SKLU-1 Cells Research Articles

Effects of the tetravanadate [V4O12]4− anion on the structural, magnetic, and biological properties of copper/phenanthroline complexes

The aim to access linked tetravanadate [V4O12]4− anion with mixed copper(II) complexes, using α-amino acids and phenanthroline-derived ligands, resulted in the formation of four copper(II) complexes [Cu(dmb)(Gly)(OH2)]2[Cu(dmb)(Gly)]2[V4O12]·9H2O (1) [Cu(dmb)(Lys)]2[V4O12]·8H2O (2), [Cu(dmp)2][V4O12]·C2H5OH·11H2O (3), and [Cu(dmp)(Gly)Cl]·2H2O (4), where dmb = 4,4′-dimethioxy-2,2′-bipyridine; Gly = glycine; Lys = lysine; and dmp = 2,9-dimethyl-1,10-phenanthroline. The [V4O12]4− anion is functionalized with mixed copper(II) units in 1 and 2; while in 3, it acts as a counterion of two [Cu(dmp)]2+ units. Compound 4 crystallized as a unit that did not incorporate the vanadium cluster. All compounds present magnetic couplings arising from Cu⋯O/Cu⋯Cu bridges. Stability studies of water-soluble 3 and 4 by UV–Vis spectroscopy in cell culture medium confirmed the robustness of 3, while 4 appears to undergo ligand scrambling over time, resulting partially in the stable species [Cu(dmp)2]+ that was also identified by electrospray ionization mass spectrometry at m/z = 479. The in vitro cytotoxicity activity of 3 and 4 was determined in six cancer cell lines; the healthy cell line COS-7 was also included for comparative purposes. MCF-7 cells were more sensitive to compound 3 with an IC50 value of 12 ± 1.2 nmol. The tested compounds did not show lipid peroxidation in the TBARS assay, ruling out a mechanism of action via reactive oxygen species formation. Both compounds inhibited cell migration at 5 µM in wound-healing assays using MCF-7, PC-3, and SKLU-1 cell lines, opening a new window to study the anti-metastatic effect of mixed vanadium–copper(II) systems.Graphical abstract

Synthesis of Dendrimer Conjugates with Naproxen and their Anticancer Activity

Abstract: Here we report the easy synthesis of dendrimer conjugates with four and eight naproxen moieties at the periphery. The synthesis involved two steps protection-deprotection sequence joined with triethylene glycol. A comparison was made between the cytotoxicity of dendron-naproxen conjugates and that of G1.0 G2.0 dendrimer-naproxen conjugates. Cytotoxicity studies showed that dendron conjugates were toxic towards all cancer cell lines studied, and their toxicity was significantly lower than that of dendrimer conjugates. However, dendrimer conjugates showed higher cytotoxicity and selectivity against K-562 and SKLU- 1 than dendron conjugates.

Phytochemical Constituents from the Aerial Parts of Acanthus ebracteatus Vahl. and Their Cytotoxic Activity

Objective Acanthus ebracteatus Vahl. (family Acanthaceae) is distributed in the mangrove areas of northern Vietnam. The whole plant has the effect of dissolving blood stasis, reducing swelling, relieving pain, dissolving phlegm, and is often used to treat back pain, numbness, cough, and asthma. In our screening program for seeking cytotoxic active compounds, the methanol extract of this plant significantly showed activity and was selected for further study. In this paper we report the chemical constituents of A. ebracteatus and their cytotoxic activity towards two cancer cell lines, HepG2 and SK-LU1 in vitro. Methods The dried and powdered aerial parts of A. ebracteatus were ultrasonically extracted with methanol. This extract was suspended in water and successively partitioned with n-hexane, dichloromethane, and ethyl acetate to give n-hexane, dichloromethane, ethyl acetate, and water-soluble extracts. These extracts were fractionated and purified by column and HPLC chromatographic methods. The chemical structures of the isolates were determined by analysis of HRESIMS, and 1D and 2D NMR, and CD spectra. The isolates were screened for their in vitro cytotoxic activity by SRB assay. Results One new flavone glycoside (1) and ten known compounds (2-11), including five flavones (2-6), three alkaloids (7-9), and two phenolic glycosides (10 and 11), were isolated from the aerial parts of A. ebracteatus. Compounds 7–9 showed moderate cytotoxic activity against HepG2 and SK-LU1 cell lines with IC50 values ranging from 28.2 to 31.1 µM. Compounds 2–6, 8, 9, and 11 are reported herein from A. ebracteatus for the first time. Conclusion One new (1) and five known flavonoids (2-6), together with three alkaloids (7-9) and two phenolic glycosides (10 and 11) were recorded from A. ebracteatus. Our results preliminarily revealed that alkaloids may be cytotoxic ingredients of A. ebracteatus.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

BackgroundCnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis.MethodsThe presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation.ResultsP. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium.ConclusionThese results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Cell growth underpinned by sepiolite

This paper reports the role of clay minerals, sepiolites, on the proliferation behaviour of human cancer cells. It reports as well on the proliferation of U251 (central nervous system, glioblastoma) and SKLU-1 (lung adenocarcinoma) cells by sepiolite bearing different extent of isomorphic substitution (IS), either because of the inclusion of Al3+, Fe3+.2+, or Ti4+ in Si structural sites (IS at the tetrahedral sheet, T) or that of Ni2+ in Mg structural sites (IS at the octahedral sheet, O). Studied sepiolites were originally from Ampandrandara, Madagascar; Cerro del Almodóvar, Spain; Deiva Forest, Italy; Eskidir, Turkey; Peguera, Falcondo Plant, Dominican Republic; Sepetciköyü, Turkey; Shimien, China; and Vallecas, Spain. Furthermore, obtained results for sepiolites were compared against those for clays (bentonites). Diffractograms showed characteristic patterns for sepiolite, with no evidence of significant accumulation of secondary phases. XRF data confirmed the incorporation of Al, Fe and, Ti; and Ni, consistent with IS at T and O. The effect of sepiolite on cellular proliferation was determined using the SRB protocol. All sepiolites induced inhibition or increment on the proliferation response of U251 or SKLU cells, depending on the sepiolite; however no correlation between proliferation against composition or microporosity properties became evident. Most notably, sepiolite from Sepetciköyü, Turkey, owning the highest microporosity (evidenced by surface area σs) of the sepiolite series, 343m2g−1, exerted the highest proliferation response for U251 and SKLU-1 cells, namely, 100% inhibition and 22.8±12.1% increase, respectively. Sepiolites from Ampandrandara, Sepetciköyü, and Deiva Forest, owing very low contents of Al (Al2O3≤0.2%) and variable σs yielded the highest inhibition in U251 cells proliferation, best accounted for by growth was limited by specific-adsorption mechanisms in which structural changes associated to Al-for-Si IS at T favoured the adsorption of metabolic growth components [epidermal growth factor receptor (EGFR)], thereby inhibiting the development of primary glioblastomas. On the other hand, increments (%) in SKLU-1 cells proliferation did not correlate with microporosity (measured σs values), yet two data clusters were identified, higher and lower data values, i.e., 22.8≤% increment≤39% (σs=83, 220, or 343m2g−1) and 6.9≤% increment≤14.2% (96≤σs≤266). The second group was composed by Ampandrandara, Sepetciköyü, and Deiva Forest, generating surface sites that catalyze the overexpression of activin A. So, the growth behaviour for both U251 and SKLU-1 cells was affected by Al at Tvia Al-for-Si IS if proceeded to a small degree. In all, however, the overall chemical composition lacked to serve as predictor for growth. Structural considerations supported the idea that controlled cell growth by sepiolite was not limited by the retention of small solutes at inner surfaces. Finally, whether variations in microporosity exerted changes in the cell proliferation behaviour was strongly dependent if the phyllosilicate was a clay mineral (sepiolite) or a clay (bentonite).

Role of bentonite clays on cell growth

Bentonites, naturally occurring clays, are produced industrially because of their adsorbent capacity but little is known about their effects on human health. This manuscript reports on the effect of bentonites on cell growth behaviour. Bentonites collected from India (Bent-India), Hungary (Bent-Hungary), Argentina (Bent-Argentina), and Indonesia (Bent-Indonesia) were studied. All four bentonites were screened in-vitro against two human cancer cell lines [U251 (central nervous system, glioblastoma) and SKLU-1 (lung adenocarcinoma)] supplied by the National Cancer Institute (USA). Bentonites induced growth inhibition in the presence of U251 cells, and growth increment in the presence of SKLU-1 cells, showing that interactions between bentonite and cell surfaces were highly specific. The proliferation response for U251 cells was explained because clay surfaces controlled the levels of metabolic growth components, thereby inhibiting the development of high-grade gliomas, particularly primary glioblastomas. On the other hand, the proliferation response for SKLU-1 was explained by an exacerbated growth favoured by swelling, and concomitant accumulation of solutes, and their hydration and transformation via clay-surface mediated reactions.

Bcl-xL Silencing Induces Alterations in hsa-miR-608 Expression and Subsequent Cell Death in A549 and SK-LU1 Human Lung Adenocarcinoma Cells

Bcl-xL is an anti-apoptotic protein that is frequently found to be overexpressed in non-small cell lung cancer leading to an inhibition of apoptosis and poor prognosis. Recently, the role of miRNAs in regulating apoptosis and cell survival during tumorigenesis has become evident, with cancer cells showing perturbed expression of various miRNAs. In this study, we utilized miRNA microarrays to determine if miRNA dysregulation in bcl-xL silenced lung adenocarcinoma cells could be involved in regulating cell death. Short interfering RNA-based transfection of A549 and SK-LU1 lung adenocarcinoma cells was successful in inducing a reduction in bcl-xL expression levels, resulting in a decrease in cell viability. A total of 10 miRNAs were found to be significantly differentially expressed when compared between siRNA-transfected and non-transfected cells including hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304 and hsa-miR-608. When overexpression studies on hsa-miR-608 was performed via transfection of miRNA mimics, cell death was found to be induced in A549 and SK-LU1 cells in comparison to untreated cells. This effect was reversed when knockdown studies involving anti-sense inhibitors were introduced. Combination of siRNA based silencing of bcl-xL (siBcl-xL) followed by anti-sense inhibitor transfection led to a decrease in the apoptotic population of A549 and SK-LU1 cells in comparison to cells only treated with siBcl-xL, illustrating the connection between bcl-xL, hsa-miR-608 and cell death. Gene target prediction analysis implicated the PI3K/AKT, WNT, TGF-β, and ERK signaling pathways as targets of bcl-xL induced miRNA alterations. We have demonstrated that bcl-xL silencing in A549 and SK-LU1 cells leads to the occurrence of cell death through the dysregulation of specific miRNAs. This study also provides a platform for anti-sense gene therapy whereby miRNA expression can be exploited to increase the apoptotic properties in lung adenocarcinoma cells.

Abstract 3124: Smoking effects on CYP24A1 in lung adenocarcinoma: Epigenetic changes by smoking

Abstract CYP24A1 is the rate limiting catabolic enzyme for 1α,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. We have previously demonstrated that the mRNA expression of CYP24A1 is an independent prognostic factor for survival in lung adenocarcinoma (AC) and that the antiproliferative effects of 1,25-D3 are inversely proportional to CYP24A1 expression. We then asked if smoking is associated with increased expression of CYP24A1 and a decrease in serum level of 1,25-D3; whether increased CYP24A1 expression was due to epigenetic changes caused by smoking. We measured CYP24A1 mRNA in resected lung AC (n=100) specimens and compared CYP24A1 in smokers (former/current) versus non-smokers. In parallel, we performed metabolism experiments in A549 (high CYP24A1) and SKLU-1 (low CYP24A1) cells to study the functional relevance of increased CYP24A1. We evaluated the promoter methylation status of CYP24A1 in the cancer cell lines and lung AC patients using pyrosequencing PCR analysis. Finally, quantitative chromatin immunoprecipitation PCR (ChIP-qPCR) analysis was performed to validate the CYP24A1 methylation was regulated by histone modifiers. Non-smokers had higher serum levels of 25-D3 compared with smokers (albeit small numbers of non-smokers, n=11). Smoking lung AC patients showed higher mRNA expression of CYP24A1 (P < 0.00374) and poorer 5-year survival compared to nonsmokers in Kaplan-Meir survival analysis (Logrank P < 0.0435). A549 cells catabolized 1,25-D3 to a greater extent compared to SKLU-1 at 48-h treatment of 100 nM 1,25-D3 (5.67 vs 103 pmole 1,25-D3 remained). Smoking increased CYP24A1 expression in lung AC patients through hypomethylation of CYP24A1 promoter. In the pyrosequencing analysis, A549 cells were unmethylated (1.01%) but SKLU-1 cells highly methylated (52.02%). Treatment with the DNA methyltransferase inhibitor 5-aza-2α-deoxycytidine (5-Aza) activates CYP24A1 expression in lung cancer cells. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in lung cancer cells. ChIP-qPCR reveals that treatment with 5-Aza and/or TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with 5-Aza and/or TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Taken together, smoking induced the expression of CYP24A1 and showed faster metabolism of 1,25-D and led to poorer survival in lung AC patients, This induction might be related to epigenetic changes in CYP24A1 gene. By increasing the substrate or silencing CYP24A1 gene, the possibility of CYP24A1 gene for chemoprevention will be investigated. Supported by NIH R21CA128193-01-A1 and VA Merit I01CX000333-02. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3124. doi:1538-7445.AM2012-3124

CYP24A1Is an Independent Prognostic Marker of Survival in Patients with Lung Adenocarcinoma

The active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25-D(3)), exerts antiproliferative effects in cancers, including lung adenocarcinoma (AC). CYP24A1 is overexpressed in many cancers and encodes the enzyme that catabolizes 1,25-D(3). The purpose of our study was to assess CYP24A1 as a prognostic marker and to study its relevance to antiproliferative activity of 1,25-D(3) in lung AC cells. Tumors and corresponding normal specimens from 86 patients with lung AC (stages I-III) were available. Affymetrix array data and subsequent confirmation by quantitative real time-PCR were used to determine CYP24A1 mRNA expression. A subsequent validation set of 101 lung AC was used to confirm CYP24A1 mRNA expression and its associations with clinical variables. The antiproliferative effects of 1,25-D(3) were examined using lung cancer cell lines with high as well as low expression of CYP24A1 mRNA. CYP24A1 mRNA was elevated 8- to 50-fold in lung AC (compared to normal nonneoplastic lung) and significantly higher in poorly differentiated cancers. At 5 years of follow-up, the probability of survival was 42% (high CYP24A1, n = 29) versus 81% (low CYP24A1, n = 57) (P = 0.007). The validation set of 101 tumors showed that CYP24A1 was independently prognostic of survival (multivariate Cox model adjusted for age, gender, and stage, P = 0.001). A549 cells (high CYP24A1) were more resistant to antiproliferative effects of 1,25-D(3) compared with SKLU-1 cells (low CYP24A1). CYP24A1 overexpression is associated with poorer survival in lung AC. This may relate to abrogation of antiproliferative effects of 1,25-D(3) in high CYP24A1 expressing lung AC.

Impact of smoking on the expression of claudins in lung carcinoma

Rationale Tight junctions regulate the paracellular permeability and orientation of cells and claudins are key components of tight junctions. Objectives To study the influence of cigarette smoke on claudin expression in vitro and in lung cancer patients. Methods We studied the effect of smoking on claudin expression by exposing a bronchial cell line (BEAS-2B) and two carcinoma cell lines (SK-LU1 and SK-MES1) to tobacco smoke for 48 h and analysed their claudin mRNA expression. The relation between smoked pack years and protein expression of claudins 1–5 and 7 in 344 lung cancer patients was determined by immunohistochemistry. Measurements and main results In BEAS-2B cells and SK-LU1 cells, an initial increase was followed by a decline in the mRNA expression of several claudins. In SK-MES1 cells, no evident elevation in claudin expression was observed. Intense claudin 1 and 4 positivity was found more often in cancer samples of smokers and ex-smokers compared to non-smokers ( p < 0.001 and p = 0.003, respectively). Heavy smokers with longer than 40 pack-years consumption had more often intense claudin 1 ( p = 0.011), 4 ( p = 0.050) or 7 ( p = 0.058) expression in squamous cell carcinoma compared to non-smokers or smokers with fewer pack-years. Claudin 1 positivity predicted a better survival in adenocarcinoma ( p = 0.044) and in squamous cell carcinoma ( p = 0.027) and claudin 4 positivity in adenocarcinoma only ( p = 0.048). In squamous cell carcinoma, claudin 7 positivity was associated with a better survival ( p = 0.011). Conclusions Bronchial BEAS-2B cells and SK-LU1 cells respond to tobacco smoke by changing their claudin mRNA synthesis and resulting tight junction permeability changes may thus contribute to tobacco induced carcinogenesis both during initiation and progression. This concept is strengthened by findings in the clinical tumour material, where tobacco consumption was associated with claudin expression.

Evaluation of CYP24A1 mRNA expression as a prognostic marker and predictor of calcitriol activity in lung adenocarcinoma.

10631 Background: 1α25,dihydroxycholecalciferol (calcitriol) is antiproliferative in cancers, including lung adenocarcinoma (AC). Calcitriol, however, rapidly induces expression of cytochrome P450 24R-hydroxylase (CYP24A1). CYP24A1 inactivates calcitriol. We examined the prognostic and predictive (of calcitriol efficacy) significance of CYP24A1 in lung AC. Methods: Tumors and corresponding normal lung from patients with resected lung AC (stages I-III) were used to examine CYP24A1 mRNA expression with Affymetrix arrays. Affymetrix SNP arrays were used to assess copy number changes. A subsequent set of 101 lung AC were used to confirm high/low CYP24A1 mRNA expression by qRT-PCR. We examined antiproliferative activity of calcitriol (using WST-1 colorimetric assay) on two lung cancer cell lines, A549 and SKLU1 (high and low CYP24A1 mRNA). siRNA against CYP24A1 was used to demonstrate effects of blocking CYP24A1 in the same cell lines. Results: CYP24A1 mRNA was elevated 8-15 fold compared to normal lung in 30% of 86 lung AC patients and higher in poorly differentiated tumors, but not associated with histology, pathological stage or gender. At 5 years the probability of survival in the high CYP24A1 group was 42% versus 87% in the low CYP24A1 group (p = 0.0008). In tumors with elevated CYP24A1, 30% showed a gain in the region of 20q (location for CYP24A1 gene). A second set of 101 tumors confirmed that higher CYP24A1 expression was independently prognostic of survival (p = 0.008) using a multivariate Cox proportional model. In vitro studies demonstrated a 30% reduction in cell growth in SKLU-1, but little effect in A549 with 100nM calcitriol. Blocking CYP24A1 expression with CYP24A1 siRNA partially restored sensitivity to calcitriol in A549 cells and to a greater extent in SKLU-1 cells. Conclusions: CYP24A1 over expression is associated with poorer survival in lung AC. Growth inhibitory action of calcitriol was observed in the low CYP24A1 but not in the high CYP24A1 cell line. The translational relevance of these findings relate to a potential role for calcitriol as adjuvant therapy in resected lung cancer, based on CYP24A1 expression in the tumor. No significant financial relationships to disclose.

Synthesis and characterization of di-phenyl-tin IV-salicyliden- ortho-aminophenols: Analysis of in vitro antitumor/antioxidant activities and molecular structures

The one pot reactions carried among salicylaldehyde 1, ortho-aminophenols 2a– 2g, and di-phenyl-tin IV oxide 3 led to seven di-phenyl-tin IV compounds 4a– 4g in good yields (97–83%). All compounds were analyzed by IR, 1H, 13C, 119Sn NMR spectroscopy, mass spectrometry and elemental analyses; furthermore, in the case of compounds 4b, 4c, 4e and 4g by X-ray diffraction. Compounds 4a– 4g were tested in vitro against six human tumor cell lines U251, PC-3, K-562, HCT-15, MCF-7 and SKLU-1 to assess their in vitro antitumor activity. The results suggest biological specificity towards U251, MCF-7 and SKLU-1 cells at doses below 2.5 μM, which are lower than cis-platin IC 50’s in the three cell lines. Since the inhibitory concentration values for the series were alike to Ph 2SnCl 2 is feasible that only the Ph 2Sn moiety is responsible for those activities, further experiments are under research. Besides, 4a– 4g were tested for their antioxidant efficiency in rat brain homogenate showing that 4g is more active (IC 50 = 3.01 μM) than the flavone quercetin (natural antioxidant, IC 50 = 4.11 μM) on inhibition of thiobarbituric acid reactive substances (TBARS). The TBARS activity (IC 50) correlates with the ortho-aminophenol substitutions and a linear combination among sigma Hammett, one bond tin coupling constants and tin chemical shifts against the measured IC 50−TBARS was found. This correlation gave basis that the implied molecular variables can become trackers for the calculation of TBARS inhibitory concentrations in similar systems. Moreover, there seemed to be an inverse structure–response behavior among activities, since the 4g derivative is the less active compound for cytotoxic assays meanwhile it is the best in antioxidant tests.

Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas

We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays. Three phosphorylated forms and one unphosphorylated form of the Op18 protein were identified and found to be overexpressed in lung adenocarcinomas as compared with normal lung. The percentage of phosphorylated to total Op18 protein isoforms increased from 3.2% in normal lung to 7.9% in lung tumors. Both the phosphorylated and unphosphorylated Op18 proteins were significantly increased in poorly differentiated tumors as compared with moderately or well differentiated lung adenocarcinomas (p<0.03), suggesting that up-regulated expression of Op18 reflects a poor differentiation status and higher cell proliferation rates. This was further verified in A549 and SKLU1 lung adenocarcinoma cell lines by examining Op18 levels and phosphorylation status following treatment that altered either cell proliferation or differentiation. The increased expression of Op18 protein was significantly correlated with its mRNA level indicating that increased transcription likely underlies elevated expression of Op18. The overexpression of Op18 proteins in poorly differentiated lung adenocarcinomas and the elevated expression of the phosphorylated forms of Op18 may offer a new target for drug- or gene-directed therapy and may have potential utility as a tumor marker.

CD40 expression by human bronchial epithelial cells.

CD40 is a 50-kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)-gamma was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)-alpha was the best cytokine able to up-regulate CD40 in SKLU1 cells. A combination of IFN-gamma and TNF-alpha was slightly more effective than the cytokine alone at up-regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell-surface-antigen expression on either cell line. The production of cytokines such as interleukin (IL)-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL-6 and GM-CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF-alpha-induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.