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Bleomycin-induced DNA damage and repair in human cells permeabilized with lysophosphatidylcholine
1Biochemistry/Biophysics Program, Washington State University, Pullman 99164-4660.
Abstract:
We have examined bleomycin-induced DNA damage and repair in confluent human fibroblasts that were reversibly permeabilized to small molecules (e.g., deoxynucleotide triphosphates and trypan blue) by a short exposure to 80 micrograms/ml lysophosphatidylcholine. We found that this treatment dramatically increases the dose effectiveness of bleomycin in inducing DNA strand breaks and DNA repair synthesis in these cells. For example, when intact cells (not treated with lysophosphatidylcholine) were incubated with 100 micrograms/ml bleomycin, only about 5% of the cell population was observed to have undergone measurable DNA repair synthesis (by autoradiography). On the other hand, when these cells were reversibly permeabilized with lysophosphatidylcholine before treatment, we observed significant repair synthesis in greater than 80% of the cells using a bleomycin dose of only 5 micrograms/ml. Furthermore, sufficient levels of single- and double-strand breaks were introduced into nucleosome linker DNA of permeabilized cells to yield a nucleosomal repeat pattern in alkaline and neutral agarose gels. However, no change in the amount of DNA less than 23 kilobases was observed on these gels when intact cells were incubated with bleomycin.
Insights
Lysophosphatidylcholine permeabilization enhances bleomycin's DNA damage and repair effects in human fibroblasts. This method significantly boosts DNA strand breaks and repair synthesis at lower bleomycin doses.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Bleomycin is a chemotherapeutic agent used to induce DNA damage.
- Understanding DNA damage and repair mechanisms is crucial for cancer treatment.
- Human fibroblasts are a common model system for studying cellular responses to DNA damage.
Purpose of the Study:
- To investigate the effect of reversible cell permeabilization on bleomycin-induced DNA damage and repair.
- To determine if lysophosphatidylcholine treatment enhances the sensitivity of human fibroblasts to bleomycin.
Main Methods:
- Human fibroblasts were treated with lysophosphatidylcholine to reversibly permeabilize cell membranes.
- Permeabilized and intact cells were exposed to varying doses of bleomycin.
- DNA damage and repair synthesis were assessed using techniques like autoradiography and agarose gel electrophoresis.
Main Results:
- Lysophosphatidylcholine treatment significantly increased the dose effectiveness of bleomycin in inducing DNA strand breaks and repair synthesis.
- Permeabilized cells showed significant repair synthesis at 5 µg/ml bleomycin, compared to 5% of intact cells at 100 µg/ml.
- Bleomycin induced single- and double-strand breaks in nucleosome linker DNA of permeabilized cells, evident in gel electrophoresis patterns.
Conclusions:
- Reversible permeabilization of human fibroblasts enhances their susceptibility to bleomycin-induced DNA damage.
- This enhanced sensitivity allows for more effective induction of DNA strand breaks and repair synthesis at lower drug concentrations.
- The findings suggest potential for improved drug delivery or efficacy studies using permeabilized cell models.