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Published on: January 11, 2019
Differential gene expression in apoptotic 32Dcl3 cells: induction of metallothionein
R Houben1, J Troppmair, J Hidalgo
1Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Germany.
Abstract:
Growth factor deprivation induced cell death of the hematopoietic cell line 32Dcl3 is widely used as a model system to study apoptotic signalling pathways. Here we show that the onset of cell death after IL-3 withdrawal can be strongly delayed by either cycloheximide or actinomycin D, indicating that de novo protein synthesis is required. Subtractive cDNA library hybridization was used to identify genes upregulated in apoptotic 32Dcl3 cells. Here we present data showing metallothionein-I (MT-I) mRNA transiently upregulated by a factor of three- to 20-fold. Increased levels of total MT-I+II protein after IL-3 withdrawal were demonstrated. An induction of MT-I RNA as well as of MT-I+II total protein was also observed in serum deprived NIH3T3 fibroblasts. Testing the effect of different inducers of apoptosis on 32Dcl3 cells we found that only IL-3 withdrawal and ethanol treatment led to an upregulation of MT-I mRNA level. Since MTs are believed to play a role in the metabolism of zinc, we tested the effect of zinc on induced cell death. When 32Dcl3 cells are treated with zinc (50-300 microM) in the absence of IL-3, loss of viability as well as degradation of the cellular DNA were delayed, indicating that zinc represses apoptosis. On the other hand zinc pre-treatment induced MT expression and accelerated the onset of apoptosis. Our data, therefore, suggest that MT exerts a proapoptotic function.
Insights
Metallothionein-I (MT-I) upregulation is required for apoptosis in 32Dcl3 cells. Zinc represses apoptosis by inhibiting MT-I, suggesting MT-I has a pro-apoptotic function in cell death signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Hematopoietic cell line 32Dcl3 is a model for studying apoptotic signaling pathways.
- Growth factor deprivation, like IL-3 withdrawal, induces cell death in 32Dcl3 cells.
- De novo protein synthesis is necessary for this IL-3 withdrawal-induced cell death.
Purpose of the Study:
- To identify genes upregulated during apoptosis in 32Dcl3 cells.
- To investigate the role of metallothionein-I (MT-I) in apoptosis.
- To explore the effect of zinc on apoptosis and MT-I expression.
Main Methods:
- Subtractive cDNA library hybridization to identify upregulated genes.
- Quantitative analysis of MT-I mRNA and MT-I+II protein levels.
- Induction of apoptosis using IL-3 withdrawal, ethanol, and serum deprivation.
- Treatment with zinc to assess its effect on cell viability, DNA degradation, and MT expression.
Main Results:
- MT-I mRNA was transiently upregulated 3- to 20-fold during apoptosis.
- Increased MT-I+II protein levels were observed after IL-3 withdrawal in 32Dcl3 cells and serum-deprived NIH3T3 fibroblasts.
- MT-I mRNA upregulation occurred only with IL-3 withdrawal and ethanol treatment.
- Zinc (50-300 microM) delayed cell death and DNA degradation in IL-3-deprived cells, indicating apoptosis repression.
- Zinc pre-treatment induced MT expression and accelerated apoptosis onset.
Conclusions:
- MT-I is upregulated during apoptosis induced by IL-3 withdrawal and ethanol.
- Zinc represses apoptosis, potentially by inhibiting MT-I function.
- Metallothioneins appear to exert a pro-apoptotic function in this cellular model.
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