Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown

Abstract

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.

Cell Cycle Dependence of Group VIA Calcium-independent Phospholipase A2 Activity

“…These results demonstrate that PC mass accumulates before cell division and are consistent with previous reports (16,26). As several reports have attributed glycerophospholipid accumulation to decreased turnover (16,18), we measured the half-life of PC to determine if this was true in the CHO cell system as well. To achieve this, CHO cells were labeled to equilibrium with [ 3 H]choline, arrested at various points in the cell cycle, and then subjected to a chase with excess unlabeled choline.…”

“…These observations indicate that PC accumulation during S phase is not solely dependent on increased synthesis. Instead, several reports suggest that a decrease in phospholipid turnover contributes to the net accumulation of PC during late G 1 and S phase (13,16,18,19). These observations suggest that one or more of the phospholipase activities must be regulated during the cell cycle.…”

“…Our data suggest that activity is down regulated through alternative splicing of the iPLA 2 message, generating truncated proteins that accumulate during G 1 . Together, these data suggest that glycerophospholipid metabolism is regulated in the context of the cell cycle and iPLA 2 It has long been established that glycerophospholipid metabolism is regulated during the cell cycle (13,16,18,19). These studies have focused on synchronized populations of cells, typically generated through starvation followed by re-addition of growth factors or serum.…”

Phosphoinositides and metabolic control: how many messengers?

“…In the first of these, quiescent serum-starved Chinesehamster ovary (CHO-K 1 ) cells were stimulated to progress synchronously through the cell cycle by addition of dialysed serum (Tones et al, 1988). Pulse-labelling with j2P showed increased synthesis of all phospholipids in the G1 phase, but there was no specific change in the phosphoinositides, nor could the release of inositol phosphates be detected using [7HJinositol.…”

4‐(Methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone promoted gastric cancer growth through prostaglandin E receptor (EP2 and EP4) in vivo and in vitro

“…A modified [ 3 H]‐thymidine incorporation assay was used to determine the amount of DNA synthesis. ( 15 ) Cells were incubated with NNK for 24 h, and incubated with 0.5 μCi/mL [ 3 H]‐thymidine (Amersham Corporation) for another 4 h followed by 10% trichloroacetic acid. After several washings, 1% sodium dodecyl sulfate (SDS) was incubated for 15 min at 37°C.…”