Development of Cell Lines from the Sheep Used to Construct the CHORI-243 Ovine BAC Library

Two cell lines, designated MARC.OVSM, and MARC.OKF, were initiated from the aorta and kidney, respectively, obtained from the Texel ram used to make the CHORI-243 Ovine BAC library. These cell lines have been submitted to the NIA Aging Cell Repository at the Coriell Cell Respositories, Camden, NJ, USA, and will be made publicly available.     

Establishment in monolayer culture and characterization of four human melanoma cell lines.

Four new human melanoma cell lines were established in monolayer culture from xenograft lines originating from different patients. Several distinct characteristics of the source xenograft lines were retained in the cell lines, e.g., number of chromosomes, DNA-index, and cell ultrastructure. Cell volume was generally larger for the cell lines than for the corresponding xenograft lines, but the differences among the lines were similar in vitro and in vivo. The cell lines showed significant differences in growth pattern, i.e., cell motility and degree of intercellular contact. Cell cycle time (Tc) during exponential growth ranged from 15 to 21 h. The differences among the lines in Tc were mainly due to differences in the duration of S. Growth fraction was close to 100% and cell loss was negligible during exponential growth. Plating efficiency was 90-100% in the presence of feeder cells. The four cell lines represent a valuable supplement to the xenograft lines for future studies of the cell biology, pathophysiology, metastatic behavior, and treatment sensitivity of malignant melanoma.     

Generation of Mouse Small Intestinal Epithelial Cell Lines That Allow the Analysis of Specific Innate Immune Functions

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.     

On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos

The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells.     

The establishment of heliothine cell lines and their susceptibility to two baculoviruses

Summary A total of eight cell lines were established from Helicoverpa armigera (3) and H. punctigera (5) embryos and ovaries. Cell lines were established and grown in TC100 and/or TC199-MK containing 10% fetal bovine serum. The serum-free medium ExCell™ 400 was also used, with and without 10% supplemental fetal bovine serum, but failed to generate cell lines from fat bodies, embryos, or ovarian tissues. Cell lines consisted of heterogenous cell types ranging from oval to fibroblast-like. This is the first report on the successful establishment of cell lines from H. punctigera. Cell lines from the two species were distinguishable from each other by DAF-PCR, and noticeable differences in minor bands were observed among cell lines from the same species. All of the established cell lines from both species were susceptible to HzSNPV but did not replicate more virus than that of a H. zea cell line (BCIRL-HZ-AM1-A11). However, an H. punctigera cell line (HP1) replicated AcMNPV to the highest titer (1.0×10⁸ 50% tissue culture infective dose/ml), and only one of the H. armigera cell lines (HA1) was susceptible to this virus.     

Establishment in monolayer culture and characterization of four human melanoma cell lines

Four new human melanoma cell lines were established in monolayer culture from xenograft lines originating from different patients. Several distinct characteristics of the source xenograft lines were retained in the cell lines, e.g., number of chromosomes, DNA-index, and cell ultrastructure. Cell volume was generally larger for the cell lines than for the corresponding xenograft lines, but the differences among the lines were similar in vitro and in vivo. The cell lines showed significant differences in growth pattern, i.e., cell motility and degree of intercellular contact. Cell cycle time (T_c) during exponential growth ranged from 15 to 21 h. The differences among the lines in T_c were mainly due to differences in the duration of S. Growth fraction was close to 100% and cell loss was negligible during exponential growth. Plating efficiency was 90–100% in the presence of feeder cells. The four cell lines represent a valuable supplement to the xenograft lines for future studies of the cell biology, pathophysiology, metastatic behavior, and treatment sensitivity of malignant melanoma.     

Establishment of cell lines from serial samples from two patients with lung tumours before and after chemotherapy

Six cell lines were derived from pleural effusions of two lung cancer patients and established in vitro in our laboratory. Cell line AE1 was obtained from a small cell lung cancer (SCLC) before the patient had received any chemotherapy; the other lines (AE2 and AE3) were established from tumour recurrences in the same patient after therapy. Cell lines DG1 and DG2 were derived from specimens of an untreated non-small cell lung cancer (NSCLC), while cell line DG3 originated from pleural effusions recurring in the same patient after therapy. The results of the present study show that: (a) the SCLC lines AE1, AE2 and AE3 are heterogeneous in their biological characteristics and in their chemosensitivity patterns. In particular lines AE2 and AE3 are less responsive to cis-Platinum (DDP) and Adriamycin (ADM) than line AE1, so that they may reflect resistant subpopulations existing within the original tumour, selected following therapy with these drugs. In contrast, however, line AE1 proved more resistant to Vepesid (VP16) than lines AE2 and AE3. (b) The three NSCLC lines are similar in various biological features as well as in their chemosensitivity to DDP and Vinblastine (VBL).Abbreviations NSCLC Non Small Cell Lung Cancer - SCLC Small Cell Lung Cancer Recipient of a Fellowship of the Italian Association for Cancer Research     

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preferential expression of a common T cell receptor structure by T cells induced to proliferate in vitro with a murine retrovirus.

We have successfully isolated continuous T cell lines from murine spleen which have been induced to proliferate after in vitro exposure to the murine leukemia virus RadLV. Cell lines isolated from several strains of mice have an "immature" phenotype and are immortalized CD4- CD8- CD3+ cell lines. Cell lines of similar phenotype have now been derived from many individual mice, after spleens have been infected with two different RadLV viruses, a leukemogenic and a nonleukemogenic isolate. Among cell lines induced with RadLV/C6VL, an unusually high proportion of cells was found to bind the 124-40 anticlonotypic antibody specific for the alpha beta TCR expressed by C6VL/1 cells which produces RadLV/C6VL. This was not reflected in cell lines induced with the RadLV/V13 isolate nor in various lymphocyte subsets freshly isolated from normal mice, or induced to proliferate in culture. Cells expressing a common TCR structure would appear to be appropriate targets for in vitro proliferation and transformation induced by RadLV.     

Marsupial cells in long-term culture

Cell lines have been developed from several species of Australian marsupials and studied during long-term growth. Cell lines developed from macropodid skin or heart tissues all had reproducible finite life-spans. However, cell lines developed from dasyurids showed bariable behavior in culture: lines developed from Antechinus stuartii and Dasyurus viverrinus had finite life-spans, while lines developed from Sminthopsis crassicaudata had indefinite life-spans. S. crassicaudata lines usually became heteroplloid, but one was still diploid after 150 population doublings, while another contained a proportion (10%) of haploid cells. Other lines were developed from the peramelid, Perameles nasuta, and the phanlngerid, Trichosurus vulpecula.     

淋巴抑瘤素对五株肿瘤细胞株的体外抑瘤效应的分析研究

淋巴细胞经刺激后分泌一种多肽类物质,这种细胞因子被称为淋巴抑瘤素。在体外培养中发现不同来源的肿瘤细胞对淋巴抑瘤素的敏感性是不同的,表现为三种类型:强反应株,此类细胞对其抑瘤效应反应强烈,抑制率达90％以上;弱反应株,此类细胞的抑制率在70％左右;另一类为负反应株,此类细胞对淋巴抑瘤素不但不表现出抑瘤效应,反而出现助长肿瘤细胞生长的效应。由于体外测定中有上述现象,所以建议在体内应用这类细胞因子时,应像抗菌素测抗菌谱一样测定其抗瘤谱,以利于对症用药。     

Development and partial characterization of heliothine cell lines from embryonic and differentiated tissues

The goal of this study was to generate cell lines from a variety of insect tissues that could be useful for developing in vitro assays with tissue-specific properties. In this article, we describe the establishment of new cell cultures from differentiated (primarily neural) and undifferentiated tissues (primarily embryonic) and their initial characterization. Cell lines were established from the following tissues of the budworm, Heliothis virescens, and the bollworm, Helicoverpa zea: larval ventral nerve cords (4 lines), larval midguts (1 line), adult ovaries (1 line), and embryonic tissues (11 lines). Cell lines were primarily characterized by morphological examination and polymerase chain reaction (PCR) (both deoxyribonucleic acid amplification fingerprinting and inter-simple sequence repeats PCR).     

A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)

Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies.     

Spontaneous cytotoxicity of human lymphoblast cell lines mediated by normal peripheral blood lymphocytes. I. Differential susceptibility of T-versus B-cell lines.

Human lymphoblast cell lines of B- and T-cell origin have been tested for their ability to serve as targets in a 4-hr ⁵¹Cr release microcytotoxicity assay using normal human peripheral blood lymphocytes as effector cells. Cell lines of T-cell origin were susceptible to lysis in this assay by effector lymphocytes from all normal donors tested. Cell lines of B-cell origin were repeatably lysed by normal lymphocytes from some, but not all donors. Spontaneous cytotoxicity of B-cell lines, when observed, was also quantitatively less than was obtained using T-cell lines as targets. One cell line (RPMI-7666), of B-cell origin, was not susceptible to spontaneous cytotoxicity by almost all of the normal lymphocyte effectors tested. Lymphocytes from patients with acute lymphoblastic leukemia in remission were less capable of effecting lysis in this assay.     

Increased G2 delay in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc

Cell cycle perturbation after irradiation was studied in five cell lines transfected with oncogenes. Two immortalized, radio-sensitive cell lines with D0s of 1.06 and 1.08 Gy were compared to three radioresistant cell lines with D0s of 1.68-2.17 Gy. The sensitive cell lines were transfected with the v-myc or c-myc oncogenes, the resistant cell lines with the v-myc plus H-ras oncogenes. Exponentially growing populations were exposed to 5, 10, or 15 Gy of orthovoltage radiation. The percentage of cells in each phase of the cell cycle was determined at various times after irradiation using flow cytometry. All cell lines underwent a dose-dependent arrest in G2 phase after irradiation, but the resistant cell lines underwent a significantly longer arrest in G2 phase after irradiation than did the sensitive cell lines. In conjunction with other results from our laboratories, we suggest that this difference in G2 arrest may be the basis for the increased resistance of cells transfected with oncogenes to irradiation.