Insulin crosslinked to the catalytic fragment A of diphtheria toxin has previously been shown to be cytotoxic to insulin-responsive mouse BALBc/3T3 cells but noncytotoxic to insulin-nonresponsive ...variant IN-2 cells Miskimins, W. K. & Shimizu, N. (1979) Biochem. Biophys. Res. Commun. 91, 143-151. We have now used this cytotoxic chimeric insulin to select resistant variants of Swiss/3T3 fibroblasts. Of eight resistant colonies isolated, two proved to be deficient in insulin receptor and exhibited altered morphologies and growth rates. These variants, however, retained the ability to bind and respond to epidermal growth factor. Thus, this selection technique is effective for the isolation of variants specifically related to cellular binding and processing of insulin. Such variants will be advantageous for the genetic and biochemical characterization of the mechanism of insulin's action.
Expression of the transferrin receptor is necessary for cells to progress through S-phase. The transferrin receptor gene promoter is activated as a delayed event following growth factor stimulation ...of quiescent fibroblasts. Serum stimulation in the presence of vanadate leads to superactivation of the transferrin receptor promoter, suggesting a role for tyrosine phosphorylation. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, a tyrosine kinase-regulated enzyme, blocks mitogen-dependent activation of the transferrin receptor promoter. Furthermore, wortmannin was able to block activation of this promoter when added several hours after serum stimulation of quiescent cells. This suggests that phosphatidylinositol 3-kinase may be required in mid to late G1 and that it is directly involved in a pathway leading to activation of the transferrin receptor promoter. This is further supported by the finding that the transferrin receptor promoter is much less responsive to mitogenic stimulation in cells that have been stably transfected with a dominant negative form of the phosphatidylinositol 3-kinase regulatory subunit. Activation of S6 kinase, an event known to be downstream of phosphatidylinositol 3-kinase activation, appears not to be involved in activation of the transferrin receptor promoter since no effect was observed by treatment of cells with rapamycin.
The intracellular translocation and processing of epidermal growth factor (EGF) in 3T3 cells has been studied utilizing Percoll density gradients. EGF is internalized and rapidly becomes associated ...with two types of intracellular compartments. The extent to which EGF is delivered to these two compartments is apparently regulated depending upon the cell's physiological condition. In growth medium, an increased proportion of EGF is taken up into a Golgi-like element. Uptake through this pathway correlates with a decrease in degradation of the ligand. In the absence of serum and amino acids, an increased proportion of EGF is taken up into a component which has a density of 1.05. Uptake through this pathway correlates with increased degradation of the ligand. The ligand taken up through both pathways is transferred to dense vesicles which comigrate with lysosomes. In the presence of growth medium, however, dense vesicles containing EGF can be shown to be lysosomal enzyme-deficient upon further fractionation. In addition, in the presence of serum, a portion of the internalized EGF is apparently released from the cells, intact, and then re-bound. The processes described may be important in the production of a mitogenic response and the ability of cells to self-regulate their responsiveness to the growth factor.
A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that ...appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-O-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-O-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.
A combination of adsorption (protamine-agarose) and gel filtration (Sephacryl S-300) chromatography was used to enrich for factor(s) in Xenopus laevis frog egg extract that induce nuclear ...swelling-chromatin decondensation in permeabilized human sperm. It was determined that a 70-fold purification of the factor(s) that induce human sperm nuclear swelling has resulted from the purification scheme. The reduced, active factor(s) have a Kav of 0.12 and an approximate molecular weight of 290,000 daltons. The extract nuclear swelling activity is sensitive to temperatures of 50 degrees C and above, as well as proteolytic treatment. RNase and cycloheximide treatments of the extracts have no effect on the nuclear swelling activity. These data suggest that the egg extract factor(s) that induce nuclear swelling in permeabilized human sperm are protein(s) that are present in the unfertilized frog egg.
Recently we have isolated six variants of Swiss/3T3 mouse fibroblasts that are resistant to the cytotoxic insulin-diphtheria toxin A fragment. All of the variants proved to have greatly reduced or no ...insulin binding capacity, and several variants showed altered morphologies and growth characteristics. We now report on the further characterization of one of these variants, CI-3, which displays a massive accumulation of membranous vesicles in its cytoplasm. By electron microscopy these vesicles resemble lysosomes. They also appear to fluoresce bright orange after treatment of viable cells with acridine orange. However, the specific activity of several lysosomal enzymes is depressed in CI-3. Additionally, there is an apparent shift in the density of vesicles containing lysosomal enzymes in this variant. These alterations may be directly related to CI-3's resistance to the cytotoxic insulin and have some important bearings on the mechanism of insulin action.
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