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

Proffered paper presentation

Introduction
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).

Methods
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.

Conclusions
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.