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Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D
K Rothbarth1, E Spiess, B Juodka
1Division Biochemistry of the Cell and Biomedical Structure Analysis Group, German Cancer Research Center, D-69120 Heidelberg, Germany.
Abstract:
Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.
Insights
Overexpressing the C1D gene triggers programmed cell death (apoptosis) in tumor cells by activating DNA-PK. This conserved gene
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The conserved gene C1D encodes a DNA-binding and DNA-PK-activating protein.
- C1D is physiologically expressed across 50 human tissues, indicating a fundamental cellular role.
- Tight regulation of C1D expression is crucial, as elevated levels induce apoptotic cell death.
Purpose of the Study:
- To investigate the role of C1D gene overexpression in inducing apoptosis in tumor cell lines.
- To characterize the morphological and molecular changes associated with C1D-induced apoptosis.
- To explore the potential bystander effect and the involvement of p53 in C1D-mediated cell death.
Main Methods:
- Vector-dependent overexpression of the C1D gene in various tumor cell lines.
- Utilizing C1D-EGFP fusion constructs for cell identification and morphological tracing.
- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay to detect DNA fragmentation.
- Co-culture experiments to assess bystander effects.
- Analysis of apoptosis in cells with functional and non-functional p53.
Main Results:
- Vector-dependent C1D overexpression induced apoptosis in tumor cell lines, evidenced by DNA end labeling.
- C1D-EGFP expression led to characteristic apoptotic morphological changes, including nuclear disintegration and cell shrinkage.
- A bystander effect was observed, where non-transfected cells co-cultured with C1D-expressing cells also underwent apoptosis.
- C1D-induced apoptosis was dependent on functional p53, suggesting a link to the DNA-PK/p53 pathway.
Conclusions:
- The conserved C1D gene, when overexpressed, can trigger apoptosis in tumor cells via DNA-PK activation.
- C1D-induced apoptosis involves distinct morphological changes and can affect neighboring non-transfected cells.
- The p53 pathway is essential for C1D-mediated apoptosis, highlighting a potential therapeutic target in cancer treatment.