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Strategy for selection of cell variants deficient in poly(ADP-ribose) polymerase
S Chatterjee1, S J Petzold, S J Berger
1Department of Medicine, R. L. Ireland Cancer Center, University Hospitals of Cleveland, Case Western Reserve University, School of Medicine, Cleveland 44106.
Abstract:
A selection strategy to obtain cells deficient in poly(ADP-ribose) polymerase was developed based on the fact that treatment with high levels of N-methyl-N'-nitro-N-nitrosoguanidine results in sufficient activation of poly(ADP-ribose) polymerase to cause NAD and ATP depletion leading to cessation of all energy-dependent processes and rapid cell death. In contrast, cells with low levels of poly(ADP-ribose) polymerase should not consume their NAD and might therefore be more likely to survive the DNA damage. Using this approach, we have cloned a number of cell lines containing 37-82% enzyme activity. The apparent decrease in poly(ADP-ribose) polymerase activity is not due to increases in NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, or phosphodiesterase activities. Further characterization of the poly(ADP-ribose) polymerase-deficient cells indicates that they have prolonged generation times and increased rates of spontaneous sister chromatid exchanges.
Insights
Researchers developed a method to select cells with low poly(ADP-ribose) polymerase activity, which are more resistant to DNA damage. These poly(ADP-ribose) polymerase-deficient cells exhibit slower growth and more genetic instability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Poly(ADP-ribose) polymerase (PARP) is a crucial enzyme in DNA repair.
- High PARP activation by DNA damaging agents like N-methyl-N'-nitro-N-nitrosoguanidine depletes cellular NAD+ and ATP, leading to cell death.
- Cells with inherently lower PARP activity may exhibit resistance to such DNA damage.
Purpose of the Study:
- To develop and validate a selection strategy for isolating cell lines with deficient poly(ADP-ribose) polymerase activity.
- To characterize the biochemical and cellular properties of these PARP-deficient cells.
Main Methods:
- Utilizing N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA damage and exploit differential PARP activation for cell selection.
- Quantifying poly(ADP-ribose) polymerase activity in selected cell lines.
- Assessing other related enzyme activities (NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, phosphodiesterase) to confirm specificity.
- Evaluating cellular characteristics such as generation time and sister chromatid exchange rates.
Main Results:
- Successfully cloned cell lines with 37-82% reduction in poly(ADP-ribose) polymerase activity.
- Confirmed that the observed decrease in PARP activity was not attributable to increased hydrolase or phosphodiesterase activities.
- Characterized PARP-deficient cells as having prolonged generation times.
- Observed increased rates of spontaneous sister chromatid exchanges in PARP-deficient cells.
Conclusions:
- A viable selection strategy for generating poly(ADP-ribose) polymerase-deficient cells was established.
- These deficient cells exhibit altered cellular kinetics and genomic instability, highlighting the role of PARP in cell cycle regulation and genome integrity.