Source, alterations, characteristics and use of a new dog cell line (Cf2Th).

A cell line derived from normal fetal canine thymus (Cf2Th) has been in culture since 1967. During cultivation the cells have changed morphologically from a fibroblast-like to flat, fusiform appearance and karyologically from diploid (2n=78) with 76 telocentric autosomes to hypodiploid with newly formed atelocentric chromosomes. The cells retain canine characteristic enzyme activity (G6PD and LDH) as well as cell membrane fluorescence and are free of mycoplasma. High passage cells produce tumors in ATST mice. No endogenous viruses have been detected in these cells. No original publication exists, to date, on the origin of this line, but seed stocks thereof have been distributed to many laboratories and the cells have served as experimental substrates in a number of published works on oncology albeit under different designations. The present information is offered in order to establish the provenance of this valuable cell line and to list characteristics which may serve to monitor for its purity and to distinguish it from other existing cell lines of dog origin also in common use.     

Chromosomes of the murine leukemia virus indicator cell line XC

A cell line derived from the Rous Sarcoma Virus induced rat tumor XC (Svoboda), which was recently utilized as an indicator for the presence of murine leukemia virus growing in mouse cells, has been examined karyologically. The cells differ considerably from each other as well as from the normal rat karyotype (Rattus norvegicus, 2n=42). The modal chromosome number is 41. All cells bear one or more chromosome markers in common as well as non-rat-like chromosomes, but rat-like chromosomes still preserve the identity of species origin.Supported by Contract No. PH 43-63-13 between the University of California and the National Cancer Institute, National Institutes of Health (Special Virus Cancer Program).     

Establishment of duck cell line derived from experimental tumor induced by 20-methylcholanthrene

Summary Experimental tumors developed in white Pekin ducks after intramuscular implantation of 20-methylcholanthrene. Cells derived from the primary tumor were adapted successfully to grow in vitro and have growth characteristics similar to that of established cell lines of mammalian origin. The cell density rises rapidly and the doubling time is approximately 19 hr. The duck cells have been cultured succesfully for at least 80 passages in vitro. The continuously cultured cells have the characteristic chromosome pattern of duck, and the DNA of the duck cell line hybridized with duck liver DNA. We believe we have established a continuous cell line of avian origin. Electron-microscopic examinations of the tumor cells and RNA-directed DNA polymerase of the cell-free supernate show no evidence of endogenous virus production. This study was supported by Public Health Service Research Grants CA 16479 and CA 20012 from the National Cancer Institute and RR 00890 from the National Institutes of Health.     

Characteristics of a new rapidly proliferating rabbit cell line

Summary A new tissue culture cell line (Calg-ARLC) has been established from an explant culture of adult rabbit lung. The Calg-ARLC line has been characterized with respect to morphology, chromosome constitution, tissue culture requirements, proliferative capacity, cell cycle, attainable synchrony, radioisotopically labeled precursor incorporation into nucleic acids and protein, and radioisotope sensitivity. The cells are fibroblast-like in appearance with a stabilized heteroploid chromosomal modal number of 35. They grow exponentially from high split ratios in several commercially available defined media with a generation time of 12 hr and are easily synchronized. Although sensitive to some isotopically labeled precursors, high specific activity nucleic acids have been isolated. The ARLC line is especially useful for the isolation of high specific activity nucleic acids and proteins of rabbit origin. The Calg-ARLC line should be invaluable in the fractionation of reiterated DNA sequences since no very rapidly reassociating DNA sequences such as those found in mouse are evident. This work was supported by operating grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Development,characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK)

Summary Cell line CRFK, derived from kidney tissue of a normal domestic kitten, was initiated in 1964. With intermittent periods of storage in the frozen state, it has been grown in vitro during more than 200 passages, without apparent loss of susceptibility to selected viruses. Various herpesviruses and feline viruses belonging to differnet virus groups grow readily and with distinct, cytopathic features. The cells now grow as a smooth monolayer of epithelial-like cells; most have 37 chromosomes (2n−1) and are thus aneuploid for cat karyotype. Three distinct marker chromosomes are identified. The cell line, which is free of mycoplasmal contaimination, is useful in feline virus research and diagnostic medicine and has become of particular interest in cancer research. Supported in part by Contract E73-2001-NO1-CP-3-3237 within the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Public Health Service and the Morris Animal Foundation and General Research Support funds of the New York State Veterinary College. CRFK cells from stock provided by C. G. Fabricant are available for distribution to investigators from the Cell Culture Laboratory, Naval Biomedical Research Laboratory.     

Rimantadine but not amantadine protects fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene

Summary The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimantadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene. This work was supported by Contract N01-CP-43240 within The Virus Cancer Program of the National Cancer Institute.     

Characterization of cells cultured from early lactation milks

Summary Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differential epithelial cells, but tissue macrophages. R. L. C. was supported by Grant No. Ca 19455 from the National Cancer Institute, a Yamagiawa-Yoshida Memorial International Cancer Study Grant, and the Imperial Cancer Research Fund. J. A. P. was supported by Grant No. CA 19455 from the National Cancer Institute.     

A new cell line derived from nickel sulfide-induced rat rhabdomyosarcoma

Summary The morphological and chromosome characteristics of a cell line derived from a nickel sulfide-induced rhabdomyosarcoma in the Fisher strain of rats have been described. At the time of this report the cell line has undergone 59 passages and continues to exhibit the heteroploid trait and has retained the malignant properties. This cell line, which lends itself to in vitro and in vivo (in rat) cultivation and carries easily recognizable chromosome markers, may prove to be useful in further research on tumor cells. This investigation was supported in part by a grant in aid of research from the National Cancer Institute of Canada. Department of Biomedical Sciences.     

Ovine cells: Their long-term cultivation and susceptibility to visna virus

Summary Sheep choroid plexus (SCP) cells have been subcultured more than 120 times and have undergone over 300 cell generations. These fibroblastic-appearing SCP II-B cells contain ovine-specific antigens, have an absolute plating efficiency of 23 to 28% and are as susceptible to visna virus infection and virus-induced cytopathology as their low passage level counterparts. Cultures of low, relatively high and high passage level SCP cells produced equivalent amounts of visna virus at similar rates when infected with equal amounts of visna virus. The passage level of the SCP II-B cells, their elapsed number of cell generations, their possession of ovine-specific antigens and their full susceptibility to visna virus allow these cells to be considered an established line of sheep cells. This work was partially supported by grants from the National Cancer Institute (CA12678) and the National Institute of Allergy and Infectious Diseases (AI12465).     

Characterization of a newly established feline lymphoma-derived cell line (BKD) lacking T and B cell surface markers

Summary A new feline lymphoma-derived cell line, designated BKD, was isolated from an anterior mediastinal tumor. Cells of this line were characterized as lymphoid based on morphology, the lack of intracellular esterase and peroxidase activity, and absence of phagocytic function. In contrast with other established feline lymphoma-derived cell lines, cells of the BKD line lack characteristics of both feline T-cells and B-cells in that they neither form rosettes with guinea pig erythrocytes nor have demonstrable surface or cytoplasmic immunoglobulin. Approximately one third of BKD cells form EAC rosettes, a significant number of rosette forming cells (p=0.0001) when compared to background sheep E-rosetting activity. In addition, a consistently titratable level of interleukin-2-like activity was produced when BKD cells were coincubated with concanavalin A and phorbol-12-myristate-13-acetate. Chromosome analysis showed that a majority of BKD cells are diploid. This new cell line has been continuously replicating in culture for over one year and produces feline leukemia virus as demonstrated by several analyses. This research was aided by grants AI-22360 and CA-34103 from the National Institutes of Health and by grant IM-298 from the American Cancer Society.     

Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells

Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.     

Characterization of a cell line (SW756) derived from a human squamous carcinoma of the uterine cervix

Summary An established cell line, SW756, derived from a primary squamous carcinoma of the uterine cervix is described by its morphology, ultrastructure, karyotype, genetic signature analysis, HLA typing, and tumorigenesis in the nude mouse. Cultured cells obtained from the SW756 derived nude mouse tumor also were studied for chromosome and isozyme markers. The original tumor was poorly differentiated carcinoma with minimal keratinization and is compared with that occurring in the nude mouse after the cultured cells were inoculated. The nude mouse tumor showed similar histological features, but better differentiation than the original tumor. Karyotype analysis of SW756 demonstrated a hyperdiploid stem line number and several marker chromosomes (MI-M6). No HeLa marker chromosomes were identified. The isozyme pattern for SW756 reported by others has been confirmed. The unique chromosome and isozyme features have been identified repeatedly in the cultured cells and, most importantly, in the post nude mouse culture. We recommend SW756 as a defined human tumorigenic cell line derived from a primary squamous carcinoma of the uterine cervix. This investigation was supported in part by Public Health Research Grant CA-06294 from the National Cancer Institute, Department of Health and Human Services.     

Peculiarities of ovine cells in culture

Summary The chromosome complement of two ovine (Ovis aries L.) kidney cell lines are described. Nuclei of MDOK, the older cell line, are generally larger than those of OK cells, they are hypo-tetraploid and show considerable chromosomal irregularities both in structure and behavior. The OK line observed since its initiation, is now hyper-diploid and exhibits a gradual accumulation of chromosomes. Both cell lines have in common the fact that they now maintain a larger proportion of chromosomes with interstitial centromeres than telocentric chromosomes. This observation parallels similar ones made earlier in bovine cells. However, the processes whereby this condition arose in the respective cultures are believed to be different and are discussed.Dedicated to Professor Dr. H. Bauer on the occasion of his 60th birthday.Supported in part by Contract No. PH 43-63-13 from the National Cancer Institute, National Institutes of Health, Public Health Service.     

Synthesis and anti-HIV activity of some non-nucleoside 2,3-disubstituted quinazoline derivatives (Part-V)

Several [2-phenyl-4(3H)-oxo-3-quinazolinylamino]-N-substituted- arylacetamides (1a-j) have been synthesized and tested at the National Cancer Institute, Bethesda, Maryland, USA, for their anti-HIV activity against susceptible human host cells (CEM cell line) over a wide range of concentrations (6.35 x 10(-8) to 2.00 x 10(-4) M). The highest protection observed is 45.67%. The structures of these compounds have been established on the basis of elemental analysis and spectral data.     

Establishment, identification and characterization of a new sheep sinus tumor cell line

A cell line designated SRT was established from a sheep sinus tumor. Following primary culture, the cells were serially passaged 40 times. SRT cells maintained an epithelioid fibroblast-like appearance and had a population doubling time of approximately 18 hr. Karyotype analysis of 14th passage cells showed the modal 2n chromosome number to be between 46 to 60, due to a large variation in acrocentric chromosome number. The electrophoretic mobilities of enzymes extracted from SRT cells were identical with those from normal sheep sinus cells. It propagated a number of ovine, bovine and canine viruses. Some virus-like particles (80-120 nm) were observed under the electron microscope. The tumor origin, good growth and wide range of virus susceptibility make SRT a highly suitable cell line for in vitro cancer research and for comparative virology studies.     

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium con- taining chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul- fate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to con- fluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical re- searches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.     

Cerebral microvessels and derived cells in tissue culture

Summary An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C₆ glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence). This work was supported in part by an Arteriosclerosis Specialized Center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL-14230, and Grant 584-127703 from the Veterans Adminsitration. This paper is dedicated to the memory of Steve Frommes, Electron Microscopist and Photographer.