Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells
G Melino1, M Annicchiarico-Petruzzelli, L Piredda
1Department of Experimental Medicine, University of Rome Tor Vergata, Italy.
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.
Insights
Overexpressing tissue transglutaminase (tTG) in neuroblastoma cells induces apoptosis, a programmed cell death. This finding reveals tTG
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- Neuroblastoma is a pediatric cancer originating from neural crest cells.
- Apoptosis, or programmed cell death, is a crucial cellular process.
- Tissue transglutaminase (tTG) is an enzyme involved in protein cross-linking.
Purpose of the Study:
- To investigate the role of tissue transglutaminase (tTG) in neuroblastoma cell apoptosis.
- To explore the relationship between tTG expression and neuroblastoma cell death.
- To elucidate the biochemical mechanisms underlying tTG-induced apoptosis.
Main Methods:
- Overexpression of full-length human tTG cDNA in SK-N-BE(2) neuroblastoma cells.
- Transfection with antisense tTG cDNA to inhibit tTG expression.
- Assessment of cell proliferation, cell death rates, and apoptotic markers.
- Analysis of Bcl-2 protein levels and neuronal differentiation markers (NCAM, neurofilaments).
- Investigation of retinoic acid (RA)-induced apoptosis.
Main Results:
- tTG overexpression drastically reduced proliferation and increased apoptosis in neuroblastoma cells.
- Dying cells exhibited characteristic apoptotic morphological changes.
- tTG-transfected cells showed increased Bcl-2 levels and neuronal differentiation markers.
- Antisense tTG transfection decreased both spontaneous and RA-induced apoptosis.
- Apoptosis was regulated by RA and occurred preferentially in substrate-adherent cells.
Conclusions:
- Tissue transglutaminase (tTG) directly promotes phenotypic maturation toward apoptosis in neuroblastoma cells.
- tTG-dependent protein cross-linking is a key biochemical event in apoptosis induction.
- tTG plays a significant role in regulating neuroblastoma cell death pathways.
