Patterns of cell death induced by eluates from denture base acrylic resins in U-937 human monoblastoid cells.

The purpose of this study was to investigate in vitro the apoptosis- and necrosis-inducing potential of eluates from three heat-polymerized and four autopolymerized poly(methyl methacrylate)-based denture base resins. Our hypothesis was that the rate of cell death by apoptosis and/or necrosis induced by such denture base resins could be an important indicator of their cytotoxicity degree. U-937 human monoblastoid cells were exposed for 24 h and 48 h to eluates of 0.1 g/ml, 0.2 g/ml, 0.4 g/ml, and 0.8 g/ml extracted for 24 h and 48 h. The characteristics of apoptosis and necrosis were evaluated by flow cytometry and light and electron microscopy. Eluates from all resins enhanced cell death by apoptosis and necrosis in U-937 cells in a dose- and time-dependent fashion. Eluates from autopolymerized resins yielded higher percentages of apoptosis and necrosis than the heat-polymerized ones. The results support our hypothesis that eluates of poly(methyl methacrylate)-based denture base acrylic resins activate death-signaling pathways, and that the extent of this process reflects their biocompatibility degree.

The effect of heat- and auto-polymerized denture base polymers on clonogenicity, apoptosis, and necrosis in fibroblasts: denture base polymers induce apoptosis and necrosis.

Eluates from poly(methyl methacrylate)-based denture base polymers have recently been found to enhance death by apoptosis and necrosis in U-937 human monoblastoid cells. The present study investigated the potential of such polymers to induce apoptosis and/or necrosis and to alter clonogenicity in L929 murine fibroblasts. A fibroblast cell line was chosen because the impairment of fibroblasts subjacent to denture bases may result in a weaker or more permeable mucosa. Two aspects were addressed: the effect of direct contact with the denture base polymers and the effect of eluates extracted from the polymers. For this purpose L929 fibroblasts were seeded on disks manufactured from three heat-polymerized and four autopolymerized denture base polymers or in different concentrations of their eluates. The effects were evaluated by light, fluorescent, confocal and electron microscopy, counting of colonies, and flow cytometry. Disks and eluates of all polymers enhanced cell death by apoptosis and necrosis in L929 cells and decreased their clonogenic potential in a dose-dependent manner. Apoptosis was the main form of cell death. In general, the deleterious effects were stronger when cells were plated directly on the polymer disks than in the eluates. The autopolymerized polymers, except one, yielded higher percentages of apoptosis and necrosis than the heat-polymerized polymers. The results of the study indicated that poly(methyl methacrylate)-based denture base polymers trigger death-signals in L929 fibroblasts and open doors for possible modulation of the cell/biomaterial interaction.

cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells.

Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis.

Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line.

The protein phosphatase inhibitors okadaic acid and calyculin A at moderate concentrations induced three types of apoptotic promyelocytic leukemia cell death, distinct with respect to ultrastructure and polynucleotide fragmentation. Calyculin A at higher concentrations (> 50 nM) induced a non-apoptotic death type with high ATP and pronounced micromitochondriosis. This suggests that protein phosphorylation pathways are involved in the triggering of several death pathways. Activation of the cAMP kinase induced yet another apoptotic death type, preferentially affecting cells in S-phase. In fact, cAMP acted in two ways to stop IPC promyelocyte proliferation: (1) block in late G1 (preventing new cells from entering DNA replication); and (2) induction of apoptosis in S-phase. cAMP and phosphatase inhibitors acted via distinct pathways. The inhibitors suppressed cAMP-induced death, but only at concentrations high enough to commit the cells to alternative, less conspicuous death types. The tumor-promoting activity of okadaic acid and calyculin A may therefore not be by protection against apoptosis. DNA fragmentation correlated with the novel feature of limited 28 S rRNA cleavage, suggesting co-ordinated polynucleotide cleavage, possibly directed against illegitimate polynucleotides, in some apoptotic death types.

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis.

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Organ culture of a glioblastoma from a patient with an unusually long survival.

Multicellular tumor spheroids were directly initiated in vitro from the biopsy specimens of a patient who is alive and who has had no neurological changes in 7 years after the gross removal of a glioblastoma. The spheroids were studied alone and in confrontation with aggregates of fetal rat brain tissue. Both in the biopsy and in the tumor spheroids, a very high proportion of cells were proliferating, as flow cytometric deoxyribonucleic acid measurements showed that 40% of the cells in the biopsy specimens and in the tumor spheroids were in the S and G2M phases of the cell cycle. Despite this high proliferation rate, the volume of the spheroids decreased, indicating an even greater cell loss. Light and scanning electron microscopic studies also indicated cell death in the spheroids. This behavior may be related to the long-time survival.