Transfer of anchorage independence by isolated metaphase chromosomes in hamster cells

The cellular property of being able to grow on agar (aga⁺) or to show anchorage independence has been transferred by means of metaphase chromosomes from CHO cells to BHK and other permanent transformed hamster lines unable to grow on agar. As with other genetic markers, the transferents are unstable when grown under non-selective conditions. The aga⁺ transferents are tumorigenic, providing further evidence for the association between the ability to grow in agar or anchorage independence and tumorigenicity. Evidence has been obtained in these experiments for the existence of at least two discrete events in the transformation of normal into tumorigenic cells. The ability to transfer and select for the aga⁺ marker in recipient cells indicates that tumorigenicity behaves dominantly phenotypically.     

Apoptotic HPV positive cancer cells exhibit transforming properties

Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+) or Ca Ski (HPV16+) cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-), were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.     

The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.     

Chemically and virally transformed cells able to grow without anchorage in serum-free medium: evidence for an autocrine growth factor

BA10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serum-free (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin. BA10-IR cells and FR3T3-Ki cells also release beta-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable beta-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors. Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980). The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Progression elevated gene-3, PEG-3, induces genomic instability in rodent and human tumor cells

Genomic instability is a fundamental component of cancer progression. Subtraction hybridization identified a novel rodent gene, progression elevated gene-3 (PEG-3) whose expression directly correlates with cancer aggressiveness and progression. Moreover, ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed phenotype. We demonstrate that PEG-3 expression in rodent tumor cells correlates directly with genomic instability as characterized by alterations in chromosome composition and structure. Additionally, elevated endogenous or ectopic expression of PEG-3 in rodent and human tumor cells, respectively, enhances gene amplification, as monitored by resistance to methothrexate (MTX) and amplification of the dihydrofolate reductase (dhfr) gene. Stable expression of PEG-3 in normal cloned rat embryo fibroblast (CREF) cells marginally elevates MTX resistance, but morphology remains unaltered and anchorage independence is not induced, suggesting that these phenotypes are separable in immortal cells and gene amplification may precede the acquisition of morphological and oncogenic transformation. The present studies document that stable, inducible, and transient expression of PEG-3 in cancer cells augments genomic instability. In these contexts, one mechanism by which PEG-3 influences cancer progression may be by preferentially facilitating the development of genomic changes in evolving cancer cells.     

Genetic analysis of tumorigenesis: I. Expression of tumor-forming ability in hamster hybrid cell lines

Two new fibroblasts cell lines from a male Chinese hamster embryo are described: one tumorigenic in nude mice (CHEF 16-2) and the other not (CHEF 18-1). Both lines have a stable diploid mode and mean of 22 with 10 pairs of homologous autosomes. The cell lines differ unambiguously in cloning ability in methylcellulose, colony morphology, and tumorigenicity; the expression of these traits was examined in a set of 18-1 X 16-2 hybrid clones. The results show initial suppression of tumorigenicity and anchorage independence, followed by growth in the nude mouse of subsets of cells with chromosomes reduced to the diploid range. Further studies are in progress to establish whether ability to grow in methylcellulose and in the nude mouse segregate coordinately at the cellular level, and whether this segregation has an identifiable chromosomal basis.     

Phenotypic and karyotypic transitions in the spontaneous transformation of a rat cell line

After 20-50 transfers, a rat myofibroblast line, Hmf-n, 'spontaneously' transforms to an established (immortalized) line of smaller, rapidly cycling fibroblastoid cells (tHmf-f). From these 1 degree transformants, colonies of larger, slower growing anchorage-independent (tHmf-e) cells of epithelioid phenotype emerge. Both transformants grow in low serum and low calcium media, but the tHmf-f cells are highly tumorigenic in nude mice, have diminished substrate adhesivity, and limited anchorage independence, whereas tHmf-e are less tumorigenic, firmly substrate adherent, and markedly anchorage independent. Most tHmf-f are trisomic; most tHmf-e transformants are hypodiploid, a third are tetraploid, and all have chromosomal abnormalities, but no trisomy. Hmf-n cells have polar stress fiber arrays terminating in vinculin adhesion plaques, colinear extracellular fibronectin matrices, and linear non-coincident deposits of fodrin. Microtubules (mt) and vimentin-intermediate filaments (IF) parallel the actin cables. Stress fibers of the tHmf-f are moderately reduced, their vinculin adhesion plaques and fibronectin matrices intact; fodrin is diffuse. Mts and IFs are normal and axial. Most epithelioid tHmf-e have no stress fibers, adhesion plaques, or extracellular fibronectin; instead, dense actin microfilament meshworks are attached to plasma membrane, as is fodrin. Mt and IF are radial. Both transformed phenotypes are stable over greater than 300 continuous passages. The differentiation-inducing agents DMSO, cyclic AMP, 5-azacytidine, and mezerein, were ineffective in normalizing shape or cytoskeleton of transformed Hmf, and butyrate was selectively toxic to 50% of tHmf-e. But hydrocortisone induced striking polarization, and increase in number, and alignment of stress fibers of both tHmf-f and tHmf-e. Growth, anchorage, cytoskeletal arrangements, and tumorigenic potential are not closely correlated in these stable, spontaneously transformed lines of distinct pheno- and karyotype originating from the same normal parental cell, suggesting independent acquisition of properties associated with transformation.     

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.     

A gene on pig chromosome 14 suppresses cellular anchorage independence of the mouse cell line GM05267.

We have generated pig-mouse somatic cell hybrids by fusing normal pig fibroblasts with an anchorage independent mouse cell line GM05267. High quality G-banding analysis was applied to a set of 18 hybrid cell lines derived from 15 independent hybrids and chromosomes were identified. Cytogenetic analysis showed that all hybrids contained one or several pig chromosomes with normal morphology devoid of any structural changes. Out of 18 hybrids tested for colony formation in soft agar, 15 were suppressed for anchorage independence while the remaining three were not suppressed. Correlation of the cellular phenotype with the pig chromosome content of the hybrids suggests that the suppressor function for anchorage independence is located on pig chromosome (SSC) 14. We have previously shown that a suppressor gene for anchorage independence (SAI1) is located on rat chromosome (RNO) 5 and another suppressor gene for the same phenotype is located on human chromosome (HSA) 9. Given the genetic homology of both RNO5 and HSA9 with two pig chromosomes including SSC14, the third suppressor gene we have mapped on SSC14 may well be a functional homologue of the previously identified rat and human genes.     

The Ha-ras-induced transformed phenotype of rat-1 cells can be suppressed in hybrids with rat embryonic fibroblasts

Somatic cell hybrids were isolated from fusions of diploid embryonic rat fibroblasts with transformed Rat-1 cells which contained 4 to 5 copies of the transforming human Ha-ras 1 gene. In contrast to their transformed parental cells four hybrid clones showed normal morphology, long latency periods of tumorigenicity in newborn rats, anchorage requirement of proliferation, and an eightfold-reduced amount of secreted transforming growth factor activity. Thus these hybrids are called suppressed with regard to expression of the Ha-ras-induced transformed phenotype. Tumorigenic derivatives of the suppressed hybrids that had segregated chromosomes were isolated. Since two of the tumorigenic hybrid clones showed the similar low level of secreted transforming growth factors as the suppressed hybrids, decreased production of transforming growth factor activity is unlikely to be a sufficient criterion for suppression of malignancy. Whereas one of the suppressed hybrids expressed the transforming gene product p21 at a level similar to that of the transformed parental cells, other suppressed hybrids expressed less p21. This suggests that the suppressed phenotype can be regulated at the posttranslational level of p21 but that additional controls of expression of p21 are likely to exist. DNA of the suppressed hybrids transformed Rat-1 cells to proliferation in the presence of semisolid agar. Thus the activated human Ha-ras gene in the suppressed hybrids retained its biological activity even though it did not transform these cells to tumorigenicity.     

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.     

Recessive (mediator-) revertants from c-H-ras oncogene-transformed NIH 3T3 cells: tumorigenicity in nude mice and transient anchorage and serum independence of the recovered tumor cells in culture.

We have reported earlier the isolation of two recessive, serum- and anchorage-dependent revertants (R116 and R260) from a c-H-ras oncogene-transformed NIH 3T3 line. In both revertants, the oncogene was fully expressed and fusion of either revertant with (untransformed) NIH 3T3 cells, or of the two revertants with one another, resulted in transformed progeny. These, and other data, indicated that the transforming activity of the oncogene was impaired in the two revertants in consequence of defects in distinct genes needed to mediate this activity. We report here that neither revertant could be re-transformed by the K-ras or N-ras oncogene (though they could be re-transformed by several other oncogenes). The two revertants turned out to be tumorigenic in nude mice (though less so than the parental transformed cells). The tumor cells, as recovered, formed foci and had a transformed morphology and a greatly diminished serum and anchorage dependence. Growth of the cells in culture (for 20 passages) resulted in their regaining the characteristics (i.e., anchorage and serum dependence) of cultured R116 and R260 cells. Proliferation of the cells in nude mice was not accompanied by a change in the level of ras oncogene expression or in gene amplification, at least as manifested in the lack of appearance of double-minute chromosomes. The addition of the growth factors TGF alpha and beta to the medium of either revertant did not support anchorage-independent growth.     

Properties of cells transformed by the middle T-antigen-coding region of polyomavirus.

A series of 10 Fischer rat transformed clonal cell lines were independently obtained in infections with a defective polyomavirus containing a scrambled genome except for an intact middle and small T-antigen-coding region. These cells synthesize middle and small T antigens; no fragment of large T antigen can be detected in any of them. The transformed phenotype of this set of cell lines (designated LT-) has been studied with respect to serum dependence, saturation density, and anchorage independence and compared with the phenotype of a set of six transformants (designated LT+) which synthesize detectable to high levels of shortened or normal-sized large T antigen. Both the LT+ and the LT- groups of polyomavirus transformants display a range of transformed phenotypes. These ranges overlap, and the variations within each group are larger than the variations between the two groups. Thus, the results suggest that, for established Fischer rat fibroblasts, the maintenance of any of the three phenotypes tested and, in particular, of serum independence is not necessarily correlated with the levels of large T antigen or fragments thereof.     

Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells

We report here on gene transfer studies designed to investigate the function of the alpha 5 beta 1 integrin and its role in transformation. Transfection of the human alpha 5 and beta 1 cDNAs into transformed Chinese hamster ovary (CHO) cells followed by methotrexate-induced amplification yielded clonal cell lines overexpression this fibronectin receptor. The overexpressors deposited more fibronectin in their extracellular matrix and migrated less than control cells. In addition, they showed reduced saturation density and reduced ability to grow in soft agar. The overexpressor cells, unlike the control CHO cells, were nontumorigenic when injected subcutaneously into nude mice. The results indicate that extracellular matrix recognition by the alpha 5 beta 1 integrin plays a role in the control of cell proliferation and suggest that a reduction of this fibronectin receptor may be responsible for the acquisition of anchorage independence by transformed cells.     

The majority of independently transformed BHK cell clones share a single functional lesion which determines anchorage independence and influences tumorigenicity

Summary Expression of the anchorage-independent transformed phenotype in BHK 21/13 cells generally behaves as a recessive trait. When chemically induced and spontaneously arising transformants are fused to the nontransformed parent line, transformation is initially suppressed, reappearing after extended growth of the hybrids. In this paper, complementation for the expression of anchorage independence was sought among a large group of such transformants, all independently derived from BHK 21/13 cells. Tumorigenicity studies on selected hybrids and parental lines indicated that the in vitro trait of anchorage independence is an accurate indicator of in vivo neoplasia for these cells. Seventeen of the 18 clones tested did not complement one or more of three tester strains. This result indicates that anchorage independence arose in these clones as a result of lesions in the same genetic function and suggests that the final step in the progressive changes of carcinogenesis may frequently be restricted to lesions at a single locus. This investigation was supported by National Institutes of Health grant CA27306.     

The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence.

Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.     

Different expression of cell surface fibronectin in spontaneously and X-ray transformed cells

The presence of cell surface fibronectin was examined by indirect immunofluorescence in 8 groups of related rat fibroblast lines expressing an in vitro transformed phenotype. The transformed cells were selected for anchorage independent growth either after X-ray treatment (X-ray transformed cells) or from control cultures (spontaneously transformed cells). All transformed fibroblasts of the latter class showed reduced expression of fibronectin at the cell surface, whereas most of the X-ray transformed derivatives exhibited a fibronectin-positive phenotype, like the untransformed parents. Moreover, from the fibronectin-negative spontaneously transformed cells, ouabain-resistant variants were isolated, the majority of which had regained the capacity to form an extracellular matrix of fibronectin. These results emphasize the variability in the properties of transformed cells and suggest that the properties of in vitro transformed cells may depend on the cause of transformation.     

Transfection and amplification of the chicken c-src proto-oncogene in Rat-1 cells

The chicken cellular proto-oncogene c-src was cotransfected into normal Rat-1 cells with the mouse dhfr gene. Selection for amplification of dhfr sequences resulted in co-amplification of the chicken c-src gene. Cell clones expressing varying levels of c-src associated kinase activity were isolated, none of these had a transformed morphology. In contrast, expression of v-src in Rat-1 cells resulted in morphological transformation and the ability to grow in soft agar in an anchorage independent way.