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Lipid signalling pathways in normal and ras-transfected NIH/3T3 cells
1Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Abstract:
The role of ras oncogenes in cellular signalling pathways involving phospholipid breakdown was studied in untransfected and proto-H-ras and mutated H-, K- and N-ras transfected NIH/3T3 cells. When the cells were grown at low cell densities, all of the ras transfected cells had 2-4 fold higher diacylglycerol (DAG) levels compared to growing NIH/3T3 cells. At high cell densities, DAG levels decreased in the former and increased in contact inhibited NIH/3T3 cells. In this regard, only cells transformed by mutated cellular and viral H-ras oncogenes (but not by the H-ras proto-oncogene) had elevated DAG levels compared to contact inhibited NIH/3T3 cells. The basal levels of inositol phosphates in ras transfected cells were not significantly different from NIH/3T3 cells and did not vary with cell density. Thus, the elevated DAG levels are not a consequence of increased phosphoinositide hydrolysis. The latter was stimulated by serum and bombesin only in normal and proto-H-ras transfected cells. In contrast, stimulation by bradykinin was observed only in cells transformed by mutated cellular ras oncogenes. Furthermore, aluminum fluoride stimulated phosphoinositide breakdown in the latter cells indicating that there was no uncoupling of the G protein from phospholipase C. Treatment of ras transfected cells with dibutyryl cyclic AMP (DB-cAMP), which causes an inhibition of growth and a reversal of the transformed morphology, did not alter the basal levels of inositol phosphates, DB-cAMP, however, did lower DAG levels in some of the transformed cell lines, but elevated DAG levels in low density NIH/3T3 cells. These findings indicate that the ras gene product p21 is not involved in phosphoinositide hydrolysis and that DAG levels do not correlate with cell growth in either normal or ras transfected NIH/3T3 cells. Thus, p21 appears to alter cell growth through mechanism(s) independent of lipid signalling pathways.
Insights
Ras oncogenes do not directly impact phospholipid breakdown in NIH/3T3 cells. Elevated diacylglycerol (DAG) levels in ras-transfected cells are independent of phosphoinositide hydrolysis, suggesting ras alters cell growth via non-lipid signaling pathways.
Area of Science:
- Cellular Biology
- Oncology
- Molecular Signaling
Background:
- Ras oncogenes play a critical role in cellular signaling pathways.
- Phospholipid breakdown is a key event in signal transduction.
- Understanding ras oncogene involvement in these pathways is crucial for cancer research.
Purpose of the Study:
- To investigate the role of ras oncogenes in phospholipid breakdown.
- To determine the relationship between ras oncogenes, diacylglycerol (DAG) levels, and inositol phosphates.
- To elucidate the signaling mechanisms by which ras oncogenes affect cell growth.
Main Methods:
- Culturing untransfected and ras-transfected NIH/3T3 cells at varying densities.
- Measuring diacylglycerol (DAG) and inositol phosphate levels.
- Stimulating cells with serum, bombesin, bradykinin, and aluminum fluoride.
- Treating cells with dibutyryl cyclic AMP (DB-cAMP).
Main Results:
- Ras-transfected cells exhibited elevated DAG levels at low densities, which decreased at high densities.
- Mutated ras oncogenes, but not proto-H-ras, led to sustained high DAG levels in transformed cells.
- Basal inositol phosphate levels were unaffected by ras transfection or cell density.
- Phosphoinositide hydrolysis was differentially stimulated by various agents depending on ras mutation status.
- DB-cAMP treatment affected DAG levels but not inositol phosphates, and did not alter basal levels.
Conclusions:
- The ras gene product p21 is not directly involved in phosphoinositide hydrolysis.
- Elevated DAG levels in ras-transfected cells are not a consequence of increased phosphoinositide hydrolysis.
- DAG levels do not correlate with cell growth in normal or ras-transfected NIH/3T3 cells.
- Ras appears to alter cell growth through mechanisms independent of lipid signaling pathways, specifically phosphoinositide hydrolysis.