Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells.

Abstract

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.The transglutaminase multigene family includes intracellular and extracellular enzymes that catalyze Ca2"-dependent reac-tions in which -y-carboxamide groups of peptide-bound glutamine residues serve as acyl donors and primary amino groups of several compounds function as acceptor substrates (16,22). The reaction results in posttranslational modification of proteins by establishing e(-y-glutamyl)lysine cross-linkages and/or covalent incorporation of biogenic amines (di-and polyamines and histamine) into proteins. Diamines and polyamines also participate in cross-linking reactions through the formation of N,N-bis(-y-glutamyl)polyamine cross-bridges (16, 40, 43). The transglutaminase-dependent formation of stable cross-linkages induces protein polymerization, which confers high resistance to mechanical breakage and chemical attack to the polypeptides involved in the linkage (17,21

Involvement of 5-lipoxygenase in programmed cell death of cancer cells

“…Neuroblastoma SK-N-BE(2) cells undergo PCD when treated with RA for 3 to 5 days (Melino et al, 1994;Piacentini et al, 1992Piacentini et al, , 1993. Table 2 shows that retinoids increased PCD in parallel to a significant enhancement of 5-lipoxygenase activity.…”

“…Treatment with retinoids significantly increased light emission from neuroblastoma cells (Table 2), although longer incubation times were needed due to the slower induction of PCD in this model (Melino et al, 1994;Piacentini et al, 1992Piacentini et al, , 1993.…”

“…In order to verify whether the activation of 5-lipoxygenase and luminescence also occurred with a different inducer of PCD, we treated neuroblastoma cells with retinoic acid for 3 or 5 days, a model well-characterised in our laboratory (Melino et al, 1994;Piacentini et al, 1992Piacentini et al, , 1993. Treatment with retinoids significantly increased light emission from neuroblastoma cells (Table 2), although longer incubation times were needed due to the slower induction of PCD in this model (Melino et al, 1994;Piacentini et al, 1992Piacentini et al, , 1993.…”

Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-β and retinoic acid-induced cancer cell death

“…27,28 IRF-1, as a tumor suppressor with antiproliferative function, can also induce expression of the death ligand TNF-related apoptosisinducing ligand (TRAIL) in cancer cells treated with RA and IFN-g. 29 In addition, both IFN-a and RA are able to increase the expression of tissue transglutaminase II (tTGase II), a multifunctional enzyme with an apoptotic role in various cells. [30][31][32] To investigate the IFN-b/RA signaling pathway in MCF-7 cells, we tested tyrosine phosphorylation of Stat1 and gene expression of the key players described above. As shown in Figure 6a, tyrosine phosphorylation of Stat1 can be detected as early as 5 min after stimulation, which reached a peak level at 30 min and decreased after 3 h. The Stat1 protein level was rather constant during the early time course, but increased gradually after treatment from 9 to 48 h. IRF-1 expression was induced from 3 to 12 h, whereas p48 expression increased at 6 h and was further enhanced at 48 h. In contrast, Stat2 and TRAIL, and tissue tTGase II expression increased at a much later time (48 and 24 h, respectively).…”

Transglutaminase 5 is regulated by guanine–adenine nucleotides

“…TGase2 is involved in both intracellular and extracellular biological processes such as receptor-mediated endocytosis, cell adhesion, apoptosis and wound healing [4][5][6]. Several reports indicate that TGase2 activation can be associated with apoptosis (although recent evidence would suggest that involvement of TGase2 in apoptosis is indirect [7,8]) or with anti-apoptotic effects mediated by Rb [9].…”