Immortalization Of Human Fibroblasts Research Articles

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

Comparison of major cytoskeletons among normal human fibroblasts, immortal human fibroblasts transformed by exposure to Co-60 gamma rays, and the latter cells made tumorigenic by treatment with harvey murine sarcoma virus

Immortally transformed human fibroblasts in general acquire an epithelial shape, while normal human fibroblasts demonstrate a spindle-shaped feature. In order to investigate this difference, three types of major cytoskeletal elements, namely, F-actin, tubulin, and vimentin of immortal human fibroblasts were morphologically compared with those of normal human fibroblasts. As a result, a significant difference was observed in the distribution and in the number of F-actin fibers between immortal and normal fibroblasts. The cells of three immortally transformed fibroblast lines, KMST-6, WI-38 VA-13, and SUSM-1, showed a striking reduction in the number, and an altered pattern of organization, of actin fibers. On the other hand, in the normal fibroblasts, actin fibers ran parallel to each other along the long axis of the cells. Tubulin and vimentin showed no significant difference between the immortal and normal cells. Our present data show that the morphological changes seen in the immortally transformed cells are due to the disorganization and the decrease in number of actin fibers. Interestingly, both the immortal cells (KMST-6), which were not tumorigenic, and the Harvey murine sarcoma virus-transformed KMST-6 cells, which were tumorigenic and demonstrated an enhanced expression of the ras gene, revealed an overall similarity in the organization pattern and the number of actin fibers. These findings seem to indicate that the immortality transformed cells have already acquired some cancer characteristics.