and its metabolites in blood, liver, and kidney are presented.

Mechanism of amyloid beta-peptide (1-42) toxicity in PC12 cells.

Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of Alzheimer's disease. There is evidence that A beta is neurotoxic. The role of signal transduction pathways on A beta-induced toxicity in PC12 cells was investigated. Our results revealed that A beta-induced arachidonic acid was released in a time-dependent manner. Inhibitors of cyclooxygenase (1 microM indomethacin) and lipooxygenase (100 microM nordihydroguairetic acid) protected PC12 cells against A beta-induced toxicity. These data suggest that A beta toxicity is mediated by activation of the arachidonic acid cascade. Furthermore, protein kinase C activators (phorbol ester and 1-oleyl-2-acetyl-glycerol) and tacrine reversed A beta-induced toxicity. These results suggest that A beta toxicity can be modulated by manipulating signal transduction pathways and may provide the basis for novel therapeutic interventions.

Interleukin 1 potentiates the secretion of beta-endorphin induced by secretagogues in a mouse pituitary cell line (AtT-20).

Previous work has shown that corticotropin releasing factor, vasoactive intestinal peptide, phorbol ester, and forskolin cause the secretion of adrenocorticotropic hormone and beta-endorphin from the AtT-20 mouse pituitary cell line. Human recombinant interleukin 1 alpha and 1 beta also stimulated adrenocorticotropic hormone and beta-endorphin secretion from AtT-20 cells in a time- and dose-related manner. The effect appeared only after pretreatment with interleukin 1 (IL-1) for at least 18 hr and was maximum at 24 hr. After pretreatment of the cells over a period of time with IL-1, the secretion induced by corticotropin releasing factor and vasoactive intestinal peptide was increased in more than an additive manner. The enhancement of corticotropin releasing factor-induced beta-endorphin release produced by IL-1 was apparent after 12 hr and reached a maximum at 24 hr. IL-1 did not affect forskolin-induced cAMP generation but enhanced the effect of forskolin on beta-endorphin secretion. This suggests that IL-1 does not induce adenylate cyclase and that forskolin causes the secretion of beta-endorphin by a mechanism independent of cAMP. IL-1 enhanced phorbol ester-induced beta-endorphin secretion. After prolonged treatment with phorbol ester (an activator of protein kinase C), the secretion induced by phorbol ester was abolished as well as the enhancement induced by IL-1. However, prolonged treatment with phorbol ester had no effect on IL-1-induced beta-endorphin secretion. These observations suggest that IL-1 enhances peptide-generated secretion of beta-endorphin by inducing protein kinase C.

Interleukin 1 potentiates agonist-induced secretion of beta-endorphin in anterior pituitary cells.

Interleukin 1 (IL-1) has been shown to potentiate the release of beta-endorphin induced by secretagogues, including corticotropin releasing factor (CRF) and phorbol ester (TPA), in the mouse AtT-20 pituitary tumor cell line (Fagarasan et al., PNAS, 1989, 86, 2070-2073). In cultured rat anterior pituitary cells, pretreatment with IL-1 caused only a small increase in beta-endorphin release but significantly potentiated CRF-and vasopressin-stimulated beta-endorphin secretion. Vasopressin stimulates the secretion of beta-endorphin in normal pituitary cells but not in AtT-20 cells. However, treatment of AtT-20 cells with IL-1 induced the expression of vasopressin-mediated beta-endorphin release; this effect of IL-1 was reduced after depletion of protein kinase C by prolonged treatment with TPA. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in AtT-20 cells after depletion of protein kinase C, and after treatment with staurosporine. These findings indicate that treatment with IL-1 amplifies receptor-mediated responses to the major physiological secretagogues in normal corticotrophs, and initiates a secretory response to vasopressin in AtT-20 cells.

GRP receptors are present in non small cell lung cancer cells.

Previously, GRP receptors were characterized in small cell lung cancer cells and here non-small cell lung cancer (NSCLC) cells were investigated: (125I-Tyr4) bombesin (BN) or 125I-GRP bound with high affinity to NCI-H720 (lung carcinoid) and NCI-H1299 (large cell carcinoma) cells. Binding was specific, time dependent, and saturable. Specific (125I-Tyr4)BN binding to NCI-H1299 cells was inhibited with high affinity by GRP, BN, GRP14-27, (D-Phe6)BN6-13methyl ester, moderate affinity by NMB, and low affinity by GRP1-16. BN (10 nM) transiently elevated cytosolic calcium in a dose dependent manner. BN caused translocation of protein kinase C from the cytosol to the membrane and the translocation caused by BN was reversed by (D-Phe6)BN6-13methylester. BN stimulated arachidonic acid release and the increase caused by BN was reversed by (D-Phe6)BN6-13methylester. Using a clonogenic assay, BN stimulated the growth of NCI-H720 cells, and the number of colonies was reduced using (D-Phe6)BN6-13methylester. These data suggest that GRP receptors that are present in lung carcinoid and NSCLC cells may regulate proliferation.

Interleukin 1 induces beta-endorphin secretion via Fos and Jun in AtT-20 pituitary cells.

Previous work had shown that interleukin 1 (IL-1), after a long period of treatment, stimulates beta-endorphin release and potentiates the effects of secretagogues in AtT-20 cells, a mouse anterior pituitary cell line. Treatment of AtT-20 cells with IL-1 induced a transient and early stimulation of mRNA expression by both immediate-early protooncogenes Fos and Jun (mouse c-fos and c-jun). The effect appeared within 30 min, and returned to basal levels after 2 hr. Desensitization of protein kinase C by phorbol ester pretreatment had no effect on the ability of IL-1 to induce Fos and Jun mRNA expression. Somatostatin, an inhibitor of cAMP and beta-endorphin secretion, did not reduce the IL-1 effect on Fos and Jun mRNA expression. Addition to AtT-20 cells of antisense oligonucleotides to Fos and Jun abolished the secretion induced by IL-1. These results indicate that immediate-early signals Fos and Jun are involved in IL-1-induced beta-endorphin secretion in AtT-20 cells.

Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of beta-endorphin in a mouse pituitary cell line.

Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates beta-endorphin release and potentiates the secretion induced by many secretagogues. Desensitization of protein kinase C (PKC) by pretreatment with phorbol ester [phorbol 12-tetradecanoate 13-acetate (TPA)] for 8 hr abolished the secretion induced by TPA as well as the enhancement of TPA-induced beta-endorphin release produced by IL-1. Desensitization of PKC only partly abolished the potentiating effects of IL-1 on corticotropin-releasing factor-induced beta-endorphin secretion. In contrast, IL-1-induced beta-endorphin release was independent of PKC. We observed that treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-, 20-, and 60-kDa proteins within minutes, presumably by early activation of protein kinases. Prolonged treatment with TPA, which was shown to desensitize an 87-kDa protein (a substrate for PKC), had no effect on IL-1-induced phosphorylation of 20-, 60-, and 87-kDa proteins, indicating that the phosphorylation of these proteins does not involve PKC. IL-1 does not generate cAMP in AtT-20 cells, suggesting that a cAMP-dependent protein kinase is also not involved. Prolonged treatment with IL-1 abolishes the capacity of cytokine to induce the phosphorylation of 20- and 60-kDa proteins. The presence of IL-1 was required initially only for a short time to induce late secretion in AtT-20 cells. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of beta-endorphin.