Tumorigenicity of human HT1080 fibrosarcoma X normal fibroblast hybrids: chromosome dosage dependency

The tumorigenic capacity of hybrids formed by fusion of the highly tumorigenic HT1080 human fibrosarcoma cell line with nontumorigenic normal fibroblasts was examined. The HT1080 also contains an activated N-ras oncogene. Near-tetraploid hybrids which contained an approximately complete chromosomal complement from both parental cells were nontumorigenic when 1 X 10(7) cells were injected s.c. into athymic (nude) mice, whereas the parental HT1080 cells produced tumors in 100% of the animals with no latency period following injection of 2 X 10(6) cells. Tumorigenic variants were obtained from these hybrids which had lost only a few chromosomes compared to cells from the nontumorigenic mass cultures. In addition, several near-hexaploid hybrids were obtained which contained approximately a double chromosomal complement from the HT1080 parental line and a single chromosomal complement from the normal fibroblasts. All of these near-hexaploid hybrids produce tumors in 100% of nude mice with no latency period. Our results indicate that tumorigenicity of these particular human malignant cells of mesenchymal origin can be suppressed when fused with normal diploid fibroblasts. In addition, the results suggest that tumorigenicity in this system is chromosomal dosage dependent, since a diploid chromosomal complement from normal fibroblasts is capable of suppressing the tumorigenicity of a near-diploid but not a near-tetraploid chromosomal complement from the tumorigenic HT1080 parent. Finally, the loss of chromosome 1 (the chromosome to which the N-ras oncogene has been assigned) as well as chromosome 4 was correlated with the reappearance of tumorigenicity in the rare variant populations from otherwise nontumorigenic near-tetraploid hybrid cultures. Our results also suggest the possibility that tumorigenicity in these hybrids may be a gene dosage effect involving the number of activated N-ras genes in the hybrids compared to the gene(s) controlling the suppression of the activated N-ras genes.     

Elevated expression of an exogenous c-myc gene is insufficient for transformation and tumorigenic conversion of established fibroblasts

Two established rat fibroblast lines, differing only by their number of generations in culture, show dramatically different responses to the elevated c-myc expression delivered by an efficient murine c-myc retrovirus vector. Thus, a late passage (60 generation) FR3T3 line acquires a transformed and tumorigenic phenotype upon introduction of this activated c-myc gene as indicated by its altered morphology, high efficiency of focus formation, soft agar clonability, saturation density in monolayer culture, and short latency of tumorigenicity in syngeneic hosts. Remarkably, none of these characteristics, except for an increased refractility in monolayers and an epidermal growth factor (EGF)-dependent agar clonability, were observed in a variety of early passage (10 generation) FR3T3 c-myc clones. BALB/c A31 fibroblasts transfected with this c-myc retroviral vector behaved essentially the same as the FR3T3 early line except for their inability to grow in suspension in response to EGF. However, transformation and tumorigenic conversion of each of these three fibroblast lines was achieved by an activated ras oncogene. Hence, elevated c-myc expression is insufficient for transformation of established fibroblasts but depends upon other acquired cooperating functions which are not necessary for ras induced transformation. We also demonstrate that endogenous c-myc expression remains unaffected even in clones expressing a 100-fold excess of exogenous c-myc RNAs demonstrating that c-myc autoregulation is not operative in these cells.     

Lymphomagenesis in E mu-myc transgenic mice can involve ras mutations

Transgenic mice bearing c-myc driven by the immunoglobulin heavy chain enhancer (E mu) initially exhibit a preneoplastic lymphoproliferative syndrome from which clonal pre-B or B lymphomas develop at random. To investigate whether this transition involves the activation of oncogenes capable of transforming fibroblasts, we transfected DNA from 14 E mu-myc lymphomas into NIH3T3 cells and tested the tumorigenicity of the transfectants in nude mice. By this assay and subsequent direct analysis of the lymphoma mRNA by cDNA cloning or the polymerase chain reaction, two independent E mu-myc lymphomas were shown to contain an N-ras or a K-ras oncogene mutated at codon 61. When incorporated into a recombinant retrovirus, the mutant N-ras allele could collaborate with myc to transform preneoplastic E mu-myc bone marrow pre-B cells. These results indicate that spontaneous mutation of ras genes is one pathway to lymphomagenesis in E mu-myc mice but that many of the lymphomas arise in response to changes that do not register in fibroblasts.     

Identification of transforming activity of free fatty acid receptor 2 by retroviral expression screening

Gallbladder cancer (GBC) is a highly fatal malignancy in humans. Genetic alterations in KRAS or TP53 as well as overexpression of ERBB2 have been shown to contribute to the development of certain types of GBC. However, many cases of GBC do not harbor such genetic changes, with other transforming events awaiting discovery. We here tried to identify novel cancer-promoting genes in GBC, with the use of a retroviral cDNA expression library. A retroviral cDNA expression library was constructed from a surgically resected clinical specimen of GBC, and was used to infect 3T3 fibroblasts in a focus formation assay. cDNA incorporated into the transformed foci was rescued by PCR. One such cDNA was found to encode free fatty acid receptor 2 (FFAR2), a G protein-coupled receptor for short-chain fatty acids. The oncogenic potential of FFAR2 was confirmed both in vitro with the focus formation assay and by evaluation of cell growth in soft agar as well as in vivo with a tumorigenicity assay in nude mice. The isolated FFAR2 cDNA had no sequence alterations, suggesting that upregulation of FFAR2 expression may contribute to malignant transformation. Indeed, all of quantitative RT-PCR, in situ hybridization, and immunohistochemical analyses showed that the amount of FFAR2 mRNA and its protein product was increased in digestive tract cancer specimens. Furthermore, short-chain fatty acids potentiated the mitogenic action of FFAR2 in 3T3 cells. Our data thus, for the first time, implicate FFAR2 in carcinogenesis of the digestive tract. ( Cancer Sci 2009)     

Permanent conversion of mouse and human cells transformed by activated ras or raf genes to apparently normal cells by treatment with the antibiotic azatyrosine

Azatyrosine [L-beta-(5-hydroxy-2-pyridyl)-alanine], an antibiotic isolated from Streptomyces chibanensis, inhibited the growth of NIH 3T3 cells transformed by the activated human c-Ha-ras gene but did not significantly inhibit the growth of normal NIH 3T3 cells. Surprisingly, upon treatment with azatyrosine most of the transformed cells apparently became normal. These apparently normal cells, named revertant cells, grew in the presence of azatyrosine and stopped growing when they reached confluency, and their normal phenotype persisted during prolonged culture in the absence of azatyrosine. The revertant cells did not grow in soft agar and scarcely proliferated in nude mice. The human c-Ha-ras gene present in transformed NIH 3T3 cells was still present in the revertant cells and was expressed to the same extent as in the original transformed cells, producing the same amount of activated p21. Treatment with azatyrosine caused similar conversion of NIH 3T3 cells transformed by activated c-Ki-ras, N-ras, or c-raf to apparently normal cells, but NIH 3T3 cells transformed by hst or ret were not exclusively converted by azatyrosine. Human pancreatic adenocarcinoma cells, which are known to contain an amplified activated c-Ki-ras gene and an amplified c-myc gene, were also converted to flat and giant revertant cells by treatment with azatyrosine.     

A threshold effect in the induction of tumorigenicity of an established human cell line by v-mos

The nontumorigenic immortal human cell line, SV80, was transfected with the v-mos gene to assess the gene's effect on tumorigenicity of cultured human cells. Two classes of cells, each containing functional v-mos, were obtained. The first class contained low levels of v-mos RNA, was morphologically transformed, but was nontumorigenic in nude mice. The second was also morphologically transformed, but contained high levels of v-mos RNA and was tumorigenic. The results indicate that SV80 cells behave similarly to murine fibroblasts in their response to v-mos in that they can be rendered tumorigenic by the viral oncogene. However, tumorigenicity was effected through a mechanism which involves different threshold doses for morphologic and tumorigenic transformation.     

Multistep process of neoplastic transformation of normal human fibroblasts by 60Co gamma rays and Harvey sarcoma viruses

As reported previously (Namba et al., 1985), normal human fibroblasts were transformed by 60Co gamma-ray irradiation into immortal cells with abnormal karyotypes. These transformed cells (KMST-6), however, showed a low cloning efficiency in soft agar and no transplantability. However, upon treatment with Harvey murine sarcoma virus (Ha-MSV), the cells acquired elevated clonability in soft agar and transplantability in nude mice. Ha-MSV alone, however, did not convert normal human fibroblasts into either immortal or tumorigenic cells. The Ha-MSV-transformed KMST-6 cells showed an enhanced expression of the ras oncogene, but normal and 60Co gamma-ray-transformed cells did not. Our current data suggest that gamma rays worked against normal human cells as an initiator, giving rise to chromosome aberrations and immortality, and that Ha-MSV, probably through its ras oncogene, played a role in the progression of the malignant cell population to a more malignant one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.     

A survey of growth in soft agar and cell surface properties as markers for transformation in adult rat liver epithelial-like cell cultures.

Continuous epithelial-like cell lines derived from normal adult rat liver and hepatocarcinomas were evaluated for their growth in soft agar and five properties of the cell membrane as markers for neoplastic transformation. A correlation of these properties was made to the tumorigenicity of the lines in nude mice. Growth in soft agar was a specific and sensitive marker, whereas the data on uptake of 2-deoxy-D-glucose were consistent, with high uptake being a specific but clearly not a sensitive marker. Agglutination and hemadsorption mediated by concanavalin A, multinucleation in the presence of cytochalasin B, and the cell membrane activity of adenosine triphosphatase did not correlate with tumorigenicity of the other markers for transformation. In addition, it is shown that Mycoplasma infection does not alter any of these properties but that infection can be eliminated by passage of cells through nude mice.     

Transformation by inorganic arsenic compounds of normal Syrian hamster embryo cells into a neoplastic state in which they become anchorage-independent and cause tumors in newborn hamsters

Arsenic is a known human carcinogen, but little evidence exists for its carcinogenicity in animals. In order to investigate the ability of inorganic arsenics to transform normal cells into a neoplastic state, mass cultures of normal, diploid Syrian hamster embryo (SHE) cells exposed to various concentrations of sodium arsenite or sodium arsenate for 48 hr were continually passaged and tested for neoplastic transformation, as determined by anchorage-independent growth in semisolid agar and tumorigenicity in newborn hamsters. Twenty-one of 22 (96%) untreated, control cultures senesced by 20 passages. While 1 culture escaped senescence, it did not acquire the ability to either grow in semisolid agar or form tumors in animals. Ten of 14 (71%) cultures exposed to sodium arsenite or sodium arsenate escaped senescence. Nine of the 10 (90%) arsenic-treated immortal cultures acquired the anchorage-independent phenotype. Five of 5 anchorage-independent cultures examined were tumorigenic. Two of 3 morphologically transformed colonies induced by sodium arsenate also acquired the ability to grow in semisolid agar when isolated. Amplification of the c-myc or c-Ha-ras oncogene was detected in 3 of 5 and 4 of 5 tumorigenic cell lines, respectively. Both c-myc and c-Ha-ras were amplified even in a preneoplastic, anchorage-dependent cell line, but neither was amplified in 6 of 9 anchorage-independent cell lines. Overexpression of c-myc and c-Ha-ras mRNA was observed in most of the neoplastically transformed cell lines but not in the preneoplastic cell line. Experiments using the methylation-sensitive restriction endonuclease isoschizomers HpaII and MspI revealed hypomethylation of c-myc and c-Ha-ras in the 5'-CCGG sequence of arsenic-exposed cell lines but not in the parental SHE cells or a spontaneously transformed cell line. Thus, inorganic arsenics induce neoplastic transformation of normal, diploid mammalian cells. Overexpression of oncogenes by DNA hypomethylation may participate in the arsenic-induced neoplastic transformation of mammalian cells.     

Identification of the transforming activity of Indian hedgehog by retroviral expression screening

To identify novel cancer-promoting genes in biliary tract cancer (BTC), we constructed a retroviral cDNA expression library from a clinical specimen of BTC with anomalous pancreaticobiliary duct junction (APBDJ), and used the library for a focus formation assay with 3T3 fibroblasts. One of the cDNAs rescued from transformed foci was found to encode Indian hedgehog homolog (IHH). The oncogenic potential of IHH was confirmed both in vitro with the focus formation assay and in vivo with a tumorigenicity assay in nude mice. The isolated IHH cDNA had no sequence alterations, suggesting that upregulation of IHH expression may contribute to malignant transformation. Quantitation of IHH mRNA among clinical specimens has revealed that the expression level of IHH in BTC with APBDJ is higher than that in BTC without APBDJ and than in non-cancerous biliary tissues. Our data thus implicate a direct role of IHH in the carcinogenesis of BTC with APBDJ. ( Cancer Sci 2009)     

Susceptibility of skin fibroblasts from individuals genetically predisposed to cancer,to transformation by the tumour promoter 12-o-tetradecanoylphorbol-13-acetate

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. Thus, it might be predicted that skin fibroblasts from these patients would exhibit increased susceptibility to in vitro transformation by tumour promoters alone. In the case of skin fibroblasts from RB patients, transformation as assessed by the ability of the cells to grow in semisolid medium and their migration in collagen gels did not occur. However, experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone, although the pattern of migration of the parent cell population in collagen gels was unchanged and they were non-tumorigenic in nude mice. The clones which grew in semi-solid medium, although stable with regard to anchorage-independent growth, were also unaltered in terms of their migration pattern in collagen gels and their tumorigenicity in nude mice, and were considered not to be completely transformed. Parallel cytogenetic analysis showed that, during the course of these transformation studies, TPA significantly increased not only tetraploidy but also the chromosome aberration frequency. Several quadriradial figures were noted.     

Neoplastic transformation induced by adeno 12-SV40 hybrid virus in skin fibroblasts from humans genetically predisposed to cancer

The DNA hybrid adeno 12-SV40 virus induced neoplastic transformation of human skin fibroblasts derived from individuals with adenomatosis of the colon and rectum (ACR), an inherited cancer syndrome. The line established from the transformed ACR fibroblasts became stable and was initially virus productive, but infectious hybrid virus gradually decreased over serial passages. Cultured skin fibroblasts from normal individuals infected with adeno 12-SV40 also underwent morphological transformation and were virus productive. Although these cells had a prolonged life as compared with uninfected controls, they eventually died. All of the transformed cells contained both adeno 12 and SV40 large tumor antigens, formed large cell aggregates, grew in liquid growth medium above an agar base, and formed colonies in soft agar. The altered ACR cells became heteroploid and produced tumors when transplanted subcutaneously into nude mice. The results suggest that ACR individuals have increased susceptibility to neoplastic transformation by oncogenic tumor viruses.     

Cell aggregation assay: a rapid means of evaluating and selecting in vitro transformed cells

A cell aggregation assay for evaluating in vitro transformation has recently been reported. Transformed cells formed larger cell aggregates than counterpart normal cells when suspended in liquid media above an agar base, a property which correlates with growth in soft agar and tumorigenicity. Using a human osteosarcoma cell line transformed by viruses and chemicals, we found increased cell growth of the transformed aggregates over untransformed cells. Moreover, certain aggregation properties (size/survival of aggregates) of transformed rat, mouse, hamster, human, dog, cat, chimpanzee, and sheep cell lines were found to be correlated with tumor potential, regardless of the method of transformation (spontaneous, chemical, or virus induced). Certain lines derived from cell aggregates growing in liquid medium above an agar layer were tumorigenic in nude mice, whereas the parent transformed line was not. Therefore, this assay can be utilized not only to evaluate tumorigenic potential, but also for selection of cells, which have undergone malignant transformation.     

Tax protein of human T-cell leukemia virus type I is required for maintenance of the transformed phenotype.

We have isolated and characterized revertants of a clonal cell line (40MRatcl-1) of human T-cell leukemia virus type I Tax-transformed Rat1 cells. The 40MRatcl-1 cells contain a single copy of tax gene, form large colonies in soft agar, elicit tumors rapidly in nude mice and revert to the normal phenotype at low frequency. From one of its subclones (B7) bearing pSV2gpt DNA as a marker gene, four morphologically reverse-transformed cell lines were isolated. They display contact inhibition at confluency, lose the ability to form colonies in soft agar, fail to form tumors in nude mice and restore the transformed phenotype similar to that of 40MRatcl-1 cells by transfection with the tax-expression plasmid. Southern blot analysis revealed that they have lost the tax gene. Our results indicate that transformation of Rat1 cells by Tax is not the consequence of secondary mutations of cellular genes and that tax functions are directly required for establishment and maintenance of the transformed phenotype.     

Stepwise neoplastic transformation of a telomerase immortalized fibroblast cell line

We have described recently a human fibroblast cell line immortalized through ectopic telomerase expression (cen3tel), in which the extension of the life span was associated with the appearance of chromosomal aberrations and with the ability to grow in the absence of solid support. As reported in this article, on further propagation in culture, cen3tel cells became neoplastically transformed, being able to form tumors in nude mice. The analysis of the cells, during the gradual transition toward the tumorigenic phenotype, allowed us to trace cellular and molecular changes associated with different phases of transformation. At the stage in which they were able to grow in agar, cen3tel cells had lost contact growth inhibition but still retained the requirement of serum to proliferate and were not tumorigenic in immunocompromised mice. Moreover, they showed a down-regulation of the INK4A locus and were resistant to oncogenic Ras-induced senescence but still retained a functional p53. Subsequently, cen3tel cells became tumorigenic, lost p53 function because of a mutation in the DNA-binding motif, and overexpressed c-myc. Interestingly, tumorigenic cells did not carry activating mutations either in the ras proto-oncogenes (H-ras, N-ras, and K-ras) or in B-raf. Cen3tel cells gradually became hyperdiploid but did not display centrosome abnormalities. To our knowledge, cen3tel is the first telomerase immortalized fibroblast line, which became neoplastically transformed. In this system, we could associate a down-regulation of the INK4A locus with anchorage-independent growth and with resistance to Ras-induced senescence and link p53 mutations and c-myc overexpression with tumorigenicity.     

A reassessment of methylcholanthrene transformation in the C3H10T1/2 cell culture system

We previously demonstrated that four tumorigenic methylcholanthrene (MCA) transformed cell lines derived from C3H10T1/2 cells each contain a common G34----T nucleotide alteration in the c-Ki-ras gene. In contrast, a non-tumorigenic MCA transformant does not contain this mutation. We have now examined 75 newly isolated MCA transformants of C3H10T1/2 cells for their degree of morphological transformation, the presence of the c-Ki-ras G34----T mutation, colony formation in soft agar, and tumorigenicity in nude mice. Although many of these new MCA transformants exhibit morphological characteristics indistinguishable from previously isolated tumorigenic MCA transformants, none contain the G34----T mutation in the c-Ki-ras gene. Only one newly isolated MCA transformant can grow in soft agar. Of 14 tested, none of the new MCA C3H10T1/2 transformants are tumorigenic in nude mice.     

Anti-tumor surveillance of non-contact-inhibited transformed cell lines

In order to determine if the correlated expression of transformation and tumorigenicity is affected by the agents used to induce transformants or by the immune status of the host used to test the tumorigenicity of transformants, we derived a series of cloned cell lines from foci of transformed cells induced by treatment of the contact-inhibited mouse cell line B/C-N7.ICI with the DNA demethylating agent 5-azacytidine (5-AZC) or the DNA demethylating and mutating agent benzo(a)pyrene dihydrodiol epoxide (BPDE). The transformed cell lines were injected into syngeneic nude and normal mice to determine their tumorigenicity. The results of this analysis showed that 93% of the transformants induced by 5-AZC treatment grew as tumors when injected into nude mice. Of those lines capable of growing as tumors in nude mice, 86% were also tumorigenic when injected into normal mice. In contrast, only 64% of BPDE-induced transformants grew as tumors in nude mice, and of those, only 44% were also tumorigenic in normal mice. The existence of non-contact-inhibited transformants that are tumorigenic only in nude mice indicates that host anti-tumor immune surveillance mechanisms are operative in normal mice. Further, the difference in both the percentage of transformed cell lines that are tumorigenic and the percentage of tumorigenic transformants that are susceptible to immune surveillance when transformants are induced by BPDE as compared to 5-AZC indicates that the transforming agent can affect both the correlation between the expression of transformation and tumorigenicity, and the interaction between the immune system and tumorigenic transformants.