Effect of calcium channel agonist Bay K 8644 on calcitonin secretion from a rat C-cell line

Bay K 8644, a novel dihydropyridine, stimulates calcitonin secretion in a dose-dependent manner from a rat medullary thyroid carcinoma cell line, rMTC 6-23, and causes an increase in cytosolic free calcium concentration, as measured by quin-2. These effects are competitively inhibited by nifedipine, and completely abolished in the absence of extracellular calcium. These data suggest that calcium influx via voltage-dependent calcium channels plays a crucial role in the regulation of cytosolic free calcium concentration and calcitonin secretion.     

Phorbol ester stimulates calcitonin secretion synergistically with A23187, and additively with dibutyryl cyclic AMP in a rat C-cell line

The mechanism of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on calcitonin secretion was studied in a rat C-cell line, rMTC 6–23. TPA stimulated calcitonin secretion at the concentration of 16nM. This effect was synergistically enhanced with calcium ionophore, A23187. Synthetic diacylglycerol, 1-oleoyl-2-acetyl-glycerol (OAG), also showed a synergism with A23187 on calcitonin secretion. When dibutyryl cyclic AMP was added with TPA, an additive effect was obtained. These data suggest that C-kinase might be a possible regulator of calcitonin secretion in addition to the cyclic AMP-mediated pathway.     

Somatostatin receptor subtype 1-selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor and so far medical therapy has provided inconclusive results. In the human MTC cell line TT, expressing all somatostatin (SST) receptor subtypes, cell proliferation decreases with SST and SST receptor subtype 2 (sst(2)), but not sst(5), selective agonist treatment, whereas calcitonin (CT) expression and secretion are reduced by SST, but not by sst(2) and sst(5) agonists. The effectiveness of two new SST analogs, BIM-23926 and BIM-23745, selectively interacting with sst(1), was investigated in the TT cell line. DNA synthesis is significantly reduced by BIM-23926 (27-40% at 10(-10)-10(-6)M) and BIM-23745 (32-90% at 10(-8)-10(-6)M). Viable cell number is also significantly reduced by both BIM-23926 (40% at 10(-12)-10(-6)M) and BIM-23745 ( approximately 40% at 10(-10)-10(-6)M). Treatment with sst(1)-selective agonists significantly reduces CT secretion and gene expression, with a reduction of CREB phosphorylation. These findings suggest that potent sst(1)-selective agonists could have a therapeutic role in MTC.     

Terminally differentiated postmitotic tumor cells in a rat rhabdomyosarcoma cell line

A permanent rat rhabdomyosarcoma cell line (BA-HAN-1C) has been established, the phenotype of which is characterized by the coexistence of undifferentiated mononuclear cells and differentiated multinuclear myotube-like giant cells. The failure of attempts to separate these two cell types by repeated recloning procedures indicates their close histogenetic relationship and suggests that differentiation in this tumor proceeds in a similar manner to that in normal striated muscle where postmitotic myotubes arise from mononuclear myoblasts by fusion. The morphologically undifferentiated mononuclear tumor cells were shown to be actively proliferating and to incorporate thymidine methyl-3H(3H-TdR). The myotube-like giant cells neither incorporated 3H-TdR nor underwent mitosis or exhibited any clonogenic potential. After retransplantation into syngenic rats, tumor growth was markedly retarded when the tumor cell inoculum contained a high percentage of myotube-like giant cells. These data show that proliferative activity in this rhabdomyosarcoma cell line is confined to the mononuclear tumor cell compartment, the multinuclear myotube-like giant cells having withdrawn from the cell cycle and represent terminally differentiated postmitotic cells. This cell line should provide a valuable tool for further investigation of coherent aspects of proliferation and differentiation using various differentiation inducers.     

Characterization of LHY-821, a novel moderately differentiated endometrial carcinoma cell line

Endometrial cancer is a major problem for women but only a small number of comprehensively characterized cell models are available for studies. Here, we established a new cell line derived from a Stage IIIc(1) Grade 2 endometrial adenocarcinoma. The cell line, designated LHY-821, was characterized using growth curve, karyotyping, immunohistochemical staining, immunoblotting, drug sensitivity assay, invasion assay, and xenografting in nude mice. LHY-821 has a doubling time of about 46?h and a colony-forming efficiency of approximately 71?%. These cells expresse high levels of progesterone receptor but not estrogen receptor and are sensitive to medroxyprogesterone acetate (MPA). LHY-821 also expresses pan-cytokeratin, PTEN, p53, β-catenin, IGF-1, and IGF-2. In addition, karyotype analysis revealed that LHY-821 possessed a near diploid karyotype including 6q-, 10p-, Xq-, 13q+, 17p+, and Triplo-12. LHY-821 showed highly tumorigenicity in nude mice (100?%) and weak invasiveness. Chemosensitivity tests showed that LHY-821 was sensitive to both carboplatin and paclitaxel. LHY-821 is an immortalized cell line which had survived more than 80 serial passages; it may provide a novel tool to study the molecular mechanism and potential treatment for endometrial cancer.     

Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line

Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.     

Inhibition of tumor cell growth by a human B-cell line

An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B-cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20⁺, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy-BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy-BM binding. Lytic activity of Hairy-BM was found only against Breast-M.Abbreviations sIg Surface immunoglobulin - CTL Cytotoxic T-cells - NK Natural killer - IL2 Interleukin 2 - LAK Lymphokine activated killer - CSN Culture supernatant - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoolium bromide - PCR Polymerase chain reaction - TIL Tumor-infiltrating lymphocytes - HCL Hairy cell leukemia - TNF Tumor necrosis factor     

Characterization of L-glutamine transport by a human neuroblastoma cell line

This study characterized theNa⁺-dependent transport of L-glutamine by ahuman neuroblastoma cell line, SK-N-SH. The Na⁺-dependentcomponent represented >95% of the total glutamine uptake. Kineticstudies showed a single saturable high-affinity carrier with aMichaelis constant (K_m) of 163 ± 23 µMand a maximum transport velocity (V_max) of13,713 ± 803 pmol · mgprotein¹ · min¹. Glutamine uptakewas markedly inhibited in the presence of L-alanine, L-asparagine, and L-serine. Li⁺ didnot substitute for Na⁺. These data show thatL-glutamine is predominantly taken up through systemASC. Glutamine deprivation resulted in the decrease of glutamine transport by a mechanism that decreasedV_max without affectingK_m. The expression of the system ASC subtypeASCT2 decreased in the glutamine-deprived group, whereas glutaminedeprivation did not induce changes in system ASC subtype ASCT1 mRNAexpression. Adaptive increases in Na⁺-dependent glutamate,Na⁺-dependent 2-(methylamino)isobutyric acid, andNa⁺-independent leucine transport were observed underglutamine-deprived conditions, which were completely blocked byactinomycin D and cycloheximide. These mechanisms may allow cells tosurvive and even grow under nutrient-deprived conditions.

     

Ultrastructure of double minutes from a human tumor cell line

Double minutes (dm) have been isolated from human tumor cells by zonal centrifugation and by differential pelleting of chromosome suspsension. These methods allowed collection of dm in sufficient quantity and purity for visualization with electron microscopy. Ultrastructurally, the chromatin fibers in dm resemble thrance fragments was found. When the two isolation protocols were compared, differential pelleting was shown to increase purity twofold to 85% dm by mass. The differential pelleting procedure enables easy collection of dm in sufficient quantity and purity for chemical analysis.     

Secretion of antileucoprotease from a human lung tumor cell line

Two human tumor cell lines were analyzed for the production of human antileucoprotease (ALP). One of them, a human squamous lung carcinoma cell line (HS-24) synthesized, as confirmed by Western blot analysis, high amounts of ALP in serum-free medium. The supernatant inhibited elastase, chymotrypsin and trypsin. Northern blot analysis with an 18-mer radiolabelled oligonucleotide, derived from an ALP specific cDNA clone, revealed a specific mRNA of about 700-800 nucleotides in HS-24 tumor cells. In contrast, a secondary human lung tumor cell line (SB-3), derived from the adrenal cortex, did not synthesize ALP when assayed under identical conditions. The supernatant inhibited only trypsin and chymotrypsin.     

Immunoextracted calcitonin in human gastric secretion

Immunoreactive calcitonin (iCT) has been demonstrated in human gastric juice after immunoextraction with immobilized antibodies and subsequent radioimmunoassay. The basal levels were 4.5 +/- 3.1 (mean +/- SD) pg-Eq/ml gastric juice; range 1.2-9.1 pg-Eq/ml; n = 7, and after stimulatory gastric secretion test with pentagastrin 0.3 +/- 0.2 pg-Eq/ml; range 0.1-0.7 pg-Eq/ml; n = 7 (p less than 0.01). The main fraction of iCT from gastric juice eluted in the same region as synthetic human calcitonin (hCT) on Sephadex G-75 gel chromatography. Reverse phase chromatography in a fast protein liquid chromatography (FPLC) system revealed a slightly less hydrophobic character of the iCT from gastric juice compared to synthetic monomeric hCT. The results were further confirmed by using an additional antiserum. In plasma, the calcitonin (CT) levels were after immunoextraction at the basal state 6.6 +/- 1.7 pg-Eq/ml (mean +/- SD); range 5.1-10.1 pg-Eq/ml; n = 7 and after pentagastrin stimulation 9.4 +/- 5.4 pg-Eq/ml; range 6.3-18.5 pg-Eq/ml; n = 7.     

Growth factor production by a human megakaryocytic tumor cell line

A recently described human megakaryocytic tumor cell line was analyzed for the presence of growth factor activity and was found to produce large quantities of transforming growth factor beta-like (TGF-beta) and basic fibroblast growth factor-like (bFGF) activities. Growth factor activities were identified using a radioreceptor assay for the TGF-beta-like activity, a heparin-binding assay for the b-FGF-like activity, and a demonstration of distinct biological activities for each type of factor. Tumor poly-A+ RNA revealed strong signals when probed with complementary DNA corresponding to bovine basic FGF and human TGF-beta and weak signals when probed with cDNA corresponding to epidermal growth factor (EGF) and TGF-alpha. The levels of EGF and TGF-alpha produced in the tumor line were too low to be detected by radioreceptor assays. Relative levels of messenger RNA encoding each of the growth factors reflected the relative levels of each of the respective factors tested. These data represent the first definitive identification of FGF-like activities in megakaryocytic-like cell lines. Interestingly, the line displayed little activity similar to platelet-derived growth factor (PDGF) when assayed either biochemically or by poly-A+ RNA analysis.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line

Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors.     

Characterization of JOK-1, a human gastric epithelial cell line

Summary Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line “JOK-1” These cells express conventional, epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherins, β-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epitheliallike barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line “MDCK” also expresses serveral epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherin, β-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.