DMS-79 Cells Research Articles

Abstract 2479: Preclinical assessment of imaging agents for identification of fucosyl-GM1 expression in vivo

Abstract Background: Fucosyl-GM1 (FucGM1) is a monosialoganglioside highly expressed in 50%-70% of small cell lung cancer (SCLC) cells, supporting it as a potential biomarker. BMS-986012 is a nonfucosylated, first-in-class, fully human immunoglobulin G1 monoclonal antibody that binds to FucGM1 with high affinity and specificity. Understanding how FucGM1+ tumor expression correlates with response to BMS-986012 may improve patient selection and therapeutic efficacy. Objective: Evaluate ability of imaging agents built on BMS-986012 to detect tumor FucGM1 expression. Methods: BMS-986012 was labeled with a near-infrared fluorophore. Binding to human H520 (FucGM1−) and DMS79 (FucGM1+) xenografts was assessed in vivo via optical imaging. BMS-986012 was labeled with deferoxamine and radiolabeled to produce 89Zr-BMS-986279, which was evaluated in vitro with H520 and DMS79 cells. Tracer distribution and tumor accumulation was assessed by positron emission tomography (PET) imaging in mice bearing bilateral H520 and DMS79 xenografts and analyzed with ex vivo autoradiography (ARG). Results: Optical imaging with fluorescent BMS-986012 showed statistically significant accumulation in DMS79 vs H520 tumors at all time points (P<.05), beginning 6 hours post administration and reaching a 3:1 maximum uptake ratio. Maximum tumor to background signal was measured at 96 hours (final imaging time point). Total radiolabeled 89Zr-BMS-986279 binding to intact DMS79 cells was ≈15-fold higher than to H520 control cells in vitro and was almost completely blocked by coincubation with a 1000-fold excess of BMS-986012. In vivo, longitudinal PET imaging of 89Zr-BMS-986279 showed significantly higher tracer accumulation in FucGM1+ DMS79 xenografts vs H520 tumors (P<.001). The difference in tracer uptake between tumor models increased with time and was highest (≈2-fold) approximately 8 days post injection. PET signal decreased over time in both FucGM1− H520 xenografts and background muscle tissues, consistent with slow accumulation of specific binding and clearance from background common with antibody-based tracers. Ex vivo ARG studies confirmed in vivo PET results and indicated that 89Zr-BMS-986279 can be used to assess FucGM1 expression throughout the tumor volume, with signal found in the periphery and interior of tumor samples. Conclusions: In vitro cell binding, in vivo fluorescence and PET imaging, and ex vivo ARG studies demonstrated that 89Zr-BMS-986279 was sensitive and specific for detecting FucGM1 expression in tumor cells. Specificity to the intended target in animal disease models coupled with kinetics typical of antibody-based tracers further support the utility of 89Zr-BMS-986279 as a potential FucGM1 imaging agent. A phase 2 study is evaluating safety and efficacy of BMS-986012 combined with carboplatin, etoposide, or nivolumab in patients with newly diagnosed extensive-stage SCLC (NCT04702880). Citation Format: R. Adam Smith, Erin L. Cole, Andrea Olga Shorts, Thomas Petrone, Joonyoung Kim, Daniel Cohen, Paul Morin, Samuel J. Bonacorsi, Patrick L. Chow. Preclinical assessment of imaging agents for identification of fucosyl-GM1 expression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2479.

Irradiation Decreases the Neuroendocrine Biomarker Pro-Opiomelanocortin in Small Cell Lung Cancer Cells In Vitro and In Vivo.

BackgroundSmall cell lung cancer (SCLC) is an extremely aggressive disease, commonly displaying therapy-resistant relapse. We have previously identified neuroendocrine and epithelial phenotypes in SCLC tumours and the neuroendocrine marker, pro-opiomelanocortin (POMC), correlated with worse overall survival in patients. However, the effect of treatment on these phenotypes is not understood. The current study aimed to determine the effect of repeated irradiation treatment on SCLC cell phenotype, focussing on the neuroendocrine marker, POMC.ResultsHuman SCLC cells (DMS 79) were established as subcutaneous xenograft tumours in CBA nude mice and then exposed to repeated 2Gy irradiation. In untreated animals, POMC in the blood closely mirrored tumour growth; an ideal characteristic for a circulating biomarker. Following repeated localised irradiation in vivo, circulating POMC decreased (p< 0.01), in parallel with a decrease in tumour size, but remained low even when the tumours re-established. The excised tumours displayed reduced and distinctly heterogeneous expression of POMC compared to untreated tumours. There was no difference in the epithelial marker, cytokeratin. However, there were significantly more N-cadherin positive cells in the irradiated tumours. To investigate the tumour response to irradiation, DMS79 cells were repeatedly irradiated in vitro and the surviving cells selected. POMC expression was reduced, while mesenchymal markers N-cadherin, β1-integrin, fibroblast-specific protein 1, β-catenin and Zeb1 expression were amplified in the more irradiation-primed cells. There were no consistent changes in epithelial marker expression. Cell morphology changed dramatically with repeatedly irradiated cells displaying a more elongated shape, suggesting a switch to a more mesenchymal phenotype.ConclusionsIn summary, POMC biomarker expression and secretion were reduced in SCLC tumours which regrew after irradiation and in repeatedly irradiation (irradiation-primed) cells. Therefore, POMC was no longer predictive of tumour burden. This highlights the importance of fully evaluating biomarkers during and after therapy to assess clinical utility. Furthermore, the gain in mesenchymal characteristics in irradiated cells could be indicative of a more invasive phenotype.

Abstract 3530: Gene expression profile of OTX015, a BET bromodomain inhibitor, in preclinical models of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) models

Abstract Background. BET bromodomain proteins recognize chromatin modifications and act as epigenetic readers contributing to gene transcription. OTX015, a novel BET bromodomain inhibitor currently in clinical Phase Ib studies in hematologic malignancies and solid tumors, including lung cancer. We have shown in vitro and in vivo activity of OTX015 in NSCLC and SCLC models (Riveiro et al; EORTC 2014), however the mechanism of action and relevant affected genes are not fully characterized. This study aimed to elucidate pathways and genes affecting response/resistance to OTX015 in lung models.Methods. OTX015 (OncoEthix SA, Switzerland), GI50 values were determined with the MTT assay after 72h exposure in 5 NSCLC cell lines (H2228, H3122, A549, HOP62, HOP92) and 4 SCLC cell lines (H82, H69, DMS79, DMS114). For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. RNA was isolated after treatment with DMSO or OTX015 (500 nM) for 2, 4, 8 or 12 and 24h and gene expression profiles (GEP) were obtained in all NSCLC and 2 SCLC (H82 and DMS114) cell lines with Illumina HumanHT-12 Expression BeadChips. Results. OTX015 displayed antiproliferative effects in 4 of the 5 NSCLC cell lines with GI50 values from 110 to 940 nM, whereas A549 cells (harboring mutations in KRAS and LKB1 genes) were resistant to OTX015. Among SLCL models, DMS114 cells showed dose-dependent sensitivity to OTX015 [GI50 = 120nM], while H82, H69, and DMS79 cells were resistant [GI50 &amp;gt;6 μM], despite overexpressing CMYC and/or NMYC. In sensitive-NSCLC models we observed an increase in cells in the S phase after OTX015 treatment, whereas an increase in the G1 phase was observed in the sensitive DMS114 cell line. GEP showed that changes in expression levels of EFR3B and genes related to RNA biogenesis were higher in SCLC cells with reduced sensitivity to OTX015 than in sensitive cells. On the other hand, genes related to histones and chromatin structure were the most differentially expressed after OTX015 exposure in both sensitive and resistant cell lines. In NSCLC models, EFR3B and FOS gene up-regulation, IL7R and IL6-related gene down-regulation were associated with higher OTX015 sensitivity Expression changes more prominent in OTX015-resistant cells affected the transcription factor FOXD1 and genes related to RNA processing. Conclusions. Our data contribute to elucidate the mechanisms associated with OTX015 sensitivity in several lung cancer in vitro models, supporting the rationale for the ongoing clinical Phase Ib study in solid tumors including NSCLC patients (KRAS+; ALK+). Citation Format: Maria Eugenia Riveiro, Ivo Kwee, Lucile Astorgues-Xerri, Mohamed Bekradda, Ramiro Vazquez, Andrea Rinaldi, Esteban Cvitkovic, Francesco Bertoni. Gene expression profile of OTX015, a BET bromodomain inhibitor, in preclinical models of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2015-3530

566 GNS396 and analogues are potent new small molecules to target and kill chemotherapy-resistant subpopulation cells in acute myeloid leukemia

Background: Standard combination chemotherapy for advanced SCLC has not changed substantially in the last two decades. The challenge when identifying novel SCLC therapies is the prevalence of multiple inactivating mutations in tumor suppressor genes (TP53, PTEN, RB1) and gain-of-function oncogenic mutations (PI3K3CA, MYC family). BET bromodomain inhibitors such as OTX015 (OncoEthix SA) which binds to human bromodomain proteins BRD2/3/4, modulate the transcription of target genes via epigenetic mechanisms. OTX015 shows preclinical activity in hematologic and solid tumors and is currently in clinical development. Here we characterized the effects of OTX015 on a series of SCLC cell lines. Material and Methods: OTX015 growth inhibition 50% (GI50) values were determined with the MTT assay after 72 h exposure in four established SCLC cell lines (H82, H69, DMS79, DMS114). Protein levels were analyzed by Western Blot using commercial antibodies and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, then 4 and 24 h post-treatment. For cell cycle analysis, cells were stained with propidium iodide and analyzed for DNA content with a FACScan flow cytometer. Results: DMS114 cells showed dose-dependent sensitivity to OTX015 [GI50 = 120 (84–172) nM], while H82, H69, DMS79 cells were resistant [GI50 >6 mM], although they overexpressed CMYC and/or NMYC proteins. While all four SCLC cell lines are KRAS wild-type and TP53 mutated, OTX015-resistant SCLC cell lines harbor a homozygous mutation in RB1, while DMS144 cells express RB1 wild-type. All cell lines exhibited similar basal mRNA levels of BRD2/3/4, CMYC, NMYC, HEXIM, BCL-2 and p21. Following OTX015 treatment at 500 nM, CMYC and NMYC mRNA levels were unchanged, while mRNA levels of HEXIM and genes coding for histones (HIST1H2BK, HIST2H2BJ) were upregulated in all four cell lines. In DMS114 cells, OTX015 caused cell cycle arrest in G1 in a timedependent manner, which may be explained by an upregulation at the protein level of the cell cycle inhibitor p27. Conclusion: Our findings suggest that the MYC family does not mediate OTX015 antitumor effects in SCLC cell lines. The presence of functional RB1 protein, controlling cell progression at G1, may explain the cytostatic effects of OTX015. Further studies investigating the therapeutic potential of OTX015 in SCLC are ongoing.

Zebrafish as an innovative model for neuroendocrine tumors

Tumor models have a relevant role in furthering our understanding of the biology of malignant disease and in preclinical cancer research. Only few models are available for neuroendocrine tumors (NETs), probably due to the rarity and heterogeneity of this group of neoplasms. This review provides insights into the current state-of-the-art of zebrafish as a model in cancer research, focusing on potential applications in NETs. Zebrafish has a complex circulatory system similar to that of mammals. A novel angiogenesis assay based on the injection of human NET cell lines (TT and DMS79 cells) into the subperidermal space of the zebrafish embryos has been developed. Proangiogenic factors locally released by the tumor graft affect the normal developmental pattern of the subintestinal vessels by stimulating the migration and growth of sprouting vessels toward the implant. In addition, a description of the striking homology between zebrafish and humans of molecular targets involved in tumor angiogenesis (somatostatin receptors, dopamine receptors, mammalian target of rapamycin), and currently used as targeted therapy of NETs, is reported.

Highly Efficient Assay of Circulating Tumor Cells by Selective Sedimentation with a Density Gradient Medium and Microfiltration from Whole Blood

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

Abstract C53: Potent CDC, ADCC, and antitumor activity of human monoclonal antibodies to Sialyl Lewisa.

Abstract The carbohydrate antigen sialyl-Lewis A (sLea), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells. sLea serves as a ligand for epithelial leukocyte adhesion molecules and over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells. Since tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting. Hybridomas were generated with human B cells isolated from individuals vaccinated with MSKCC's cancer vaccine. Antibody producing cells were identified by ELISA using sLea-HSA and synthetic sLea -PAA-biotin conjugates. Binding to the native antigen expressed on the cell surface was tested by FACS analysis on sLea positive DMS-79 cells and others and the biological activity was measured in CDC and ADCC assays. The binding affinity was determined by Surface Plasmon Resonance and the specificity of the carbohydrate binding was evaluated by ELISA and by glycan array analysis (CFG, Array 4.1). Recombinant antibodies were generated in CHO cells and purified on Protein A (IgG) or hydroxyapatite (IgM), respectively. We report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea -KLH vaccine. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by BiaCore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). Both antibodies were highly specific for Neu5Acα2–3Galβ1–3 (Fucα1–4) GlcNAcβ and did not bind to sialyl-Lewisx, Lewisa, and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 E/T ratio). These antibodies were tested in two xenograft models: 1) Treatment of animals with 5B1 on the day of engraftment with DMS-79 cells in a subcutaneous model completely prevented tumor growth. 2) Delayed treatment with various doses of 5B1 showed dose dependent protection up to complete cure in SCID mice engrafted (IV) with Colo205 cells. 7E3 antibodies did not show higher protection despite increased apparent affinity. The specificity and potency of 5B1 antibodies in CDC and ADCC assays translates into good activity in xenograft models. Further studies are warranted to explore the activity against additional cancer specific cell lines in animal models and to pursue preclinical development of 5B1 antibodies. Funding support by NCI #R42CA128362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C53.

Loss of Glucocorticoid Receptor Expression by DNA Methylation Prevents Glucocorticoid Induced Apoptosis in Human Small Cell Lung Cancer Cells

Human small cell lung cancer (SCLC) is highly aggressive, and quickly develops resistance to therapy. SCLC cells are typically insensitive to glucocorticoids due to impaired glucocorticoid receptor (GR) expression. This is important as we have previously shown that expression of a GR transgene induces cell death in-vitro, and inhibits tumor growth in-vivo. However, the underlying mechanism for loss of GR expression is unknown. The SCLC cell line, DMS79, has low GR expression, compared to non-SCLC cell lines and normal bronchial epithelial cells. Retroviral GR expression in DMS79 cells caused activation of the apoptotic pathway as evidenced by marked induction of caspase-3 activity. Methylation analysis of the GR promoter revealed some methylation in the 1D, and 1E promoters of the GR gene, however the ubiquitous constitutively active 1C promoter was heavily methylated. In the 1C promoter there was a highly significant increase in DNA methylation in a panel of 14 human SCLC cell lines compared to a mixed panel of GR expressing, and non-expressing cell lines, and to peripheral blood mononuclear cells. Furthermore, within the panel of SCLC cell lines there was a significant negative correlation seen between methylation of the 1C promoter, and GR protein expression. Reversal of GR gene methylation with DNA methyltransferase inhibition caused increased GR mRNA and protein expression in SCLC but not non-SCLC cells. This resulted in increased Gc sensitivity, decreased Bcl-2 expression and increased caspase-3 activity in SCLC cells. These data suggest that DNA methylation decreases GR gene expression in human SCLC cells, in a similar manner to that for conventional tumor suppressor genes.

ASCL1-coexpression profiling but not single gene expression profiling defines lung adenocarcinomas of neuroendocrine nature with poor prognosis

BackgroundLung adenocarcinoma is heterogeneous regarding histology, etiology and prognosis. Although there have been several attempts to find a subgroup with poor prognosis, it is unclear whether or not adenocarcinoma with neuroendocrine (NE) nature has unfavorable prognosis. Materials and methodsTo elucidate whether a subtype of adenocarcinoma with NE nature has poor prognosis, we performed gene expression profiling by cDNA microarray for 262 Japanese lung cancer and 30 normal lung samples, including 171 adenocarcinomas, 56 squamous cell carcinomas and 35 NE tumors. A co-expression gene set with ASCL1, an NE master gene, was utilized to classify tumors by non-negative matrix factorization, followed by validation using an ASCL1 knock-down gene set in DMS79 cells as well as an independent cohort (n=139) derived from public microarray databases as a test set. ResultsThe co-expression gene set classified the adenocarcinomas into alveolar cell (AL), squamoid, and NE subtypes. The NE subtype, which clustered together almost all the NE tumors, had significantly poorer prognosis than the AL subtype that clustered with normal lung samples (p=0.0075). The knock-down gene set also classified the 171 adenocarcinomas into three subtypes and this NE subtype also had the poorest prognosis. The co-expression gene set classified the independent database-derived American cohort into two subtypes, with the NE subtype having poorer prognosis. None of the single NE gene expression was found to be linked to survival difference. ConclusionCo-expression gene set with ASCL1, rather than single NE gene expression, successfully identifies an NE subtype of lung adenocarcinoma with poor prognosis.

Human Monoclonal Antibodies to Sialyl-Lewisa (CA19.9) with Potent CDC, ADCC, and Antitumor Activity

The carbohydrate antigen sialyl-Lewis(a) (sLe(a)), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract and breast and on small-cell lung cancers. Since overexpression of sLe(a) appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe(a) is an attractive molecular target for tumor therapy. We generated and characterized fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe(a)-KLH vaccine. Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe(a) (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC(50) 0.1 μg/mL vs. 1.7 μg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells engrafted into SCID (severe combined immunodeficient mice) mice. Treatment during the first 21 days with four doses of r5B1 (100 μg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. On the basis of the potential of sLe(a) as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility.

Abstract B122: Human monoclonal antibodies to sialyl-Lewis-A with potent CDC and ADCC activity

Abstract The carbohydrate antigen sialyl-Lewis A (sLea) is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells, but is expressed minimally or not at all on normal tissues. sLea serves as a ligand for epithelial leukocyte adhesion molecules and higher expression of sLea was observed in patients with greater node involvement. Since over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells and tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting. Here we report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea vaccine at MSKCC. Antibodies were identified by ELISA using sLea-HSA conjugates and binding to the native antigen was verified by FACS analysis on DMS-79 cells. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by Biacore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). These antibodies did not bind to sialyl-Lewis X, Lewis A and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 ratio). These results are very encouraging and we believe that further studies to scale up and test these antibodies in various tumor challenge models are warranted. Since sLea is widely expressed on human cancers, such antibodies could eventually find utility in approximately half of the new cancer cases occurring each year. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B122.

Synthesis of sialyl Lewisa (sLea, CA19-9) and construction of an immunogenic sLea vaccine

Sialyl Lewis(a) (sLe(a)), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe(a) is known to be the ligand for endothelial cell selectins suggesting a role for sLe(a) in cancer metastases and adhesion. For these reasons, sLe(a) may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe(a) is structurally similar to sLe(x) and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe(a) will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe(a) hexasaccharide and its conjugation to KLH to construct a sLe(a)-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe(a)-HSA by ELISA and against sLe(a) positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe(a) plus GPI-0100 or unconjugated sLe(a) mixed with KLH plus GPI-0100 failed to produce antibodies against sLe(a). However, mice immunized with sLe(a)-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe(a)-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe(a) by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe(a) positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le(y), Le(x), and sLe(x) by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe(a) positive cancer in clinical settings.

Characterization of the Effects of Cyclooxygenase-2 Inhibition in the Regulation of Apoptosis in Human Small and Non–Small Cell Lung Cancer Cell Lines

Cyclooxygenase-2 enzyme (COX-2) is overexpressed in human non-small cell lung cancer (NSCLC) but is not expressed in small cell lung cancer. Selective COX-2 inhibitors have been shown to induce apoptosis in NSCLC cells, an effect which is associated with the regulation of intracellular MAP kinase (MAPK) signal pathways. Our aims were to characterize the effects of COX-2 inhibition by rofecoxib on apoptosis in human NSCLC and small cell lung cancer cell lines. The human NSCLC cell line NCI-H2126 and small cell lung cancer cell line DMS-79 were used. Constitutive COX-2 protein levels were first determined by Western blot test. Levels of apoptosis were evaluated by using propidium iodide staining on FACScan analysis after incubation of NCI-H2126 and DMS-79 with p38 MAPK inhibitor SB202190 (25 ?microM), NF-kappaB inhibitor SN50 (75 microg/mL), and rofecoxib at 100 and 250 microM. All statistical analysis was performed by analysis of variance. Western blot test confirmed the presence of COX-2 enzyme in NCI-H2126 and absence in DMS-79. Interestingly, rofecoxib treatment demonstrated a dose-dependent increase in apoptosis in both cell lines. Given this finding, the effect of rofecoxib on NF-kappaB and p38 MAPK pathways was also examined. Apoptosis in both cell lines was unaltered by SN50, either alone or in combination with rofecoxib. A similar phenomenon was observed in NCI-H2126 cells treated with SB202190, either alone or in combination with rofecoxib. In contrast, p38 MAPK inhibition greatly upregulated DMS-79 apoptosis in a manner that was unaltered by the addition of rofecoxib. Rofecoxib led to a dose-dependent increase in apoptosis in both tumor cell lines. This effect occurred independently of COX-2, NF-kappaB, and p38 MAPK pathways in DMS-79 cells. As such, rofecoxib must act on alternative pathways to regulate apoptosis in human small cell lung cancer cells.

Gene expression phenotyping of an ACTH-producing small cell lung cancer line

DNA microarray techniques were used to compare gene expression in an adrenocorticotropin (ACTH)-producing human small cell lung carcinoma line (DMS-79) with six other small cell lung cancer (SCLC) lines that do not produce ACTH. Twelve genes were expressed at more than five-fold higher levels in DMS-79 cells. Two transcription factors were the genes that exhibited the most remarkable over-expression: T-box 3 mRNA was detected at levels 19.31±3.78 times those observed in the SCLCs. Thyroid transcription factor (TTF-1, T/ebp, Nkx2.1) was expressed at 14.24±3.41-fold higher in DMS-79 cells. Seven genes were identified whose expression levels were at least five-fold lower in the ACTH-producing cell line. Variation in culture medium formulation did not significantly affect the gene expression profile of DMS-79 cells and expression data observed in microarray experiments were corroborated by northern blot analysis of RNA from the same cell lines. These experiments reveal new candidate genes that could be involved in the dysregulation of POMC gene expression manifested by ACTH-producing nonpituitary tumors.

Proopiomelanocortin gene expression and DNA methylation: implications for Cushing's syndrome and beyond.

Proopiomelanocortin gene (POMC) is recognised as playing an important role in the regulation of the hypothalamo-pituitary-adrenal axis, adrenal development and obesity. POMC is activated in ACTH-dependent Cushing's syndrome. The syndrome may occur when the highly tIssue-specific 5' promoter of human POMC is activated in pituitary and non-pituitary sites. Whilst the factors involved in transcription in the corticotrophs of the anterior pituitary gland are becoming well delineated, the mechanism of activation in non-pituitary sites is not fully understood. This promoter is embedded within a defined CpG island, and, in contrast to somatically expressed CpG island promoters reported to date, is methylated in normal non-expressing tIssues, but is specifically unmethylated in expressing tIssues, tumours and the POMC-expressing DMS-79 small-cell lung cancer cell line. Methylation in vitro is sufficient for silencing of expression. In particular, methylation near the response element for the tIssue-specific POMC activator PTX1, diminishes POMC expression. Sites outside the PTX1 response element may be important for binding, and this may have implications for pituitary development. DMS-79 cells lack POMC-demethylating activity, implying that the methylation and expression patterns are likely to be set early or prior to neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy. It is conceivable that in POMC neurons of the hypothalamus the POMC promoter is subject to a variable density of methylation with clear implications for the signalling of satiety and obesity.

The CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression.

Ectopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing's syndrome. ACTH is cleaved from POMC. The syndrome may occur when the highly tissue-specific promoter of the human POMC gene (POMC) is activated. The mechanism of activation is not fully understood. This promoter is embedded within a defined CpG island, and CpG islands are usually considered to be unmethylated in all tissues. We demonstrate that much of this CpG island is methylated in normal nonexpressing tissues, in contrast to somatically expressed CpG island promoters reported to date, and is specifically unmethylated in expressing tissues, tumors, and the POMC-expressing DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in all tissues. E2F factors binding to the upstream domain IV region of the promoter have been shown to be involved in the expression of POMC in SCLC. We show that these sites are methylated in normal nonexpressing tissues, which will prevent binding of E2F, but are unmethylated in expressing tissue. Methylation in vitro is sufficient for silencing of expression, which is not reversed by treatment with Trichostatin A, suggesting that inhibition of expression may be mediated by means other than recruitment of histone deacetylase activity. The DMS-79 cells lack POMC demethylating activity, implying that the methylation and expression patterns are likely to be set early or before neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy.

DIFFERENTIAL REGULATION OF CALCITONIN SECRETION IN NORMAL AND NEOPLASTIC PULMONARY NEUROENDOCRINE CELLS IN VITRO

Within the mammalian lung, cells with a neuroendocrine phenotype are few in number and are sparsely distributed. In contrast, neuroendocrine neoplasms represent a major group of lung cancers. The aim of this study was to develop a model of mammalian PNECs and to compare glucocorticoid regulation of calcitonin secretion in normal and neoplastic cells with neuroendocrine differentiation. Cell cultures of PNECs were initiated after the disaggregation of neonatal hamster lungs with 0.1% collagenase and fractionation of the resultant cell suspension on a gradient of iodixanol (1.320 g/mL). Cell fractions enriched in PNECs were identified by positive staining for 5-hydroxytryptamine and the presence of calcitonin. Calcitonin secretion was investigated after exposure to hydrocortisone (0 to 1000 nM). A dose-dependant inhibition of calcitonin secretion was seen after 7 days between 10 nM (55% of control), and 1000 nM (29%) hydrocortisone. Cell cultures grown in the presence of hydrocortisone also contained significantly fewer PNECs between 10 nM (90% of control), and 1000 nM (45%). Human bronchial carcinoid cells (NCIH727) cultured under identical conditions showed a similar inhibition of calcitonin secretion between 10 nM (53%) and 1000 nM (52%), although at these concentrations, no reduction in cell number was seen. In contrast, 2 human small cell lung cancer cell lines (DMS-79 and COR-L24 cells) showed no dose-dependent inhibition of calcitonin secretion and no effect on cell proliferation in response to hydrocortisone. These results show that enriched cultures of mammalian PNECs can be used to investigate functional aspects of their biology, including peptide secretion in response to potential regulators. Furthermore, calcitonin secretion is inhibited in normal PNECs and bronchial carcinoid cells at physiological concentrations of glucocorticoids, but this feature appears not to be present in the 2 more invasive neuroendocrine neoplasms (small cell lung cancer cells) investigated in this study.

Analysis of the human proopiomelanocortin gene promoter in a small cell lung carcinoma cell line reveals an unusual role for E2F transcription factors.

The small cell lung carcinoma (SCLC) cell line DMS-79 has been used as a model for studying the molecular mechanism underlying the ectopic ACTH syndrome. We previously showed that two domains of the human Proopiomelanocortin (POMC) gene promoter were specifically active in DMS-79 cells. The present study focuses on the more distal one, Domain IV (-376/-417). DNaseI footprinting experiments identified a single binding site for DMS-79 cell proteins in this domain. Gel-shift and sequence analysis indicated that E2F proteins might bind this site. Indeed, proteins from DMS-79 cells which bind this site (i) have in vitro DNA binding properties indistinguishable from those of E2F proteins (ii) form, like E2F proteins, multiprotein complexes which can be dissociated by sodium deoxycholate and (iii) are recognized by antibodies directed against E2F proteins. Further, we show that the rat POMC distal promoter domain contains a homologous sequence which constitutes a natural mutant of the human POMC E2F binding site, since it does not bind E2F. We show by transient transfection that this natural mutant, in the context of the rat POMC promoter, is not active in DMS-79 cells by contrast to the human POMC E2F binding site. We conclude that E2F binding is required for the activity of Domain IV in DMS-79 cells and contributes to the expression of the POMC gene in SCLC. Further studies are required to elucidate the role of E2F factors in POMC gene transcription in SCLC cells, but our results have identified mechanisms different from those in pituitary corticotroph cells that are used by these SCLC tumor cells.

Analysis of proopiomelanocortin gene transcription mechanisms in bronchial tumour cells

The ectopic ACTH syndrome results from the transcription of the proopiomelanocortin (POMC) gene in non pituitary tumors. To determine its mechanisms, we examined in the human bronchial carcinoma cell line DMS-79 transacting factors binding to the human POMC gene promoter. Three binding sites were identified in the proximal promoter and proteins were studied by gel-shift assays. One of them is a binding site for Nur77/Nurr1 proteins in corticotroph cells but is bound in DMS-79 cells by factor(s) distinct from these proteins. The remaining two binding sites bound yet unidentified proteins and were both functionally active in DMS-79 cells. We also showed that DMS-79 cells lacked a factor required for tissue-restricted POMC gene expression in corticotroph cells. Altogether, our results indicate that POMC gene expression in DMS-79 cells is achieved without several of the corticotroph factors and provide a preliminary characterization of some factors involved in this process. They also reveal that DMS-79 cells are deficient in proteins involved in the regulation by cAMP and glucocorticoids.

Expression of 11β-hydroxysteroid dehydrogenase type 2 in an ACTH-producing small cell lung cancer

Non-pituitary tumors that produce adrenocorticotropic hormone (ACTH) exhibit resistance to the normal feedback effects of glucocorticoids on proopiomelanocortin (POMC) gene expression. This glucocorticoid resistance is typically complete, although some tumors show only relative glucocorticoid resistance in the clinical setting. The molecular mechanisms responsible for these clinical pathophysiologic observations are unknown, but might include glucocorticoid receptor defects or aberrant expression of enzymes or transporters that exclude glucocorticoids from access to their intracellular receptors. We examined whether ACTH-producing non-pituitary tumor cells might express 11β-hydroxysteroid dehydrogenase (11β-HSD), the principal ‘gatekeeper’ enzyme known to metabolize glucocorticoids. 11β-HSD mRNA and enzyme activity were assessed in DMS-79 cells, a line derived from an ACTH-producing small cell lung cancer. RT-PCR studies showed expression of mRNA encoding 11β-HSD2 but not 11β-HSD1 in DMS-79 cells. Control human fibroblasts expressed predominantly 11β-HSD1 but also had detectable 11β-HSD2 mRNA, while HepG2 hepatoma cells also expressed only 11β-HSD2 mRNA. Whole cell assays in DMS-79 cells revealed 11β-HSD activity with a K m for cortisol of 26.1 ± 9.0 nM and V max of 57.0 ± 5.9 pmol/h/mg protein. HepG2 cells expressed a similar high affinity enzyme activity, while control fibroblasts expressed 11β-HSD activity with a K m for cortisol of 652 nM. Conversion of cortisol to cortisone in DMS-79 cells was inhibited to 7% of baseline by addition of 10 μM glycyrrhetinic acid. Dexamethasone (20 nM) was converted to a single product in DMS-79 cells at a rate of 17.2 pmol/h/mg protein; this activity was also inhibited by glycyrrhetinic acid. We conclude that DMS-79 cells express 11β-HSD2. While DMS-79 cells harbor additional defects in glucocorticoid signaling, these data suggest that expression of 11β-HSD2 might contribute to the development of the glucocorticoid-resistant phenotype of some ACTH-producing tumors.