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Tumor Progression of Skin Carcinoma Cells in Vivo Promoted by Clonal Selection, Mutagenesis, and Autocrine Growth Regulation by Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor

2001; Elsevier BV; Volume: 159; Issue: 4 Linguagem: Inglês

10.1016/s0002-9440(10)62541-2

1525-2191

Margareta M. Mueller, Wolfgang Peter, M. Mappes, Andrea Huelsen, Heinrich Steinbauer, Petra Boukamp, Michael Vaccariello, Jonathan A. Garlick, Norbert E. Fusenig,

Cancer-related Molecular Pathways

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo- progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations. Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo- progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations. The development of cancer is a multistep process, in which cells accumulate genetic alterations thereby gradually progressing from a normal to a malignant phenotype. The stepwise evolution from premalignant lesions to malignant and finally metastasizing tumors is understood as the result of an increasing dysregulation of endogenous growth control mechanisms and independence of environmental growth regulatory factors.1Aaronson SA Growth factors and cancer.Science. 1991; 254: 1146-1153Crossref PubMed Scopus (1147) Google Scholar One of the best studied models for tumor development and progression is that proposed by Fearon and Vogelstein2Fearon ER Vogelstein B A genetic model for colorectal carcinogenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9839) Google Scholar for colon carcinogenesis. Detailed genetic analysis of different stages in the process of tumor development revealed that an accumulation of multiple changes in tumor suppressor genes as well as oncogenes is necessary for the evolution of malignant tumors. To date it is not clear whether a specific number of genetic alterations or a defined sequence in which these alterations occur is prerequisite for a malignant tumor to develop. The understanding of the molecular mechanisms underlying tumor progression has been greatly improved by the development of in vitro model systems such as that for colon carcinoma.3Paraskeva C Corfield AP Harper S Hague A Audcent K Williams AC Colorectal carcinogenesis: sequential steps in the in vitro immortalization and transformation of human colonic epithelial cells (review).Anticancer Res. 1990; 5: 1189-1200Google Scholar However, the specific role of the in vivo microenvironment in controlling or facilitating the transition to a more advanced stage of cell transformation is still poorly understood because of the complexity of the tissue influences. We have previously developed an in vitro cell transformation system of human skin keratinocytes and characterized the genetic and phenotypic alterations associated with the subsequent stages of carcinogenesis to squamous cell carcinomas (SCCs).4Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar In parallel, we have studied the tissue-related changes occurring with tumorigenesis and malignant progression and have begun to characterize the accompanying alterations induced by transformed epithelia in the neighboring connective tissue, using a matrix-inserted surface transplantation assay.5Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar, 6Fusenig NE, Skobe M, Vosseler S, Hansen M, Lederle W, Airola K, Tomakidi P, Stark HJ, Boukamp P, Breitkreutz D: Tissue models to study tumor-stroma interactions. Proteases and Their Inhibitors in Cancer Metastasis Edited by RJ Muschel, J-M Foidart. Kluwer, Academic Press, (in press)Google Scholar In these studies, we have demonstrated the critical role of the tissue microenvironment in controlling and enhancing distinct stages in skin carcinogenesis both by intraepithelial7Javaherian A Vaccariello M Fusenig NE Garlick JA Normal keratinocytes suppress early stages of neoplastic progression in stratified epithelium.Cancer Res. 1998; 58: 2200-2208PubMed Google Scholar and epithelial-mesenchymal interactions.5Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar, 6Fusenig NE, Skobe M, Vosseler S, Hansen M, Lederle W, Airola K, Tomakidi P, Stark HJ, Boukamp P, Breitkreutz D: Tissue models to study tumor-stroma interactions. Proteases and Their Inhibitors in Cancer Metastasis Edited by RJ Muschel, J-M Foidart. Kluwer, Academic Press, (in press)Google Scholar, 8Airola K Fusenig NE Differential stromal regulation of MMP-1 expression in benign and malignant keratinocytes.J Invest Dermatol. 2001; 116: 85-92Crossref PubMed Scopus (35) Google Scholar In vitro transformation of human cells requires, as a first step, the loss of cell senescence and acquisition of immortality, leading to permanently growing but nontumorigenic cell populations.9Rhim JS Yoo JH Park JH Thraves P Salehi Z Dritschilo A Evidence for the multistep nature of in vitro human epithelial cell carcinogenesis.Cancer Res. 1990; 50: S5653-S5657PubMed Google Scholar, 10Boukamp P Peter W Pascheberg U Altmeier S Fasching C Stanbridge EJ Fusenig NE Step-wise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, H-ras oncogene activation and additional chromosome loss.Oncogene. 1995; 11: 961-969PubMed Google Scholar, 11Trott DA Cuthbert AP Overell RW Russo I Newbold RF Mechanisms involved in the immortalization of mammalian cells by ionizing radiation and chemical carcinogens.Carcinogenesis. 1995; 16: 193-204Crossref PubMed Scopus (71) Google Scholar This immortalization process is frequently induced by transfection or infection with oncogenic viruses such as simian virus 40 or human papilloma virus-16, -18, or -31.12Durst M Dzarlieva-Petrusevska RT Boukamp P Fusenig NE Gissmann L Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA.Oncogene. 1987; 1: 251-256PubMed Google Scholar, 13Kaur P McDougall JK Characterization of primary human keratinocytes transformed by human papillomavirus type 18.J Virol. 1988; 62: 1917-1924Crossref PubMed Google Scholar, 14Fusenig NE Boukamp P Breitkreutz D Huelsen A Altered regulation of growth and differentiation at different stages of transformation of human skin keratinocytes.in: Rhim JS Dritschilo A Neoplastic Transformation in Human Cell Culture. Humana Press, Totowa1991: 235-250Crossref Google Scholar Tumorigenic conversion of these immortalized cells can then be induced by chemical and physical carcinogens or by specific oncogenes.15Durst M Gallahan D Jay G Rhim JS Glucocorticoid-enhanced neoplastic transformation of human keratinocytes by human papillomavirus type 16 and an activated ras oncogene.Virology. 1989; 173: 767-771Crossref PubMed Scopus (125) Google Scholar, 16Bookland EA Swaminathan S Oyasu R Gilchrist KW Lindstrom M Reznikoff CA Tumorigenic transformation and neoplastic progression of human uroepithelial cells after exposure in vitro to 4-aminobiphenyl or its metabolites.Cancer Res. 1992; 52: 1606-1614PubMed Google Scholar, 17Klein-Szanto AJ Iizasa T Momiki S Garcia-Palazzo I Caamano J Metcalf R Welsh J Harris CC A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells.Proc Natl Acad Sci USA. 1992; 89: 6693-6697Crossref PubMed Scopus (107) Google Scholar, 18Rhim JS Neoplastic transformation of human cells in vitro.Crit Rev Oncog. 1993; 4: 313-335PubMed Google Scholar In addition, long-term passage of some of these virally immortalized cells leads to spontaneous progression to a tumorigenic phenotype, suggesting a certain degree of genetic instability in these cells.19Brown KW Gallimore PH Malignant progression of an SV40-transformed human epidermal keratinocyte cell line.Br J Cancer. 1987; 56: 545-554Crossref PubMed Scopus (45) Google Scholar, 20Fusenig NE Boukamp P Breitkreutz D Huelsen A Karjetta S Stanbridge E Transformation of human skin epithelial cells in vitro: concepts and stages of transformation.in: Chadwick KH Seymour C Barnhard B Cell Transformation and Radiation-Induced Cancer. Adam Hilger IOP Publishing Ltd., Bristol1989: 45-56Google Scholar, 21Hurlin PJ Kaur P Smith PP Perez-Reyes N Blanton RA McDougall JK Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.Proc Natl Acad Sci USA. 1991; 88: 570-574Crossref PubMed Scopus (171) Google Scholar, 22Chen TM Pecoraro G Defendi V Genetic analysis of in vitro progression of human papillomavirus-transfected human cervical cells.Cancer Res. 1993; 53: 1167-1171PubMed Google Scholar, 23Reddel RR Salghetti SE Willey JC Ohnuki Y Ke Y Gerwin BI Lechner JF Harris CC Development of tumorigenicity in simian virus 40-immortalized human bronchial epithelial cell lines.Cancer Res. 1993; 53: 985-991PubMed Google Scholar, 24Durst M Seagon S Wanschura S zur Hausen H Bullerdiek J Malignant progression of an HPV16-immortalized human keratinocyte cell line (HPKIA) in vitro.Cancer Genet Cytogenet. 1995; 85: 105-112Abstract Full Text PDF PubMed Scopus (32) Google Scholar We have established a multistage skin carcinogenesis model system based on the spontaneously immortalized human keratinocyte line HaCaT.4Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar, 25Boukamp P Petrussevska RT Breitkreutz D Hornung J Markahm A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3395) Google Scholar This cell line does not harbor any viral sequences that might add to genetic instability and thus has maintained its nontumorigenic phenotype for extended culture passages.26Boukamp P Popp S Altmeyer S Hulsen A Fasching C Cremer T Fusenig NE Sustained non-tumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte line HaCaT.Genes Chromosom Cancer. 1997; 19: 201-214Crossref PubMed Scopus (110) Google Scholar Moreover, HaCaT cells closely resemble normal human keratinocytes in their growth and differentiation potential25Boukamp P Petrussevska RT Breitkreutz D Hornung J Markahm A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3395) Google Scholar, 27Ryle CM Breitkreutz D Stark HJ Leigh IM Steinert PM Roop D Fusenig NE Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HaCaT and in ras-transfected tumorigenic clones.Differentiation. 1989; 40: 42-54Crossref PubMed Scopus (138) Google Scholar, 28Breitkreutz D Boukamp P Ryle CM Stark HJ Roop DR Fusenig NE Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line.Cancer Res. 1991; 51: 4402-4409PubMed Google Scholar, 29Breitkreutz D Schoop VM Mirancea N Baur M Stark HJ Fusenig NE Epidermal differentiation and basement membrane formation by HaCaT cells in surface transplants.Eur J Cell Biol. 1998; 75: 273-286Crossref PubMed Scopus (118) Google Scholar, 30Schoop VM Mirancea N Fusenig NE Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts.J Invest Dermatol. 1999; 112: 343-353Crossref PubMed Scopus (256) Google Scholar and have thus become a frequently used model system to study keratinocyte biology and transformation. Immortalization of HaCaT cells was most likely a consequence of UV-type mutations in both alleles of the p53 tumor suppressor gene31Lehman TA Modali R Boukamp P Stanek J Bennett WP Welsh JA Metcalf RA Stampfer MR Fusenig NE Rogan EM Harris CC p53 mutations in human immortalized epithelial cell lines.Carcinogenesis. 1993; 14: 833-839Crossref PubMed Scopus (379) Google Scholar similar to those found in a high percentage of skin carcinomas and premalignant lesions.32Brash DE Sunlight and the onset of skin cancer.Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (265) Google Scholar In addition, HaCaT cells show a loss of chromosomes 3p, 4p, and 9p, areas in which senescence genes have been identified or postulated.33Boukamp P Bleuel K Popp S Vormwald-Dogan V Fusenig NE Functional evidence for tumor-suppressor activity on chromosome 15 in human skin carcinoma cells and thrombospondin-1 as the potential suppressor.J Cell Physiol. 1997; 173: 256-260Crossref PubMed Scopus (15) Google Scholar Despite these genetic alterations, HaCaT cells retain a stable chromosome content and remain nontumorigenic throughout 320 passages.26Boukamp P Popp S Altmeyer S Hulsen A Fasching C Cremer T Fusenig NE Sustained non-tumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte line HaCaT.Genes Chromosom Cancer. 1997; 19: 201-214Crossref PubMed Scopus (110) Google Scholar Because of their genetic make-up and primarily maintained phenotypic normality, they are considered to represent a very early stage in skin tumorigenesis.4Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar Their tumorigenic conversion was achieved by transfection with the mutated val-12 Harvey-ras oncogene34Boukamp P Stanbridge EJ Foo DY Cerutti PA Fusenig NE c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo, but lacks correlation with malignancy.Cancer Res. 1990; 50: 2840-2847PubMed Google Scholar but also by growth under elevated temperature (sunburn conditions)35Boukamp P Popp S Bleuel K Tomakidi E Burkle A Fusenig NE Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature.Oncogene. 1999; 18: 5638-5645Crossref PubMed Scopus (57) Google Scholar or extremely stressful culture conditions such as repeated single cell cloning36Huelsen A: In-vitro-Analysen von Proliferations- und Differenzierungseigenschaften humaner Keratinozyten in unterschiedlichen Transformationsstadien. Doctorate thesis 1990, University Kaiserslautern, Department of BiologyGoogle Scholar or adaptation to autotrophy.37Hill M Hillova J Mariage-Samson R Malignant transformation of human keratinocytes during adaptation to autotrophy.In Vitro Cell Dev Biol. 1991; 27: A270-A272Crossref Scopus (10) Google Scholar In epithelial skin tumors, an activated ras oncogene has been detected in squamous and basal cell carcinomas, suggesting its causal role at least in a subset of epithelial skin tumors.38Ananthaswamy HN Pierceall WE Molecular alterations in human skin tumors.Prog Clin Biol Res. 1992; 376: 61-84PubMed Google Scholar, 39Taguchi M Tsuchida T Ikeda S Sekiya T Alterations of p53 gene and Ha-ras gene are independent events in solar keratosis and squamous cell carcinoma.Pathol Int. 1998; 48: 689-694Crossref PubMed Scopus (3) Google Scholar Tumorigenic conversion of the HaCaT cells resulted in clones growing as epidermoid cysts (benign tumors) after subcutaneous injection.34Boukamp P Stanbridge EJ Foo DY Cerutti PA Fusenig NE c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo, but lacks correlation with malignancy.Cancer Res. 1990; 50: 2840-2847PubMed Google Scholar Additional loss of copies of chromosome 15 in the ras-transfected HaCaT cells was correlated with malignant tumor cells, forming well-differentiated SCCs in nude mice.33Boukamp P Bleuel K Popp S Vormwald-Dogan V Fusenig NE Functional evidence for tumor-suppressor activity on chromosome 15 in human skin carcinoma cells and thrombospondin-1 as the potential suppressor.J Cell Physiol. 1997; 173: 256-260Crossref PubMed Scopus (15) Google Scholar, 40Bleuel K Popp S Fusenig NE Stanbridge EJ Boukamp P Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.Proc Natl Acad Sci USA. 1999; 96: 2065-2070Crossref PubMed Scopus (105) Google Scholar Here we report that further progression of the malignant HaCaT-ras clones to even more aggressive and eventually metastatic phenotypes was not observed after extended passaging in vitro or after culture stress such as repeated single cell cloning. In contrast, their late passage cells exhibited reduced tumorigenicity when injected subcutaneously into nude mice, resulting in diminished tumor growth rates. On the other hand, cells recultured from established heterotransplants showed an increased malignant potential when reinjected into animals, indicating that the in vivo microenvironment exerted a selective pressure and favored malignant progression. We have studied this phenomenon in detail and observed a stepwise tumor progression to highly malignant and metastasizing tumor cell lines obtained through sequential in vivo passages. Characterization of the accompanying genetic and phenotypic changes strongly indicates both clonal selection and further genetic alterations as causal mechanisms for the development of advanced tumor stages. This tumor progression, shown to be stably maintained in recultured tumor cells, is associated with distinct genetic alterations and is phenotypically characterized by an altered growth regulation.41Mueller MM Fusenig NE Constitutive expression of G-CSF and GM-CSF in human skin carcinoma cells with functional consequence for tumor progression.Int J Cancer. 1999; 83: 780-789Crossref PubMed Scopus (69) Google Scholar Cell lines used in this study are the benign HaCaT-ras clone A-5, the malignant HaCaT-ras clones II-3 and II-4,34Boukamp P Stanbridge EJ Foo DY Cerutti PA Fusenig NE c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo, but lacks correlation with malignancy.Cancer Res. 1990; 50: 2840-2847PubMed Google Scholar as well as the benign tumorigenic HaCaT line HaCaTp36Huelsen A: In-vitro-Analysen von Proliferations- und Differenzierungseigenschaften humaner Keratinozyten in unterschiedlichen Transformationsstadien. Doctorate thesis 1990, University Kaiserslautern, Department of BiologyGoogle Scholar and the malignant line HaCaT40°35Boukamp P Popp S Bleuel K Tomakidi E Burkle A Fusenig NE Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature.Oncogene. 1999; 18: 5638-5645Crossref PubMed Scopus (57) Google Scholar (Figure 1). The latter two cell lines had been converted to tumorigenicity by culture stress. The stress was exerted either by repeated single-cell cloning (HaCaTp) or by continued growth (>10 passages) at 40°C instead of 37°C (HaCaT40°). Under both stress conditions HaCaT cells were converted to tumorigenicity. The tumors formed were either noninvasive, slowly growing benign cysts HaCaTp36Huelsen A: In-vitro-Analysen von Proliferations- und Differenzierungseigenschaften humaner Keratinozyten in unterschiedlichen Transformationsstadien. Doctorate thesis 1990, University Kaiserslautern, Department of BiologyGoogle Scholar or locally invasive SCCs HaCaT40°.35Boukamp P Popp S Bleuel K Tomakidi E Burkle A Fusenig NE Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature.Oncogene. 1999; 18: 5638-5645Crossref PubMed Scopus (57) Google Scholar The following cell lines were established after in vivo passage by recultivation of subcutaneously transplanted tumors: HaCaTpT, HaCaT40°RT, II-3RT, II-4RT, A-5RT1, and A-5RT3 (Figure 1). Cell lines were cultivated in 4× modified Eagle's medium with 5% fetal calf serum (FCS) following routine protocols.25Boukamp P Petrussevska RT Breitkreutz D Hornung J Markahm A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3395) Google Scholar Both benign and malignant HaCaT clones were serially passaged at a split ratio of 1:5 to 1:10 and tested for tumorigenicity at different passage levels. The cell lines A-5 and II-4 were cloned by ring isolation and clonal populations were again tested for tumorigenicity. For recultivation of tumor cells, subcutaneous tumors were surgically removed from the nude mouse, separated from stroma and necrotic areas, and proliferative tumor margins were minced into 1-mm3 pieces. These tumor fragments were transferred to 10-cm tissue culture plates containing 6 × 106 lethally irradiated (70 Gy) human fibroblast feeder cells in standard media. When islands of tumor cells had formed (after 5 to 10 days) the pieces of tumor tissue were removed by aspiration and the cells were further cultivated according to standard protocols.25Boukamp P Petrussevska RT Breitkreutz D Hornung J Markahm A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3395) Google Scholar Cells were routinely tested for mycoplasma contamination by standard procedures42Stacey A Doyle A Routine testing of cell cultures and their products for mycoplasma contamination.Methods Mol Biol. 1997; 75: 305-311PubMed Google Scholar and always found to be negative. Their origin from transfected HaCaT-ras cells (containing the neo-resistance gene34Boukamp P Stanbridge EJ Foo DY Cerutti PA Fusenig NE c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo, but lacks correlation with malignancy.Cancer Res. 1990; 50: 2840-2847PubMed Google Scholar) as well as the absence of contaminating mouse cells were proved by the resistance of the recultivated cells to G418 (400 μg/ml) treatment. For HaCaT40° and HaCaTp cells elimination of mouse cell contamination was done as described.35Boukamp P Popp S Bleuel K Tomakidi E Burkle A Fusenig NE Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature.Oncogene. 1999; 18: 5638-5645Crossref PubMed Scopus (57) Google Scholar Growth curves were recorded from cells seeded at a density of 4 × 103 cells/cm2 in 4× modified Eagle's medium with 5% FCS by daily counting of the cells in three independent cultures with an electronic counter (Casy, Schaerfe System, Reutlingen Germany). For determination of colony-forming efficacy, cells were plated at a density of 100 cells/6-cm dish in 4× modified Eagle's medium containing 1% FCS. After 24 hours, allowing for cell attachment, medium was shifted to 5% FCS or no FCS, respectively. After 10 days the medium was removed, cells were fixed in 3.7% formaldehyde, stained with Mayer's hematoxylin, and colonies were counted. Colony-forming efficacy was calculated as the ratio of colonies to the number of cells seeded. The colony-forming efficacy of A-5 cells was arbitrarily set to 100%. Semiconfluent cultures were harvested by trypsinizing with 0.1% trypsin/ethylenediaminetetraacetic acid in phosphate-buffered saline without Ca2+ and Mg2+containing 4 mg/ml colcemid, centrifuged, and the cell pellet resuspended in hypotonic solution of 70 mmol/L KCl. After incubation at 37°C for 15 to 25 minutes cells were fixed by three changes of methanol/acetic acid 3:1 and spread on glass slides. G-banding was performed after the slides had been aged for several days as described.43Boukamp P Tilgen W Dzarlieva RT Breitkreutz D Haag D Riehl RK Bohnert A Fusenig NE Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin.J Natl Cancer Inst. 1982; 68: 415-427PubMed Google Scholar To analyze numerical chromosome alteration, at least 50 metaphases were counted and 25 karyotypes were arranged and analyzed for structural changes. Tumor formation was assayed after subcutaneous injection of 5 × 106 and 1 × 105 cells in 100 μl of culture medium into the interscapular region of 4- to 6-week-old athymic nude (Swiss nu/nu) mice and tumor size was measured in two axes throughout an observation period of 6 months. The tumor volume was calculated following published procedures.40Bleuel K Popp S Fusenig NE Stanbridge EJ Boukamp P Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.Proc Natl Acad Sci USA. 1999; 96: 2065-2070Crossref PubMed Scopus (105) Google Scholar The metastatic potential of tumor cells was assayed using two separate transplantation systems. Experimental metastasis was assayed by injecting 1 × 106 cells into the tail vein of athymic nude mice and lung colony formation was monitored for a period of 18 weeks. Spontaneous metastasis from tumor cells grafted as skin-like surface transplants was determined after the transplantation of organotypic cultures as described previously.7Javaherian A Vaccariello M Fusenig NE Garlick JA Normal keratinocytes suppress early stages of neoplastic progression in stratified epithelium.Cancer Res. 1998; 58: 2200-2208PubMed Google Scholar Briefly 7-day-old organotypic co-cultures of tumor cells were trimmed using a surgical punch (1.4 cm in diameter) and transplanted onto the back muscle fascia of nude mice. Animals were sacrificed at 9 weeks after transplantation, and grafts as well as regional lymph nodes were dissected and prepared for histology. Successive in vivo passages of tumor cells without intermittent in vitro growth were performed by subcutaneous transplantation of tumor fragments from the previous in vivo passage. Tumors were surgically removed, separated of stroma and necrotic areas, and 0.5 mm3 pieces of the proliferative tumor margins were subcutaneously transplanted via skin incision. Tumor growth was observed for up to 6 months and monitored by weekly measurements of two tumor diameters. Genomic DNA of cultured tumor cells was isolated by the sodium dodecyl sulfate lysis procedure,44Saxon PJ Srivatsan ES Leipzig GV Sameshima JH Stanbridge EJ Selective transfer of individual human chromosomes to recipient cells.Mol Cell Biol. 1985; 5: 140-146PubMed Google Scholar and 10 mg of DNA were digested overnight at 37°C with the restriction endonucleases Bam HI and Eco RI, respectively. Hybridization conditions, electrophoresis, and autoradiographic procedures were as described previously.45Geiser AG Der CJ Marshall CJ Stanbridge EJ Suppression of tumorigenicity with continued expression of the c-Ha-ras oncogene in EJ bladder carcinoma-human fibroblast hybrid cells.Proc Natl Acad Sci USA. 1986; 83: 5209-5213Crossref PubMed Scopus (68) Google Scholar Cellular proteins were extracted from 107 trypsinized cells and the ras proteins immunoprecipitated as described previously.10Boukamp P Peter W Pascheberg U Altmeier S Fasching C Stanbridge EJ Fusenig NE Step-wise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, H-ras onco