Increased Saturation Density Research Articles

Morphological and neoplastic transformation of C3H/10T1/2 Cl 8 mouse embryo cells by insoluble carcinogenic nickel compounds

We studied induction of cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts by soluble and insoluble carcinogenic nickel compounds. Soluble nickel sulfate and nickel chloride caused dose-dependent cytotoxicity in the concentration range from 0.5 microM to 100 microM after 48 hr treatments, but neither compound induced morphological transformation even at concentrations causing up to 94% cytotoxicity. Insoluble nickel subsulfide, nickel monosulfide, and nickel oxide caused dose-dependent cytotoxicity and a low, dose-dependent frequency of morphological transformation in the concentration ranges from 0.5 to 40 microM, 5 to 50 microM, and 50 to 400 microM, respectively, after 48 hr exposure of cells to these compounds. Foci were predominantly of type II morphology; type III foci were rare. The insoluble nickel compounds studied caused no induction of base substitution mutations to ouabain resistance in 10T1/2 cells over concentration ranges that induced morphological transformation. Nickel subsulfide and nickel monosulfide were taken into cells by phagocytosis, since particles were visible in intracytoplasmic vacuoles. Numerous nickel oxide particles were found associated with cells, but true phagocytic uptake was difficult to detect since no vacuoles were observed. We twice cloned type II and type III foci induced by insoluble nickel compounds, established independent cell lines, and characterized their phenotypes. Four of seven of these cell lines had three- to fourfold increased saturation densities compared to 10T1/2 cells, formed type II and type III foci in reconstruction assays, and grew in soft agarose. One cell line induced by nickel oxide formed tumors in nude mice. These data indicate that insoluble carcinogenic nickel compounds induced type II foci in 10T1/2 cells, some of which were tumorigenic, and that the 10T1/2 cell system is suitable for studying mechanisms of nickel compound-induced morphological transformation in mammalian cells.

Isolation of Swiss 3T3 cell variants with altered heparan sulfate

The replica filter technique has been used to isolate variants of Swiss mouse 3T3 cells which produce heparan sulfates with altered levels of sulfation. These changes in the extent of sulfation correlate with alterations in cell morphology, in the organization of cytoskeletal elements, focal contacts, and the extracellular matrix, and in the growth regulation of cells, as expressed by saturation density. An increase in the extent of heparan sulfate sulfation occurs concomitantly with a decreased saturation density and enhanced focal contact formation. In contrast, graded decreases in sulfation correlate with graded increases in saturation density and losses of cytoskeletal and extracellular matrix organization. These graded responses appear very similar to those which have been reported for the transformation of cells with fusiform mutants of Rous sarcoma virus or the adenovirus type 2 E1a transforming gene and suggest that the morphological changes observed in the transformed cells can be controlled by cellular systems.

The study of multi-stage carcinogenesis in retinoblastoma and familial polyposis coli patient-derived skin fibroblast cell culture systems

Carcinogenesis is considered to be a multi-step process comprising ‘initiation’, ‘promotion’ and ‘conversion’ events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited ‘initiation’ event which is present in every cell. Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl- N′-nitrosoguanidine (MNNG) and the tumourpromoter 12- O-tetradecanoypphorbol-13-acetate (TPA) alone. When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice. Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before. Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies. However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines.

Characterization of mutant human fibroblast cultures transformed with simian virus 40.

Fibroblast cell strains derived from a normal individual and from eight patients with various genetic mutations were transformed by a small-plaque variant of simian virus 40 (SV40, strain 776), cloned and studied after long-term in vitro maintenance. Seven of the cultures continued to express the mutant phenotype. Cultures derived from a patient with phosphoglycerate kinase I deficiency exhibited reappearance of normal enzyme activity after transformation. Compared to untransformed controls, all transformed cultures displayed decreased population doubling times, an increase in the relative number of cycling cells and increased saturation density on solid substrates, and did not show evidence of cellular senescence after long-term cultivation. Unlike previous studies on wild-type SV40-transformed human fibroblasts, the majority of cultures transformed by the small-plaque variant of SV40 did not exhibit signs of crisis. The cells also exhibited a decreased dependence on serum and were able to grow in semi-solid medium. The different transformed cultures expressed variable levels of SV40 large T-antigen, synthesized some infectious SV40 virus, and contained both unique arrangements and quantities of covalently integrated and episomal SV40 DNA. No correlation was observed between the rate of growth and synthesis of infectious virus in the different transformed clones. These studies indicate that this small-plaque variant of SV40 can be used effectively to generate long-lived human cultures, which generally retain their mutant phenotype. Transformation with this SV40 variant permits the generation of large quantities of clonal cell cultures for the biochemical and molecular analysis of their genetic defects.

Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro.

Isolated rat calvaria cells plated at low density in medium supplemented with ascorbic acid and organic phosphate form discrete three-dimensional mineralized nodules having the characteristics of bone. We have studied the effects of glucocorticoids on the formation of bone nodules by these cell populations. Cells isolated from 21-day-old fetal rat calvaria were maintained in vitro for up to 27 days. Dexamethasone (Dex) induced a dose-related increase in the number of nodules formed, with a peak at 10 nM and a half-maximal response at about 1 nM. Dex (10 nM) also significantly increased the size of bone nodules formed (P less than 0.002). High concentrations of Dex (1 microM) did not increase nodule number. In cells in primary culture maintained in medium containing 10 nM Dex, the increase in nodule number was 50-100% over the control value. The effect of Dex was much greater in first subculture cells, where the number of nodules was 600-800% higher than the control value. Dishes collected and quantitated from 12-27 days showed that nodule formation ceased between 15 and 18 days in cultures without Dex, whereas in the presence of Dex the number of nodules increased up to 27 days. Addition of 10 nM Dex only during specific periods resulted in significantly more nodules than in control cultures, but significantly fewer nodules than in cultures constantly exposed to Dex. Cell population doubling times during log phase growth were unaltered, but a significant increase in saturation density (P less than 0.001) was observed with 10 nM Dex. Hydrocortisone also caused an increase in the number of nodules formed, with a maximal effect of 50 nM and a half-maximal response at 8 nM. The results indicate that physiological levels of glucocorticoids stimulate bone nodule formation in long term cell culture by increasing the number of cells forming bone nodules and that maximization of the stimulatory effect of glucocorticoids on bone formation may require constant exposure to low levels of the hormone.

Inhibition of phorbol ester-mediated phenotypic changes in cultured cells by hypoxanthine.

Hypoxanthine induces the differentiation of certain transformed cells in vitro, so analyses were undertaken to determine whether this purine metabolite might influence the expression of transformed phenotypes induced in normal cells by chemical agents. Chinese hamster embryo cells and human skin fibroblasts in culture were treated with the promoting agent phorbol-12,13-didecanoate (PDD) with or without prior treatment with 3-methylcholanthrene (MCA), and various phenotypic effects were monitored. Hypoxanthine was found to inhibit significantly the formation of type III foci and the increase in saturation density observed for Chinese hamster cells treated with MCA plus the phorbol ester. Inosine and the hypoxanthine analogue allopurinol could also mediate the effect on saturation density, while xanthosine could not. An increase in the saturation density of human skin fibroblasts, which can be induced by the phorbol ester alone, was also inhibited by hypoxanthine. There was no significant effect on the growth rate or the intracellular nucleotide pools with hypoxanthine-treated cells. The results suggest that a normal purine metabolite, hypoxanthine, can modulate the expression of transformed phenotypes induced in vitro by the known tumor promotor PDD. These observations could help in elucidating the cellular basis for promotion of carcinogenesis.

Modulation of WI‐38 cell proliferation by elevated levels of CaCl2

Elevating the level of extracellular calcium (CaEx2+) increases the saturation density achieved by the normal human diploid cell line, WI-38, but does not change the growth rate. Day 7 cell yields remain unchanged when [CaEx2+] is between 0.5 mM and 3.0 mM, decrease when [CaEx2+] less than 0.5 mM, and increase when [CaEx2+] greater than 3.0 mM. Combining hydrocortisone with additional CaCl2 results in an additive effect on the saturation density relative to that obtained with each treatment separately. The stimulatory effect of elevated [CaCl2] is independent of serum concentration but is lost when WI-38 cells are grown in conditioned medium. Stimulation is recovered when conditioned medium is diluted with serum-free medium. In the case of young cultures grown in conditioned medium, stimulation can also be recovered when higher than usual levels of additional CaCl2 are used (2-3 mM). A glutamine supplementation to the conditioned medium potentiates cell response to elevated [CaCl2]. These results indicate that the loss of an enhanced saturation density when cells are grown in conditioned medium is not due to serum depletion but is more likely the effect of metabolites and/or nutrient depletion. When older or less vigorously growing cultures are grown in conditioned medium, additions of up to 3 mM CaCl2 only lead to inhibition, suggesting an age-related change in proliferative regulation. Elevated levels of CaEx2+ also enhance the proliferative response of quiescent monolayers to serum stimulation. This finding, along with the increase in saturation density of Ca2+-treated cultures, suggests that an elevated level of CaEx2+ affects cell entry into and exit from quiescence brought on by density-dependent inhibition.

Characterization of Fischer rat embryo (CREF) cells transformed by bovine papillomavirus type 1

Transformation of an established Fischer rat embryo (CREF) cell line by bovine papillomavirus type 1 (BPV-1), in contrast to transformation by type 5 adenovirus or the T24 (Ha-ras) oncogene, resulted in transformants which did not exhibit a major increase in saturation density or a decrease in 125I-epidermal growth factor binding. BPV-1-transformed CREF clones did, however, grow in agar suspension culture and were tumorigenic in both nude mice and Fischer rats. The majority of transformed clones contained multiple extrachromosomal copies of BPV DNA. One transformed CREF clone contained integrated BPV-1 DNA which underwent sequence rearrangements following tumor formation in a Fischer rat and reestablishment in cell culture. BPV copy number varied in subclones of transformants isolated from the same monolayer focus, in agar-derived subclones of the same transformed focus, in tumors and in tumor-derived BPV-transformed CREF subclones. The degree of expression of specific transformation-related phenotypes, i.e., saturation density, growth in agar, and tumorigenicity, was not correlated with BPV copy number in transformed clones. Analysis of the biological properties of tumor-derived BPV-1-transformed CREF subclones indicated that certain transformants developed a stable increase in expression of transformation-related properties, a process termed “progression.” TPA did not enhance the frequency of BPV-1 transformation or BPV DNA copy number in transformed CREF cells. The present study demonstrates that the CREF-transformation system can be utilized to study the molecular basis of BPV-1 transformation and should prove useful in studying the role of specific BPV-1 transforming proteins in regulating expression of the transformed phenotype and the mechanism by which transformed cells undergo progression of the transformed state.

Influence of increased extracellular calcium concentration and donor age on density-dependent growth inhibition of human fibroblasts.

Elevated calcium chloride concentration [( CaCl2]) has been shown to increase saturation density for an established mouse fibroblast line and for human fetal lung fibroblasts (WI-38). In order to examine the effect of increased [CaCl2] on human fibroblasts from donors of varying age, fibroblasts were grown in medium (basal level of 1.8 mM CaCl2) supplemented with fetal bovine serum (FBS) until confluent. Compared to controls in basal medium, newborn foreskin fibroblasts exposed to additional CaCl2 had a 110-450% increased cell yield that was independent of [CaCl2] within the range of an additional 1.5-5.0 mM. The effect was maintained over an eightfold range of FBS concentration. Initial growth rate was unaffected, but a prolongation of exponential phase occurred for cultures exposed to increased [CaCl2]. Confluent cultures refed medium with increased [CaCl2] were stimulated 5- to 10-fold more than cultures refed basal medium. An additional 2 mM CaCl2 resulted in a 210% increase for young adult-derived fibroblasts versus a 29% increase for old adult-derived fibroblasts (P less than 0.001). These data indicate that increased [CaCl2] decreases density-dependent growth inhibition of postnatal human dermal fibroblasts in vitro and that this effect is donor age dependent.

Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation.

We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)1] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.

Familial polyposis coli: growth characteristics of karyotypically variable cultured fibroblasts, response to epidermal growth factor and the tumour promoter 12-0-tetradecanoyl phorbol-13-acetate.

Growth in low serum and cell saturation density was investigated in 20 skin fibroblast cultures from 17 patients with the autosomal dominant cancer prone condition, familial polyposis coli (FPC). Compared with non-fetal control cultures, the grouped FPC cultures showed significantly better growth in low serum and approximately 30% increase in saturation density. Neither of these properties was correlated with high tetraploidy or clonal rearrangement of the chromosomes. No difference in response to epidermal growth factor was demonstrable between cultures from normal and affected subjects. The tumour promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), had no differential effect on growth in high and low density cultures of FPC and normal cells in short term experiments; both cell types displayed a biphasic response to the agent at low cell density. However, in long term experiments FPC skin cultures showed growth stimulation and greater resistance to the toxic effects of TPA than normal cells. Cells from both fetal and non-fetal controls as well as from FPC subjects displayed anchorage independent growth after treatment with TPA, but in general FPC cultures from skin and colon responded to a greater extent than non-fetal controls. Marked change in tetraploidy after treatment was evident only in those FPC and control cultures which were highly chromosomally abnormal. Both groups showed a slight increase in stable and unstable chromosome rearrangements with treatment but one FPC culture became totally chromosomally abnormal and cloned in agar with high efficiency, as did one of the treated fetal controls which, however, had normal chromosomes.

Density-dependent expression of keratins in transformed rat liver cell lines

Immunomorphological examination of the distribution of three keratins in cultured rat liver-derived epithelial cell lines of the IAR series was performed in order to find out the effects of neoplastic evolution on the expression of these epithelium-specific markers. Specific monoclonal antibodies were used to reveal various intermediate filament proteins: keratins with molecular masses of 55, 49 or 40 kD (K55, K49 or K40), and vimentin. The expression of keratins was negligible in sparse and dense cultures of nontransformed lines, which had typical epithelial morphology. The examined keratins were also absent in the sparse cultures of transformed lines, which have lost partially or completely the morphological features of epithelia. However, cells in dense cultures of most transformed lines contained numerous keratin filaments. It is suggested that the paradoxical increase of keratin expression after transformation is due to increased saturation density of transformed cultures; this high density favours the expression. As shown by the experiments with culture wounding, the effects of density are local and reversible. While K55 was present in all the cells of dense cultures, the expression of the other two keratins was dependent on the cell position within these cultures. It is suggested that the expression of the latter two keratins, besides high cell density, also requires the presence (K40) or the absence (K49) of cell-substratum contacts. Possible mechanisms of the effects of cell density on the expression of keratins are discussed.

Establishment and Characterization of the Human Cholangiocarcinoma Cell Line HChol-Y1 in a Serum-Free, Chemically Defined Medium

A human cholangiocarcinoma cell line, designated as HChol-Y1, was established in a protein-free, chemically defined medium after a very short period of primary culture in 0.1% fetal bovine serum (FBS)-containing medium. The cell line has been propagated in this medium for 2 years. The cells grew as a monolayer and the doubling time was about 52 hours. Addition of FBS did not stimulate cell growth (population-doubling time = 50 hr) or increase saturation density. The cells grown in a protein-free medium secreted small amounts of carcinoembryonic antigen (CEA) and large amounts of carbohydrate antigen (CA) 19/9 (CEA: 12.5 +/- 2.1 ng/10(6) cells/48 hr; CA 19/9: 760 +/- 52 IU/10(6) cells/48 hr); these tumor markers were immunohistochemically demonstrated in HChol-Y1 cells. Addition of FBS slightly stimulated the production of CEA and CA 19/9. The HChol-Y1 cell line was xenotransplantable in athymic nude mice and increased the serum CEA and CA 19/9 levels in the tumor-bearing nude mice. For determination as to whether a human carcinoma cell line can proliferate and secrete CEA and CA 19/9 in synthetic medium without any protein supplements, the cells were cultivated long term (2 yr) in a protein-free, chemically defined medium. When this method of cultivation is used, it is easy to purify these substances from spent medium, because contaminating antigens such as FBS or other substances usually added to cultures are absent.

Enhanced proliferation of WI38 cells in the presence of glucocorticoid-conditioned medium

The addition of hydrocortisone (HC) or dexamethasone (DEX) to WI38 cells at subcultivation is known to result in increased saturation densities (20–40%). We report that maximal increases in saturation density are, however, only observed if HC is added to the culture shortly after subcultivation. We have found that the proliferative response of WI38 cells to glucocorticoids is mediated by a secondary stimulatory factor(s) present in medium-conditioned by cells in the presence of the hormone. Control cultures refed with medium conditioned in the presence of HC for the first 24 h after seeding (24 h HC-CM) achieve saturation densities 20–40% higher than cultures refed with either medium conditioned in the absence of the hormone (24 h CM) or 24 h CM supplemented with fresh HC. Furthermore, WI38 cultures remain responsive to the stimulatory activity present in 24 h HC-CM throughout the growth cycle. The stimulatory effects of 24 h HC-CM are enhanced by repeatedly refeeding cultures; WI38 cells refed every 2 days with 24 h HC-CM demonstrate an extended period of logarithmic growth and achieve densities 2–3 times higher than controls. A preliminary characterization of this activity shows it to be of low molecular weight (MW) (dialyzable using 12 000 MW cut-off tubing) and heat-stable (75°C).

Subclone heterogeneity in a clonally-derived osteoblast-like cell line

To analyze the phenotypic diversity of a clonal rat osteosarcoma cell line (ROS 17/2) we have subcloned the cell line and characterized four sub-clones, ROS 17/2-A.II, A.III, A.V, and A.XIV. The subclones retained many of the characteristics of the parent clone that are considered typical of normal osteoblast-like cells; they responded to parathyroid hormone and isoproterenol, and had a negligible response to prostaglandin E 2 as measured by their respective changes in cyclic AMP concentration. In addition up to a 75% decrease in 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) binding was observed over a four-fold increase in cell density. The morphologies of the subclones varied from spindle-shaped, fibroblast-like to cuboidal. Doubling times varied from 24 to 48 hours, and basal alkaline phosphatase (AP) levels differed by as much as 10 times over the initial 3 months in culture. After 6 months (~ 100 PDL), the population doubling time of subclone A.XIV decreased from ~ 48 to ~ 20 hrs and there was a 2.5 to 3-fold increase in saturation density. This cell line was designated A.XIV.1 and was compared to a thawed sample from frozen stock of the original A.XIV isolate, designated A.XIV.2. These two populations, the parent cell line (ROS 17/2) and subclone A.V had similar growth properties, but differed with respect to changes in their alkaline phosphatase activity (AP) with time in culture: that is, all clones increased AP with time but there was a three to five-fold difference in their respective AP levels at various times in culture. All clones except A.V exhibited decreased AP activity upon reaching their saturation densities. The observed subclone diversity could provide a basis for creating a dynamic heterogeneous cell population in the parent cell line and thus alter its expressed phenotype with time in culture or with changing culture conditions.

Transforming potency of flavoring agents in Chinese hamster cells.

To investigate the transforming potency of flavoring agents, Chinese hamster (CH) B241 cells were treated with various concentrations of flavoring agents for 24 hours during their exponential growth period. Surviving cells were cultivated for generations without the agents to observe the appearance of growth properties characteristic of transformed cells. Highly increased rates of structural and numerical chromosome abnormalities were seen in the cells treated with a high concentration of the agents, especially with allylisothiocyanate (AITC) or tr-cinnamic aldehyde (CA). During subsequent passages of the treated cells, the survivors from treatment with sublethal doses of AITC or CA showed a significant increase in cloning efficiency in soft agar medium, especially after passage in soft agar. Also noted was an increase in saturation density in monolayer culture, though a significant increase in plating efficiency at low serum level was not observed. These characteristic changes in AITC- or CA-treated cells were associated with the significant increase in frequencies of cells containing almost 3n to 4n chromosomes.

DNAase I encapsulated in liposomes can induce neoplastic transformation of syrian hamster embryo cells in culture

We have used liposomes to deliver DNAase I inside normal Syrian hamster embryo (SHE) cells. We showed the entrance of DNAase I inside the cell by dose-dependent cytotoxicity; and the entrance of DNAase I into the nucleus by the induction of chromosomal aberrations and somatic mutation at the HPRT locus (but not at the Na +/K + ATPase locus). The induction of neoplastic transformation in cultures treated by DNAase I-in-liposomes was manifested by increased saturation density, colony formation at low seeding density, colony formation in 1% serum and 0.3% agar, and tumorigenicity in 100% of injected animals. The acquisition of anchorage-independent growth became apparent only after 39–57 posttreatment population doublings. Thus damage to DNA alone can initiate the neoplastic transformation process; but for full expression of the neoplastic phenotypes, a long progression time is required for the acquisition of anchorage-independent growth and tumorigenicity.

Altered growth properties of normal human cells induced by phorbol 12,13-didecanoate.

The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cells with 10(-7) or 10(-8) M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4 alpha-phorbol 12,13-didecanoate failed to elicit similar cellular responses.

Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine cytomegalovirus

Semipermissive, primary hamster embryo (HE) cells were morphologically transformed in vitro by infection with UV-irradiated equine cytomegalovirus (equine herpesvirus type 2; ECMV). Cell lines (designated EC-1-3) were established independently from foci and were shown to exhibit growth and biological properties typically associated with transformed cells: altered morphology, loss of contact inhibition, increased saturation density, decreased generation time, immortality in culture, normal growth in low concentrations of serum, colony formation in soft agar, and resistance to ECMV superinfection. All ECMV transformed cells were restrictive for the replication of equine herpesvirus type 1(EHV-1) which shares 2.9% homology with ECMV and replicated to high titers in normal HE cells. All EC cell lines were oncogenic in immunocompetent syngeneic LSH hamsters. Tumor cell lines were established from selected malignant fibrosarcomas that developed at the site of injection. Two of the transformed cell lines (EC-2 and EC-3) were found to be persistently infected and to release infectious ECMV from 0.5 to 2% (EC-2) and 0.8 to 5% (EC-3) of the total cell populations. The transformed cell line EC-1 as well as all tumor cell lines were virus nonproducers. Data from DNA-DNA reassociation analyses indicated the presence of 8–32 ECMV genome equivalents per productive EC-2 cell and greater than 300 ECMV genome equivalents per productive EC-3 cell. Small amounts of subgenomic ECMV DNA sequences were detected in the nonproducer EC-1 transformed cells and in all tumor cell lines (EC-1T, -2T, -3T). Some of these DNA sequences must be expressed since ECMV-specific polypeptides were demonstrated in all transformed and tumor cell lines by indirect immunofluorescence using antiserum to ECMV-infected cell extracts and since the sera of tumor-bearing hamsters contained ECMV antibody as detected by an ECMV plaque reduction assay.

In vitroevaluation of the tumor‐promoting potential of diesel‐exhaust‐particle extracts

Diesel‐exhaust‐particle extracts at nontoxic doses were evaluated in BALB/c mouse embryo 3T3 cells and Chinese hamster V‐79 lung cells for their ability to mimic the actions of the known tumor promoter tetradecanoyl phorbol acetate (TPA). The extracts were tested in a two‐stage model of carcinogenesis in 3T3 cells and found to marginally promote the occurrence of transformed foci over a concentration range of 2–10 μg/ml. The particle extracts also caused a loss of cell‐surface fibronectin and produced an increased saturation density of 3T3 cells. Using V‐79 cells, the extracts were found to inhibit metabolic cooperation between cells. Based on these results, it was concluded that diesel‐exhaust particles contain compounds that act as weak tumor promoters.