The effect of cell cycle regulation on cardiolipin metabolism

Hauff, K.1, Linda, D.3, Xu F.1, Hatch G.1-4. 1Departments of Pharmacology and Therapeutics, 2Biochemistry and Medical Genetics, and 3Internal Medicine, Faculty of Medicine, 4Center for Research and Treatment of Atherosclerosis, University of Manitoba, Winnipeg, Canada.

Cardiolipin (CL) is a major phospholipid integral for proper cardiac cell and mitochondrial function (Hatch, 2004). CL is an important mediator in ATP energy production and mitochondrial mediated apoptosis, and therefore plays an important role in growth, division and regulation of the eukaryotic cell (Hatch, 1998; Hatch, 2004). Production of CL is dependent on the presence of certain precursors, and has been shown to utilize glycerol, linoleic acid and glucose. Differential incorporation of these precursors may pose a possible form of regulation over CL metabolism (Xu et al., 2003). We hypothesize that CL biosynthesis is regulated as a normal function in cell cycle control.

In order to determine the differences in CL synthesis levels during cell regulation, HeLa cells were synchronized for 24 hours in media containing 0% fetal bovine serum (FBS). This was followed by a period of incubation for up to 24 hours, with or without 10% FBS to stimulate a return to cell division or maintenance of quiescence. Induction of DNA synthesis as a surrogate for cell division was confirmed by methyl-3HThymidine incorporation into DNA. Cells were radiolabeled with CL precursors, D-14C(U)glucose, 1-14Clinoleic acid or 1,3-3Hglycerol. The cells were then harvested and compared for total biosynthesis of CL from each precursor by thin layer chromatographic separation of lipid products, followed by measurement of radioactivity incorporation. Mitochondrial extracts were isolated and assayed, in vitro, for activity of the enzymes involved in CL synthesis, cytidinediphosphate-1,2-diacyl-sn-glycerol (CDP-PG) synthetase, and phosphatidylglycerolphosphate (PGP) synthase. Semi-quantitative RT-PCR of these enzymes was used to indirectly measure transcriptional regulation of CL synthesis.

Actively dividing HeLa cells produce more CL from glucose or linoleic acid than their quiescent counter parts (N=2), however, glycerol utilization is decreased. This increase of CL synthesis in proliferating cells was associated with a 30% increase in CDP-DG synthetase activity (49±6 vs. 63±4 pmol·min-1/mg protein) as well as a 22% increase in PGP synthase activity (373±22 vs. 457±13 pmol·min-1/mg protein) after 24 hours (N=3; p<0.05). RT-PCR of CDP-DG synthetase and PGP synthase indicates an increase in enzyme transcription 12 and 24 hours after release from synchronization.

A differential metabolic preference in CL synthesis during induction of cell division has never been described in any organism. A better comprehension of this process and its regulation will help us to better understand the disease processes CL is known to play a role in, and may lead to future targets for therapy. This metabolic preference, specifically in cancer (HeLa) cells, may also provide insight into development of potential chemotherapeutic agents.

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