Polyamine depletion switches the form of 2-deoxy-D-ribose-induced cell death from apoptosis to necrosis in HL-60 cells.

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy- -ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. -Rib-treated cells with normal polyamine levels, progressing through the G1 into the S and G2/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G1 phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus

Effect of Spermine on Membrane-Associated and Membrane-Inserted Forms of Protein Kinase C

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form). In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on N(max); spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes. Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca++, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected

Effect of spermine on association of protein kinase C with phospholipid vesicles.

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action

Relationship Between Spermine and Calcium on the Activation Process of Protein Kinase C.

Relationship Between Spermine and Calcium on the Activation Process of Protein Kinase C

The effect of spermine on calcium requirement for protein kinase C association with phospholipid vesicles.

We have previously reported that polyamines interfere with protein kinase C-membrane interactions. ..

Differential induction of spermidine/spermine N1-acetyltransferase activity in cisplatin-sensitive and -resistant ovarian cancer cells in response to N1,N12-bis (ethyl)spermine involves transcriptional and post-transcriptional regulation.

The growth inhibition that occurs in cisplatin-sensitive 2008 human ovarian cancer cells in response to the spermine analog, N1,N12-bis(ethyl)spermine (BESpm), is associated with a potent induction of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism. Conversely, in cisplatin-resistant C13* cells, which are less responsive to BESpm, enzyme induction does not occur at comparable levels after exposure to the bis(ethyl)-derivative. In this study we have investigated the molecular mechanisms underlying the differential induction of SSAT activity in cisplatin- sensitive and -resistant cells. Northern blot analysis revealed a difference in the level of SSAT mRNA expression in the two cell lines; in particular, 2008 cells treated with 10 µM BESpm for progressively increasing periods of time accumulated more heteronuclear (3.5 kb) and mature (1.3/1.5 kb) SSAT mRNAs than its resistant variant. SSAT mRNA accumulation paralleled enzyme activity and both were almost completely prevented in the two lines by co-treatment with 5 µg/ml Act-D, suggesting that transcription plays a major role in the analog-mediated induction of SSAT. Moreover, when Act-D was added 48 h after BESpm exposure, SSAT mRNA and enzyme activity were stabilized in both cell lines. Therefore, the marked difference in the induction of SSAT activity seems to be related to increased enzyme synthesis, particularly in sensitive cells, whose SSAT protein turnover was also greatly reduced (half-life >12 h in 2008 cells versus 5 h in C13* cells) in the presence of BESpm. These findings suggest that cisplatin-resistance modulates the SSAT response to BESpm at transcriptional and post-transcriptional levels

Elf electromagnetic fields affect gene expression of hegenerating rat liver following partial hepatectomy

Pulsed extremely-low-frequency magnetic fields (ELF-PEMFs) influence the expression of oncogenes c-myc and c-ras and of ornithine decarboxylase (ODC) in the regenerating rat liver following partial hepatectomy. In fact, while the mRNA's encoding both oncogenes are present in very low amounts in the normal liver, their concentration is dramatically increased during regeneration. Ornithine decarboxylase and c-myc mRNA's reach a maximum during the early phases of regeneration (3 hours after surgery) and decrease thereafter. c-ras mRNA reaches a maximum 40 hours after the operation. Treatment with ELF-PEMFs delivered to the animals immediately after the operation and every 12 hours thereafter increases the concentration of both oncogenes and of ornithine decarboxylase mRNA's at 3 hours (c-myc and ODC) and at 40 hours (c-ras) respectively.Pulsed extremely-low-frequency magnetic fields (ELF-PEMFs) influence the expression of oncogenes c-myc and c-ras and of ornithine decarboxylase (ODC) in the regenerating rat liver following partial hepatectomy. In fact, while the mRNA's encoding both oncogenes are present in very low amounts in the normal liver, their concentration is dramatically increased during regeneration. Ornithine decarboxylase and c-myc mRNA's reach a maximum during the early phases of regeneration (3 hours after surgery) and decrease thereafter. c-ras mRNA reaches a maximum 40 hours after the operation. Treatment with ELF-PEMFs delivered to the animals immediately after the operation and every 12 hours thereafter increases The concentration of both oncogenes and of ornithine decarboxylase mRNA' s at 3 hours (c-myc and ODC) and at 40 hours (c-ras) respective-ty. © 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

Spermine protects protein kinase C from phospholipid-induced inactivation

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca2+. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca2+. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca2+ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca2+ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca2+ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation

Cisplatin-resistance modulates the effect of protein synthesis inhibitors on spermidine/spermine N-1-acetyltransferase expression

Cisplatin (DDP)-resistance confers a deficient expression of spermidine/spermine N-1-acetyltransferase (SSAT) gene in response to the spermine analog N-1,N-12-bis(ethyl)spermine (BESpm) in the DDP-resistant human ovarian carcinoma cell line (C13*), compared with their parental DDP-sensitive 2008 cells. This SSAT gene deficiency is correlated with a reduced growth sensitivity to spermine analogs. This study was performed to determine whether SSAT gene expression of resistant cells was kept suppressed by labile repressor proteins developed during resistance selection. We show here that inhibitory concentrations of cycloheximide (CHX) and anisomycin (ANISO) differentially affect BESpm-induced SSAT activity in 2008 and in C13* cells in a concentration-dependent manner and allow resistant cells to reach activation levels comparable to those of the sensitive cells. Northern blot analysis revealed that both CHX and ANISO in combination with BESpm caused a synergistic BESpm-mediated accumulation of SSAT mRNA in C13* cells, with respect to each drug alone, while in 2008 cells only a slight increase was observed. The more pronounced effect of inhibitors on the SSAT activity induced by BESpm in the resistant cells was also the result of a more prolonged stabilization of SSAT mRNA and enzyme protein. By contrast, sub-inhibitory concentrations of CHX and ANISO did not significantly stimulate BESpm-induced SSAT transcription and activity. These results suggest that labile repressor proteins, related to DDP-resistance phenotype, play a regulatory role in SSAT gene expression, and further indicate that by overcoming this inhibitory control it is possible to recover BESpm response

2-Deoxy-D-ribose-induced apoptosis in HL-60 cells is associated with the cell cycle progression by spermidine

The presence of polyamines is required for the apoptotic program triggered by a-deoxy-D-ribose (dRib) in HL-60 cells, but their oxidative metabolites does not appear to be involved in the oxidative stress caused by the sugar. The present study points to a relationship between spermidine-induced G(1) to S phase transition and the onset of dRib-induced apoptosis. Conversely, the G(1) block induced by alpha-difluoromethylornithine (DFMO) is associated with a protective effect against dRib-induced cell suicide. Replenishment of the intracellular spermidine pool by exogenous putrescine and spermidine induces cell cycle progression and restores apoptotic levels. The present data indicate that the induction of cell cycle progression by spermidine is a condition facilitating the activation of the apoptotic process by dRib