Tumorigenicity of partial transformation mutants of Rous sarcoma virus.

Chicken embryo cells infected with partial transformation mutants of Rous sarcoma virus were tested for tumor-forming ability in chickens and in nude mice. Cells transformed by each of these partial transformation mutants display different combinations of transformation parameters. They therefore present a potentially favorable system for analyzing which properties of transformed cells are necessary for tumor formation. We found that the relative tumorigenicity of the virus mutants was generally similar in chickens and in nude mice, except that certain temperature-conditional mutants appeared to be sensitive to the differences in body temperature of the two experimental animals. (The body temperature of nude mice is 4 to 5 degrees C lower than that of chickens). Thus, the nude mouse appears to be a suitable system for testing the tumorigenicity of transformed chicken cells. Because mice are nonpermissive for Rous sarcoma virus infection and replication, it was possible to recover the transformed chicken cells from the tumors in this host and to determine what phenotypic changes they had undergone during tumor development. We also examined the relationship between various cellular properties of the virus-infected chicken cells in vitro and their tumorigenicity in nude mice. The combined results of these two studies indicated that anchorage independence and plasminogen activator production were highly correlated with the tumor-forming ability of these cells, whereas loss of fibronectin did not correlate with tumorigenicity. Furthermore, the inability of the least tumorigenic virus mutant to stimulate the phosphorylation of a 36,000-Mr target of pp60src raises the possibility that the 36,000-Mr protein plays a role in tumor formation.     

Enhance anchorage independence and tumorigenicity of aneuploid Chinese hamster cells with nearly doubled chromosome complements.

The role of a cell's chromosome complement in its tumorigenic and anchorage-independent growth properties in vitro was investigated by injecting a Chinese hamster cell line and its subclones into immunodeficient nude mice and by plating the cells in a semisolid medium containing methylcellulose. The parental WOR-6 cell clone originally consisted of 89% 1s cells and 11% cells with a nearly double (2s) complement. Tumors that developed from WOR-6 were found to consis entirely or primarily of cells with near 2s chromosome complements. Subclones of WOR-6 that contained only 1s cells rarely produced tumors in nude mice, even at high inoculum doses, whereas clones containing a high fraction of 2s cells were consistently tumorigenition, serial passage of WOR-6 cells in semisolid medium resulted in selective enrichment for near 2s cells and, concomitantly, greatly enhanced tumorigenicity. Analyses of G-banded chromosomes revealed that the 1s cells of the WOR-6 parental clone, which has a modal chromosome number of 21 and a range of 18 to 23, is completely or partially monosomic for some chromosomes and trisomic for others. The 2s cells, selected both in vivo through growth as tumors in nude mice and in vitro in semisolid medium, appeared to have resulted from preferential duplication of certain chromosomes of the 1s cells. Our results therefore suggest that cells which develop multiple copies of selected genes, while remaining functionally hemizygous for other loci, acquire an enhanced anchorage-independent growth potential in vitro and increased tumorigenicity. This conclusion is consistent with the observation that cellular tumorigenicity is correlated with anchorage independence (Rreedman and Shin, 1974) and leads support to OHNO'S (1974) suggesting that aneuploidy is a possible means employed by cells to express recessive phenotypes and increase their tumorigenicity.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Genetic analysis of tumorigenesis. VIII. Suppression of SV40 transformation in cell hybrids and cytoplasmic transferants

Intraspecies somatic cell hybrids of BALB/c mouse 3T3 and SV40-transformed embryonic fibroblast (SVT2) cells were analyzed for transformation-associated properties and their tumorigenic potential in nude mice. In confirmation of our earlier findings, hybrids expressing the viral T-antigen were not suppressed for the ability to clone in medium with 1% serum. In contrast, division rate in medium with 1% or 10% serum, anchorage independence, cytochalasin-sensitive growth control, and tumorigenicity were suppressed noncoordinately, and the extent of suppression varied from one hybrid to another. Suppression was not simply determined by the increased chromosome content of the hybrid cells, nor was suppression correlated with rearrangements of the integrated viral sequence (SAGER et al., 1981a, b). Similar results were found in cytoplasmic transferants expressing T-antigen. Four independent transferants and subclones derived from them varied in the extent of suppression of anchorage independence and tumorigenicity. In both hybrids and transferants, a low serum requirement for clonal growth apparently was determined solely by expression of SV40 T-antigen, but other transformation properties, as well as tumorigenicity, appeared to require multiple changes in the cellular genome for their expression. These changes must occur during or after viral integration, since they are not expressed in uninfected 3T3 cells.     

Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.     

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.     

Decreased tumorigenicity correlates with expression of altered cell surface carbohydrates in Lec9 CHO cells.

To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice.     

Microfilament bundles in cultured cells : Correlation with anchorage independence and tumorigenicity in nude mice

The distribution of actin microfilament bundles in cell lines 3T3B, CHO, HeLa and CLID extracted with 0.1% Triton X-100 was examined by indirect immunofluorescence using human actin antibodies and by electron microscopy of whole cells grown directly on support grids. Anchorage dependence as determined by growth in soft agar and tumorigenicity in nude mice was also investigated. Immunofluorescent staining showed that CHO and HeLa cells have normal numbers and distributions of actin microfilament bundles as compared with similarly spread control 3T3B cells. A significant fraction of the mouse CLID cells showed comparable numbers of microfilament bundles as 3T3B cells but their distribution was markedly different. In many cases the bundles radiated from a region close to the cell's centre or near its projections and usually penetrated the projections. The presence of diffuse staining in 4% of the cell population also indicated the existence in these cells of disorganized actin. Electron microscope studies of well spread regions of negatively-stained, Triton-extracted cells corroborated the observations made with the immunofluorescence technique. In 3T3B, CHO and CLID cells abundant microtubules were found, colinearly arranged with actin filaments in the thin cytoplasmic extensions. While CLID, CHO and HeLa cells showed the capacity to grow in soft agar, only CLID and HeLa cells produced tumours in athymic nude mice. The observations suggest that a reduction or disorganisation of the actin microfilament bundles may not in itself be essential at least for the non-virally transformed cells studied to show anchorage independence or to produce tumours in nude mice.     

Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability

Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell- surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.     

Alterations in growth requirements of kidney epithelial cells in defined medium associated with malignant transformation

The possibility has been investigated that (1) the supplements required for the growth of the Madin Darby Canine Kidney (MDCK) cell line in serum-free Medium K-l are indeed requirements for the growth of normal kidney cells in vitro, and (2) that alterations in these growth requirements are associated with malignant transformation. Consistent with the hypothesis that MDCK cells resemble normal kidney cells in culture, primary cultures of baby mouse kidney epithelial cells grow in Medium K-l and respond to the 5 components in the-medium. The growth properties of Moloney sarcoma virus (MSV)-transformed MDCK cells in defined media have been examined. Unlike MDCK cells, MSV-transformed MDCK cells form tumors in adult nude mice. Although they still respond to the 5 factors in Medium K-l, the optimal dosage for insulin is lower for the MSV transformants than for MDCK cells. The MSV transformants also have an additional requirement for growth in Medium K-l – fibronectin. Variants of MDCK cells have been isolated that have lost the PGE₁ requirement for growth in defined medium. These variant cells have acquired (1) the ability to form tumors in adult nude mice and (2) an alteration affecting cAMP metabolism, in addition to PGE₁ independence.     

Epstein-Barr virus in nontumorigenic and tumorigenic nasopharyngeal carcinoma (NPC) somatic cell hybrids

Somatic cell hybrids between mouse fibroblasts and human cells derived from nasopharyngeal carcinoma (NPC) biopsies or NPC tumors propagated in nude mice were examined for the expression of the Epstein-Barr nuclear antigen (EBNA), retention of Epstein-Barr viral (EBV) DNA, and tumorigenicity in nude mice. In all hybrids the expression of EBNA correlated with the detection of EBV-DNA. After more than 2 years in culture, the hybrids examined retained similar amounts of EBV-DNA when compared to previously published data. Retention of EBV-DNA did not correlate with the presence of any particular human chromosome. Use of either rodent cell lines, clone 1D or IT-22, did not affect the retention nor loss of EBV-DNA. For tumorigenicity studies, NPC cells were fused with IT-22 cells and injected into nude mice. Tumor formation did not depend on the presence or absence of EBNA and detectable EBV-DNA sequences; tumorigenicity in these studies could not be correlated with the presence of any particular human chromosome or the origin of the NPC biopsy.     

Tumorigenicity of revertants from an SV40-transformed line

A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.     

Chemical carcinogen-mouse mammary tumor virus interactions in cell transformation

We have studied the process of mammary cell transformation in vitro using a single cell clone (Clone 18) from a presumptive epithelial cell line, C57MG, derived from a normal mammary gland; a mouse mammary tumor virus (MMTV) host-range variant (RIII)vp4; and the potent initiating carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). After several serial subcultures, cells treated with virus and then with carcinogen exhibited an altered (transformed) morphology, a dramatic increase in anchorage independence, an increase in multinucleation after exposure to cytochalasin B, an enhanced ability to proliferate in low Ca2+ (0.01 mM) medium, and tumorigenicity when inoculated subcutaneously into athymic (nude) mice. Although some of these phenotypic alterations were observed also in cultures treated singly with MMTV or DMBA and in cultures exposed to DMBA before infection with MMTV, enhanced cytochalasin B multinucleation and tumorigenicity were properties observed only in mass cultures of cloned cells first infected with MMTV and then exposed to DMBA. This demonstrates for the first time that exposure of presumptive mammary epithelial cells to MMTV followed by DMBA, but not to either agent alone or to DMBA followed by MMTV, results in malignant transformation of these cells.     

Suppressor genes for malignant and anchorage-independent phenotypes located on human chromosome 9 have no dosage effects

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) human chromosome (HSA), derived from human fibroblast strain GM0705, into the Syrian hamster cell line BHK-191-5C produced only near-tetraploid hybrids, although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells. However, the tumorigenicity and the anchorage independence could be suppressed in the near-tetraploid hybrids with one copy of the der(9)t(X;9) chromosome. The introduction of an HSA X chromosome did not suppress either of these phenotypes. We concluded that in addition to two suppressor genes, one for tumorigenicity and another for anchorage independence, HSA 9 might carry a third gene capable of inhibiting cellular growth in vitro, which had dosage effects. In the present study, keeping one copy of the der(9)t(X;9) chromosome, we have increased the hamster background chromosome number beyond hexaploid level by fusing two microcell-generated hybrid cell lines, where both malignant and anchorage-independent phenotypes were suppressed, with the parental malignant BHK-191-5C cell line. Tests with nude mice showed that hybrids containing one copy of the der(9)t(X;9) chromosome against the increased background of chromosomes of malignant parental origin were still suppressed for both phenotypes. These results suggest that the suppressor genes for malignancy and for anchorage independence have no dosage effects, in contrast to the suppressor gene(s) for cellular growth.     

Malignant transformation of p53-deficient astrocytes is modulated by environmental cues in vitro.

The early incidence of p53 mutation in astrocytomas suggests that it plays an important role in astrocyte transformation. Astrocytes isolated from homozygous p53 knockout mice grow rapidly, lack contact inhibition, and are immortal. Here we tested whether the loss of p53 is sufficient for progression to tumorigenicity of astrocytes. We grew primary astrocytes under three conditions for over 120 population doublings and assessed their antigenic phenotype, chromosome number, and expression of glioma-associated genes as well as their ability to form colonies in soft agarose and tumors s.c. and intracranially in nude mice. Under two conditions (10% FCS and 0.5% FCS plus 20 ng/ml EGF), cells acquired the ability to form colonies in soft agarose and tumors in nude mice, and this was accompanied by the expression of genes, including epidermal growth factor receptor, platelet-derived growth factor receptor alpha and beta, protein kinase Cdelta, and vascular endothelial growth factor, which are known to be aberrantly regulated in human astrocytomas. Under the third condition (0.5% FCS plus 10 ng/ml basic fibroblast growth factor), astrocytes gained the ability to form colonies in soft agarose and had abnormal chromosome numbers similar to cells in the first two conditions but did not form tumors in nude mice or overexpress glioma-associated genes. These data provide experimental evidence for the idea that the malignant progression initiated by the loss of p53 may be subject to modulation by extracellular environmental influences.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Carcinogenesis or tumourigenicity testing of animal cell lines for vaccine preparation by colony formation on soft agar and by agglutination under plant lectins

The carcinogenic or tumourigenic testing of seven animal kidney cell lines (F-81, CRFK, MDCK, Vero, Vero-2 cell line, MA-104 and BHK-21) established in China, were carried out in more than 700 nude mice for colony formation in soft agar and for agglutination under different density of plant lectins. Tests showed that there were correlation between cell line chromosome number variations and anchorage independence in soft agar, agglutinability under lectins and tumour-forming ability in nude mice. Since testing in vitro was more economical, simpler and faster and thus thought to be more reliable, we recommend measuring agglutinability, followed by anchorage independence or analysis of karyotype as the initial means for monitoring tumourigenicity of animal cell lines in nude mice.     

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.     

High recurrence of rearrangements involving chromosome 14 in an ataxia telangiectasia lymphoblastoid cell line and in its mutagen-treated derivatives

Summary The cytogenetic characterization of CH cell line obtained by Epstein-Barr-virus transformation of the lymphocytes of a patient affected by ataxia telangiectasia is reported. Control CH cells and 2 subcultures treated with the mutagens R7000 or NQO were developed in parallel and studied. A common chromosome anomaly, a der(14) t(11;14) (q13.2;q32), was found in all the studied karyotypes, indicating that it occurred either in vivo or early in vitro. In non-treated cultures, additional anomalies were present in 6 derived subclones. All R-7000 treated cells had the same karyotype corresponding to one of the subclones observed without prior treatment. All NQO-treated cells acquired 2 common anomalies, and could be differentiated into 2 subclones because of the addition of a t(7;14) or a t(11;14). Chromosome 14 was involved in various rearrangements after breakage in band q11.2 or q12 in 6/8 subclones. This was not correlated with tumorigenicity, which was clearly increased in mutagen-treated cells as tested by in vitro growth in semi-solid medium and in vivo by grafts into nude mice or growth on the chorio-allantoic membrane of chick embryos. The CH cell line and its derivatives appear to be a promising in vitro system, showing various stages progressing towards malignancy, and reproducing a number of chromosome anomalies spontaneously occurring in AT patients.     

Early passage primate cell immortality is independent of tumorigenicity

Summary Seven continuous primate cell lines were tested in three systems (nude mice, muscle organ culture, and soft agarose) for their ability to express characteristics usually associated with malignant cell lines. Five of the seven cell lines failed to produce tumors in nude mice, failed to show a tumor-like pattern of growth in muscle organ culture, and failed to produce colonies in soft agarose. The remaining two cell lines showed different degrees of tumorigenicity in nude mice, and gave frankly positive results in the twoin vitro assays. In addition, one of these lines appeared to progress from potential to overt tumorigenicity. We conclude that acquisition of infinite life in primate cell lines is not invariably equivalent to the ability to form tumors.