Immortalization Of Fibroblasts Research Articles

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.

Partial transformation of mouse fibroblastic and epithelial cell lines with the v- myc oncogene

To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v- myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse fibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v- myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors, v- myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v- myc-transformed fibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v- myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2–3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v- myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v- myc oncogene induces morphological changes and anchorage independence in immortal mouse fibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic.

Hypersensitivity of Cultured Ataxia-Telangiectasia Cells to Etoposide<xref ref-type="fn" rid="FN2">2</xref>

Fibroblasts from patients with ataxia-telangiectasia (A-T) were found to be hypersensitive to killing by the antineoplastic agent etoposide. The A-T fibroblast strains GM5823, GM367, and GM2052 were twofold to threefold more sensitive to killing by etoposide than fibroblasts from normal controls (AG1521, AG1522, and IMR90). A simian virus 40 (SV40)-transformed, immortal human fibroblast line (GM5849) derived from the A-T cell line GM5823 was also studied. GM5849 retained the unusual sensitivity of nontransformed A-T fibroblast lines to x-irradiation, bleomycin, and neocarzinostatin (zinostatin). GM5849 was also more sensitive to etoposide than were SV40-transformed fibroblasts from normal controls. M1, and SV40-transformed fibroblast line derived from a patient with xeroderma pigmentosum, had the same sensitivity to etoposide as SV40-transformed fibroblasts derived from normal controls.

Changes in activity and amount of active elongation factor 1α in aging and immortal human fibroblast cultures

Stoichiometrically estimated amounts of active elongation factor, EF-1α, remain constant in serially passaged Phase II cultures of human fibroblasts, MRC-5, but decrease by 45% towards the end (Phase III) of their lifespan. Catalytic activity of EF-1α is also reduced by 35% in Phase III old cells. The SV40 transformed immortal cell line MRC-5V2 has 30% higher levels of active EF-1γ without significant increase in its catalytic activity. Low-serum-associated G 1 arrest of normal and transformed cells reduces amounts of active EF-1α by 35% and 20%, respectively. Catalytic activity, however, is reduced rapidly only in G 1 arrested normal cells and not in transformed cells. Even though the cell cycle-related changes are reversible both in normal and transformed cells, the age-related decline in amounts of active EF-1α and its activity are irreversible and, most probably, crucial.

Isolation of a line of immortal chicken embryo fibroblasts after transfection with the nuclei of Rous sarcoma virus-transformed Chinese hamster cells

Secondary cultures of chicken embryo fibroblasts were transfected with purified nuclei from lysed cells of a clonal line of temperature-sensitive Rous sarcoma virus ( tsRSV)-transformed Chinese hamster fibroblasts. After propagation for 3 months an established cell line designated ChR32 was obtained in one chicken cell culture. The cells of this line have been propagated so far for 18 months, whereas normal chicken embryo fibroblasts died after 2 months. The established cells were heteroploid with a diploid modal number of macrochromosomes and two Z chromosomes. No Chinese hamster chromosomes could be identified. Southern blot analysis of DNA from the uncloned ChR32 cells and the clones provided evidence that these established cells were, in fact, clonal in origin and contained full-length RSV proviruses and no defective proviruses. Furthermore, they contained, at the 3′ end proviral-cellular junction, BglII, HpaI, KpnI, SacI, and XbaI fragments of the same size as the Chinese hamster donor cells, suggesting that the cellular sequence adjacent to the provirus is of Chinese hamster origin. The cells after establishment were able to grow continuously at 37 ° or 41 °C and produce a large amount of ts sarcoma virus particles. A corollary finding was that these virus particles were non-leaky for the transforming function at the non-permissive temperature.

Immortal fibroblasts?

Uncommitted fibroblasts may exist ( Kirkwood & Holliday, 1975; Holliday, Huschtscha, Tarrant & Kirkwood, 1977). I show that, even if such cells divide significantly faster than committed ones, the uncommitted cells would be overlooked if one uses current culture techniques. Some general guidelines for testing the model are outlined. Uncommitted cells would be useful in studying the biochemical mechanisms of aging and development.

Afferent projection of the superior laryngeal nerve in the brainstem.

The splicing factor SF2/ASF is an oncoprotein that is up-regulated in many cancers and can transform immortal rodent fibroblasts when slightly overexpressed. The mTOR signaling pathway is activated in many cancers, and pharmacological blockers of this pathway are in clinical trials as anticancer drugs. We examined the activity of the mTOR pathway in cells transformed by SF2/ASF and found that this splicing factor activates the mTORC1 branch of the pathway, as measured by S6K and eIF4EBP1 phosphorylation. This activation is specific to mTORC1 because no activation of Akt, an mTORC2 substrate, was detected. mTORC1 activation by SF2/ASF bypasses upstream PI3K/Akt signaling and is essential for SF2/ASF-mediated transformation, as inhibition of mTOR by rapamycin blocked transformation by SF2/ASF in vitro and in vivo. Moreover, shRNA-mediated knockdown of mTOR, or of the specific mTORC1 and mTORC2 components Raptor and Rictor, abolished the tumorigenic potential of cells overexpressing SF2/ASF. These results suggest that clinical tumors with SF2/ASF up-regulation could be especially sensitive to mTOR inhibitors.