sq-23377 and Lung-Neoplasms

Neutrophils play a major role in tumor biology. Among other functions, neutrophils can release extracellular traps (NETs), mesh-like structures of decondensed chromatin fibers, in a process termed NETosis. Originally characterized as an antimicrobial mechanism, NETosis has been described in cancer, but cancer-related predisposition is not clear. In the current study, we investigated the predisposition of circulating neutrophils to release NETs in lung cancer and the impact of G-CSF on this function, comparing circulating neutrophils isolated from cancer patients to the LLC and AB12 mouse models. We find that neutrophils from both healthy donors and cancer patients display high NETotic potential, with 30-60% of cells undergoing NETosis upon PMA stimulation. In contrast, neutrophils isolated from tumor-bearing mice displayed only 4-5% NETotic cells, though significantly higher than naive controls (1-2%). Despite differential mechanisms of activation described, Ionomycin and PMA mainly triggered suicidal rather than vital NETosis. G-CSF secreting tumors did not increase NETotic rates in murine neutrophils, and direct G-CSF stimulation did not promote their NET release. In contrast, human neutrophils strongly responded to G-CSF stimulation resulting also in a higher response to PMA + G-CSF stimulation. Our data show clear differences in NETotic potentials between human and murine neutrophils. We do not find a predisposition of neutrophils to release NETs in lung cancer patients compared to healthy controls, whereas cancer may modulate neutrophils' NETotic potential in mice. G-CSF secreted from tumors differentially affects murine and human NETosis in cancer. These important differences should be considered in future studies of NETosis in cancer.

Topics: Animals; Cell Line, Tumor; Extracellular Traps; Granulocyte Colony-Stimulating Factor; Humans; Ionomycin; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neutrophils; Tetradecanoylphorbol Acetate

We have previously reported that chronic, recurrent and low-dose exposure to asbestos fibers causes a reduction in antitumor immunity. Investigation of natural killer (NK) cells using an in vitro cell line model and comprising in vitro activation using freshly isolated NK cells co-cultured with chrysotile fibers, as well as NK cells derived from asbestos-exposed patients with pleural plaque (PP) or malignant mesothelioma (MM), revealed decreased expression of NK cell activating receptors such as NKG2D, 2B4 and NKp46. An in vitro differentiation and clonal expansion model for CD8+ cytotoxic T lymphocytes (CTLs) showed reduced cytotoxicity with decreased levels of cytotoxic molecules such as granzyme B and perforin, as well as suppressed proliferation of CTLs. Additionally, analysis of T helper cells showed that surface CXCR3, chemokine receptor, and the productive potential of interferon (IFN)γ were reduced following asbestos exposure in an in vitro cell line model and in peripheral CD4+ cells of asbestos-exposed patients. Moreover, experiments revealed that asbestos exposure enhanced regulatory T cell (Treg) function. This study also focused on CXCR3 expression and the Th-17 cell fraction. Following activation with T-cell receptor and co-culture with various concentrations of chrysotile fibers using freshly isolated CD4+ surface CXCR3 positive and negative fractions, the intracellular expression of CXCR3, IFNγ and IL-17 remained unchanged when co-cultured with chrysotile. However, subsequent re-stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin resulted in enhanced IL-17 production and expression, particularly in CD4+ surface CXCR3 positive cells. These results indicated that the balance and polarization between Treg and Th-17 fractions play an important role with respect to the immunological effects of asbestos and the associated reduction in antitumor immunity.

Topics: Asbestos; CD4-Positive T-Lymphocytes; Coculture Techniques; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Interleukin-17; Ionomycin; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Phorbol Esters; Receptors, Antigen, T-Cell; Receptors, CXCR3; T-Lymphocytes, Cytotoxic; Th17 Cells

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.

Topics: ADAM Proteins; Animals; Apoptosis; Calcium; Calpain; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Enzyme Activation; Epithelial Cells; HEK293 Cells; Hepatocyte Growth Factor; Humans; Ionomycin; Lung Neoplasms; Mice; Necrosis; Neoplasm Metastasis; Proto-Oncogene Proteins c-met; RNA Interference; RNA, Small Interfering; Signal Transduction

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apoptosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-gamma release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a "per-cell" basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Bryostatins; Cell Proliferation; Flow Cytometry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; Interleukin-2; Interleukin-7; Interleukins; Ionomycin; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tumor Cells, Cultured

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.

Topics: Actins; Adenocarcinoma; Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Cytochalasin D; Cytoskeleton; Digitonin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Ionomycin; Lung Neoplasms; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Paclitaxel; Thapsigargin; Time Factors

T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12-myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappa B nuclear translocation and diminished I kappa B alpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic I kappa B alpha persisted as a result of alterations in I kappa B kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.

Topics: Adenocarcinoma; Apoptosis; Carcinoma, Non-Small-Cell Lung; CD3 Complex; Humans; I-kappa B Kinase; I-kappa B Proteins; Ionomycin; Jurkat Cells; Lung Neoplasms; NF-kappa B; Protein Serine-Threonine Kinases; T-Lymphocytes; Tetradecanoylphorbol Acetate

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

Topics: Apoptosis; Calcium; Calpain; Carcinoma, Large Cell; Cell Nucleus; Cell Separation; Cells, Cultured; Cytochrome c Group; Cytoplasm; DNA; Enzyme Activation; Flow Cytometry; Humans; Ionomycin; Ionophores; Lung Neoplasms; Mitochondria; Models, Molecular; Oligopeptides; Poly(ADP-ribose) Polymerases; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured

The intracellular pathway of polymeric immunoglobulin receptor (pIgR) is governed by multiple signals that lead to constitutive transcytosis. In addition, in transfected polarized MDCK cells, polymeric immunoglobulin A (pIgA) binding stimulates rabbit pIgR-transcytosis, owing to phospholipase-C gamma 1 activation and increase of intracellular calcium. Transcytosis of rat pIgR across hepatocytes is similarly accelerated by pIgA injection. In contrast we show here that human Madrin-Darby Canine Kidney (pIgR)-transcytosis, in human Calu-3 and human pIgR-transfected MDCK cells, is not promoted by pIgA, as monitored by a continuous apical release of its secreted ectodomain. However, the incubation of cells expressing human or rabbit pIgR with pIgA induces a comparable IP3 production, and pIgR-transcytosis of either species is accelerated by the protein kinase C (PKC)-activator phorbol myristate acetate. Without pIgA, mimicking phospholipase-C activation by combining low concentrations of phorbol myristate acetate with ionomycin, or high concentrations of ionomycin alone, stimulates the rabbit, but not the human, pIgR transcytosis. These data suggest that the species difference in pIgA-induced pIgR-transcytosis does not stem from the defective production of second messengers, but from a different sensitivity of pIgR to intracellular calcium. Our results outline the danger of extrapolating to humans the abundant data obtained from mucosal vaccination of laboratory animals.

Topics: Adenocarcinoma; Animals; Calcium Signaling; Cell Line; DNA, Complementary; Dogs; Enzyme Activation; Humans; Immunoglobulin A; Inositol 1,4,5-Trisphosphate; Ionomycin; Ionophores; Kidney Tubules, Proximal; Lung Neoplasms; Protein Kinase C; Protein Transport; Rabbits; Rats; Receptors, Polymeric Immunoglobulin; Recombinant Fusion Proteins; Second Messenger Systems; Signal Transduction; Species Specificity; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured; Vaccination

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines.. TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry.. The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3(+) leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3(+) TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3(+) TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3(+) TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3(+)CD8(+) component of the TIL synthesized only type-1 cytokines, whereas the CD3(+)CD4(+) component synthesized both type-1 and type-2 cytokines.. These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; CD3 Complex; Cytokines; Gene Expression Regulation; Humans; Interleukin-2; Ionomycin; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Muromonab-CD3; T-Lymphocytes; Tetradecanoylphorbol Acetate

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenocarcinoma; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Glucosamine; Humans; Ionomycin; Leukocyte Elastase; Lung Neoplasms; Mucins; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured

Human small-cell lung carcinoma (SCLC) cells express neuronal-like voltage-operated calcium channels (VOCCs) and release mitogenic hormones such as serotonin (5-HT). Opioid peptides, on the other hand, have been shown to reduce SCLC cell proliferation by an effective autocrine pathway. Here we show that in GLC8 SCLC cells, only delta-opioid receptor subtype mRNA is expressed. Consistently, the selective delta-opioid agonist [D-Pen2-Pen5]-enkephalin (DPDPE), but not mu and kappa agonists, potently and dose-dependently inhibits high-threshold (HVA) VOCCs in these cells. As in peripheral neurons, this modulation is largely voltage-dependent, mediated by pertussis toxin (PTX)-sensitive G-proteins, cAMP-independent, and mainly affecting N-type VOCCs. With the same potency and selectivity, DPDPE also antagonizes the Ca(2+)-dependent release of [3H]serotonin ([3H]5-HT) from GLC8 cells. However, DPDPE inhibits not only the depolarization-induced release, but also the Ca(2+)-dependent secretion induced by thapsigargin or ionomycin. This suggests that besides inhibiting HVA VOCCs, opioids also exert a direct depressive action on the secretory apparatus in GLC8 cells. This latter effect also is mediated by a PTX-sensitive G-protein but, contrary to VOCC inhibition, it can be reversed by elevations of cAMP levels. These results show for the first time that opioids effectively depress both Ca2+ influx and Ca(2+)-dependent hormone release in SCLC cells by using multiple modulatory pathways. It can be speculated that the two mechanisms may contribute to the opioid antimitogenic action on lung neuroendocrine carcinoma cells.

Topics: Analgesics; Base Sequence; Calcium; Calcium Channel Blockers; Calcium Channels; Carcinoma, Small Cell; Cyclic AMP; Electrophysiology; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Ion Channel Gating; Ionomycin; Ionophores; Lung Neoplasms; Membrane Potentials; Molecular Sequence Data; Opioid Peptides; Pertussis Toxin; Potassium Chloride; Receptors, Opioid, delta; Serotonin; Terpenes; Thapsigargin; Tritium; Tumor Cells, Cultured; Virulence Factors, Bordetella

Lymphocytes obtained from tumor-draining lymph nodes (DLN) can have potent in vivo antitumor activity after in vitro activation with bryostatin 1 and ionomycin. However, the presence of visceral metastases in the donor can inhibit the effectiveness of such lymphocytes. In the present study, we tested the ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression in a murine model.. Mice were injected with MCA-105 sarcoma cells in the footpad alone or in the footpad and the tail vein to establish lung metastases. Cyclophosphamide was given i.p. 1 day before harvesting the draining popliteal lymph nodes. For all donor groups, DLN cells were activated with 5 nM bryostatin 1 and 1 microM ionomycin and cultured for 7 days in 20 U/ml IL-2. Activated DLN cells were then adoptively transferred to syngeneic mice with 3-day lung metastases.. The adoptive transfer of DLN cells from mice with footpad tumors only significantly reduced the number of lung metastases compared to untreated mice. However, activated DLN cells obtained from mice with both footpad and lung tumors were significantly less effective. Treatment of similar donor mice with 10 mg/kg cyclophosphamide significantly improved the anti-tumor activity of adoptively transferred cells. This dose of cyclophosphamide did not reduce the number of cells obtained from each lymph node or the expansion of cell numbers in vitro.. These results suggest that the administration of low-dose cyclophosphamide prior to harvesting DLN cells may improve the success of adoptive immunotherapy in cancer patients.

Topics: Animals; Bryostatins; Cyclophosphamide; Female; Immune Tolerance; Immunotherapy, Adoptive; Ionomycin; Lactones; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Macrolides; Mice; Mice, Inbred C57BL; Mitogens; Neoplasm Metastasis; Neoplasm Transplantation; Sarcoma, Experimental

Transcriptional regulation of the human parathyroid hormone-related protein (PTHrP) gene by calcitonin was examined in a lung cancer line (BEN cells). Northern analysis demonstrated that calcitonin caused a rapid 4.5-fold elevation in PTHrP mRNA. Transient transfection of a construct containing 1119 base pairs of the human PTHrP gene 5' to the ATG start site of translation, fused to the CAT reporter sequence, was used to demonstrate a five-fold increase in transcription by calcitonin. Similar increases were also observed when transfected cells were exposed to a number of cAMP agonists including forskolin, as well as isobutyl-methylxanthine. A putative cAMP responsive element (5'-TGACTTCA-3') present within exon 4 was placed upstream of the heterologous SV40 promoter. Expression of this construct was elevated 4.5-fold in response to calcitonin and 7-fold in response to forskolin. Similar responses to calcitonin occurred with a smaller construct (pZMR30) containing 530 bp of sequence upstream of the ATG start site. Thus we postulate that calcitonin acts at least partially via cAMP through this element in exon 4 of the human PTHrP gene.

Topics: 1-Methyl-3-isobutylxanthine; Base Sequence; Bucladesine; Calcitonin; Colforsin; Cyclic AMP; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Proteins; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; RNA, Neoplasm; Second Messenger Systems; Transcription, Genetic; Tumor Cells, Cultured

Small cell lung carcinoma is an aggressive neuroendocrine tumor that secretes several hormones, some of which act as autocrine growth factors. In order to obtain more information on the process of hormone secretion from this tumor, we have studied the role of intracellular free Ca2+ concentrations and voltage-operated calcium channels in the control of [3H]serotonin release from in vitro growing cell lines. We found that the Ca2+ ionophore ionomycin and the Ca(2+)-ATPase antagonist thapsigargin induced a dose-dependent increase of intracellular Ca2+ and a parallel enhancement of [3H]serotonin release. KCl-induced depolarization also stimulated a dose- and Ca(2+)-dependent [3H]serotonin release that in the GLC8 cell line was effectively inhibited by Ca2+ channel antagonists (Cd2+, nitrendipine, verapamil, omega-conotoxin GVIA, and omega-agatoxin IVA) and potentiated by the Ca2+ channel agonist BayK8644. Autoantibodies against Ca2+ channels present in the sera of Lambert-Eaton myasthenic patients antagonized KCl- but not ionomycin-induced [3H]serotonin release. Polymerase chain reaction analysis indicated that GLC8 cells express L-, N-, and P-type neuronal Ca2+ channel alpha 1 subunits, together with two types of Ca2+ channel beta subunits. The presence of three functionally distinct high threshold Ca2+ channels was also revealed by patch clamp experiments; high threshold Ca2+ channels were identified as dihydropyridine-sensitive (L-type), omega-conotoxin GVIA-sensitive (N-type), and omega-agatoxin IVA-sensitive (P-type). Our data demonstrate that [3H]serotonin is released by small cell lung carcinoma cells in a Ca(2+)-dependent manner and that depolarization-induced [3H]serotonin release is mediated by Ca2+ influx through distinct, neuron-like, Ca2+ channel subtypes.

Topics: Autoantibodies; Base Sequence; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Carcinoma, Small Cell; Dihydropyridines; DNA Primers; Fura-2; Humans; Ionomycin; Lambert-Eaton Myasthenic Syndrome; Lung Neoplasms; Molecular Sequence Data; omega-Conotoxin GVIA; Peptides; Potassium Chloride; Serotonin; Terpenes; Thapsigargin; Tritium; Tumor Cells, Cultured

Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Bryostatins; Female; Immunoglobulin G; Immunotherapy, Adoptive; Interferon-gamma; Ionomycin; Lactones; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Macrolides; Methylcholanthrene; Mice; Mice, Inbred C57BL; Sarcoma, Experimental; T-Lymphocytes; Tumor Necrosis Factor-alpha

Simultaneous protein kinase C stimulation with phorbol 12,13-dibutyrate (PDBU) and calcium mobilization with ionomycin (Io) trigger cellular events leading to expression of proliferation-associated genes in human lymphocytes. The effect of a 16-hr exposure to PDBU and Io on the growth and cytotoxic activity of murine splenocytes and tumor-infiltrating lymphocytes (TIL) cocultured with interleukin-2 (IL-2) was studied. PDBU + Io increased the number of cytotoxic effector cells that could be generated in lymphokine-activated killer cells (LAK) cultures (40-fold) and to a lesser extent in TIL (10-fold). DNA synthesis of TIL increased significantly when exposed to PDBU + Io. Also, TIL stimulated with PDBU + Io demonstrated in vitro tumor-specific lytic activity significantly greater than that of control TIL (500 lytic units vs 50). The cell-surface phenotype of TIL treated with PDBU + Io was identical to that of control TIL (> 95% CD-3+, CD-4-, CD-8+). Results of adoptive immunotherapy using splenocytes stimulated by PDBU + Io and cultured in IL-2 were identical to those achieved when standard LAK cultures were used. However, treatment using TIL stimulated by PDBU + Io led to a significant reduction in the number of pulmonary nodules compared to standard TIL. In addition, PDBU + Io-stimulated TIL maintained significant in vivo activity without the need for systemic IL-2 administration. Pharmacologic manipulation of cytotoxic precursor cells is a useful strategy for improving the generation of murine cytotoxic effector cells. By using PDBU + Io, cytotoxic lymphocytes could be generated with improved in vitro and in vivo activity.

Topics: Animals; Cell Division; Cytotoxicity, Immunologic; Female; Immunotherapy, Adoptive; Ionomycin; Killer Cells, Lymphokine-Activated; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Transplantation; Phorbol 12,13-Dibutyrate

To define the role of neuropeptides in lung cancer biology, we evaluated the effect of seven peptide classes on signal transduction and growth in human lung and breast cancer cell lines. Flow cytometric methods were used to quantitate the calcium response in individual cells produced by these peptides alone or in combination. The effects on growth were assessed by [3H]thymidine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and soft agarose colony assays. All lung cancer cells demonstrated calcium responses to one or more peptides with classic small cell lines displaying the greatest responsiveness, followed by variant small cell lines and non-small cell lines. Breast cancer cell lines demonstrated little or no response. There was great variability in the magnitude of calcium response and pattern of response between lung cancer cell lines to individual neuropeptides. Bradykinin was the most potent peptide and produced responses in the highest fraction of lung cancer cell lines. Combinations of peptides produced greater intracellular calcium release than the single peptides, although in less than a quantitatively additive manner. Each peptide produced a refractory period which was peptide class specific. The growth stimulating effects of these neuropeptides were absent or small in magnitude and did not correlate with calcium signal transduction. These results imply that lung cancer cells display a wide sensitivity to neuropeptides but in a very heterogeneous manner. Knowledge of this heterogeneity should be incorporated into the design of antitumor strategies based on this autocrine pathway.

Topics: Bombesin; Bradykinin; Calcium; Carcinoma, Small Cell; Cell Division; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance; Drug Synergism; Humans; Ionomycin; Lung Neoplasms; Neuropeptides; Signal Transduction; Time Factors; Tumor Cells, Cultured