Increased Saturation Density Research Articles

A novel cell transformation with DNA-containing cytoplasmic yaba tumor poxvirus

Monkey kidney cells were morphologically transformed in vitro with uv-irradiated Yaba tumor poxvirus. Cell lines established were shown to be virus nonproducers and exhibited biological characteristics typical of transformed cells. These characteristics included increased saturation density, reduced serum requirements for growth, and ability to grow in soft agar. The morphological alterations of transformed cells were similar to Yaba virus-induced tumor cells and were characterized by loss of contact inhibition, multinucleated cells, and cytoplasmic lipid droplets. Southern blot hybridization revealed that sequences homologous to low-molecular-weight viral DNA (5.1, 4.8, 3.9 kbp) were present in the transformed cells. Yaba virus-specific antigens detected by immunofluorescence assays were found to be in the cytoplasm of transformed cells. Four virus-specific proteins, with molecular weights of 160,000, 140,000 107,000, and 74,000 daltons, were detected in transformed cells immunoprecipitated with sera from tumor-bearing monkeys. These results indicate that DNA-containing Yaba tumor virus, which replicates exclusively in the cytoplasm, is capable of inducing cell transformation in vitro.

Growth Properties and Susceptibility to Viral Transformation of Skin Fibroblasts From Individuals at High Genetic Risk for Colorectal Cancer2, 3

Growth characteristics and susceptibility to viral transformation were compared in cultured skin fibroblasts from patients with Gardner's syndrome (GS), those with familial polyposis coli (FP), asymptomatic family members, and unrelated controls. Compared to cells from unrelated controls, cells from 4 of 5 GS patients were transformed by Kirsten murine sarcoma virus at 100- to 1,000-fold increased efficiency. The transformation efficiencies of fibroblasts from 2 of 3 FP patients were 10- to 100-fold greater than those of unrelated controls. However, because the fibroblasts from some of the young asymptomatic GS and FP family members also transformed at higher efficiency than did cells from unrelated controls, long-term observation of these families is required. This would determine whether or not these members develop clinical manifestations of GS or FP and thus establish the specificitiy of this assay for detection of individuals bearing the mutant gene. Compared to fibroblasts from unrelated controls, GS and FP fibroblasts showed a twofold to threefold increased saturation density and plating efficiency, but this difference was not noted with fibroblasts from most of the asymptomatic family members.

Oncogenic transformation by by equine herpesviruses. II. Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine herpesvirus type 1 preparations enriched for defective interfering particles.

Infection of permissive hamster embryo cells with virus preparations enriched for defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) resulted in persistent infection and oncogenic transformation. Six cell lines, designated DI-5 to -10, exhibited biological properties (immortality, increased saturation density, growth in soft agar, etc.) inherent to transformed cells, but 2 to 18% of the total cells in these cell lines were shown to release virus as judged by electron microscope studies and infectious center assays. The released virus was shown to be standard EHV-1 and not to contain DI particles as determined by density measurements of the viral DNA in the analytical ultracentrifuge and by interference assays using the released virus. Tumorigenicity studies revealed that inoculation of these persistently infected cells into newborn LSH inbred hamsters resulted in a lethal, fulminating hepatitis, whereas inoculation into older immunocompetent hamsters (+4 weeks) led to the development of metastatic fibrous sarcomas. Tumor cell lines (DI-5T to -10T) established from these sarcomas were shown to be transplantable and virus nonproducers. Hybridization analyses of cellular DNAs from DI transformed and tumor cell lines using 32P-labeled genomic EHV-1 DNA as probes indicated that the whole virus genome was detectable in multiple copies (23 to 45) in the transformed cells and that DNA sequences representing only 43.5 to 56.6% of the virus genome were present in amounts of 2 to 4 copies per cell in the DI tumor cells. Expression of these viral DNA sequences as demonstrated by the detection of virus-neutralizing antibodies, 50% neutralizing dose titers ranging from 1:50 to 1:1,000, in the sera of animals inoculated with either the virus-producing transformed cells or the virus-nonproducing tumor cells. Further, EHV-1-specific proteins were detected in the membrane and the perinuclear region of bothDI transformed and tumor cells by indirect immunofluorescent assays using antisera against EHV-1 structural antigens, EHV-1 nonstructural antigens, or preparations of EHV-1 DI particles. The roles of DI particles in mediating persistent infection and cellular transformation are discussed.

Tumorigenicity and in vitro characteristics of rat liver epithelial cells and their adenovirus-transformed derivatives.

Cloned and uncloned epithelial cultures were established from the liver of a 3-week-old AS rat. These epithelial cultures were neither tumorigenic nor did they display anchorage-independent growth. One of the clones was cytogenetically normal after 53 in vitro passages (approximately 200 population doublings after cloning). Eight transformed lines were isolated from the liver epithelial cells after infection with adenovirus type 12 (Ad-12). Five of these produced typical Ad12 T-antigen, whereas three appeared to be T-antigen-negative. All were tumorigenic in newborn syngeneic rats. The T-antigen-positive transformed lines produced anaplastic-epithelial tumors, whereas the T-antigen-negative transformed lines produced adenocarcinomas. Although all the transformed lines were tumorigenic, some were fibronectin-positive while others produced no detectable fibronectin. The normal (untransformed) epithelial cells produced fibronectin. These results are interesting for two reasons: (1) there are relatively few reports of fibronectin on epithelial cells and (2) they emphasize the view that there is no absolute correlation between reduced fibronectin and tumorigenicity in transformed cells. The transformed lines displayed in vitro characteristics similar to those of transformants derived from embryonic and fibroblastic cell strains, notably, increased saturation density and changes in cellular morphology. Some of the transformed cell lines, but not all, displayed anchorage-independent growth. All the transformed cell lines were picked from multi-layered foci so that morphological criteria (i.e. piling-up focus) for isolating transformants from the epithelial cultures were similar as in embryonic and fibroblastic transforming cell systems. With the new cell system we have developed we can, using the same epithelial cell line (clone C3), study both virus transformation and virus mutagenesis.

Oncogenic transformation by equine herpesviruses (EHV) I. Properties of hamster embryo cells transformed by ultraviolet-irradiated EHV-1

Permissive primary hamster embryo cells (HEF) were morphologically transformed in vitro by uv-irradiated standard equine herpesivrus type 1 (EHV-1). Cell lines (LSEH) were established and were shown to exhibit growth and biological properties typical of transformed cells. These properties included an altered, network-like cellular morphology, loss of contact inhibition, increased saturation density, decreased generation time, immortality in culture, formation of colonies in soft agar, growth at low concentrations of serum, and resistance to EHV-1 superinfection. These LSEH transformed cell lines were oncogenic in both newborn and immunocompetent inbred LSH syngeneic hamsters, and LSEH tumor cell lines were established from the tumors which were identified as malignant fibrous sarcomas. Both the LSEH transformed and tumor cell lines were determined to be virus nonproducers, since released virus could be neither detected by electron microscopy, infectious center assays, or cocultivation assays nor induced by iododeoxyuridine (IUdR) or growth at higher temperature. Analysis of the hybridization kinetics of 125I- or 32P-labeled EHV-1 DNA with DNA from low-passaged LSEH transformed cell lines, tumor tissues, or LSEH tumor cell lines revealed that viral DNA sequences were present in all cells and that the amount of these viral DNA sequences varied from one cell line to another. In the transformed cell lines, these values ranged from 3.3 to 20.9% of the EHV-1 genome in amounts of 0.3 to 2.2 copies/cell, whereas the DNA of tumor tissues and low-passaged LSEH tumor cell lines contained from 2.2 to 12.2% of the EHV-1 genome in amounts of 1.2 to 4.0 copies/cell. These viral DNA sequences were expressed since viral antigens were detected in LSEH transformed cells, tumor tissues, and tumor cell lines. Neutralizing antibody, ND 50 titers ranging from 1 8 to 1 100 , was detected in the sera of hamsters that had been inoculated with either LSEH transformed or LSEH tumor cells. In addition, EHV-1-specific speypeptides were demonstrated in the cell membrane, cytoplasm, and perinuclear region of both LSEH transformed and tumor cell lines by indirect immunofluorescence using antisera to purified EHV-1 virions or antisera to EHV-1 infected HEF cell extracts.

Action of phorbol esters in cell culture: Mimicry of transformation, altered differentiation, and effects on cell membranes

The carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast to initiating carcinogens, the action of the tumor-promoting phorbol esters does not appear to involve covalent binding to cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose-glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor-promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane-related effects include enhanced uptake of 2-deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor to cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of "two stage" carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.

“Spontaneous” neoplastic transformation in vitro of epithelial cell strains of rat liver: Cytology, growth and enzymatic activities

We can follow in culture the spontaneous neoplastic transformation of one epithelial cell strain derived from normal rat liver and determine, by comparison with other liver cell strains, the divers modifications which appear before or during transformation. A decrease in adhesiveness seems to be one of the first manifestation. Increase in saturation density, ability to grow in soft agar and acquisition of tumorigenicity appear much later. Moreover, in contrast to the other cell strains, in this transformed cell strain, some enzymatic activities were increased and tyrosine aminotransferase was induced by dexamethasone.

Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells

Mutants of SV40 with deletions of various sizes mapping between 0.54 and 0.59 on the genome grow at a rate equal to or slightly slower than that of wild-type virus, in a range of host cells. Their ability, however, to induce transformation in several mouse, rat and rabbit cell lines is impaired. The extent of transformation observed is dependent upon the assay used to measure it, but in general, the ability of the mutants to transform falls as the size of the deletion increases. In addition, rat embryo fibroblasts transformed by deletion mutants have fewer of the characteristics of a fully transformed phenotype (for example, growth in low serum, increased saturation density, growth in semi-solid medium) than those transformed by wild-type virus. During lytic infection, immunoprecipitable T antigen produced by the deletion mutants is of the same size as that seen during infection with wild-type virus, and is present at a similar level. Mutant virus-coded small t protein, however, is reduced in size compared with that from wild-type virus. For each mutant, the reduction in protein size is dependent upon the amount of DNA deleted, but not on the relative position of the deletion in the genome. These results demonstrate that the DNA sequences mapping between 0.54 and 0.59 on the viral genome code for the small t protein, and that SV40-induced transformation is at least partially dependent upon the expression of this protein.

Human placental cells transformed by tsA mutants of simian virus 40: a model system for the study of placental functions.

Human placental cells were transformed with wild-type simian virus 40 (SV40) and temperature-sensitive SV40 mutants of the A and B classes. Four criteria for transformation were used: decreased generation time, increased saturation density, increased efficiency of growth on plastic, and ability to overgrow a nontransformed monolayer. Cell lines transformed by tsA mutants lost the transformed phenotype at the restrictive temperature (40 degrees); therefore, the A function of SV40 is required for the maintenance of the transformed phenotype activity, an inhibition of human chorionic gonadotropin synthesis, and an increase in thymidine kinase activity were seen when human placental cells transformed by wild-type or tsB mutants of SV40 were grown at 33 degrees or 40 degrees and when tsA transformants were grown at 33 degrees. When tsA transformants were grown at 40 degrees, alkaline phosphatase activity and human chorionic gonadotropin synthesis were greatly stimulated and thymidine kinase activity was greatly reduced, approximating their levels in the placenta.

Induction of some transformation-related properties by a transformation-defective mutant of avian sarcoma virus

Abstract A transformation defective mutant ( TY 9) was obtained from the Prague strain of RSV by ultraviolet irradiation. It is a deletion mutant with slightly longer genomic RNA than ordinary td mutants isolated from the same strain by Vogt [ Virology 46 , 939–946 (1971)] . After several passages of cells infected with TY 9, partial expression of some characteristics of the transformed state was observed; these were fusiform shape, increased saturation density and hexose uptake, production of plasminogen activator, and colony-forming ability in soft agar medium.

Temperature Sensitivity of Cyclic-Adenosine-3':5'-Monophosphate-Binding Proteins, Activity of Protein Kinases and the Regulation of Cell Growth

A clone of neuroblastoma cells has been selected for its ability to survive and multiply at 40 degrees C. This temperature-resistant clone, like clones of neuroblastoma cells selected for resistance to dibutyryladenosine 3':5'-monophosphate (Bt2-Ado-3':5'-P) showed an increased tumorogenicity in animals and an increased saturation density at 37 degrees C. The Ado-3':5'-P-binding proteins and Ado-3':5'-P-dependent protein kinases from the temperature-resistant and non-resistant cells have been partially purified by chromatography on a DEAE-cellulose column. The Ado-3':5'-P-binding proteins from temperature-resistant cells were more sensitive to temperature than the binding proteins from non-resistant cells. After incubation of binding proteins from resistant cells at 37 degrees C, the specific activity of Ado-3':5'-P-binding to proteins was decreased about 50% and the apparent association constant (Ka) for Ado-3':5-p-binding was decreased from 7.4 X 10(7)M-1 to 4.4 x 10(7)M-1. There was no such decrease with binding proteins from non-resistant cells. A decrease in the activity of binding proteins from the temperature-resistant cells, but not of those from non-resistant cells, was also found when the proteins were stored at 2 degrees C. Treatment with 2-mercaptoethanol made binding proteins from the resistant cells less temperature-sensitive. In the absence of added Ado-3:5-P the protein kinase activity from the temperature-resistant cells was about 50% of the activity from non-resistant cells. Kinase activity was increased by addition of Ado-3:5-P and there was a greater increase with kinases from resistant cells. The maximum protein kinase activity was found in the presence of 10muM Ado-3':5'-P for the temperature-resistant cells and 0.1 muM Ado-3':5'-P for the non-resistant cells. The results indicate that the temperature sensitivity of Ado-3':5'-P-binding proteins, and the activity of protein kinase from cells selected for resistance to high temperature, are similar to those of cells selected for resistance to Bt2-Ado-3':5'-P. It is suggested that the temperature sensitivity of Ado-3':5'-P-binding proteins and the activity of Ado-3':5'-P-dependent protein kinases are involved in the regulation of malignancy and of cell growth at different temperatures.

Lack of correlation between growth characteristics, agglutinability by plant lectins and the malignant phenotype.

Wheat germ agglutinin and Concanavalin A were found to agglutinate a variety of normal hamster cells as well as a number of lines of transformed or tumor cells. The normal cells included preparations from embryos and neonatal organs, and the spontaneous line, BHK-21 C13. In order to explore more fully the situation in regard to the BHK cells, three sublines were isolated and studied. A "flat revertant" selected by FUdR treatment grew to diminished saturation density and failed to produce colonies in soft agar in contrast to the parent line, which did. Yet, it was only minimally less tumorigenic and less agglutinable than the parent line. Lines derived from one and four consecutive in vivo tumor passages showed increased tumorigenicity and growth in soft agar when compared to the parent line. Although the first passage line showed increased saturation density, the saturation density of the fourth passage line was similar to or less than that of the parent cells. Agglutinability of these lines was not significantly increased. These results indicate that, in contrast to widely accepted beliefs, tumorigenicity may correlate poorly with growth characteristics and agglutination by plant lectins.

Cyclic AMP and the Transformation of Fibroblasts

Cyclic AMP is found in a wide variety of organisms and cells. It is a chemical switch that regulates the activity of existing enzymes and also can induce the synthesis of new proteins. This chapter reviews the role of cyclic AMP in fibroblasts, especially the role of cyclic AMP in the transformation of these cells. The transformed fibroblasts grow faster than normal cells and have a greatly increased saturation density. The transformed cells look different, generally they are less spindly and more refractile in appearance; they are less adhesive to the substratum on which they are growing and will grow in soft agar, whereas normal cells will not; they have altered surface molecules and are readily agglutinated by plant lectins; they often secrete abnormal amounts of macromolecules; and have abnormal tRNA and increased aerobic glycolysis. Cyclic AMP has an important role in regulating many properties of normal fibroblasts. It is involved in controlling cell shape, adhesiveness to the substratum, motility, growth, agglutinability by lectins, and the synthesis of certain macromolecules. As a result of the transformation by chemical carcinogens, X-rays, oncogenic viruses, or spontaneous selection, cyclic AMP levels are decreased and this decrease produces some of the abnormal properties of transformed cells. Transformation by different viruses decreases adenylate cyclase activity by diverse mechanisms because a variety of transformation factors exist.

GANGLIOSIDES IN DNA VIRUS-TRANSFORMED AND SPONTANEOUSLY TRANSFORMED TUMORIGENIC MOUSE CELL LINES

In mouse cell lines transformed by SV40 virus, a decrease was observed in the higher ganglioside homologues disialo-ceramidetetrahexoside and monosialo-ceramidetetrahexoside. Such change was observed only in cells which carry the virus genome, and it correlated with increased saturation density in tissue culture and with rejection in immunologically competent syngeneic host. This indicates that the change is one of the virus-regulated functions, and it is postulated that it relates to the rejection of the virus-transformed cells.