Angelo Spinedi

Tissue transglutaminase-dependent posttranslational modification of the retinoblastoma gene product in promonocytic cells undergoing apoptosis.

The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program.

Inhibition of “Tissue” Transglutaminase Increases Cell Survival by Preventing Apoptosis

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of “tissue” transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca2+-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.

Membrane Lipid Alterations and Na+-Pumping Activity in Erythrocytes From IDDM and NIDDM Subjects

The Na+-pumping activity of the erythrocyte plasma membrane in diabetic subjects was studied together with the lipid composition. Insulin-dependent diabetes mellitus (IDDM) patients (n = 25) were divided into young (28.1 ± 7.4 yr old, mean ± SD; n = 16) and old (71.7 ± 9.8 yr old; n = 10) subjects; the age of non-insulin-dependent (NIDDM) patients was 70.7 ± 11.5 yr (n = 10). The Na+-pumping activity, estimated from both Na+-K+-ATPase and ouabain binding, was significantly decreased in IDDM and NIDDM subjects, but its insulin sensitivity was retained only in young IDDM subjects. The total cholesterol and phospholipid content of the erythrocyte plasma membrane was lowered in IDDM subjects, and cholesterol-to-phospholipid molar ratio was significantly decreased. In NIDDM subjects the significant decrease of the two lipid components did not alter their ratio. The analysis of major phospholipid components of erythrocyte membranes revealed that only phosphatidylcholine is significantly increased in young diabetic subjects. The fatty acid composition of major phospholipid classes was significantly altered in all cases: the unsaturation index appeared to be increased in phosphatidylserine and sphingomyelin for both IDDM and NIDDM subjects and was also increased in phosphatidylcholine in the latter group.

Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis.

We report that apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells associates with accumulation of monohexosylsphingolipids produced not only by short-chain ceramide glycosylation but also through glycosylation of a ceramide pool endogenously produced. By high-performance thin layer chromatography on borate silica gel plates, newly formed monohexosylsphingolipids were identified as glucosylceramides (GluCer); however, accumulation of lactosylceramide or higher-order glycosphingolipids was not observed. GluCer accumulation was fully suppressed by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; moreover, while this inhibitor had no effect on cell viability when administered alone, it markedly potentiated the apoptotic effect of C6-Cer. These results provide evidence that activation of GluCer synthesis is an important mechanism through which CHP-100 cells attempt to escape ceramide-induced apoptosis.

Tamoxifen and somatostatin affect tumours by inducing apoptosis.

Tamoxifen is a commonly used chemotherapeutic agent in human breast cancer, although some tumours develop resistance. Somatostatin is also being introduced as an anti-tumour agent. Here we show that the action of these drugs is, at least partly, due to their induction of apoptosis. Both 50 nM somatostatin, and 60 nM tamoxifen significantly enhanced the percentage of cells undergoing apoptosis, when compared to untreated or oestrogen treated control cells. This effect was observed in SK-N-BE(2) human neuroblastoma cells and in MCF-7G human breast cancer cells but not in their drug-resistant counterpart MCF-7A which showed a very low rate of spontaneous programmed cell death. Finally, we propose a simple test of the sensitivity and resistance of individual tumours to these agents by assessing their ability to induce apoptosis in vitro as measured by flow cytometry.

Apoptosis induced by doxorubicin in neurotumor cells is divorced from drug effects on ceramide accumulation and may involve cell cycle-dependent caspase activation

Doxorubicin (0.5 μg/ml) induced caspase‐dependent apoptosis in SH‐SY5Y neuroblastoma and CHP‐100 neuroepithelioma cells. The apoptotic response started to be evident ~15 h after drug administration and, as monitored over a 48‐h period, was more pronounced in CHP‐100 than in SH‐SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B1 (25 μM) ; this compound, however, did not prevent drug‐induced apoptosis. Untreated cells from both lines expressed negligible p53 levels ; on the other hand, whereas p53 and p21Cip1/Waf1 were rapidly up‐regulated in doxorubicin‐treated SH‐SY5Y cells, such a response was not observed in CHP‐100 cells. Doxorubicin induced a G2/M phase block in both cell lines, but whereas the G1 phase was markedly depleted in CHP‐100 cells, it was substantially retained in SH‐SY5Y cells. In the latter system, double G1 and G2/M block largely preceded cell death ; however, as apoptosis underwent completion, it selectively targeted late S and G2/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G1 cell population. These results support the notion that doxorubicin‐induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G1 cells appear to be refractory to doxorubicin‐induced apoptosis, p53 up‐regulation and p21Cip1/Waf1 expression may provide an unfavorable setting for the apoptotic action of the drug.

Arachidonic acid incorporation and redistribution in human neuroblastoma (SK-N-BE) cell phospholipids

Abstract: The incorporation and redistribution of [1‐14C]arachidonic acid in SK‐N‐BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum‐enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse‐labelled with [1‐14C]arachidonic acid and then reincubated in label‐free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid‐stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid‐stable PtdEtn and alkenylacyl‐PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid‐stable PtdCho to both alkenylacyl‐PtdEtn and acid‐stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.

Calpain involvement in calphostin C-induced apoptosis

A major problem in assessing the role of calpains in apoptosis induction concerns the fact that calpain inhibitors can also impair the activity of the proteasome, also reported to be involved in apoptosis. Herein we showed that apoptosis induced by calphostin C in U937 human promonocytic leukemia cells was associated, at its onset, with enhanced protein (poly)ubiquitination. This observation prompted us to study whether protein degradation through the ubiquitin/proteasome pathway was involved in apoptosis induction. We found that N-acetyl-Leu-Leu-norleucinal (50 microM), a proteasome as well as a calpain inhibitor, was able to reduce calphostin C-induced apoptosis by approximately 60%, whereas lactacystin (10 microM), a specific proteasome inhibitor, was ineffective. These results suggest that calphostin C-induced apoptosis is partly calpain-mediated, but does not require protein degradation through the ubiquitin/proteasome pathway.

A possible growth factor role of IL-6 in neuroectodermal tumours

Preliminary data have shown that IL-6 may act as an autocrine growth factor to control proliferation. We further characterised the role of IL-6 in tumour growth as an autocrine/paracrine growth factor in neuroectodermal tumours. We evaluated the production and secretion of IL-6 by seven human melanoma, five neuroblastoma and one glioblastoma cell lines. Moreover, we determined their IL-6-dependent growth in serum free-medium or under minimal growth-supplement conditions: IL-6 dependent growth was observed in two non-IL-6 producing melanoma and in one neuroblastoma cell lines. In addition, expression of IL-6 mRNA and peptide was increased by retinoic acid. The data support the hypothesis that IL-6 contributes to neuroectodermal tumour growth, even though it shows a less potent effect than other reported growth factor such as IGF-II.

Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10–100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 μM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20–100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50–100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 μM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 μM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10–20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.