The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion.

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy.

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.

Establishment and characterization of continuous cell line MTC-SK derived from a human medullary thyroid carcinoma.

Tumor cells of a human medullary thyroid carcinoma were isolated and propagated in tissue culture. Several cell lines with different morphology developed from the primary culture, among others a fibroblast-like growing cell line (MTC-F) and a cell line growing as a suspension of single cells and spherical cell clusters (MTC-SK). The MTC-SK cell line was serially propagated for 90 passages, over 3 years. When examined at different times throughout the in vitro period, MTC-SK exhibited properties characteristic of medullary thyroid carcinomas: the cells maintained their epithelioid morphology; endocrine granules were demonstrated in the cytoplasm by electron microscopy; in situ hybridization confirmed the production of calcitonin- and bombesin-mRNA (gastrin releasing peptide); the cells revealed positive immunoreactivity with antibodies to calcitonin, calcitonin gene-related peptide, and bombesin. The in vitro properties of the MTC-SK cells corresponded to the results obtained from the tissue of origin. Cytogenetic studies of the MTC-F cell line revealed a supernumerary metacentric chromosome (20?). In the MTC-SK cell line the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16.

Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region.

Establishment of tumor cell lines as model systems for studying tumor biology or as a part of immunotherapeutic anti-cancer strategies is of high importance, whereby the highest possible preservation of the original tumor cell phenotype is a prerequisite for these aims. Since overexpression of the catalytic subunit of human telomerase (hTERT) is known to minimally alter the cellular phenotype, we focused on the establishment of cell lines derived from human fibroma from a MEN1 patient by ectopic expression of hTERT. Additionally, a cell line was generated by introduction of the early region of SV40 (SV40 ER). Both approaches resulted in continuous cell lines, and neither T1-LOHG (hTERT) nor SV1-LOHG (SV40 ER) showed a transformed phenotype. While SV40 ER-transfected cells underwent dramatic changes in morphology and growth characteristics, hTERT-expressing cells indeed retained a phenotype highly similar to the parental cells. Nevertheless, hTERT overexpression resulted in increased growth rates after about 70 population doublings (PD) and alterations of mRNA levels of genes associated with tumor pathogenesis. Thus, our data suggest that ectopic hTERT expression leads to immortalization of LOHG-F, sustaining many characteristics of the non-transfected counterparts, but continuous growth in vitro is associated with changes of the cellular phenotype.

Stimulation of autologous antitumor T-cell responses against medullary thyroid carcinoma using tumor lysate-pulsed dendritic cells.

Dendritic cells (DCs) have attracted wide interest because of their unique capacity to elicit primary and secondary antitumor responses. We have generated autologous tumor lysate-pulsed DCs from three patients with medullary thyroid carcinoma (MTC) and tested them for their ability to stimulate cytotoxic T-cell responses against autologous MTC tumor cells in vitro. The aim of our investigations was to evaluate the potential efficacy of DC-based immunotherapy in patients with MTC. DCs were generated from peripheral blood monocytes using GM-CSF and IL-4 (immature DCs) or GM-CSF, IL-4, and TNFalpha (mature DCs). Our results indicate that mature tumor lysate-pulsed DCs are able to elicit a human leukocyte antigen class I-restricted cytotoxic T-cell response against autologous MTC tumor cells, whereas immature tumor lysate-pulsed DCs do not stimulate significant antitumor activity. We feel that our data may be relevant for future clinical trials of active immunotherapy using tumor lysate-pulsed DCs in patients with MTC who have residual or distant disease after surgical treatment. The fact that mature DCs displayed a substantially higher capacity to stimulate autologous antitumor T-cell responses than immature DCs underlines the importance of a maturation step in immunotherapy protocols based on DCs.

Cytotoxic activity of camptothecin and paclitaxel in newly established continuous human medullary thyroid carcinoma cell lines.

At the time of diagnosis, more than one quarter of patients with medullary thyroid carcinoma (MTC) has distant metastases. Only few of these patients can be cured by surgery. Standard chemotherapy is characterized by low response rates and short response time. The establishment of eight human MTC cell lines provides a new basis for in vitro investigation of cytotoxic drugs. Camptothecin (CPT) and paclitaxel, which never have been investigated in the treatment of MTC, were tested for their cytotoxic profile in comparison with the clinically ineffective dacarbazine. Eight MTC cell lines were established from seven patients with MTC. IC(50) values were calculated from dose-response relationships using cell counts and a formazan dye assay (WST-1). IC(50) values were 3.5 +/- 1.2 nmol/liter for CPT and 8.2 +/- 1.9 nmol/liter for paclitaxel. Dacarbazine showed no reduction of cell proliferation at concentrations 10-fold higher than clinically achievable. Given peak plasma concentrations of 65 +/- 20 nmol/liter for CPT and 1 micro mol/liter for paclitaxel, these promising in vitro results provide a basis for the performance of clinical trials in patients with advanced MTC.

Comparative genomic hybridization and cytogenetic analysis in a bronchial adenoma: three clones with different chromosomal aberrations.

Classical chromosomal analysis and comparative genomic hybridization (CGH) were performed in a tubular bronchial gland adenoma. Trisomy of chromosomes 2, 11, 18 and 20 and clonal loss of Y were found in cultured cells derived from two different kryotubes; this was also confirmed by CGH from one of these tubes. Cells from two other tubes, investigated by CGH only, showed gains and losses of parts of chromosome 11q in one, and in the second additional gain of the distal portion of 9q and 17q, respectively. CGH analysis of tumor DNA extracted from paraffin-embedded sections showed no chromosomal imbalances. In cell culture growth the advantage of specific clones probably altered the clone distribution. This study highlights the risk of cytogenetic analysis based on cell cultures only.

New continuous cell-line from human medullary-thyroid carcinoma - sinj - phenotypic analysis and invivo carcinogenesis.

SINJ is a new continuous human cell line derived from a lymph node metastasis of a probably sporadic medullary thyroid carcinoma. It is compared to MTC-SK, another medullary thyroid carcinoma cell line, established earlier (1). SINJ has been continuously cultivated in vitro for two years. The cells grow as a suspension of single cells and cell clusters. Repeated immunocytochemistry showed positive immunoreactivity with antibodies to CT, CGRP and GRP. The maintenance of NSE and chromogranins were proved. Northern blot analysis confirmed endocrine activity at mRNA level. Flow cytometry of 27 SINJ - clones showed 25 diploid and two tetraploid subpopulations. Cytogenetic analyses strengthened these findings. According to DNA analysis the cells are free of SV40 sequences. Tumorigenicity was proved in nude mice. The new cell line SINJ has potential use for in vitro studies of medullary thyroid carcinomas in sporadic as well as hereditary forms - the MEN2A syndrome.

The affinity of MCF7 breast cancer cells to hyaluronan substrates of different molecular weight and concentrations in an in vitro model.

The affinity of MCF7 breast cancer cells to hyaluronan (HA) was investigated in an in vitro model. The cells form a tightly adhering monolayer on native HA with a concentration of 5 mg/ml. On native HA at higher concentrations the cells reduce their adhesion to the substrate in favor of increased intercellular bonds, resulting in a cluster-like aggregate that tends to detach from the substrate. Aggregate formation is accomplished after 12 h incubation. The phenomenon is independent of the CD44 receptor. Degradation of native HA by hyaluronidase abolishes aggregate formation even at high HA concentrations in favor of formation of a firmly adhering monolayer. This model may help to understand tumor spread on HA tissue structures and may explain therapy successes with hyaluronidase in tumor patients.

Cytogenetic and CGH studies of four neuroendocrine tumors and tumor-derived cell lines of a patient with multiple endocrine neoplasia type 1.

A malignant insulinoma (LOHG-I), a carcinoid of the lung (LOHG-L), a parathyroid adenoma (LOHG-NSA), and a fibroma (LOHG-F) were obtained from a patient with multiple endocrine neoplasia type 1 (MEN1). Long-term cultures were established. Essential neurobiological properties of the cell lines were proven immunocytochemically and by electron microscopy. Molecular analysis of the germline DNA showed a 4 bp deletion in exon 3 of the MEN1 gene. Cytogenetic and CGH analyses of the tumors/tumor cell lines revealed diploidy and balanced and unbalanced structural aberrations different for each tumor. Chromosomes 6q21, 11q and 17q were most frequently involved in clonal structural aberrations.

Establishment of a continuous cell line from a human carcinoid of the small intestine (KRJ-I).

A new continuous cell line from a human malignant carcinoid of the small intestine (KRJ-I) was established. The cells showed morphological and immunocytochemical features of the tumor of origin and expressed estrogen receptors. The cells are growing as a suspension, forming multicellular aggregates and spheroids. Electron microscopy confirmed the presence of neuroendocrine granules. Dose-dependent growth inhibition was observed after incubation with 5-azacytidine. Cytogenetic analyses of the tumor of origin, the cell line KRJ-I and a liver metastasis KRJ-II revealed clonal tetraploidy and clonal loss of the Y-chromosome and chromosome 19.

Vitamin E binding protein afamin protects neuronal cells in vitro.

Afamin, an 87 kDa human plasma glycoprotein with specific binding properties for vitamin E (alpha-tocopherol) was recently characterized (Jerkovic, 1997; Vögele, 1999). In the present study the in vitro effects on neuronal cells of native human Afamin, of Afamin pre-loaded with vitamin E (Afamin+), and of vitamin E were investigated. Isolated cortical chicken neurons were maintained either under apoptosis-inducing low serum conditions or exposed to oxidative stress by the addition of H2O2 or beta-amyloid peptide(25-35). Afamin and vitamin E synergistically enhance the survival of cortical neurons under apoptotic conditions. Furthermore, Afamin alone protects cortical neurons from cell death in both experimental settings. Therefore, the plasma glycoprotein Afamin apparently displays a neuroprotective activity not only by virtue of binding and transporting vitamin E but also on its own.

[Flow cytometric measurements of in vivo incorporated bromodeoxy- uridine into the DNA of experimental and human tumors]. / Flusszytometrische Messungen von in vivo inkorporiertem Bromodeoxyuridin (BUdR) in die DNA von experimentellen und menschlichen Tumoren.

A new cell kinetic method was used in vivo in an experimental mouse tumour and in human tumours of the oral cavity for the first time. The incorporation of BUdR into cells in the S-phase that are actively synthesizing DNA enables the determination of the rate of DNA synthesis from the amount of BUdR-related fluorescence using monoclonal antibody and the proportion of S-phase cells which are actively engaged in synthesis. The application of BUdR to the patients and the preparation of the tumour is reported.

Development of standardized cell culture conditions for tumor cells with potential clinical application.

BACKGROUND: Tumor cell lines have enormous value for the study of different aspects of cancer biology and have also recently gained great importance in autologous cell-based anti-tumor therapies. However, the use of these cells is still limited because in vitro growth is hampered by suboptimal culture conditions and current media contain fetal bovine serum (FBS), which poses serious safety concerns regarding clinical application. METHODS: To address this drawback, we aimed to develop a strategy for optimization of the culture medium for human medullary thyroid carcinoma (MTC) cell lines as a model system. We combined the general cell screening system (GCSS), which continuously measured the growth behavior of cells in a 96-well plate format, with statistically based experimental designs. RESULTS: The results obtained clearly demonstrated that, just by changing the composition of the basal medium, a significantly enhanced growth rate could be observed, and by subsequent addition of several substances a serum-free cell culture medium could be developed. This medium allowed the propagation of two MTC cell lines comparable with conventionally used serum-supplemented medium. DISCUSSION: We present a fast and easy way to screen for substances that are essential for tumor cell growth in vitro. Furthermore, these tumor cells can be adapted to culture conditions that allow the use of the cells in safe cell-based therapies. This is of utmost importance because of increasing regulatory requirements.

Long-term tissue culture of human pheochromocytomas.

Two human pheochromocytomas were cultured for long periods. One was cultured for a year, the other is still growing after a cultivation time of seven months. Though decreasing with the age of cultures the cells contained catecholamines all the time. Catecholamines were demonstrated by electron microscope, by fluorescence histochemistry and by Vulpian's and Da Prada's methods. Both pheochromocytomas were responsive to Nerve Growth Factor and showed dose-dependent development of neurite-like processes.