Cell cycle lipidomics

CELL CYCLE LIPIDOMICS LIM JING YAN BSc (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Lim Jing Yan 11 Jan 2013 Acknowledgements I would like to express my heartfelt gratitude to my supervisor, A/Prof. Markus R Wenk, and co-supervisor, Prof Ong Choon Nam, for their encouragement, guidance and support throughout the whole of my PhD. I appreciated the freedom they gave me to explore in my study. I would also like to offer my sincere gratitude to A/Prof Philipp Kaldis, who has given me timely advice and invaluable suggestions throughout my PhD. I also thank Dr Aaron Z Fernandis for showing me the ropes when I first started out in the lab. I am extremely grateful to Dr Shui Guanghou, whose advice, encouragement, guidance and scientific excellence have made this thesis possible. I thank my dear lab mates, especially Husna, Sudar, Jacklyn, Charmaine and Lissya, who have kept me sane and made this journey an interesting one. I am also grateful to Dr Federico Torta and Madhu who had given me useful suggestions and inputs in my thesis. I am thankful to Huimin and Dorothy for helping me out with administrative work. Thank you, NUS Graduate School for Integrative Sciences and Engineering (NGS), for the generous funding through a research scholarship and wonderful student support. Finally, I would like to express my special appreciation to my mother, father and brother for their unconditional care and love. And special thanks to my husband, Jiunn Siong, who has been my constant source of love, strength and faith. i Table of Contents Acknowledgements i Summary . v List of Tables vii List of Figures viii List of Abbreviations xii 1. INTRODUCTION . 1.1 Lipidomics . 1.1.1 Lipids 1.1.2 Mass spectrometry 1.2 Cell cycle and lipids 1.3 Rationale and objectives of this study . 20 2. MATERIALS AND METHODS 21 2.1 Materials 21 2.2 Cell culture 21 2.3 Cell synchronisation 22 2.4 Flow cytometric analysis . 23 2.5 Immunoblot analysis . 23 2.6 Lipid analysis 24 2.6.1 Lipid extraction . 24 2.6.2 HPLC-MS profiling of diverse lipids . 25 2.7 Data analysis . 26 3. CELL CYCLE SYNCHRONISATION AND LIPID PROFILE . 28 3.1 Cell cycle synchronisation 28 3.2 Platform for cellular lipidomics setup and analysis 32 3.2.1 Cell number versus total lipid . 32 3.2.2 FBS effect on general lipid profile . 35 3.3 Materials and Methods 37 3.4 Results . 38 3.4.1 Phospholipid profile of the cell cycle . 38 3.4.2 Neutral lipid profile 61 ii 3.5 Discussion . 70 4. CHOLESTEROL ESTERS’ ROLE IN CELL CYCLE 75 4.1 Introduction . 75 4.2 Materials and Methods 79 4.2.1 Cell culture . 79 4.2.2 RNAi transfection of HeLa cells 79 4.2.3 Fluorescence Imaging of lipid droplets 79 4.2.4 AlamarBlue assay for cell viability 80 4.2.5 Analysis of kinetics of cell cycle progression 80 4.2.6 Analysis of ACAT1 and cyclin levels using western blot 81 4.2.6 MS analysis of phospholipids, sphingolipids and cholesterol in ACAT1 and negative control cells 82 4.2.7 Time-lapse Imaging of ACAT1 KD and negative control cells . 82 4.3 Results . 83 4.3.1 Fluorescent imaging of lipid droplets at different cell cycle phases and MS analysis of individual cholesterol ester species 83 4.3.2 Determination of ACAT1 protein expression, cell viability and lipid profile of ACAT1 KD cells . 86 4.3.3 Cell cycle progression of ACAT1 KD cells upon release from aphidicolin synchronisation . 89 4.3.4 Cell cycle progression of ACAT1 KD cells upon release from hydroxyurea synchronisation . 93 4.3.5 Cell cycle progression of ACAT1 KD cells upon release from nocodazole synchronisation . 95 4.3.6 Time lapse observation of cell division in ACAT1 KD and negative control cells 97 4.4 Discussion . 102 5. BREAST CANCER LIPIDOMICS 108 5.1 Introduction . 108 5.2 Materials and Methods 113 5.2.1 Breast cancer samples . 113 5.2.2 Lipid analysis 113 5.2.3 Statistical analysis . 113 iii 5.3 Results . 114 5.3.1 Phospholipid and sphingolipid profiles of breast tumor vs control 114 5.3.2 Sterol profile of breast tumor versus control 119 5.4 Discussion . 120 6. CONCLUSION . 125 7. FUTURE WORK 127 8. BIBLIOGRPAHY . 130 iv Summary Regulation of cell cycle is crucial for cell survival and function. Dysregulated cell division can result in multiple disorders, including cancer, neurological, renal and vascular proliferative diseases. Changes in cellular lipidome during the cell cycle has become an increasingly important research area. Here, we investigated the lipid composition of cells in different stages of the cell cycle using mass spectrometry-based lipidomics approaches. We used two complementary synchronisation methods (G1/S by aphidicolin and G2/M by nocodazole) with two different cell lines (HeLa- human cervical cancer cell line and MCF7- human breast cancer cell line). Among the lipid classes analysed, which included phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), sphingomyelin (SM), ceramide (Cer), glucosylceramide (GluCer), triacylglycerol (TAG), diacylglycerol (DAG) and sterols, cholesterol esters showed a significant increase in G2/M phase. This highlights that cholesterol esters may be a fundamental lipid class for cell division, as they could act as a store for cholesterol and fatty acids, which are essential components of phospholipids and are critical for membrane biogenesis in cell division. Acyl-coA:cholesterol acyltransferase (ACAT1) is the main intracellular enzyme involved in cholesterol esterification. We confirmed the importance of cholesterol esters in cell cycle using ACAT1 knocked-down (KD) cells which have lower levels of cholesterol esters. Cell cycle kinetics of ACAT1 KD and negative control cells were compared. A prolonged G2/M phase in ACAT1 KD cells was v observed, indicating that cholesterol esters metabolism is crucial especially for G2/M progression. One of the consequences of cell cycle dysregulation is the development of cancer. The connection between cell cycle and cancer is critical. The cell cycle machinery controls cell proliferation and cancer is a condition of uncontrolled cell division. Lipids have been reported to play a role in cancer. Hence, using breast cancer as a model, we conducted a pilot study to profile and compare lipids in human breast tumor and control tissues. Among other findings, we observed an increase in cholesterol esters in the tumor samples when compared to control. This further supports our previous results, where cholesterol esters were found to be important for active cell division. vi List of Tables Table 3.1 A summary of four common methods used in cell synchronisation, and the advantages and problems faced while trying each method during this experimental work. . 31 Table 3.2 In depth analysis of the fold change of lipids in both cell lines for both synchronisation methods . 58 vii List of Figures Figure 1.1 Categories of lipids with examples Figure 1.2 Schematic diagram of MRM in a triple quadrupole Figure 1.3 The stages of the cell cycle Figure 1.4 Summary of genes of lipid related proteins that are found to be regulated in the cell cycle in two published cell cycle genomics study 10 Figure 1.5 Main functions of lipids in the cell cycle. . 19 Figure 3.1 Graph representing the relationship between cell number and log value of total normalised lipid signal intensities 34 Figure 3.2 Heatmap of lipid fold changes in HeLa cells cultured in media with different FBS percentage for 24, 48 and 72 hours, as compared to the starting point (FBS 10% 0h). . 36 Figure 3.3 Schematic diagram of the work flow of cell cycle synchronisation and sample collection 37 Figure 3.4 Cell cycle analysis of MCF7 cells synchronised at G1/S by aphidicolin and then released into G2/M . 39 Figure 3.5 Heatmap representation of individual polar lipid species changes in MCF7 cells released from aphidicolin synchronisation 41 Figure 3.6 Bar graph of fold change in lipids that were significantly (p[...]... 1.2 Cell cycle and lipids The eukaryotic cell division cycle is one of the most rigorously studied biological processes It is divided into the sequential G1 (Gap 1), S (Synthesis) and G2 (Gap 2), collectively known as interphase, and M (mitotic) phases In appropriate cues, cells may exit from the cell cycle and enter quiescent state (G0) or proceed into the next cycle The two main events in the cell cycle. .. cell cycle have been unfulfilling, with only a subset of genes of lipid related proteins being found to be regulated cyclically along with human cell cycle (Figure 1.4) 9 Figure 1.4 Summary of genes of lipid related proteins that are found to be regulated in the cell cycle in two published cell cycle genomics study Since lipids are the major constituent of cellular membranes, one would expect cells... 2009; Malumbres, 2011) Different phases of the cell cycle are controlled mainly by a unique pair of Cdk-cyclin While Cdk protein levels remain consistent throughout the cell cycle, protein levels of their activating partners cyclins fluctuate during the cell cycle This 7 results in the periodic activation of Cdk, which leads to the progression of cell cycle (Figure 1.3) (Vermeulen et al., 2003) Cdk... negative control cells Error bars represent + SD 88 Figure 4.7 Cell cycle kinetics of ACAT1 KD and negative control cells which were released from G1/S synchronisation by aphidicolin 90 Figure 4.8 Western blots for ACAT1 KD and negative control cells released from aphidicolin synchronisation 92 Figure 4.9 Cell cycle kinetics of ACAT1 KD and negative control cells which were... polyploidy cells (Fernándeza et al., 2004) Besides their relative concentrations in the cell, cellular localization of phospholipids may also play essential roles in the regulation of cell cycle For instance, levels of total cellular inositol lipids including phosphatidylinositol (PI), phosphoinositide phosphate (PIP), and PI(4,5)P2 relative to the total cellular phospholipids did not vary throughout the cell. .. events in the mammalian cell cycle It is needed for many events involved in cell division, like cyclin/Cdk regulation, translation initiation etc It cooperates with other signaling pathways to control many cellular events (Garcia et al., 2006) Inhibition of PI3K has been observed to delay cell cycle progression of cells into G2/M phase (Shtivelman et al., 2002) Inactivated in quiescent cells, the first activity... of lipid changes in cell division Hence, the main objectives of this project are: (i) to obtain a comprehensive lipid profile of mammalian cells in different stages of the cell cycle, using an advanced, sensitive and accurate approach based on mass spectrometry; (ii) to identify lipid species which change in their abundance during the cell cycle and to analyse their role in cell cycle; (iii) to relate... the process from ACAT1 knockdown to cell cycle synchronisation and cell collection 81 Figure 4.2 Fluorescent microscope images of cells in different stages of the cell cycle 84 Figure 4.3 Fold change of each cholesterol ester species in G2/M as compared to G1/S 85 Figure 4.4 Effects of ACAT1 KD by RNAi transfection in HeLa cells 86 Figure 4.5 Fold change in... expect cells to double their phospholipid mass during cell cycle in order for membranes to be distributed evenly between the two daughter cells Several groups have attempted to understand how membrane phospholipid metabolism is regulated within the cell cycle Findings are so far contradictory, and seem to be cell type specific PCs are the main component of cellular membrane phospholipids (Jackowski, 1996)... two main events in the cell cycle are DNA synthesis in S phase, and the division of the parent cell into two daughter cells in the M phase G1 and G2 are periods in the cell cycle where the cell prepares to replicate its genomic material and then to divide respectively The transition from one phase of the cell cycle to another occurs in a unidirectional fashion and is traditionally known to be regulated . Data analysis 26 3. CELL CYCLE SYNCHRONISATION AND LIPID PROFILE 28 3.1 Cell cycle synchronisation 28 3.2 Platform for cellular lipidomics setup and analysis 32 3.2.1 Cell number versus total. protein expression, cell viability and lipid profile of ACAT1 KD cells 86 4.3.3 Cell cycle progression of ACAT1 KD cells upon release from aphidicolin synchronisation 89 4.3.4 Cell cycle progression. of the consequences of cell cycle dysregulation is the development of cancer. The connection between cell cycle and cancer is critical. The cell cycle machinery controls cell proliferation and