Higher Saturation Density Research Articles

Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential

Cloned DNA from the P155 mutant of polyoma virus transforms cells in culture as efficiently as wild-type DNA, but has a much lower tumorigenic potential when injected into newborn rats. Like cells transformed by wild-type DNA, cells transformed by the mutant DNA grow in low serum concentrations, form colonies in agar suspension, and grow to high saturation densities compared with untransformed cells. They are, however, much less tumorigenic since they transplant 100- to 2,000-fold less efficiently than cells transformed by wild-type DNA. Substitution of the region between 89.7 and 1.8 map units by the corresponding region of P155 DNA decreased the tumorigenicity of wild-type DNA. When this region was isolated from wild-type DNA and substituted in P155 DNA, the tumorigenicity of the latter increased to values comparable to those of wild-type DNA. This showed that the lesion affecting tumorigenicity occurred between 89.7 and 1.8 map units on the polyoma virus genome. Sequence analysis in this region revealed a 12-base-pair deletion between nucleotides 1,347 and 1,360. This identified P155 as an mlt mutant, i.e., a mutant with a deletion from a region which encodes parts of the large and middle T antigens.

Establishment and characterization of human neuroblastoma and ganglioneuroblastoma cell lines.

The establishment in culture and characterization of 4 human neuroblastoma (NB) cell lines and 1 human ganglioneuroblastoma cell line are described. Each cell line fulfilled at least 2 of 4 criteria for malignant or transformed cells: viz., subcultured more than 70 times, high saturation density with absence of contact inhibition, population-doubling time within a range of 10-40 h, and tumour formation in nu/nu mice. Each cell line also fulfilled at least 2 of 3 criteria for neuroblastoma cells: viz., humoral and cell-mediated immune reactivity toward NB-associated cell-surface antigen, intracellular storage and extra-cellular secretion of catecholamines, and characteristic neuroblast and ganglion-cell morphology.

Transformation of canine kidney cells by SV40

Canine kidney cells infected with SV40 virus led to the development of a transformed cell line (DKSV40). The transformed cells contained the nuclear T antigen of SV40 and grew in a disoriented pattern to a high saturation density. However, at 40°C the cells exhibited density-dependent inhibition of growth.

Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.

Neoplastic transformation of chimpanzee cells induced by adenovirus type 12--simian virus 40 hybrid virus.

The adenovirus 12--simian virus 40 hybrid virus produced neoplastic transformation of chimpanzee skin fibroblasts in vitro. The transformed fibroblasts showed morphological alteration and became permanent lines. The transformed cells contained both adenovirus 12 and simian virus 40 large tumor antigens and were virus producers. However at passage 9, one line (WES) was found to be a nonproducer, producing neither infectious virus nor virus-specific antigen detectable by the complement fixation test. Virus particles were not detected nor could infectious hybrid virus be rescued from this line by cocultivation with Vero cells. The transformed cells formed large cell aggregates and grew in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities; the normal chimpanzee skin fibroblasts did not. One transformed WES line produced tumors when transplanted subcutaneously into newborn nude mice, thus providing an important tool for studying tumor immunity in the chimpanzee.

Chemical transformation of cultured human skin fibroblasts derived from individuals with hereditary adenomatosis of the colon and rectum.

Chemical transformation of cultured human skin fibroblasts (PF) derived from individuals with hereditary adenomatosis of the colon and rectum is reported. Cells treated only with various levels of N-methyl-N'-nitro N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities. One altered (MNNG, 1.0 microgram/ml) cell culture formed colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (l microgram/ml) and showed a more prolonged life-span compared to the untreated cells. Altered cells became heteroploid cells. However, no progressively growing tumors were produced when cells were inoculated subcutaneously into nude mice. The data suggest that chemical carcinogens alone may not induce neoplastic transformation of fibroblasts from humans genetically predisposed to cancer and that neoplastic transformation of these skin cells by chemical carcinogens might require the presence of a tumor promotor and the use of an immuno-privileged site in the nude mouse system.

Growth potential of sheep and sea mammal cells transformed by SV40 early region DNA.

AbstractNormal fibroblasts of bearded seal, northern fur seal, sea lion, domestic sheep, and normal kidney cells of spotted dolphin and Pacific common dolphin have been transformed by fragments of SV40 DNA containing the entire early region. The transformed, T-antigen-positive cell strains grew to higher saturation densities and, except for the transformed dolphin kidney cell strains, were able, albeit poorly, to form colonies in soft agar medium. The transformed northern fur seal, spotted dolphin and Pacific common dolphin cell strains have remained diploid, the transformed sheep cells have become hypodiploid due to Robertsonian translocations, and the transformed bearded seal and sea lion cells had become hypotetraploid. Although all the transformed cells have acquired increased divisional potential and prolonged lifespan in cell culture, only the hypotetraploid cell strains of bearded seal and sea lion have evolved into continuous cell strains.

Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region.

Recombinant plasmids containing either the entire polyoma viral genome or one or the other of the two HindIII fragments of polyoma virus DNA were constructed and cloned in Escherichia coli X1776, and their DNAs were individually tested for the capacity to transform an established line of rat cells. The recombinant plasmids containing the entire polyoma genome and those containing the HindIII-1 fragment of polyoma DNA (45-1.4 map units) efficiently transform rat cells, whereas the plasmids containing the HindIII-2 fragment (1.4-45.0 map units) do not. The properties of many independent transformed cell lines established by infection with the cloned HindIII-1 fragment were determined. In contrast to the parent cell line, rat cells transformed with the cloned HindIII-1 fragment grow to high saturation densities, form colonies with high efficiency in dilute agar suspension, produce high levels of plasminogen activator, and display a disorganized arrangement of actin cables. By all criteria examined, these cells transformed by fragments are indistinguishable from cells transformed by whole polyoma viral DNA. Cellular DNA prepared from many HindIII-1 fragment-transformed cell lines was analyzed for the presence and arrangement of polyoma viral sequences by Southern blot-hybridization. In all cases examined, only those viral sequences contained within the HindIII-1 fragment of polyoma DNA were detected. These data establish a strong correlation between polyoma DNA sequences mapping within a restricted portion of the early region and the induction and maintenance of the transformed phenotype.

Cytochalasin B-induced multinucleation of human tumor and normal cell cultures

Twelve human cell cultures derived from tumors, normal tissues, and derivative cultures transformed by either a RNA tumor virus or chemical carcinogen were examined for their response to cytochalasin B (CB) and the expression of this marker was correlated with growth in soft agar and saturation density in monolayer culture. Cell lines derived from carcinoma of the bladder (T24 and RT4), kidney (Caki-1), prostate (DU 145), and breast (MCF-7) multinucleated when grown in CB-supplemented medium, whereas cell cultures derived from benign bladder epithelium (HCV-29), and normal kidney (Flow 4000) and skin (GM10) remained binucleate under comparable conditions. Human osteosarcoma (HOS) cells transformed by murine sarcoma virus (MSV) or a chemical carcinogen extensively multinucleated in response to CB treatment, relative to a morphological revertant of MSV-transformed HOS and parental HOS cells. CB-induced multinucleation was consistently correlated with the ability of cells to form colonies in soft agar but inconsistently correlated with growth to high saturation densities. These results demonstrate a differential response to CB by normal and transformed human cells and that CB-induced multinucleation provides a convenient and useful in vitro marker for neoplastic transformation.

Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome.

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.

Further characterization of a thermosensitive transformation variant of mouse fibroblasts.

Temperature-sensitive (ts) variants that express phenotype at low (33 degrees) but not at high (38.5 degrees) temperature were isolated from a mouse fibroblast strain C3H2K cells. Among these variants, cloned ts-12B cells showed at 38.5 degrees a density-dependent inhibition of growth typical of normal fibroblasts cultured in vitro. However, at low temperature they lost this capacity and grew to a higher saturation density. The ts variant was also temperature-sensitive as regards serum requirement: it required a higher concentration of serum for growth at 38.5 degrees than at 33 degrees. However, the cells behaved at both temperatures like the parent strain, possessing anchorage-dependence for growth and fibronectin, whereas they were like transformed cells with respect to release of high fibrinolytic activity. No type-C virus core protein p30 was detected at either temperature. Thus, various parameters of transformation in vitro were independently regulated in these variant cells.

Continued in vitro growth of fibroblast-like cells (RBCF-1) derived from the caudal fin of the fish, Carassius auratus

Fibroblast-like cells derived from primary culture of the caudal fin of a 2-year old goldfish were used to find out whether or not fish cells have a limited in vitro lifespan during the observation period of 22 months. The cells cultivated at 37°C (RBCF-1 line) attained exponential growth from the beginning of culture and reached to 200 population doubling number ( PDN) at the 310th day. The growth rate of the cells cultured at 30°C (RBCF-2 line) was almost the same as that of RBCF-1 cells except for a temporal decline between the 80 and 160th day of culture. The plating efficiency of RBCF-1 cells, as determined by colony formation, increased with passage and reached around 20% after 183 PDN. Subcutaneous injection of RBCF-1 cells into nude mice failed to develop any tumour so far as examined up to 5 months after injection. Feulgen-DNA cytofluorometry simultaneously combined with tritiated thymidine autoradiography revealed that, in both RBCF-1 and RBCF-2 lines, unlabelled cells which escaped from 48 hours' labelling gradually diminished from each population as PDNs increased and also that labelling indices for each line were 83 and 90% at 113 and 91 PDNs, respectively. The distribution of DNA content per cell was almost the same irrespective of the line and PDN. The modal number of RBCF-1 chromosomes was 100 at both 62 and more than 200 PDNs, and no significant change in the distribution of chromosome number could be found. The saturation cell density of RBCF-1 cells was almost identical at 20, 30 and 37°C, while extremely high saturation density was observed at 27°C. These results were discussed from a viewpoint of ageing characteristics of fish cells in culture.

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

Studies on Chinese hamster ovary mutants showing multiple cross-resistance to oxidative phosphorylation inhibitors.

Several stable Chinese hamster ovary (CHO) mutants were selected after ethylmethane sulfonate mutagenesis for resistance to oligomycin, ruatmycin, venturicidin, or antimycin. These mutants shared a number of common properties. They exhibited cross-resistance to those drugs which act on oxidative phosphorylation, irrespective of the structure and site of action of the drug. All the mutants showed a reduced ability to grow in suspension and to reach high saturation densities. They were also unable to use galactose as a carbon source. The short lag period required for selection (10-15 days), the similarity of the mutation rates for resistance to each of the four drugs, the high variance/mean ratios in fluctuation tests, and the recessive behavior of the resistance marker in hybrids suggest that the mutations responsible for resistance to oxidative phosphorylation inhibitors in CHO cells are coded by nuclear DNA. Segregation experiments indicated no linkage between the oligomycin-resistant marker (OLG) AND Thg (thioguanine resistance). Oxidative phosphorylation, as measured by the rate of respiration coupled to phosphorylation in whole cells remained as sensitive to the drugs in the mutants as in the parental cell line. Glucose transport and the overall Krebs' cycle activities also appeared similar in the mutants and the wild type. All the mutants had an increased rate of lactic acid production (up to twofold), associated with increased specific activities for several glycolytic enzymes when assayed in cell-free extracts.

Transformation of feline embryo cells in culture by a chemical carcinogen.

A feline embryo cell line was treated in vitro with various levels of 7,12-dimethylbenz[a]anthracene (DMBA) or dimethyl sulfoxide (control). Repeat treatment of DMBA only induced in vitro transformation of feline embryo cells following clonal growth and selection. The morphologically altered cells formed large cell aggregates and grew in this aggregate form when suspended in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities. However, no progressively growing tumors were produced when cells were inoculated into nude athymic mice. The transformed lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA).

Reversion of the transformed phenotype to the parental phenotype by subcultivation of X-ray-transformed C3H/10T1/2 cells at low cell density.

AbstractTransformed cells induced by X‐irradiation of a stable contact‐inhibited line of C3H mouse embryo cells (10T½) display an altered morphology (fibroblastic), a decreased sensitivity to contact inhibition, a high cell saturation density, a decreased sensitivity to interferon and a capacity to form colonies in agar and malignant tumors in mice. Under certain experimental conditions, serial passage of transformed cells (from cloned lines) at low cell density (i.e., 400 cells per 25 cm2 flask) was accompanied by a reversion from the transformed phenotype to a phenotype characteristic of the parental untransformed 10T½ cells: epithelioid morphology, low cell saturation density, increased sensitivity to interferon and failure to form colonies in agarose or tumors in mice. The revertent phenotype was stable as long as these cells were passed at low cell density. Passage at high cell density, however, resulted in a back reversion to the transformed phenotype. When transformed cells were seeded at a much lower cell density, so that only a few colonies appeared per Petri dish, reversion to the parental phenotype occurred at the first passage and appeared stable. Back reversion was no longer observed even when these cells were passed at high cell density. The experimental results suggested that reversion of the transformed to the parental phenotype was not due to a selection of a pre‐existing population of cells with the parental phenotype but rather to a change in the phenotype of the entire cell population.

Oncogenic transformation of rat lung epithelioid cells by SV 40 DNA and restriction enzyme fragments

Rat epitheloid lung cells were transformed with various preparations of SV40 dna using the Ca2+-precipitation technique. The amount of SV40 genetic information integrated into transformed clones was evaluated by DNA-DNA renaturation kinetics. The growth properties on plastic and in soft-agar were examined, as well as the ability to induce tumors in syngeneic new-born animals or in adult nude mice. One particular transformed line, which had received the Hpa II/BamH I A (59 per cent) fragment, was found to contain about 3 integrated copies of this fragment per cell and no significant amount of the Hpa II/BamH I B (41 per cent) fragment. This line which grew to high saturation densities and efficiently formed clones in low serum on plastic, produced tumors in both syngeneic rats and nude mice. Thus the Hpa II/BamH I A fragment, which mainly includes early viral information, was sufficient to impart these properties to rat epitheloid lung cells.

Variant Lines of Mouse Kidney Cells Transformed by an SV40tsA Mutant with Growth Properties of Wild-Type Transformed Cells at Nonpermissive Temperature

MKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the 'standard transformed phenotype'. At the permissive temperature (33.5 degrees C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 degrees C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 degrees C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 degrees C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 degrees C in liquid medium and methylcellulose suspension culture. The heat adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 degrees C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 degrees C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 10(6) cells) of heat-adapted lines grown at 39.8 degrees C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 degrees C.

Expression of transformation in cell hybrids. II. Nonsuppression of the transformed phenotype in hybrids between a chemically transformed and nontransformed derivatives of Balb/3T3.

Hybrid clones derived from a nitrosocarbaryl-transformed Balb/3T3 cell line, Clone H, and a nontransformed cell line THO2 resemble the transformed parent in the clone morphology, higher saturation density, colony formation in medium with reduced serum concentration, growth in agarose and ability to form clones on Balb/3T3 monolayer. Results are discussed in the framework of genetic models which permit or require dominant mutations for the expression of transformed phenotype.

Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome

Isolated restriction endonuclease fragments of the herpes simplex virus type 1 (HSV-1) genome were introduced into hamster embryo cells to identify DNA sequences capable of transforming the cells with respect to acquisition of properties correlated with tumorigenicity. One of the fragments generated by cleavage of HSV-1 DNA with the restriction endonuclease Xba I was found to induce transformation at a frequency of about 10 colonies per quantity of fragment recovered from 1 μg of uncut DNA; fractions containing the other Xba I fragments failed to induce transformation reproducibly, although occasional colonies were detected. The fragment with transforming activity (Xba I-F) is 15.5 × 10 6 daltons in molecular weight and is located between 0.30 and 0.45 map units on the HSV-1 genome. The Xba I-F transformants obtained were selected for their ability to replicate in low concentrations of serum; in addition, they were found to attain high saturation densities in the presence of 10% serum and to form colonies in semisolid medium. Moreover, the transformed cells produced at least one of the viral gene products (a membrane glycoprotein) encoded in the fragment used for transformation, indicating not only that viral DNA was incorporated into the cells, but also that viral genes were expressed.