Most mammalian cells require serum for sustained proliferation. In the presence of low amounts of serum these cells become quiescent. Re-addition of serum to deprived cells initiates a series of intracellular events that culminate in DNA replication, mitosis and cell division. Some of the earliest events in this """"""""activation sequence"""""""" involve ions: increased intracellular pH and Ca++ and increased Na+ flux. Inhibiting the changes in pH, Ca++ or Na+ will prevent activation by serum; conversely, mimicking the Ca++ or pH change will artificially activate, in some cases. There are dramatic changes in translation which occur upon serum addition after ionic changes, yet prior to the initiation of DNA synthesis. The absolute rate of translation increases about 3-fold in response to serum. Preventing this increase will inhibit stimulation. The identity of translated products in serum-stimulated cells is also distinct from cells in the absence of serum. The purpose of the proposed study is to investigate the relationships between the ionic events and the changes in translation that occur upon stimulation of quiescent cells. This investigation will be performed by determining the translation profile and translation rate of cells that have been stimulated to proliferate by a number of treatments: serum, pure growth factors, alkaline pulse, LiCl and AlCl3. Translation profile will be determined by gel fluorography of pulse-labeled proteins. It is our hypothesis that proteins whose synthesis is necessary for stimulation will be synthesized in response to all of the above stimuli. It is also hypothesized that all treatments will result in, at least a transient, increase in translation rate. The effects of artificially perturbed intracelullar pH and Ca++ levels on translation rate and profile will also be investigated. It is our hypothesis that agents which raise these ions from quiescent to stimulated levels will effect changes in protein synthesis. Agents which prevent the serum-induced changes in pH and Ca++ are likewise hypothesized to prevent the serum-induced changes in protein synthesis. At the conclusion of these studies, we will have more fully defined the interaction(s) between the ionic and translation changes that occur upon stimulation of serum-deprived cells. It is also likely that we will be able to identify proteins whose de novo synthesis is necessary for initiation of DNA replication. Finally, it is possible that these studies will determine the mechanism of alkaline pH mitogenesis.
| Martinez, R; Gillies, R J; Giuliano, K A (1988) Effect of serum on the intracellular pH of BALB/c-3T3 cells: serum deprivation causes changes in sensitivity of cells to serum. J Cell Physiol 136:154-60 |
| Lucas, C A; Gillies, R J; Olson, J E et al. (1988) Intracellular acidification inhibits the proliferative response in BALB/c-3T3 cells. J Cell Physiol 136:161-7 |
| Gillies, R J; Cook, J; Fox, M H et al. (1987) Flow cytometric analysis of intracellular pH in 3T3 cells. Am J Physiol 253:C121-5 |
| Giuliano, K A; Gillies, R J (1987) Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine. Anal Biochem 167:362-71 |
| Gillies, R J; Didier, N; Denton, M (1986) Determination of cell number in monolayer cultures. Anal Biochem 159:109-13 |
