Putrescine‐stimulated Intracellular Ca²⁺ Release for Invasiveness of Rat Ascites Hepatoma Cells

Abstract

Our previous study showed that treatment of highly invasive rat ascites hepatoma (LC-AH) cells with α α α α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, decreased both their intracellular level of putrescine and their in vitro invasion of a monolayer of calf pulmonary arterial endothelial (CPAE) cells, and that both these decreases were completely reversed by exogenous putrescine, but not spermidine or spermine. Here we show that all adhering control (DFMOuntreated) cells migrated beneath CPAE monolayer with morphological change from round to cauliflower-shaped cells (migratory cells ] i by inhibiting endoplasmic Ca 2+ -ATPase, indicating that thapsigargin mimics the effects of putrescine. These results support the idea that putrescine is a cofactor for Ca 2+ release through the Ca 2+ channel in the endoplasmic reticulum that is inhibited by ryanodine, this release being initiated by cell adhesion and being a prerequisite for tumor cell invasion. Key words: Putrescine -Tumor invasion -Difluoromethylornithine -Ryanodine -ThapsigarginThe polyamines spermidine and spermine, and the diamine putrescine, as well as normal constituents of all animal cells. On the basis of their metabolism under various physiological conditions as well as the effects of specific inhibitors of polyamine biosynthesis, polyamines have been suggested to be important in various cellular functions such as proliferation, differentiation, 1-4) cytoskeleton construction, 5) membrane transport 6, 7) and receptor responses.8, 9) However, their physiological roles are not yet well defined at the molecular level.We previously reported that pretreatment of a highly invasive tumor cell line, LC-AH cells, with DFMO, a specific irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), decreased both their intracellular level of putrescine and their invasion beneath a CPAE cell monolayer, without change in their viability, proliferative activity or levels of spermidine and spermine. 10) We also showed that these DFMO-induced decreases were completely reversed by putrescine added to the culture medium during pretreatment with DFMO or during invasion assay in which LC-AH cells were co-cultured with a CPAE cell layer for 24 h.Moreover, these reverses were observed only with putrescine, not with 1,3-diaminopropane, spermidine or spermine. 10)The LC-AH cells used in these studies have a very high cellular putrescine level (73 nmol/mg DNA) which is similar to their spermidine level (70 nmol/mg DNA). 10) This high putrescine level is a characteristic of these highly invasive LC-AH cells, the putrescine levels in a variety of other tumor cells such as Ehrlich ascites, 11) B16 melanoma, 12) Lewis lung carcinoma, 13) and leukemia 14) cells being less than 30% of their spermidine levels. Pretreatment with 0.5 mM DFMO for 5 h induced a 64% decrease in their putrescine level without significant changes in their intracellular levels of spermidine and spermine. 10) Although 0.5 mM DFMO completely inhibited the ornithine decarboxylase ...

Putrescine Activates Oxidative Stress Dependent Apoptotic Death in Ornithine Decarboxylase Overproducing Mouse Myeloma Cells

“…Previous studies have demonstrated that exogenous putrescine induced transformation of rat ascites hepatoma cells from nonmigratory to migratory cells and that this change in their behavior was preceded by an increase in the level of their intracellular Ca 2ϩ [10]. The effect of the added putrescine was suppressed by ryanodine, a blocker of cyclic ADP-ribose-triggered Ca 2ϩ release from the ER.…”

“…Polyamines were demonstrated to block and modulate several types of ion channels. In rat ascites hepatoma cells, exogenous putrescine induces an increase in their intracellular Ca 2ϩ level [10]. In contrast, in CD4 but not in CD8 T-cells, putrescine inhibits concavalin-A-induced Ca 2ϩ influx [11], demonstrating that the effect of polyamine can be cell dependent.…”

Protein Kinase C-Mediated in vitro Invasion of Human Glioma Cells through Extracellular-Signal-Regulated Kinase and Ornithine Decarboxylase

“…Notably, these effects were suppressed by antisense oligonucleotides of ODC [32]. Also, treatment with DFMO inhibited the in vitro invasion of LC-AH rat ascites hepatoma cells [34], and that of GaMg, U-251 and U-87 human glioma cell lines [35].…”