Neoplastic transformation of immortalized human keratinocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most powerful carcinogen ever tested in animals. Recent epidemiological studies have suggested its carcinogenic potential in humans. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12-simian virus 40 (Ad12-SV40) were transformed by exposures of TCDD equal to or greater than 0.1 nM for 2 wk. These transformed cells showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas no such transformation phenotypes were observed with exposures of less than 0.1 nM for 2 wk. Primary human epithelial keratinocytes exposed to various concentrations of TCDD failed to show any evidence of transformation. Induction of aryl hydrocarbon hydroxylase activity was dose dependent, as was transformation. Thus, the carcinogenicity of TCDD in this human cell system appears to be an Ah receptor-mediated process. The present study represents the first evidence of neoplastic conversion of human cells exposed to this environmentally important chemical.     

Effects of specific fatty acids on cell transformation induced by an activated c-H-ras oncogene

An increase in dietary lipid has been associated with an increase in the development of certain forms of cancer, notably breast and colon cancer, both in experimental animal studies and in human epidemiology studies. The underlying mechanisms are not, however, known with certainty. In the present studies we have examined whether certain specific fatty acids (FA) might act by enhancing the role of an activated oncogene in a model cell culture system. We found that when the rat fibroblast cell line Rat 6 was transfected with an activated human c-H-ras oncogene and the cells subsequently grown in medium supplemented with myristic acid, palmitic acid or stearic acid (20-80 microM) there was a marked enhancement of the number of transformed foci obtained. On the other hand arachidonic acid had a marked inhibitory effect in this transformation assay. However, this inhibitory effect can be partially reversed by indomethacin, an inhibitor of cyclo-oxygenase, at dose response manner. Control studies indicated that these results were not simply due to the effects of the FAs on growth of the Rat 6 cells or the process of transfection per se. Lipid analyses of cells grown in the presence of stearic acid indicated that the added FA was extensively incorporated into the major lipid classes of the cell and produced transient changes in lipid composition. This simple cell culture system may be useful for elucidating the mechanisms by which various dietary lipids and nutritional factors influence the carcinogenic process.     

Effects of triiodothyronine and tamoxifen on cell transformation induced by an activated c-Ha-ras oncogene

We have found that the thyroid hormone 3,5',3'-triiodo-L-thyronine stimulates the transformation of Rat 6 fibroblasts when these cells are transfected with an activated human c-Ha-ras oncogene (T24). 3,5',3'-Triiodo-L-thyronine did not further augment the stimulation of oncogene-induced transformation obtained with a phorbol ester tumor promoter (12-O-tetradecanoylphorbol-13-acetate) or a factor from fetal calf serum. On the other hand, tamoxifen, an antiestrogen that also inhibits protein kinase C, markedly inhibited Ha-ras-induced cell transformation in the presence of 12-O-tetradecanoylphorbol-13-acetate or fetal calf serum. Time course studies and Southern blot analyses of DNAs isolated from transformed foci provided evidence that 3,5',3'-triiodo-L-thyronine and tamoxifen do not exert their effects simply by enhancing or inhibiting integration of the transfected oncogene into cellular DNA. These findings indicate that hormonal factors can modulate the ability of an activated Ha-ras oncogene to transform cells. They may be relevant to the process of multistage carcinogenesis in vivo.     

High efficiency DNA-mediated transformation of human diploid fibroblasts

Normal human diploid fibroblasts (HDF) have only rarely been used as recipients in DNA transfection experiments even though they have the potential to add to our knowledge about the genes that control normal cell proliferation. A major impediment to the use of HDF has been their poor transfection frequency. In this paper, we show that IMR-90 human diploid fibroblasts can be stably transfected at high efficiency with pRSVneo at an average frequency of 2 x 10(-3) (0.2%) using a modified calcium phosphate-DNA coprecipitation transfection protocol. Prior to this report, the best transfection frequencies of HDF were on the order of 10(-5)-10(-4). In the present protocol it is very important to maintain relatively low cell densities, particularly during the selection process post-transfection. Using this protocol other neo-containing plasmids also yield high transfection frequencies in IMR-90. IMR-90, which are readily available from the NIH Aging Cell Repository, may be one of the best HDF to use as recipients because they are five times as efficient as are WI-38, which is another well characterized HDF.     

Effects of various chemical agents on the transformation of rat fibroblasts by an activated c-Ha-ras oncogene

We have previously reported that the potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) and a factor from fetal calf serum (FCS) markedly enhance the transformation of mouse C3H 10T1/2 and Rat 6 fibroblasts, when added to cultures following transfection with plasmid pT24 DNA that contains an activated c-Ha-ras oncogene. In the present study, we examined possible enhancing or inhibiting effects of various chemicals on the transformation of Rat 6 fibroblasts by T24 DNA when tested in the presence of calf serum, calf serum plus TPA or FCS. We found that, like TPA, the chemicals mezerein, 1-oleoyl-2-acetylglycerol, and phospholipase C increased the yield of T24-induced foci, thus further implicating protein kinase C as a critical constituent in this process. Low concentrations (10(-6)-10(-7)M) of retinoic acid (both trans and 13-cis) also stimulated cell transformation. Several compounds inhibited T24-induced transformation. These included nontoxic concentrations of the calcium ionophore A23187, indomethacin, and epsilon-amino-n-caproic acid. Compounds that failed to exert a significant reproducible effect included vasopressin, vitamin D3, selenium, antipain, Bowman-Birk inhibitor, vitamin B12, epidermal growth factor, platelet-derived growth factor, insulin, and transferrin. These findings suggest that this simple in vitro system might be useful for detecting enhancers and inhibitors of ras oncogene-induced cell transformation and also elucidating their mechanisms of action.     

Introduction of the activated N-ras oncogene into human fibroblasts by retroviral vector induces morphological transformation and tumorigenicity

The introduction of activated N-ras cDNA into normal diploid human skin fibroblast cell cultures using the retroviral vector pZIPneo results in a spectrum of morphologies ranging from near normal to, in rare instances, dense piled-up colonies of morphologically transformed cells. However, none of the clones isolated were transformed as assessed by growth on agar or tumorigenicity in nude mice. Introduction of both c-myc and N-ras oncogene cDNAs into normal skin fibroblasts failed to produce transformation as assessed by growth on agar and tumorigenicity in nude mice, although c-myc infection alone conferred immortality and the resultant doubly infected cell line was immortal. Using the same construct, activated N-ras cDNA was shown to transform immortalized human fibroblasts to tumorigenicity. However, immortalization per se was shown not to guarantee 'co-operation' with an activated N-ras gene to give malignant transformation. Although numerical and structural chromosome aberrations (clonal and non-clonal) were observed in some of the cell strains isolated after retroviral infection, these were not directly associated with viral infection, the presence of the oncogenes or with the morphologically transformed phenotype.     

Neoplastic transformation of human epithelial cells in vitro

Efforts to investigate the progression of events that lead human cells of epithelial origin to become neoplastic in response to carcinogenic agents have been aided by the development of tissue culture systems for propagation of epithelial cells. We have recently developed an in vitro multistep model suitable for the study of human epithelial cell carcinogenesis. Primary human epidermal keratinocytes acquired indefinite lifespan in culture but did not undergo malignant conversion in response to infection with Adl2-SV40 virus. Subsequent addition of Ki-MSV, which contains a K-ras oncogene, to these cells induced morphological alterations and the acquisition of neoplastic properties. Nontumorigenic human epidermal keratinocytes immortalized by Adl2-SV40 virus (RHEK-1) were also transformed by treatment with chemical carcinogens (MNNG or 4NQO) and by X-ray irradiation. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice. This in vitro system may be useful in assessing environmental carcinogens for human epithelial cells and in detecting new human oncogenes since ras oncogenes were not activated in these chemical--or X-ray--transformed RHEK-1 lines. Subsequently, it was found that this line could be transformed neoplastically by a variety of retroviruses containing H-ras, bas, fes, fms, erbB and src oncogenes. In addition, our recent results indicate that nontumorigenic RHEK-1 cells can be transformed following transfection with an activated human oncogene. Thus, this in vitro system may be useful in studying the interaction of a variety of carcinogenic agents and human epithelial cells. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of tumor viruses and chemical carcinogens or X-ray irradiation and support a multistep process for neoplastic conversion. Further, evidence for the multistep nature of neoplastic transformation of human epithelial cells in vitro using other model systems is presented.     

Malignant transformation of human fibroblasts by a transfected N-ras oncogene

The only ras oncogene as yet identified in cells from human fibrosarcomas is N-ras, but the relationship between N-ras oncogene expression and the malignant state of these cell lines is not known. To determine if expression of an N-ras oncogene causes human cells to become malignant, we transfected the N-ras oncogene from human leukemia cell line 8402, cloned into a high expression vector pSV N-ras, into MSU-1.1 cells, a nontumorigenic, infinite life span fibroblast cell strain with a normal morphology and a stable near-diploid karyotype. The transformants formed distinct foci composed of morphologically transformed cells. Cells from such foci expressed higher than normal levels of N-ras protein, exhibited growth factor independence, and formed large colonies in soft agar at a high frequency. Injection of progeny of these focus-derived cells s.c. into athymic mice resulted in progressively growing, invasive malignant tumors (round cell, spindle cell, or giant cell sarcomas) which reached a diameter of 6 mm in 3 to 4 weeks. Injection of focus-derived or tumor-derived cells i.v. resulted in tumors in various organs of the mice. The focus-derived cell strain tested, as well as the majority of the cells derived from the tumor it produced, exhibited the same near-diploid karyotype as the parental MSU-1.1 cells. Cells transfected with an N-ras oncogene that was expressed at a normal level formed only a single, indistinct focus, and cells from that focus were not malignant.     

Neoplastic transformation by TERT in FGF-2-expanded human mesenchymal stem cells

The low percentage of human mesenchymal stem cells (hMSCs) in bone marrow necessitates their in vitro expansion prior to clinical use in regenerative medicine. We evaluated the effect of long-term culture of hMSCs on telomere length and transformation capacity by TERT transfection. hMSCs were isolated from the bone marrow aspirates of 24 donors and cultured with fibroblast growth factor-2 (FGF-2). Six cell lines with >500 population doubling levels were considered immortalized. TERT was transfected into two of the six lines for a comparison of telomere length, telomerase activity, differential capacity, colony formation capacity in soft agar and tumorigenicity in immunodeficient (NOD-SCID) mice. hMSC lines exhibited elongated telomeres without the activation of telomerase and retained multi-lineage differentiation potential upon chondrogenic or adipogenic differentiation, while non-immortalized hMSCs showed a marked reduction in telomere length in the differentiation process. Immortalized hMSCs showed anchorage-independence and formed tumors in NOD-SCID mice. Histologically, these tumors consisted of differentiated cells such as fat tissue and cartilage. Two TERT-transfected hMSC lines showed high rates of tumor formation in NOD-SCID mice. These tumors were histologically similar to teratocarcinoma without differentiated cells. These cells may provide a model for the origin of cancer stem cells from adult stem cells, and indicate the possibility that telomerase activation has a major role in the malignant transformation of human stem cells. These data suggest that adult hMSCs have a potential for neoplastic transformation and have implications for the use of hMSCs in tissue engineering and regenerative medicine.     

Neoplastic transformation of human diploid fibroblasts after long-term serum starvation

Serum starvation for several days has been considered as a positive effect on the efficiency of nuclear transfer using donor cells. The effects of longer period serum starvation are not clear while similar starvation might occur in vitro maintained cells (i.e. tissue engineering products) and in vivo such as ischemia of human tissues or organs. We found human dermis fibroblasts were transformed for about 70 days caused by serum starvation (0.5% serum). The transformed cells became round and had more than one nucleolus. In 0.5% serum medium they kept almost constant growth rate as the normal fibroblasts in 10% serum medium. Abnormal karyotype including aneuploidy and structural aberrations was observed. The transformed cells had high telomerase activities, in contrast, normal fibroblasts had no detectable telomerase activities. C-myc was up-regulated while cdk2, cyclin A, p21 were down in transformed cells. Cell transplantation into SCID nude mice confirmed that the cells had the capacity of forming solid tumors. The results indicated that long-term serum starvation could lead to cell chromosomal instability and transformation.     

Partial transformation of human thyroid epithelial cells by mutant Ha-ras oncogene.

We have previously shown that activation of ras oncogenes by mutation is a frequent early event in human thyroid neoplasia. Using amphotropic retroviral vectors to achieve gene transfer, we demonstrate here that human primary thyroid epithelial cells can be partially transformed by an activated cellular or viral Ha-ras oncogene, in the absence of a cooperating oncogene. The transformation event induced by ras involves temporary rescue from senescence for up to 20 rounds of cell division together with morphological alteration, growth factor independence and anchorage independence. It has therefore been possible to reconstruct in vitro a key early event in the genesis of human epithelial neoplasia.     

Neoplastic transformation of rabbit cells by murine sarcoma viruses

Neoplastic transformation of rabbit cells by Kirsten murine sarcoma virus (Ki-MSV), the Ki-MSV pseudotype of baboon endogenous virus (Ki-MSV[BaEV]) and the Moloney-MSV pseudotype of feline leukemia virus (M-MSV[FeLV]) is reported. Rabbit cells can be readily transformed by Ki-MSV, Ki-MSV(BaEV) and M-MSV(FeLV). Rabbit cells transformed by Ki-MSV and M-MSV(FeLV) were found to be virus producers, whereas those transformed by Ki-MSV(BaEV) were nonproducers (NP). The NP cells were obtained by simply infection rabbit cells with Ki-MSV(BaEV) and subculturing the infected cells. Although the morphologically altered NP cells did not produce infectious virus or murine leukemia virus antigen, they did contain a rescuable MSV genome. All of the transformed cells formed colonies in soft agar, grew to high saturation densities and produced tumors when transplanted into nude mice. The Ki-MSV and M-MSV(FeLV)-transformed cells produced tumors in newborn WH/J rabbits, thus providing an important tool for studying tumor immunity in the rabbit.     

Causal role for an activated N-ras oncogene in the induction of tumorigenicity acquired by a human cell line

ras oncogenes have been found in approximately 15% of the human tumors analyzed. However, a causal role for these genes in the tumorigenesis of human cells has yet to be shown. Tumorigenic late-passage PA-1 human teratocarcinoma cells (E-PA-1) contain an activated N-ras gene. In this report evidence is presented that nontumorigenic early passage revertant PA-1 cells (E-PA-1) contain only the germ-line protooncogene. Introduction by gene transfer of the activated L-PA-1 oncogene induces E-PA-1 cells to form tumors, suggesting that the activated N-ras oncogene has a causal role in the tumorigenesis of these cells.     

Neoplastic transformation of breast epithelial cells by genotoxic stress

Background  

Exposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation and progression of breast cancer. The combination of these two genotoxic insults can induce significant damage to the genetic material of the cells resulting in neoplastic transformation.     

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.     

Malignant transformation of human bladder epithelial cells by DNA transfection with the v-raf oncogene

Transfection of the v-raf oncogene into immortalized, nontumorigenic human bladder epithelial cells resulted in the isolation of two tumorigenic transformants. Both were identified as human and of the same origin as the parent cell line by human leukocyte antigen typing and Southern blot analysis. Both the primary tumorigenic transfectants and the cell lines established from the induced tumors expressed v-raf mRNA and v-raf protein. In both tumorigenic transformants the level of c-myc mRNA was enhanced compared with that of the parent cell line.