Natural mineral fibers: conducting inhalation toxicology studies-part B: development of a nose-only exposure system for repeat-exposure in vivo study of Libby amphibole aerosol.

BACKGROUND: Exposure to asbestos is associated with malignant and nonmalignant respiratory disease. To strengthen the scientific basis for risk assessment on fibers, the National Institute of Environmental Health Sciences (NIEHS) has initiated a series of studies to address fundamental questions on the toxicology of naturally occurring asbestos and related mineral fibers after inhalation exposure. A prototype nose-only exposure system was previously developed and validated. The prototype system was expanded to a large-scale exposure system in this study for conducting subsequent in vivo rodent inhalation studies of Libby amphibole (LA) 2007, selected as a model fiber. RESULTS: The exposure system consisting of six exposure carousels was able to independently deliver stable LA 2007 aerosol to individual carousels at target concentrations of 0 (control group), 0.1, 0.3, 1, 3, or 10 mg/m3. A single aerosol generator was used to provide aerosol to all carousels to ensure that exposure atmospheres were chemically and physically similar, with aerosol concentration as the only major variable among the carousels. Transmission electron microscopy (TEM) coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis of aerosol samples collected at the exposure ports indicated the fiber dimensions, chemical composition, and mineralogy were equivalent across exposure carousels and were comparable to the bulk LA 2007 material. CONCLUSION: The exposure system developed is ready for use in conducting nose-only inhalation toxicity studies of LA 2007 in rats. The exposure system is anticipated to have applicability for the inhalation toxicity evaluation of other natural mineral fibers of concern.

Natural mineral fibers: conducting inhalation toxicology studies - part A: Libby Amphibole aerosol generation and characterization method development.

BACKGROUND: Asbestos has been classified as a human carcinogen, and exposure may increase the risk of diseases associated with impaired respiratory function. As the range of health effects and airborne concentrations that result in health effects across asbestos-related natural mineral fiber types are not fully understood, the National Institute of Environmental Health Sciences has established a series of research studies to characterize hazards of natural mineral fibers after inhalation exposure. This paper presents the method development work of this research project. RESULTS: A prototype nose-only exposure system was fabricated to explore the feasibility of generating natural mineral fiber aerosol for in vivo inhalation toxicity studies. The prototype system consisted of a slide bar aerosol generator, a distribution/delivery system and an exposure carousel. Characterization tests conducted using Libby Amphibole 2007 (LA 2007) demonstrated the prototype system delivered stable and controllable aerosol concentration to the exposure carousel. Transmission electron microscopy (TEM) analysis of aerosol samples collected at the exposure port showed the average fiber length and width were comparable to the bulk LA 2007. TEM coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis further confirmed fibers from the aerosol samples were consistent with the bulk LA 2007 chemically and physically. CONCLUSIONS: Characterization of the prototype system demonstrated feasibility of generating LA 2007 fiber aerosols appropriate for in vivo inhalation toxicity studies. The methods developed in this study are suitable to apply to a multiple-carousel exposure system for a rat inhalation toxicity testing using LA 2007.

Editor's Highlight: PPARß/δ and PPARγ Inhibit Melanoma Tumorigenicity by Modulating Inflammation and Apoptosis.

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-ß/δ (PPARß/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARß/δ or PPARγ enhanced ligand-induced expression of a PPARß/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARß/δ, or vice versa. Stable overexpression of either PPARß/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARß/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARß/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

Peroxisome proliferator-activated receptor-ß/δ modulates mast cell phenotype.

The peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) is known to have multiple anti-inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARß/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARß/δ modulates mast cell phenotype. Bone-marrow-derived mast cells (BMMCs) and peritoneal mast cells from Pparß/δ+/+ mice expressed higher levels of high-affinity IgE receptor (FcεRI) compared with Pparß/δ-/- mice. BMMCs from Pparß/δ+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparß/δ-/- mice. Resting BMMCs from Pparß/δ+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal-regulated kinases compared with Pparß/δ-/- mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARß/δ expression. This study demonstrates that PPARß/δ is an important regulator of mast cell phenotype.

Peroxisome proliferator-activated receptor-ß/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-ß/δ, (PPARß/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARß/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARß/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARß/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARß/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARß/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARß/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients.

Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-ß/δ- and retinoic acid receptor-dependent mechanisms.

Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARß/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARß/δ. Expression of PPARß/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent growth, and also reduced tumor mass and volume of ectopic xenografts derived from NT2/D1 cells compared to controls. The changes observed in xenografts were associated with decreased PPARß/δ-dependent expression of proliferating cell nuclear antigen and octamer-binding transcription factor-3/4, suggesting suppressed tumor proliferation and induction of differentiation. Inhibition of migration and invasion was mediated by PPARß/δ competing with formation of the retinoic acid receptor (RAR)/retinoid X receptor (RXR) complex, resulting in attenuation of RARα-dependent matrix metalloproteinase-2 expression and activity. These results demonstrate that PPARß/δ mediates attenuation of human testicular embryonal carcinoma cell progression through a novel RAR-dependent mechanism and suggest that activation of PPARß/δ inhibits RAR/RXR dimerization and represents a new therapeutic strategy.

Peroxisome Proliferator-activated Receptor-D (PPARD) Coordinates Mouse Spermatogenesis by Modulating Extracellular Signal-regulated Kinase (ERK)-dependent Signaling.

Ppard(-/-) mice exhibit smaller litter size compared with Ppard(+/+) mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard(-/-) mice compared with Ppard(+/+) mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard(+/+) mice as compared with Ppard(-/-) mice, and these were associated with decreased Sertoli cell number in Ppard(+/+) mice. Cyclin D1 and cyclin D2 expression was lower in Ppard(+/+) as compared with Ppard(-/-) mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard(+/+) mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.

Targeting Peroxisome Proliferator-Activated Receptor-ß/δ (PPARß/δ) for Cancer Chemoprevention.

The role of peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in cancer remains contentious due in large part to divergent publications indicating opposing effects in different rodent and human cell culture models. During the past 10 years, some facts regarding PPARß/δ in cancer have become clearer, while others remain uncertain. For example, it is now well accepted that (1) expression of PPARß/δ is relatively lower in most human tumors as compared to the corresponding non-transformed tissue, (2) PPARß/δ promotes terminal differentiation, and (3) PPARß/δ inhibits pro-inflammatory signaling in multiple in vivo models. However, whether PPARß/δ is suitable to target with natural and/or synthetic agonists or antagonists for cancer chemoprevention is hindered because of the uncertainty in the mechanism of action and role in carcinogenesis. Recent findings that shed new insight into the possibility of targeting this nuclear receptor to improve human health will be discussed.

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid.

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1mg/kg PFOA or 17ß-estradiol (E2, 0.5mg/kg) from PND18-20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER.

Targeting estrogen receptor-ß for the prevention of nonmelanoma skin cancer.

The potential for targeting estrogen receptor (ER)-ß in various cancer models has been gaining considerable attention in recent years. In this issue of the journal, Chaudhary and colleagues demonstrate markedly decreased ultraviolet B (UVB)-induced skin cancer in a mouse model using a highly specific ER-ß agonist, ERB-041. The mechanisms that underlie this strong inhibitory effect are mediated by inhibition of proinflammatory signaling and epithelial-mesenchymal transition (EMT). The changes in EMT were due in part to modulation of WNT/ß-catenin signaling. Collectively, the results from these studies provide important new insights into the mechanisms by which the ER-ß agonist ERB-041 inhibits UVB-induced skin cancer and opens the door for future studies that could examine combinatorial approaches for UVB-dependent skin cancer chemoprevention.

Activation of peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ) inhibits human breast cancer cell line tumorigenicity.

The effect of activation and overexpression of the nuclear receptor PPAR-ß/δ in human MDA-MB-231 (estrogen receptor-negative; ER(-)) and MCF7 (estrogen-receptor-positive; ER(+)) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-ß/δ was increased by overexpression of PPAR-ß/δ compared with controls. Overexpression of PPAR-ß/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-ß/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-ß/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-ß/δ in response to ligand activation of PPAR-ß/δ as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-ß/δ were significantly smaller, and ligand activation of PPAR-ß/δ caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-ß/δ and ligand activation of PPAR-ß/δ correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-ß/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-ß/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-ß/δ in breast cancer and evaluating the effects of PPAR-ß/δ agonists.

Mono-(2-ethylhexyl) phthalate (MEHP) promotes invasion and migration of human testicular embryonal carcinoma cells.

Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNA-binding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.

Transcriptional suppression of Sertoli cell Timp2 in rodents following mono-(2-ethylhexyl) phthalate exposure is regulated by CEBPA and MYC.

Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5'-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells.

FasL gene-deficient mice display a limited disruption in spermatogenesis and inhibition of mono-(2-ethylhexyl) phthalate-induced germ cell apoptosis.

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene-deficient mice (FasL(-/-)) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL(-/-) mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 +/- 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 +/- 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL(-/-) mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL(-/-) mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL(-/-) mice. However, PND 44 FasL(-/-) mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL(-/-) mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury.

TNF alpha-mediated disruption of spermatogenesis in response to Sertoli cell injury in rodents is partially regulated by MMP2.

Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.

Transcriptional regulation of FasL expression and participation of sTNF-alpha in response to sertoli cell injury.

The Fas/FasL signaling pathway has previously been demonstrated to be critical for triggering germ cell apoptosis in response to mono-(2-ethylhexyl)phthalate (MEHP)-induced Sertoli cell injury. Although Sertoli cells ubiquitously express the FasL protein, MEHP-induced germ cell apoptosis appears to tightly correlate with increased levels of Sertoli cell FasL. Here we characterize the transcriptional regulation of the murine FasL gene in Sertoli cells after MEHP exposure. A serial deletion strategy for 1.5 kb of the 5'-upstream activating sequence of the FasL promoter was used to determine transcriptional activity in response to MEHP. Luciferase activity of the FasL promoter in the rat Sertoli cell line ASC-17D revealed that two regions, -500 to -324 and -1250 to -1000, were necessary to drive the inducible transcription of FasL. Sequence analysis of these two regions revealed two cis-regulatory elements, NF-kappaB and Sp-1. By site-directed mutagenesis, electrophoretic mobility shift and chromatin immunoprecipitation assays, it was confirmed that MEHP-induced FasL expression is enhanced through the transcriptional regulation of both NF-kappaB and Sp-1. Experiments performed both in vitro and in vivo revealed that MEHP exposure results in an increased production of sTNF-alpha and that sTNF-alpha-mediated NF-kappaB activation causes robust increases in FasL levels in both the ASC-17D Sertoli cell line and in primary rat Sertoli cell/germ cell co-cultures. In the seminiferous epithelium, Sertoli cells express TNFR1, whereas germ cells produce TNF-alpha. Therefore, sTNF-alpha released by germ cells after MEHP-induced Sertoli cell injury acts upon Sertoli cell TNFR1 and activates NF-kappaB and Sp-1 that consequently causes a robust induction of FasL expression. These novel findings point to a potential "feed-forward" signaling mechanism by which germ cells prompt Sertoli cells to trigger their apoptotic elimination.

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation.

BACKGROUND: Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline. METHODS: Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4. RESULTS: We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression. CONCLUSION: Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.

Autocrine and paracrine regulation of interleukin-8 expression in lung cancer cells.

We had previously demonstrated that lung cancer cells, upon contact with macrophages, could be induced to secrete angiogenic factors to promote tumor angiogenesis. In this study, we focused on the paracrine and autocrine regulation of interleukin (IL)-8 expression in sensitized lung cancer cells after interacting with macrophages. We found that the IL-8 mRNA expression in lung cancer cells significantly increased after coculture with phorbol myristate acetate-treated THP-1 cells and human primary lung macrophages. Fresh lung cancer CL1-5 cells cocultured with macrophage-sensitized lung cancer cells still had a 35% of increase in IL-8 mRNA expression. The addition of anti-inflammatory agents pyrrolidine dithiocarbamate, pentoxifylline, aspirin, and dexamethasone could completely suppress the expression of IL-8 mRNA in fresh/sensitized lung cancer cell cocultures. Human recombinant tumor necrosis factor (TNF)-alpha and IL-1alpha could induce IL-8 expression in lung cancer cells in a dose-dependent manner. Neutralization with TNF-alpha and IL-1alpha antibodies in cocultures decreased the levels of IL-8 expression in sensitized lung cancer cells. Nuclear factor-kappaB transcriptional activity was also suppressed by the same antibodies, as confirmed by a reporter gene assay and the electrophoretic mobility shift assay. Our results highly suggest that both autocrine and paracrine regulation are involved in IL-8 expression of lung cancer cells cocultured with macrophage. Also, the regulations of IL-8 expression in lung cancer cells were through the nuclear factor-kappaB pathway and modulated by TNF-alpha and IL-1alpha.

Tumor-associated macrophages: the double-edged sword in cancer progression.

PURPOSE: Inflammation plays a critical role in cancer progression. In this study we investigate the pro-tumorigenic activities and gene expression profiles of lung cancer cells after interaction with macrophages. MATERIALS AND METHODS: We measured intratumoral microvessel counts and macrophage density in 41 lung cancer tumor specimens and correlated these with the patients' clinical outcome. The interaction between macrophages and cancer cell lines was assessed using a transwell coculture system. The invasive potential was evaluated by in vitro invasion assay. The matrix-degrading activity was assayed by gelatin zymography. The microarray was applied to a large-scale analysis of the genes involved in the interaction, as well as to monitor the gene expression profiles of lung cancer cells responding to anti-inflammatory drugs in cocultures. RESULTS: The macrophage density positively correlated with microvessel counts and negatively correlated with patient relapse-free survival (P < .05). After coculture with macrophages, lung cancer cell lines exhibited higher invasive potentials and matrix-degrading activities. We identified 50 genes by microarray that were upregulated more than two-fold in cancer cells after coculture. Northern blot analyses confirmed some gene expression such as interleukin-6, interleukin-8, and matrix metalloproteinase 9. The two-dimensional hierarchical clustering also demonstrated that the gene expression profiles of lung cancer cells responding to various anti-inflammatory drugs in cocultures are distinct. CONCLUSION: The interaction of lung cancer cells and macrophages can promote the invasiveness and matrix-degrading activity of cancer cells. Our results also suggest that a great diversity of gene expression occurs in this interaction, which may assist us in understanding the process of cancer metastasis.

The role of interleukin-8 in cancer cells and microenvironment interaction.

Abstract Interleukin (IL)-8, a cytokine of the CXC chemokine family that was originally classified as a neutrophil chemoattractant, is now reported to play an important role in tumor progression and metastasis in a variety of human cancers, including lung cancers. IL-8 biologic activity in tumors and the tumor microenvironment may contribute to tumor progression through its potential function in the regulation of angiogenesis, cancer cell growth and survival, tumor cell motion, leukocyte infiltration and modification of immune responses. Recently, infiltrating macrophages in tumor stroma have been considered to be able to stimulate cancer growth, enhance angiogenesis and promote metastasis, and has prognostic significance in several human cancers. Accumulating evidence also shows that cancer cells and stromal cell interaction can stimulate cancer cells, as well as stromal cells in the expression of IL-8 and other growth factors. Here, we summarize current information about IL-8 biology in human lung cancers and focus on its effect on tumor angiogenesis, regulation of IL-8 expression in tumors, its prognostic significances, the role of tumor infiltrating macrophages in the production of IL-8 in cancer cells and the tumor microenvironment, gene expression profiles after cancer cell-stromal cell interaction, and the effect of a variety anti- inflammatory drugs on the modification of IL-8 and other gene expressions in cancer cells and the tumor microenvironment in lung cancers.