Monia Fattori

Caspase activation in etoposide-treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion.

Activation of the extracellular signal‐regulated kinases (ERKs) 1 and 2 is correlated to cell survival, but in some cases ERKs can act in signal transduction pathways leading to apoptosis. Treatment of mouse fibroblasts with 20 μM etoposide elicited a sustained phosphorylation of ERK 1/2, that increased until 24 h from the treatment in parallel with caspase activity. The inhibitor of ERK activation PD98059 abolished caspase activation, but caspase inhibition did not reduce ERK 1/2 phosphorylation, suggesting that ERK activation is placed upstream of caspases. Both ERK and caspase activation were blocked in cells depleted of polyamines by the ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO). In etoposide‐treated cells, DFMO also abolished phosphorylation of c‐Jun NH2‐terminal kinases triggered by the drug. Polyamine replenishment with exogenous putrescine restored the ability of the cells to undergo caspase activation and ERK 1/2 phosphorylation in response to etoposide. Ornithine decarboxylase activity decreased after etoposide, indicating that DFMO exerts its effect by depleting cellular polyamines before induction of apoptosis. These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide.

Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells.

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.

NF-κB and ERK cooperate to stimulate DNA synthesis by inducing ornithine decarboxylase and nitric oxide synthase in cardiomyocytes treated with TNF and LPS

We previously reported that tumor necrosis factor‐α (TNF) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CM) via nitric oxide and polyamine biosynthesis. Here we show an involvement of nuclear factor‐κB (NF‐κB) in the induction of nitric oxide synthase (NOS) and ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis. In addition NF‐κB activation appears to favor survival of CM by reducing caspase activation. TNF and LPS also stimulate phosphorylation of extracellular signal‐regulated kinase (ERK), which is required for the changes in ODC and caspase activity, but not for NOS induction or NF‐κB activation. In conclusion, these results indicate that NF‐κB, in cooperation with ERK, plays a pivotal role in the growth stimulating effects of TNF and LPS, leading to the induction of both ODC and NOS and to the reduction of caspase activity.

Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes

OBJECTIVE We have recently shown that tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. METHODS CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFalpha and LPS, and/or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [3H]-thymidine. RESULTS Treatment of CMs with TNFalpha and LPS increased cell number and [3H]-thymidine incorporation. Addition of TNFalpha and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFalpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent pathways are required for the mitogenic action of TNFalpha and LPS. Moreover, addition of exogenous polyamines to untreated cells raised the cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator, also promoted DNA synthesis. Furthermore, putrescine and NO donor can additively activate sGC in cell-free extracts. CONCLUSION TNFalpha and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP.

Polyamine depletion inhibits etoposide-induced NF-κB activation in transformed mouse fibroblasts

Summary.In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-κB activation. The DNA binding activity of p65 NF-κB subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with α-difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-κB inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-κB activation. Moreover, pharmacological NF-κB inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-κB is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-κB and caspases.

Signaling Pathways Involved in the Stimulation of DNA Synthesis by Tumor Necrosis Factor and Lipopolysaccharide in Chick Embryo Cardiomyocytes

Treatment of confluent chick embryo cardiomyocytes (CM) with tumor necrosis factor-α (TNF) and lipopolysaccharide (LPS) increased cell number and [3H]-thymidine incorporation. Addition of TNF and LPS provoked an early increase in active, phosphory-lated extracellular-signal regulated kinase (ERK), an induction of ornithine decarboxylase (ODC), and a more delayed enhancement of nitric oxide synthase (NOS) activity, which appeared to be independent on the activation of the ODC/polyamine system. A number of inhibitors of signal transduction pathways involved in mitogenesis inhibited [3H]-thymidine incorporation as well as ODC and/or NOS induction. In particular, PD98059, a specific inhibitor of the ERK pathway, prevented the induction of ODC, but not that of NOS, whereas chelerythine, a protein kinase C inhibitor, reduced NOS, but not ODC activity. TNF and LPS treatment also enhanced the cGMP level in CM and both polyamine and nitric oxide (NO) biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of soluble guanylate cyclase (sGC) and cGMP dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent path-ways are required for the mitogenic action of TNF and LPS. In conclusion, the present results suggest that the mitogenic effect of TNF and LPS on CM involves both the induction of ODC, supported by ERK activation, and NOS activity. In turn, polyamine and NO biosyntheses may cooperate to enhance cGMP level, resulting in stimulation of DNA synthesis.