A9
Choline metabolism in cellular senescence, quiescence and transformation: a 1H NMR based metabolomics study
Madhu Basetti, Masako Narita, Masashi Narita, John Griffiths
Cancer Research UK Cambridge Research Institute, Cambridge, UK
Background
Senescence, which is a permanent cell cycle arrest, is thought to act as a fail-safe mechanism to prevent the malignant transformation of pre-neoplastic cell populations. Oncogene-induced senescence (OIS) is a tumour suppressor mechanism1. There is little information available about the metabolism of senescent, quiescent and transformed cells. Hence we have undertaken a 1H NMR based metabolomics study of Ras, Mek and DNA damageinduced senescence, along with replicative senescence, quiescence and malignant transformation (by E1a/Ras) in human diploid fibrobalsts (HDFs).
Method
IMR90 HDFs were cultured in DMEM, 10% FBS. OIS was induced using tamoxifen regulatable ER-ras (H-RasV12) or ERMek (MEKQ56P), while addition of viral E1A caused transformation. For DNA damage-induced senescence, cells were treated with 100M etoposide for 2 days. Cells of around passage number 37 became replicative senescent. Quiescence was induced by 3 days serum starvation. Each cohort of HDFs was grown to a population of 106 cells before harvesting with perchloric acid. After neutralisation and lyophilisation samples were re-suspended in D2O for 1H NMR analysis. TSP was used for quantitation and cell number for normalisation of the metabolite data.
Results
In general, the NMR spectral changes induced by the OIS, replicative senescence, quiescence and transformation showed little resemblance. Of the choline metabolites, phosphocholine/glycerophosphocholine (PC/GPC) showed a significant reduction in OIS and replicatively senescent cells whereas it is increased significantly in quiescent and transformed cells and showed no significant change in etoposide- treated cells.
Conclusion
We found no evidence of a common metabolic perturbation in senescence, quiescence or transformation. In particular, the changes induced in the choline metabolites by the different treatments were markedly varied. 1H NMR based metabolomics is a valuable tool to analyse the phenotypic effects of gene perturbations or other insults in cells, such as OIS, DNA damage or malignant transformations.
