Comparative studies on cell lines established from normal and radiation-exposed miniature swine

Summary Cloned cell lines were established from two swine with radiation-induced myeloproliferative disorders, including one cell culture from an animal with myelogenous leukemia and one from an animal with myeloid metaplasia. A third cloned cell line with similar morphology was established from pooled normal fetal swine cornea to compare the growth characteristics of cells from normal and irradiated swine. All three cell lines grew as foci of aggregated cells and were able to form macroscopic colonies in semisolid agar medium. The lack of normal mechanisms of contact inhibition and the observed aneuploidy indicated that these cells were morphologically transformed. Further, the cloned cells caused tumors in nude mice, clearly indicating that these cells were also malignantly transformed. A major difference between these cell lines was that type C viruses were observed only in the cells derived from swine with myeloproliferative disorders. Work supported by the U.S. Department of Energy under Contract EY-76-C-06-1830.     

In vivo or in vitro selection for resistance to natural cytotoxic cell lysis selects for variants with increased tumorigenicity

Experiments were designed to test the hypothesis that transformed cells that are NC sensitive must escape NC activity if they are to grow as tumors in normal individuals. NC-resistant variants were selected either in vivo or in vitro from NC-sensitive cell lines that grow as tumors in immunodeficient mice but not in syngeneic normal mice. The tumorigenicity of cloned NC-resistant variants was compared with the parental cell lines and to cell lines that went through the selection procedure, but after cloning remained NC sensitive. Cloned NC-resistant cell lines derived from tumors that developed in x-irradiated nude mice after the injection of an NC-sensitive cell line are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines derived from the same tumors are unable to grow as tumors in normal mice. Similarly, six of seven NC-resistant cloned cell lines independently isolated after in vitro selection for NC-resistance are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines isolated from the same in vitro selected populations are not tumorigenic in normal mice. Thus, either the in vivo or in vitro selection of NC-resistant cells selects for cells tumorigenic in normal mice; these findings, along with our previous observations that selection for cells tumorigenic in normal mice selects for NC resistance, provide compelling evidence that escape from NC activity is required before some transformed cells can grow as tumors in normal mice.     

Radiation-induced genetic instability: no association with changes in radiosensitivity or cell cycle checkpoints in C3H 10T1/2 mouse fibroblasts

We investigated various phenotypic characteristics of radiation-induced morphologically transformed C3H 10T1/2 mouse fibroblasts. The cells were treated with 8 Gy x-rays, and type II/III foci were isolated. Cell lines were developed from these foci, and subsequently clones were established from these focal lines. The clones were examined for DNA content, radiosensitivity and inducible cell cycle arrests. Besides the morphological changes associated with the transformed state, the major difference between the isolated focal lines or derived clones and the parental C3H 10T1/2 line was one of ploidy. The transformed cells often displayed aneuploid and multiple polyploid populations. No change in the radiosensitivity of the transformed cells was observed. Furthermore, the two major radiation- and staurosporine-induced G1 and G2 cell cycle arrests observed in the parental cell line were also observed in the morphological transformants, suggesting that checkpoint function was normal. Received: 15 May 1997 / Accepted in revised form: 21 October 1997     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Establishment of immortal swine kidney epithelial cells

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.     

Bovine endothelial cells transformed in vitro by benzo(a)pyrene

A cloned strain of bovine vascular endothelial cells with a finite in vitro lifespan was treated with benzo(a)pyrene (BP) after approximately 75% of its lifespan was completed. Untreated cultures of this strain senesced upon serial subcultivation and contained large, nondividing cells. In three out of seven trials, BP treatment produced transformed cells appeared in the cultures concomitant with the senescence of the parent cells. All transformed cell lines examined exhibited indefinite lifespans and altered karyotypes. Two of the lines retained most of the characteristics of normal endothelial cells, except that one became aneuploid and the other polyploid, Neither of these lines formed tumors when inoculated into nude mice. The remaining two lines retained mostly diploid kayotypes, but a high percentage of cells contained Robertsonian translocations. In one line cell volume was markedly reduced. In addition, these lines grew in multilayers, were anchorage independent, and proliferated in medium containing 0.5% serum. When 10⁷ cells of these lines were injected into nude mice, tumors appeared within 1 week and were identified as malignant hemangioendotheliomas of bovine origin.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

Growth of AKR T cell leukemia lymphoblasts in medium containing interleukin 2 (IL-2)

Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteristics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quantify IL-2 activity.     

FBJ osteosarcoma virus in tissue culture. III. Isolation and characterization of non-virus-producing FBJ-transformed cells.

Hamster and rat cell lines have been established that have been transformed by FBJ murine sarcoma virus (FBJ-MuSV) but that do not produce virus. The hamster cell line originated from an osteosarcoma that appeared in a hamster inoculated at birth with an extract of a CFNo1 mouse FBJ-osteosarcoma. The rat cell line was obtained by transferring the FBJ-MuSV genome to normal rat kidney cells in the absence of the FBJ type C virus (FBJ-MuLV), which, usually in high concentration, accompanies the FBJ-MuSV. Both transformed hamster and rat cell lines contain the FBJ-MuSV genome, which can be rescued by ecotropic and xenotropic murine type C viruses. This rescued genome produces characteristic FBJ-MuSV foci in tissue culture and, in appropriate animal hosts, induces osteosarcomas typical of those induced by FBJ-MuSV. FBJ-MuSV was isolated originally from a parosteal osteosarcoma that occurred naturally in a mouse. Since there was no previous history of passage of the agent through any other animal species, these non-virus-producing hamster and rat cells transformed by FBJ-MuSV should be very helpful in molecular studies examining the origin of spontaneous sarcoma genomes in mice.     

Chemical and immunological studies of cell surfaces from normal and transformed cells

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/c hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-¹²⁵ I or [³H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G-50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.     

Co-transfection of normal NIH/3T3 DNA and retroval LTR sequences: a novel strategy for the detection of potential c-onc genes.

Morphologically transformed, tumorigenic cell lines were obtained after co-transfecting normal NIH/3T3 DNA and cloned 3'-long terminal repeat sequences of Moloney leukemia virus (Mo-LTR) onto NIH/3T3 recipient cells. In four such cell lines the malignant phenotype was found to be associated with single and specific Mo-LTR integration sites that were retained after serial passages through NIH/3T3 and rat 208F cells, indicating that Mo-LTR sequences are linked to the activated oncogenes. In one of these clones the activated transforming gene was identified as c-raf, the cellular homologue of a recently described retroviral oncogene. This finding not only demonstrates that the mouse c-raf gene can be activated to exhibit an oncogenic potential but also that the approach chosen in this study is suitable for the detection of potential c-onc genes. In contrast to this clone, the activated transforming genes in other cell lines appear to be different from 19 previously isolated v-onc and c-onc genes. These results demonstrate the potential of the established transformation system for the detection and isolation of previously unidentified c-onc genes.     

Species-specific differences in the toxicity of rhodamine 123 towards cultured mammalian cells

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10-fold and 70-fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species-related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.     

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.     

Establishment of a swine monocyte cell line

A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig. The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation. The plating efficiency is more than 95%. Densely grown cells in flasks show an epithelioid morphology. The fundamental properties of the cells were examined for cytological definition as monocytes. A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase. A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells.     

Formation and utilization of methionine by rat liver cells in culture

The enzyme N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.     

Morphological changes in frog pronephric cell surfaces after transformation by herpes virus.

A scanning electron-microscope study has established that there are major modifications in the surface membranes of malignant cells transformed from the pronephric kidney of the frog. Tumours were induced in vivo after exposure of embryos Lucké Herpes virus. A pronephric cell line from normal embryos and 3 tumour cell lines derived from experimentally induced adenocarcinomas of the pronephros were observed. The normal embryonic kidney cells have their surfaces covered with long fingerlike microvilli throughout the cell cycle. In contrast, the surface of the pronephric tumour cells are mainly covered by multiple, ruffled or parallel straight microridges and short, stubby microvilli. These comparisons were made on cells subject to similar culture conditions. The topographic changes in the tumour cells are considered significant since the characteristic microridges and stubby villi were consistently found in all 3 pronephric tumour lines.     

Inducible transformation of cells from transgenic mice expressing SV40 under lac operon control.

If it were possible to clone in vitro cells of any type, at any stage of differentiation, from an extensively characterized animal such as the mouse, many areas of cell biology would benefit. Indeed, it would be even more helpful if these cells could subsequently be restored to their normal in vivo phenotype whenever required. Here, we describe a step on the pathway to such an idealized "clonable" mouse. In principle, it seeks to link a "universal" transforming agent to a regulatory system that is relatively simple, yet quite foreign to the mouse. A plasmid containing the bacterial lac operator/promoter region linked to the SV40 large T antigen and a vector containing the lac repressor that can be expressed in mammalian cells were coinjected into fertilized mouse oocytes utilizing the standard techniques for generating transgenic mice. Two progeny were obtained that express large T antigen in the presence, but not the absence, of the nonmetabolizable lac inducer, isopropyl-beta-thio-D-galactoside. This report characterizes fibroblast cell lines established from these transgenics that are readily transformed in vitro with isopropyl-beta-thio-D-galactoside. A significant proportion of the cells are restored to their "normal" (nontransformed phenotype) when isopropyl-beta-thio-D-galactoside is removed.     

Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.     

Tumorigenicity of herpesvirus-transformed cells correlates with production of plasminogen activator.

Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [125I]fibrin lysis assay, and high- and low-activity subclones were isolated; these retained high- or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.     

Properties of permissive monkey cells transformed by UV-irradiated simian virus 40.

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.