Neoplastic transformation of human osteoblast cells to the tumorigenic phenotype by heavy metal-tungsten alloy particles: induction of genotoxic effects

Heavy metal-tungsten alloys (HMTAs) are dense heavy metal composite materials used primarily in military applications. HMTAs are composed of a mixture of tungsten (91-93%), nickel (3-5%) and either cobalt (2-4%) or iron (2-4%) particles. Like the heavy metal depleted uranium (DU), the use of HMTAs in military munitions could result in their internalization in humans. Limited data exist, however, regarding the long-term health effects of internalized HMTAs in humans. We used an immortalized, non-tumorigenic, human osteoblast-like cell line (HOS) to study the tumorigenic transforming potential of reconstituted mixtures of tungsten, nickel and cobalt (rWNiCo) and tungsten, nickel and iron (rWNiFe). We report the ability of rWNiCo and rWNiFe to transform immortalized HOS cells to the tumorigenic phenotype. These HMTA transformants are characterized by anchorage-independent growth, tumor formation in nude mice and high level expression of the K-ras oncogene. Cellular exposure to rWNiCo and rWNiFe resulted in 8.90 +/- 0.93- and 9.50 +/- 0.91-fold increases in transformation frequency, respectively, compared with the frequency in untreated cells. In comparison, an equivalent dose of crystalline NiS resulted in a 7.7 +/- 0.73-fold increase in transformation frequency. The inert metal tantalum oxide did not enhance HOS transformation frequency above untreated levels. The mechanism by which rWNiCo and rWNiFe induce cell transformation in vitro appears to involve, at least partially, direct damage to the genetic material, manifested as increased DNA breakage or chromosomal aberrations (i.e. micronuclei). This is the first report showing that HMTA mixtures of W, Ni and Co or Fe cause human cell transformation to the neoplastic phenotype. While additional studies are needed to determine if protracted HMTA exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized HMTAs exposure may be comparable with the risk from other biologically reactive and insoluble carcinogenic heavy metal compounds (e.g. nickel subsulfide and nickel oxide).     

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.     

Effects of increased expression of protein kinase C on radiation-induced cell transformation

The in vitro oncogenic transformation of C3H 10T1/2 cells byionizing radiation is known to be enhanced by the tumor promoter12-O-tetradecanoylphorbol-13-acetate (TPA). It is also knownthat the activation of protein kinase C (PKC) by TPA is an importantstep in its tumor-promoting effect. In the present study, weexamined the effects of overexpression of a specific isoformof PKC, PKC_ß1 on 7-ray-induced transformation of 10T1/2cells. In addition, the effects of overexpression of PKC_ß1on the malignant phenotype of a previously transformed 10T1/2cell line were also evaluated. Derivatives of 10T1/2 cells thatstably overexpress PKC_ß1 were obtained by transductionwith the retroviral expression vector pMV7 carrying the ratPKC_ß1 cDNA sequence. We found that the parental 10T1/2cells and a control cell line 10T1/2 MV7, which carried onlythe pMV7 vector without the cDNA insert, expressed dose-dependenttransformation frequencies when exposed to 7-rays. On the otherhand, concurrently treated PKC-overexpressing cells that hadan 11-fold increase in enzyme activity (PKC-4 cells) failedto yield any morphologically identifiable foci. Cell lines thatexpressed lower levels of PKC_ß1 were partially resistantto transformation by     

Transformation of non-tumorigenic osteoblast-like human osteosarcoma cells by hexavalent chromates: alteration of morphology, induction of anchorage-independence and proteolytic function.

Soluble chromium (VI) compounds either alone or in combination with 3-methylcholanthrene (MC) were used to transform non-tumorigenic osteoblast-like human osteosarcoma cells (HOS TE85). The Cr(VI) compounds were highly toxic to these cells with LC50 values in the range of approximately 0.5-1.0 microM. Continuous passaging of the treated cells resulted in sustained increase in anchorage-independent (AI) colony formation. Treatment with Cr(VI) and MC resulted in substantial increase in AI growth. At the XVth passage, a number of individual AI colonies were expanded in culture and used for further studies. The cells are refractory in appearance and grow as 'nests' rather than as monolayers. The cell lines have relatively high plating efficiency (PE) in soft agar and respond to promotional effect of phorbol-12-myristate-13-acetate by an increase in PE and in the size and number of AI colonies. While the isolated cells are not tumorigenic when tested in athymic nude mice, most of the lines possess higher levels of plasminogen activator (PA) activity, considered as one of the markers of transformation. This is also reflected in the increase in the steady state level of urokinase type PA mRNA. These results show that Cr(VI) compounds are capable of promoting human cells to an altered phenotype characteristic of a stage in the carcinogenesis cascade.     

Malignant transformation of mouse BALB/c3T3 cells induced by NaNO2

The addition of sodium nitrite (NaNO₂; 5–20 mM) for 72h to mouse BALB/c3T3 cells resulted in the induction of transformedfoci (type III foci) in a dose-dependent manner. The cells isolatedfrom the NaNO₂-induced transformed foci produced progressivelygrowing tumors when inoculated into nude mice subcutaneouslyat an inoculum size of 1 x 10⁶ cells per site. In contrast,the original untreated cells did not take even at an inoculumsize of 1 x 10⁶ cells per site. The possibility that NaNO₂ mightreact with cellular or medium components to make carcinogenicN-nitrosamines and that these might induce cell transformationwas examined and almost excluded. Thus, nitrite itself seemsto have a cell transforming activity. Recent evidence suggeststhat NO₂^– is produced by the activated macrophage of mammals.We also detected NO₂^– production in culture media in themouse macrophage-like cell Line J774–A1 after lipopolysaccharide(LPS) treatment, and also in the human promyeloleukemia cellline HL60 after differentiation into macrophage-like cells by12-O-tetradecanoyl phorbol-13-acetate and further activationby LPS.     

Induction of morphological transformation, anchorage-independent growth and plasminogen activators in non-tumorigenic human osteosarcoma cells by lead chromate

The possible carcinogenicity of insoluble chromium (VI) compound, PbCrO4, in human cells has been tested using a nontumorigenic human osteosarcoma cell line (HOS, TE 85). Electron microscopic studies show that PbCrO4 is phagocytosed by HOS cells and accumulates within the vacuoles in the cytoplasm. A number of cell lines have been isolated following multiple treatment of HOS cells with PbCrO4. These cell lines are morphologically different from HOS cells, form anchorage-independent colonies in soft agar and form quickly regressing small tumor nodules in athymic nude mice. The cellular and secreted plasminogen activator (PA) levels of 5 cell lines isolated after PbCrO4 treatment are increased up to 8 fold and up to 10 fold respectively as compared to untreated HOS controls. SDS-PAGE analysis in the presence of copolymerized substrates is consistent with increase in 55 kDa urokinase-type PA (u-PA) and 68 kDa tissue-type PA (t-PA). These results show that PbCrO4 treatment leads to stable phenotypic changes indicative of the transformation of HOS cells.     

Biological and biochemical characterization of cell lines derived from initiation-promotion transformed C3H/1OT1/2 cells

A two-stage transformation protocol was used to chemically transformthe mouse embryo fibroblasts, C3H/10T1/2 Cl 8. To initiate thecells 0.37 µM 20-methyicholanthrene was used and 0.17µM of the tumor promoter 12-O-tetradecanoylphorbol-13-acetatewas employed to complete the transformation process. Six weekslater transformed foci were identified and isolated by the ring-cloningtechnique. Altogether eight different foci were trypsinizedresulting in a total of 12 morphologically transformed subclones.Three of these clones, designated TPA 41, TPA 42 and TPA 482,have been characterized in detail. Their growth morphologieswere different. The TPA 482 cells grew in a criss-cross patternwith piled up foci, thus showing a characteristic type III morphology.The TPA 482 clone did not show cell-density growth inhibitionand grew in soft agar. The TPA 41 and TPA 42 clones exhibitedcell-density growth inhibition, grew as monolayers and formedonly few colonies in soft agar. Late passages of the TPA 42clone acquired growth characteristics similar to TPA 482. TheC3H/1OT1/2 Cl 8 and the TPA 41 cells were not tumorigenic whentransplanted into syngeneic mice. TPA 482 cells were stronglytumorigenic, producing tumors in 6/6 mice in 21 days. The TPA42 cells were also tumorigenic, the first tumors appearing after4 weeks; all animals injected with TPA 42 cells had tumors after8 weeks. All tumors observed appeared to be fibrosarcomas. Flowcytometric analysis indicated differences in DNA distributionsbetween tumor cells grown in vitro and the tumors in vivo. Two-dimensionalgel analysis of the total cellular and the nuclear proteinsshowed an increase in the TPA 42 and TPA 482 cells of an acidic48 000 and a basic 83 000 mol. wt polypeptides, and a decreaseof a neutral polypeptide of mol. wt 46 000, located in the nucleusof TPA 482 cells.     

Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes on in vitro transformation of human mammary epithelial cells.

MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-alpha (TGF-alpha) gene in MCF-10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF-10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-10A Ha-ras and MCF-10A TGF-alpha cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-10A TGF-alpha/c-erbB-2 (MCF-10A TE) and 10 MCF-10A Ha-ras/c-erbB-2 (MCF-10A HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-10A TE and MCF-10A HE clones expressed a 10-fold to 40-fold increase in p185 erbB-2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF-alpha compared with MCF-10A cells. Moreover, both MCF-10A TE and MCF-10A HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-10A Ha-ras, MCF-10A c-erbB-2, or MCF-10A TGF-alpha clones. However, neither MCF-10A TE nor MCF-10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erbB-2, and TGF-alpha genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

Malignant progression of papilloma-derived keratinocytes: differential effects of the ras, neu, and p53 oncogenes

The p117 keratinocyte cell line was derived in culture from chemically induced mouse papillomas. The benignly transformed nature of these cells was demonstrated by their ability to re-form benign papillomas when grafted back onto the animal. Retroviral vectors were used to introduce into the p117 cells three distinct oncogenes: v-Ha-ras, p53, and neu. All three oncogenes were able to induce tumorigenic conversion of the p117 keratinocytes when assayed by subcutaneous injection into nude mice. However, grafting the oncogene-transformed cells onto the back of the mouse revealed important differences in the ability of the three oncogenes to induce a fully malignant phenotype. While the ras-transformed papilloma cells formed aggressive carcinomas, p53 and neu transformation yielded an intermediate phenotype, with formation of large exophytic tumors, not yet invasive but with highly dysplastic features remarkably similar to those of in situ carcinomas. These findings establish a homologous, genetically modifiable cell system in which various stages of malignant transformation can be directly compared.     

Characterization of human cells transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine.

Human osteosarcoma (HOS) clonal cells transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were characterized, and compared to non-producer HOS cells transformed by Kirsten murine sarcoma virus (Ki-MSV). The MNNG- and virus-transformed cells grew in the aggregate form above an agar base, grew in soft agar, and had a high fibrinolytic activity. When inoculated into nude mice, all the chemically or virally altered cells produced tumors or tumor nodules. When transplanted into ATS-treated hamsters, the cells transformed by MNNG (0.01 mug/ml) and Ki-MSV produced tumors but MNNG (0.1 mug/ml) transformed cells did not produce tumors. The control HOS cells did not grow in the aggregate form but formed colonies in soft agar, and had low fibrinolytic activity and no capacity to form tumors in nude mice and ATS-treated hamsters. However, one of the control clonal lines had a high level of fibrinolytic activity. Cellular aggregation properties of human transformed cells did appear to correlate with tumorigenicity in nude mice.     

Human herpesvirus-6: tumorigenicity and tumor infiltrating lymphocytes.

We previously reported that human herpes-virus-6 (HHV-6) genome (strain GS) and a cloned subfragment (pZVH14) transfected NIH 3T3 cells, induced foci of transformation with a frequency significantly above the background level. The transformed cells produced tumors in nude mice and immunocompetent (Swiss) mice. In the current study, nude mice tumors were passed into Swiss mice and more aggressive tumors (G-2TS and 14-2TS derived from HHV-6 genome and pZVH14 DNA, respectively) were produced. 14-2TS tumors caused lung metastasis upon intravenous injection. In the case of subcutaneously growing aggressive tumors, tumor infiltrating lymphocytes (TIL) were isolated and characterized as T cells but lacked tumor specific killing as monitored by 51Cr-release assays. TIL lost activity at day 52 in a 4 h assay (but not in a 19 h assay) against the autologous tumor, but not a Maloney virus induced tumor (Yac-1). These studies indicate that an established nontumorigenic fibroblast cell line, when transfected with pZVH14 DNA of HHV-6, acquires both tumorigenic and metastatic potential and tumor bearing hosts mount immune response against such tumors.     

Primary transfection as a mechanism for transformation of host cells by human tumor cells implanted in nude mice

Establishment of cell lines in vitro from a human lung cancer xenograft in nude mice resulted in transformed mouse cell lines. The transformed mouse cell lines expressed both mouse-specific and human-specific histocompatibility antigens. Of 3 cell lines, 2 were tumorigenic in BALB/c nude mice but not in normal mice. Tumors formed by the transformed mouse cell lines were fibroblastoid and epithelioid by histology. In addition, tumors exhibited neuroepithelial differentiation by ultrastructural and immunohistochemical analysis. Phenotypically they were similar to the original patient and human xenograft tumor. These data suggest that previous reports of host cell transformation and induction of fibrosarcomas may not be true fibrosarcomas. Human DNA sequences were present in the tumorigenic cell lines, indicating that spontaneous transfection of human tumor DNA into host cells had occurred. The implication of these findings is that human genetic information has been transferred to primary mouse host fibroblasts, which resulted in a transformed as well as a differentiated phenotype.     

Growth control of heterologous tissue culture cells in the congenitally athymic nude mouse.

A variety of heterologous mammalian cells were inoculated into nude mice and scored for tumorigenicity. The cells tested were from primary cell cultures, established cell lines of neoplastic origin, established cell lines of nontumor origin, and primary cell cultures transformed by oncogenic viruses. Regardless of the animal species of origin, every cell line that was tumorigenic in some other animal host and every cell line of neoplastic origin was tumorigenic in nude mice. Several tissue culture cells lines capable of indefinite growth in vitro failed to form tumors in nude mice, and the basis of this growth suppression was investigated. The findings suggest that the failure of an established cell line to form tumors in nude mice is an authentic response to host-mediated growth-regulatory signals.     

Collateral expression of proangiogenic and tumorigenic properties in intestinal epithelial cell variants selected for resistance to anoikis

Although in vitro anchorage-independent growth is widely used as a marker of cell transformation, the biological implications of this trait are poorly understood. We previously demonstrated that enforced anchorage-independent growth of a nontumorigenic, immortalized epithelial cell line (IEC-18) in multicellular spheroid culture results in massive apoptotic cell death. This death process, termed anoikis, is prevented by expression of transforming oncogenes, which also confer tumorigenic competence. This study examines whether acquisition of an anoikis-resistant phenotype is causally related to the tumorigenic capacity of transformed epithelial cells. Parental IEC-18 cells were subjected to 10 cycles of selection for survival in speroid culture. Unlike parental cells, the resulting anoikis-resistant variants (AR1.10 and AR2.10) formed relatively large tumors in nude mice. Both anoikis-resistant sublines displayed upregulated expression of vascular endothelial growth factor (VEGF), a potent angiogenesis stimulator. VEGF121 overexpression alone did not induce tumorigenic conversion of parental IEC-18 cells, which remained highly susceptible to anoikis. We postulate that both anoikis-resistance and angiogenic-competence contribute to tumor formation. Development of anoikis-resistance can be then viewed as a precondition for expression of the tumorigenic phenotype. Our results suggest that even when angiogenesis is not a rate limiting factor (e.g. in vitro) the selective pressures of solid tumor-like, 3-dimensional growth conditions favoring anoikis resistance result in collateral induction of a proangiogenic phenotype.     

Study of nm23 gene expression in an N-methyl-N-nitrosourea transformed human breast epithelial cell line

In the present study we pinpoint the time of nm23 down-regulation during the chemical transformation of the human breast epithelial cell line HBL100. The non-malignant HBL100 was transformed by exposing it to N-methyl-N-nitrosourea (MNU). We subsequently injected the transformed cells (HBL-T-MNU) into nude mice, resulting in tumor growth. With a second passage of these tumors in mice we observed lung and extra-regional node metastases. The expression of nm23 in the non-tumorigenic HBL100 cells was compared to the tumorigenic KBL-T-MNU cells as well as to the metastatic cell line HBL-T2(MNU) derived from the tumor induced by HBL-T-MNU cells. By using immunohistochemistry and Western blot analysis we documented the downregulation of nm23 expression in the tumorigenic HBL-T-MNU and metastatic HBL-T2(MNU) cell lines, as compared to the parental line HBL100. Once downregulated, nm23 expression in this model remains constant during the subsequent progression. These results suggest that nm23 down-regulation may indeed be associated with early neoplastic transformation and is maintained throughout neoplastic progression and metastatic stage.     

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.     

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.