Nuclear translocation of MAPK in cell lines, expressing normal and mutant forms of EGF-receptors (EGFR), was investigated. Using immunoblotting and immunofluorescence, EGF-induced MAPK transport was discovered in cell lines, expressing both normal receptor and one with deletion of major autophosphorylation sites. The dynamics of MAPK nuclear translocation in these cell lines was alike. Cells bearing EGFR with inactive tyrosine kinase showed no ability to EGF-dependent activation and nuclear translocation of MAPK. A suggestion is made that tyrosine kinase is needed for EGF-induced activation and nuclear import of MAPK.
Certain changes in human carcinoma A-431 are found by scanning electron microscopy. The early cell response to growth factor (after 10 minutes) involves a disappearance of microvilli, an appearance of ruffles and rounded cells, along with a decrease in cell attachment to the substrate. The cell surface changes correlate with the state of cytoskeleton elements: the material stained with iron hematoxylin is accumulated in ruffle formation sites. Retractional fibrils filled with the cytoskeleton material result from a decrease in the cell area.
Mouse epithelial mammary cell line HC11, expressing about 30 thousands receptors for epidermal growth factor (EGF) per cell, is a physiological target for this growth factor. It is found that EGF behaves as a strong mitogen for HC11 cells: EGF (10 ng/ml) produced more than a 6-fold increase in 14C-thymidine incorporation into DNA of the cells, while 10% serum stimulated only a 2.5-fold increase, compared to control. It was shown that about 60-80% of surface-bound 125I-EGF were internalized within the first 5 min upon stimulation of endocytosis, regardless of cultivation conditions. However, the serum and EGF affected on later steps of endocytosis in different way. A prolonged presence of EGF in the growth medium before the experiment led lo a significant increase in transition of internalized 125I-EGF from light to heavy endosomes. At the same time, I 0 % serum caused a dramatic decrease if the rate of 125I-EGF degradation, which was resulted in accumulation of un-degraded growth factor in lysosomes. However, serum did not influence the earlier steps of endocytosis. A conclusion has been made that it is the efficiency of EGF transition from light to heavy endosomes (which means sorting of EGF-receptor complexes for degradative pathway) that is regulated by EGF-dependent manner in HC11 cells.
A431 epidermoid carcinoma cells have an increased expression of EGF receptor. In contrast to many other cell lines and primary cells, these respond to EGF in high (more than 1 ng/ml) concentrations by cell cycle arrest, apoptosis and detachment. Clonal variants of A431 (1a and 8a), able to grow in the presence of EGF in high concentration, were previously developed in our laboratory (Gudkova, Sorokin, 1989). Here we tested upper pathways of signal transduction from EGF receptor in the clonal variants, as compared to A431. We found no reasonable difference in the expression of EGF receptor, as well as in its EGF-induced phosphorylation in A431 and clonal variants. There were also no changes in the amount and activation of ERK MAP kinase in different cell lines. In contrast, the amount of STAT1 transcription factor, known to play pro-apoptotic and antiproliferative roles, was strictly diminished in both the clonal variants tested (1a and 8a), as compared to the parental line A431. However, EGF-induced tyrosine phosphorylation of STAT1 decreased only in 8a. Increased phosphorylation of Akt protein kinase, the key component of PI-3 kinase of the anti-apoptotic and proliferative signaling pathway, was also observed in clonal variants. The data obtained demonstrate that resistance to EGF can be acquired in cells having similar levels of EGF receptor expression and phosphorylation, but different in STAT1 or PI-3 kinase signal transduction pathways. These pathways may presumably represent two antagonistic key elements regulating A431 proliferation and apoptosis.
By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.
Primary reactions on the addition of the epidermal growth factor (EGF) were investigated for the strains of A-431 cells, resistant to the antiproliferative effect of EGF. In spite of differences of EGF reception in the obtained strains, the EGF receptors in membrane preparations of these strains maintain the phosphorylating ability after addition of EGF. The rate of internalization of 125I-EGF in normal and resistant cells is the same. The production of a secreted fragment of the EGF receptor in resistant cells is lower than in normal ones. Questions of regulation of production of the normal receptor and of its shortened secreted fragment are discussed.
The response of human endometrial stem cells (hESCs) to oxidative stress has been investigated by flow cytometry. Two terminally differentiated cell lines were used for the comparison: human embryonic lung fibroblasts and human dermal fibroblasts. The oxidative stress was designed by hydrogen peroxide (H2O2) action in the wide range of concentrations (50-1500 microM) during 24 h. It has been shown that the H2O2 amount per one cell (pM/cell), but not H2O2 concentration in the growth medium, should be taken into account for the accurate evaluation of H2O2 effect on different cell lines. Therefore, in our experiments LD50 reflects the amount of H2O2 per cell, at which 50% cells survived after 24 h. We have demonstrated that hESCs are more resistant to H2O2 than embryonic lung fibroblasts, but less resistant than dermal fibroblasts.
