The growth-inhibition effect of tamoxifen in the combination chemotherapeutics on the human cholangiocarcinoma cell line QBC939

In the individual application of adriamycin, mitomycin, vindesine and their combined application with tamoxifen for the pre-treatment of the human cholangiocarcinoma cell line QBC939, QBC939 was determined by MTT assay to investigate the inhibitive effect and its initial mechanism of TAM on cell growth. Growth cycle and apoptosis of each group were determined by flow cytometry. Concentration of ADM in QBC939 was detected by flow cytometry. The levels of their P-glycoprotein were detected by immunohistochemistry. The mRNA and protein levels of apoptotic-associated genes Bcl-2 and Bax were determined by western blot and real-time PCR. The inhibitive rates of adriamycin, mitomycin, vindesine to QBC939 and the apoptosis rates of QBC939 were enhanced after the pre-treatment of tamoxifen. Influence of tamoxifen in their growth cycle was not so obvious except vindesine group because of the increasing cell numbers of G ₂/M phase in which cells may be blocked. The contents of adriamycin in cells rose after the pre-treatment of tamoxifen. Expression level of the multi-drug resistant protein on cell surface was shown as (+). Furthermore, real-time PCR and Western blot analysis revealed an upregulation of Bcl-2 and a downregulation of Bax in QBC939 after the pre-treatment of tamoxifen. Therefore, tamoxifen may have the ability to enhance the relative sensitivity of QBC939 to chemotherapeutics.     

Characterization of plasminogen activator produced by an established cell line from human ovary

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.     

Radiosensitivity of the cells of an established human melanoma cell line and the parent melanoma xenograft

Survival curves of cells from a human melanoma xenograft (E.F.) and a cell line (FME) established from this xenograft were determined. The cells of the established line were harvested from exponentially growing cultures, plateau phase cultures or solid tumours in athymic mice (FME-X) before irradiation. During irradiation the cells were kept suspended in culture medium. The colony forming ability of the cells was assayed in soft agar. The Do-value was significantly higher for the parent xenograft than for the established line, whether grown in vitro or in vivo (p less than 0.0001). In addition, the Dq-value was significantly lower for the xenograft than for exponentially growing cultures of the established line (p less than 0.05). Thus the radiation response of the cells of the established line was not representative for that of the cells from the parent xenograft. It is concluded that survival curves for established cell lines should be used with great caution in attempts to predict the radiocurability of human tumours of corresponding histological type.     

The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.     

The growth response of the established human cell line, KB, in an acidified medium.

Supplementing Eagle's Minimal Essential Medium with pyruvic acid extended the pH range for maximum growth of KB cells from pH 7.0–7.4 to pH 6.6–7.2. The stimulatory effect of pyruvic acid was independent of pH in the range 6.6–7.2. In contrast to WI-38, KB cells are not adversely effected by the addition of glucose to Eagles Essential Medium containing galactose.     

Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

Spontaneous chromosome rearrangements in an established cell line of Drosophila melanogaster

The chromosomal situation of the GM₃ line of Drosophila melanogaster was observed over a period of one year. From an initial homogeneous condition a karyotypic polymorphism evolved; four different karyotypes, identified by fluorescence patterns, emerged in the population and continued to multiply. The chromosomal rearrangements giving rise to the new karyotypes involved only heterochromatic sections.     

Giemsa banding in an established line of a human malignant meningioma

Summary Giemsa banding confirms the loss of a No. 22 chromosome in an established line of a human malignant meningioma previously demonstrated by fluorescence on short term cultures.

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Zusammenfassung Der Verlust eines Chromosoms Nr. 22 wurde durch Analyse der Giemsa-Bandmuster an einer Langzeit-Zell-Linie eines menschlichen malignen Meningeoms bestätigt. Dieser Befund war schon zuvor durch Fluorescenzfärbung an Kurzzeit-Kulturen erhoben worden.

     

Use of an established human hepatoma cell line with endogenous bioactivation for gene mutation studies

Genetic toxicology assays that rely on S9 microsomal mixes are subject to artifacts related to the generation of mutagenic metabolites by acidic pHs, variation in individual isolations of microsomes and the failure of subcellular fractions to faithfully produce metabolites generated in intact cells. We have developed a gene mutation assay utilizing the human hepatoma cell line HepG2, which has been shown to metabolize a broad spectrum of promutagens. Optimal conditions for assaying the induction of 6-thioguanine-resistant mutants in this cell line include: 1) growth of colonies for three weeks on lethally irradiated feeder layers of 10⁶ thioguanine-resistant HepG2 cells (average plating efficiency = 60–80%); 2) a thioguanine concentration in selection dishes of 10^–4M with a maximum seeding density of 2.5 × 10⁵ cells per 100 mm culture dish; and 3) a minimum expression time of 6 days. In addition to ultraviolet light C (254 nm), a cytochrome P₄₅₀ (cyclophosphamide)-dependent and a cytochrome P₄₄₈ (aflatoxin B₁)-dependent promutagen were shown to induce cytotoxicity and mutations in this test system. The present studies, therefore, suggest that the HepG2 cell line may be useful for a variety of assays in genetic toxicology.Abbreviations HAT hypoxanthine, aminopterin, thymidine - TG 6-thioguanine     

Ultrastructural localization of polysaccharides in the vacuolar system of an established cell line

Polysaccharides in the vacuolar system of an established line of monkey kidney epithelial cells, were investigated by high resolution radioautography following incorporation of 3H-fucose and by the periodic acid-thiosemicarbazide-silver proteinate staining method. Significant amounts of glycogen and glycoproteins were found in different lysosomes and prelysosomes. The radioautographic labeling of these substances correlated with the cytochemical observations. Analysis of the 3H-fucose pattern of labeling suggests that glycoproteins are transported through the different components of the vacuolar system of the cell in a sequential fashion rather than via independent pathways. The possible functional significance of polysaccharides in lysosomes is discussed. It is suggested that glycogen is taken up by lysosomes through autophagic segregation and through infolding and vesiculation of the lysosomal surface, and that the majority of glycoprotein in the lysosomal membranes does not have acid hydrolase activity.     

A hypotetraploid human T lymphoid cell line established by cell fusion.

A human T lymphoid cell line was established by cell hybridization technique from peripheral blood leucocytes of a patient with Sezary syndrome. The cells beared the surface antigens of human T lymphocyte specificity as demonstrated by immune cytolysis tests, but did not form E rosettes with sheep red blood cells. Isozyme patterns of enzymes in this line such as lactate dehydrogenase, glucose 6-phosphate dehydrogenase and esterase were of human type. The line had 79 chromosomes in modal number. This case supports the proposal that the production of tetraploids is favourable for establishment of cell lines.     

Proteomic analysis of cholangiocarcinoma cell line

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countries, such as Thailand. Distinguishing CCA from hepatocellular carcinoma (HCC) of the liver often requires the use of histochemistry, so molecular markers for diagnosis and prognosis are still required. In this study, the two-dimensional (2-D) protein map of a Thai human bile duct epithelial carcinoma cell line (HuCCA-1) has been compared to human hepatocellular carcinoma cell lines (HepG2 and HCC-S102) and a human breast epithelial cancer cell line (MCF-7). Our results show that HuCCA-1 expressed a unique pattern of proteins. Forty-three major proteins were identified by matching to the map of MCF-7, and by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). Cytokeratins CK8 and CK18 were overexpressed in both HuCCA-1 and HCC, while CK7 and CK19 were only expressed in HuCCA-1. Four specific proteins with MW/pI 57.2/5.21 (U1, vimentin), 42.2/6.20 (U2), 43.2/6.20 (U3, EF-TU), and 42.2/6.40 (U4, unidentified) were absent from HepG2. U2 showed high expression in HuCCA-1, while U1 and U4 showed high expression in HCC-S102. U2 could be separated in 2 proteins, U2/1 (alpha-enolase) and U2/2 (not identified) by using IPG pH 4-7. Galectin-3 showed high expression level in HuCCA-1 by 1-DE immunodetection, and gave only one spot with MW 32.9 kDa and pI 8.29 on 2-DE immunoblotting, Thus, certain proteins, namely CK7, CK19, U2/2 and galectin-3, may be good markers useful for differential diagnosis of cholangiocarcinoma compared to hepatocellular carcinoma.     

Characterization of newly established human osteosarcoma cell line,LM-1

Summary The characteristics of Cell Line LM-1, established from a human osteosarcoma, have been studied extensively. The cell produced both bone-specific and placental-like alkaline phosphatases when treated with hydrocortisone 21-phosphate; they had specific membrane antigens that reacted with sera from osteosarcoma patients. Injection of LM-1 cells into newborn hamsters treated with antilymphocyte serum produced nodular tumors. The characteristics of LM-1 suggest that this tumor cell line has unique features that may be useful in a variety of studies of human and animal osteosarcoma. This research is supported in part by USPHS Grants HD-09938 and CA-25746.