Malignant Transformation Of Human Cells Research Articles

Telomere elongation observed in immortalized human fibroblasts by treatment with 60Co gamma rays or 4-nitroquinoline 1-oxide

Telomeres are the tandemly repeated (TTAGGG)n sequences that make up the structural and functional ends of all chromosomes in mammals. Many lines of evidence indicate that telomeres stabilize chromosomes, prevent aberrant recombination, and direct chromosome attachment to the nuclear membrane. Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' DNA synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this end-replication problem. To overcome this problem, most eukaryotic cells use telomerase, an enzyme that elongates telomeres. However, this enzyme has not been detected in normal human cells, and these cells lose telomeres with cell division. Cellular senescence might be the result of this loss. Thus, activation of telomerase seems to be critical for the immortalization of human cell lines. In addition, substantial evidence indicates that immortalization in itself is a rate-limiting step for the malignant transformation of human cells. We have treated normal human fibroblasts (AD387, KMS-6, and OUMS-24 lines) intermittently with either 60Co gamma rays or 4-nitroquinoline 1-oxide (4NQO) during serial subcultivations, and have obtained three immortalized cell lines, SUSM-1, KMST-6, and OUMS-24F. In KMS-6 and OUMS-24, the mean terminal restriction fragment length significantly decreased as the population-doubling level increased. The rate of telomere loss was 40 and 50 bp/population doubling in the KMS-6 and OUMS-24 cell lines, respectively. Once these normal cell lines were immortalized, their telomeres became elongated. Similar data were obtained for AD387 cells and their immortalized SUSM-1 cells. These results suggest that telomeres play a critical role in cellular senescence and in the immortalization processes of human cells.

Malignant transformation of human fibroblasts by ionizing radiation.

As one step in developing an assay for quantifying the induction of malignant transformation of human cells by ionizing radiation, we exposed cells from a non-tumorigenic, infinite life span, near-diploid fibroblast strain MSU-1.1 to 4.35 Gy 60Co radiation and assayed them for focus formation. The mean frequency of foci in the irradiated population was 6 x 10(-7) cells assayed. No foci were found in the control cells. Of four focus-derived cell strains studied in detail, two produced malignant tumours within 3-7 weeks. The other two did not produce tumours during the 12-month period of study. The tumours from one strain were classified as sarcomas composed exclusively of spindle-shaped cells. Tumours from the other strain were sarcomas consisting of a mixed population of round and spindle cells. Immunoprecipitation analysis of the status of the p53 gene in the focus-derived strains, using a mutant-specific anti-body (Pab240) and an antibody that recognizes both mutant and wild-type p53 protein (Pab421), showed that the tumorigenic strains were completely devoid of p53 protein. One non-tumorigenic strain expressed wild-type p53 protein, and the other expressed a lower molecular weight form of the protein. Karyotypic analysis showed that the tumour-derived cells from one tumorigenic strain had lost one copy of chromosome 6, 14, 16 and 17. The tumour-derived cells from the second strain had lost one copy of chromosome 7, 13, 14 and 17 and part of chromosome 6, as well as part of the other copy of chromosome 7 and 17. These results suggest that the common loss of one copy of chromosome 14, 17 and part of 6 plays a causal role in the malignant transformation of these cells. Furthermore, the results indicate that it will be possible to develop a system that uses near-diploid human fibroblasts to quantify radiation-induced malignant transformation.

Malignant transformation of human fibroblasts previously immortalized with 60Co gamma rays.

Normal human fibroblasts obtained from a whole embryo were malignantly transformed with only 60Co gamma rays, but after extensive passage. The fibroblasts were exposed intermittently to 60Co gamma rays, 13 times, 2,800 rads in total, from the primary culture to the 40th passage level. At the 50th passage level the cells became morphologically changed, showing chromosomal abnormalities, and serial passages showed them to be immortalized. They were not tumorigenic on transplantation into nude mice, but became malignant after extensive passage; i.e., at the 547th passage level and 2,800 days after initiation of the culture. Our results indicate that several different genotypic changes are necessary for malignant transformation of human cells over long periods. No mutation at codons 12 and 61 of H-, K- and N-ras was detected in the malignant cells. Thus, this system may be useful to detect other cellular genes that may contribute to the malignant phenotypes of human fibroblasts.

Extended life span of human endometrial stromal cells transfected with cloned origin-defective, temperature-sensitive simian virus 40

Human endometrial stromal cells transfected with an origin-defective, temperature-sensitive simian virus 40 recombinant plasmid are dependent on T-antigen function for proliferation and at the permissive temperature have an extended life span in culture. Southern blot analysis indicates that the transfected gene is present in low copy number, possibly at a single integration site. Normal stromal cells are capable of 10 to 20 population doublings in culture. Transfected cultures have been carried at the permissive temperature to 80 population doublings before crisis. In the multistep model of malignant transformation of human cells, these cells represent one of the earliest stages: extended but finite life span. We have used these cells to investigate alterations in signal transduction that may be responsible for this early stage of transformation caused by the large T antigen. Temperature shift experiments indicate that the expression of ornithine decarboxylase (ODC) but not of c-fos is altered by the large T antigen. Induction of c-fos by serum or 12-O-tetradecanoylphorbol-13-acetate is independent of temperature. However, in the transfected cells, the induction of ODC by asparagine or serum is greatly enhanced at the permissive temperature. This result indicates that the large T antigen acts downstream of c-fos but upstream of ODC expression in the signal-transducing cascade.

Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells.

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, we have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and greater than 200 tumor specimens. Differential patterns of F19 (Mr, 120,000/95,000 glycoprotein), F24 (Mr, 95,000 glycoprotein), G171 (Mr, 75,000 glycoprotein), G253 (Mr, 90,000 glycoprotein), S5 (Mr, 120,000 glycoprotein), and Thy-1 (Mr, 25,000 glycoprotein) antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.