Caractérisation des kératinocytes immortalisés par les virus SV40, HPV (human papillomavirus) ou spontanément (original) (raw)
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Cytogenetic analysis of eight human papillomavirus immortalized human keratinocyte cell lines
International Journal of Cancer, 1989
Cytogenetic analysis was performed on human papillomavirus (HP-immortalized cell lines. Lines were established by co-transfection of primary human keratinocyte cells with HPV type 16 or 18 DNA and pSV2neo. The resulting clonal lines contained integrated HPV DNA and exhibited extended life spans in culture but were non-tumorigenic in nude mice. Two HPVl6-immortalized lines (FEPE I UI and FEPEILP) and 4 HPVIB-immortalized lines (FEA, FEHIBL, FEP18-5 and FEPI8-I I) were established. Two additional lines were derived by subsequent treatment of the FEA line with TPA and by further transfection with HSVll DNA. Cytogenetic analysis revealed that all lines were abnormal, containing a variety of numerical and structural aberrations. Six of the 8 lines were hyper-triploid and 2 were near-diploid. Examination of lines FEA, FEH18L and FEP18-I I at multiple passages in culture revealed that the lines were clonal and chromosomally stable over extended passage in culture. Structural rearrangements were most common in chromosomes I and 3 but also occurred in chromosomes 5, 7, 8, 12, 16 and 22. Marker chromosomes were present in all cell lines. A small metacentric marker, possibly an isochromosome for the short arm of chromosome 5, was consistently present in the FEA line and its derivatives (FEAB 10 and FEAT) as well as the FEM I8L line. A loss or reduction in copy number of chromosome 13 was seen in 5 of the 8 cell lines.
Proceedings of the National Academy of Sciences, 1991
We have developed a model system for progression ofhuman epithelial cells to malignancy, using a human papillomavirus type 18 (HPV-18)-immortalized human keratinocyte cell line. Cells of cell line FEP-1811 were nontumorigenic in athymic mice through at least 12 passages in culture, but after 32 passages were weakly tumorigenic, producing tumors that regressed. After 62 passages they produced invasive squamous cell carcinomas that grew progressively. The progression to malignancy was associated with an increase in the efficiency of forming colonies in soft agar and with altered differentiation properties. In an organotypic culture system, FEP-1811 cells at passages 12 and 32 exhibited features typical of premalignant intraepithelial neoplasia in vivo, and cells at passage 68 exhibited features consistent with squamous cell carcinomas. No change in copy number of the transfected HPV-18 genome or in the level of expression of the viral transforming gene products E6 and E7 was detected between tumorigenic and nontumorigenic cells. Cytogenetic analysis of cells at early, middle, and late passage levels and cells cultured from tumors revealed that several chromosomal abnormalities segregated with the tumorigenic cell populations.
Evidence for the multistep nature of in vitro human epithelial cell carcinogenesis
Cancer Research
In keeping with the multistep development of human cancer in vivo, a stepwise approach to neoplastic transformation in vitro presents a rea sonable strategy. \Ve have recently developed an in vitro multistep model suitable for the study of human epithelial cell carcinogenesis. Upon infection with the adenovirus 12-simian virus 40 hybrid virus, primary human epidermal keratinocytes acquired an indefinite life span in culture but did not undergo malignant conversion. Subsequent addition of Kirsten murine sarcoma virus and human ras oncogene or chemical carcinogens (/V-methyl-Af'-nitro-Ar-nitrosoguanidine or 4-nitroquinoline 1-oxide) to these cells induced morphological alterations and the acquisition of neoplastic properties. Subsequently it was found that this line could be transformed neoplastically by a variety of retrovirus-containing li-ras, bas, fes, fins, erhU. and src oncogenes. In addition, we found that the immortalized human epidermal keratinocyte (RHEK-1) line can be trans formed neoplastically by exposure to ionizing radiation. Thus, this in vitro system may be useful in studying the interaction of a variety of carcinogenic agents and human epithelial cells. These findings demon strate the malignant transformation of human primary epithelial cells in culture by the combined action of viruses, oncogenes, chemical carcino gens, or X-ray irradiation and support a multistep process for neoplastic conversion.
Mutation research, 1988
Mutagenesis of a diploid human fibroblast strain, KD, with the chemical carcinogen 4 nitroquinolin-1-oxide led to the isolation of stably immortalized neoplastic substrains. Four of these transformed strains, HuT-11, -12, -13, and -14, have been characterized in great detail with regard to morphology and changes in gene expression from the parental KD strain. The HuT-11, -12 and -13 substrains are immortalized and non-tumorigenic, in contrast to HuT-14 which is both immortalized and tumorigenic. The HuT-14 substrain expresses a defective beta-actin as a consequence of a point mutation in 1 of the 2 functional beta-actin alleles. All 4 HuT strains have induced expression of the phosphoprotein plastin and 2 EGF-related polypeptides, and down-regulated expression of the transformation-sensitive tropomyosin isoforms. KD and HuT cells expressing high levels of exogenous mutant beta-actin after gene transfection show morphological alterations. HuT-12 transfectants with excessive mutant be...
Immortalization of human fibroblasts transformed by origin-defective simian virus 40
Molecular and Cellular Biology, 1987
Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS...
Cell, 1977
Hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait, is associated with a predisposition to neoplasia. The present study describes the differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Primary cutaneous outgrowths were derived from normal appearing subepidermal biopsies of ACR phenotypes and appropriate controls. Exponentially growing cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny subjected to the viral probe were 100-1000 fold more susceptible to transformation than were normal skin fibroblast cultures. The virally transformed human skin fibroblasts showed a loss of anchorage dependency in carboxymethylcellulose suspension and formed tumors in athymic mice. The results suggest that skin fibroblasts obtained from individuals genetically predisposed to colon neoplasia are preferentially transformed by the Kirsten murine sarcoma virus.
Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line
The Journal of Cell Biology, 1988
In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a p...
Simple epithelial nature of some simian virus-40-transformed human epidermal keratinocytes
Cancer research, 1984
Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These result...