Regulation of signal transduction

1. 1. A systematic study is reported on the control of 1-phosphatidylinositol 4-kinase (PI kinase) and PI 4-phosphate 5-kinase (PIP kinase), enzymes of the phosphatidylinositol phosphorylation pathway which leads to the production of second messengers, IP3 and DAG. In liver of normal male, adult, fed Wistar rats the steady state activity of PI kinase was 0.5 ± 0.01 and that of PIP kinase was 0.046 ± 0.003 nmol/hr/mg protein. The concentration of IP3 was 1.8 ± 0.1 pmol/mg protein. 2. 2. That the two kinases have short half-lives was observed in starvation, where in the rat liver or bone marrow activities rapidly decreased and on refeeding were restored in a day. Injection to rats of the protein synthetic inhibitor, cycloheximide, yielded t12 = 8 min for the two enzymes in bone marrow and t12 = 80 min in liver. 3. 3. Linkage of the signal transduction enzymes with proliferation was shown by the high activities as compared to liver of these enzymes in rat organs of high cell renewal capacity, e.g., thymus, bone marrow, spleen and testes. 4. 4. Linkage with malignant proliferation was indicated by the observation that in rat hepatomas the enzyme activities increased 5- to 9-fold and were highest in rapidly growing hepatoma 3924A (29- and 45-fold). 5. 5. In human primary ovarian carcinoma PI and PIP kinase activities were elevated 4.4 and 2.9-fold, respectively, and in OVCAR-5 cells, 32- and 11-fold, respectively. Similar increases were observed in MDA-MB-435 human breast carcinoma cells in comparison with normal breast parenchymal cells. 6. 6. The linkage of signal transduction enzyme activities with malignant proliferation was also observed in experiments when human breast carcinoma cells were plated in flasks and expressed their proliferative capacity in the log phase. PI and PIP kinase activities steadily and coordinately increased to a peak 11-fold rise in mid-log phase. In late log and plateau phases the kinase activities gradually declined to the starting level. Similar observations were made for the two enzymes in human ovarian carcinoma OVCAR-5 cells and in rat hepatoma 3924A cells in tissue culture. 7. 7. In animals injected with cycloheximide the bone marrow PI and PIP kinase activities exhibited t12 = 0.12 hr, the shortest decay rate in comparison with 8 enzymes of purine and pyrimidine biosynthesis with t12 = = 0.6 to 4.3 hr. 8. 8. Injection of tiazofurin decreased PI and PIP kinase activities in the bone marrow with t12 = 82 and 78min, respectively. IP3 concentration decreased with a t12 = 23 min. 9. 9. Injection of quercetin decreased PI kinase activity in the bone marrow with t12 = 17 min; however, PIP kinase activity only decreased to 70% and activity leveled off at this value for 24 hr. The IP3 concentration followed the decline in PI kinase activity with t12 = 40 min. 10. 10. Addition of quercetin to OVCAR-5 cells in culture showed that different concentrations of this drug have little effect on PIP kinase activity; however, PI kinase activity decreased in a dose-dependent fashion with IC50 = 44 μm. The IP3 concentration sharply declined with an IC50 of 46 μm and correlated best with the decrease in PI kinase activity. 11. 11. Quercetin inhibited the proliferation of OVCAR cells with an IC50 of 63 μm and of human breast carcinoma cells with IC50 = 55 μm. The decline of PI kinase activity preceded the inhibition of cell proliferation whereas PIP kinase activity was inhibited only to a minor extent. 12. 12. In vivo studies showed that in the bone marrow IP3 concentration could be depleted by only 40–60% by injection of cycloheximide, tiazofurin or quercetin. Therefore, we postulated that there might be a salvage pathway that could replenish or maintain IP3 concentration in a homeostatic adaptation. This hypothesis is strengthened by reports in the literature indicating that IP3 may be generated by degradation of IP6 and observations that addition of IP6 to cancer cells yields IP3 provide support for this hypothesis for a salvage pathway for IP3 production.