Phospholipid metabolism in the MCF-7 cell cycle, regulation of phosphatidylcholine accumulation

• Main Author: Lin, Weiyang
• Language: en_US
• Published: 2007
• Online Access: http://hdl.handle.net/1993/1809

Cells reproduce by duplicating their components, doubling their size and dividing in two. Since phospholipids are major components of cell membranes, cells must double their phospholipid mass so that the daughter cells formed have the same membrane composition as the parent. However, the mode of phospholipid accumulation and how this is regulated within the cell cycle remained largely unknown. The objective of this project is to investigate how membrane phospholipid accumulation is coordinated and regulated in the cell cycle, with special emphasis on the accumulation of phosphatidylcholine (PC). Two synchronization protocols were developed that synchronized approximately 90% of the cells at specific points in the cell cycle. The double hydroxyurea treatment synchronized MCF-7 cells at the G1/S boundary while the combination of hydroxyurea and nocodazole blocked the cells at mitosis. Synchronized cells at different stages of the cell cycle were obtained by harvesting cells at various times after removal of the cell cycle blocking agents. Measurement of phospholipid mass at different stages of the cell cycle revealed that the contents of PC, phosphatidylethanolamine (PE) and phosphatidylinositol (PI) declined during S phase followed by a net accumulation in G2/M phase. The relative composition of PC, PE and PI remained unchanged throughout the cell cycle, suggesting that the e might be coordinated metabolism of these lipids in the cell cycle. Further studies demonstrated that the rate of incorporation of [3H]choline into PC was low in early S phase, increased from late S and throughout G2/M. The fluxes in PC synthesis correlated with changes in the activities of CTP:phosphocholine cytidylyltransferase (PCCT), cholinephosphotransferase (CPT) and the levels of membrane-associated PCCT. The results suggest that PC synthesis contributes to the fluctuation in PC levels observed during the MCF-7 cell cycle. On the other hand, there was little change in the rate of incorporation of [14C]ethanolamine into PE during S and G2/M phases. The activity of CTP:phosphoethanolamine cytidylyltransferase (PECT), the rate limiting enzyme in PE synthesis, showed little variation in the cell cycle, suggesting that the rate of PE catabolism is the dominant factor in regulating PE levels during the cell cycle. No evidence was obtained to implicate changes in cellular CTP levels in the cell cycle regulation of phospholipid synthesis. Studies on PC catabolism revealed that the rate of PC catabolism was enhanced during S phase but terminated in the G2/M phase. Taken together, our studies demonstrated that the decrease in PC content during S phase resulted from enhanced PC catabolism, while PC accumulation in G2/M was attributed to both enhanced PC biosynthesis and cessation of PC catabolism.