For in vivo PLD assays, cells were metabolically labelled with [C]lysophosphatidylcholine (0.5 μCi/1 × 10 cells; GE Healthcare, Buckinghamshire, UK) for 18 h in the absence of serum.
Quantification of drug-induced PLD: H4 cells were seeded in 96-well white-well dishes (Nunc, Langenselbold, Germany) at a density of 4×10 cells per well.
Cells derived from 3 tumours considered non-radiocurable (1 osteosarcoma, 2 melanoma) repaired significantly more X-ray PLD than cells from 3 tumours considered radiocurable (2 breast, 1 neuroblastoma).
Among all these effects, one stands out, that is the role of PLD in cell migration.
In the past, various experimental methods have been proposed to measure PLD using cell-free,[ 28] in vitro,[ 29] and in vivo[ 30] approaches.
These PLD isoform-downregulated cells showed lower macropinocytotic activity compared with cells treated with control siRNA, whereas PLD1-downregulated cells exhibited stronger inhibition than PLD2-downregulated cells.
A strategy is proposed to design a safe and simple amphiphilic prodrug delivery system, based on the elevated expression of phospholipase D (PLD) in cancer cells.
PLD regulation in cells occurs via two different signaling pathways.
The addition of a cloned and purified recombinant PLD to HeLa cells resulted in substantial remodeling of the host membrane architecture, as measured by lipid raft formation.
Moreover, gelsolin overexpression has recently been reported to suppress bradykinin-induced PLC and phospholipase D (PLD) in NIH3T3 cells (Banno et al, 1999).
In mammalian cells, PLD is a potent regulator of the Ras signaling pathway, which is strongly implicated in cancer and well known to regulate cell proliferation, differentiation and apoptosis [ 10, 11].
