Repair of trans-Pt(II) diamminedichloride DNA-protein crosslinks in normal and excision-deficient human cells
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Abstract:
In this investigation the persistence of DNA-protein crosslinks (DPC) induced by trans-Pt was determined in normal and excision-deficient xeroderma pigmentosum (XP) group A fibroblasts. After exposure to 50 microM trans-Pt for 2 h, the level of DPC increased in both cell types for several hours but by 12 h it was significantly less in normal cells. By 18 h it was approximately half the maximal value in normal cells but had decreased little in XP cells. When cells were incubated with trans-Pt and polymerase inhibitor, DNA single-strand breaks accumulated in normal but to a much lower level in XP cells. These single-strand breaks were presumably produced during excision repair and were roughly comparable in frequency to the number of DPC removed in normal cells during the same interval. By colony survival, trans-Pt was more toxic to XP cells. These results indicate that DPC were recognized in normal cells and repaired by the excision repair pathway.
Insights
Normal cells repair DNA-protein crosslinks (DPC) induced by transplatin, unlike xeroderma pigmentosum group A cells. This DNA repair pathway is crucial for mitigating transplatin toxicity and preventing cell death.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA-protein crosslinks (DPC) are cytotoxic DNA adducts.
- Xeroderma pigmentosum (XP) is a genetic disorder characterized by deficient DNA repair.
- Understanding DPC repair mechanisms is vital for cancer therapy.
Purpose of the Study:
- To investigate the persistence and repair of transplatin-induced DNA-protein crosslinks (DPC).
- To compare DPC repair in normal versus excision-deficient xeroderma pigmentosum (XP) group A fibroblasts.
- To elucidate the role of the excision repair pathway in DPC removal.
Main Methods:
- Exposure of normal and XP group A fibroblasts to transplatin.
- Quantification of DNA-protein crosslinks (DPC) over time.
- Assessment of DNA single-strand breaks in the presence of a polymerase inhibitor.
- Colony survival assays to determine cellular toxicity.
Main Results:
- Transplatin induced DPC in both normal and XP cells, but DPC levels decreased significantly in normal cells within 18 hours.
- XP cells showed minimal DPC reduction compared to normal cells.
- DNA single-strand breaks, indicative of repair, accumulated in normal cells but not in XP cells.
- Transplatin exhibited higher toxicity in XP cells, as evidenced by colony survival assays.
Conclusions:
- Normal human fibroblasts possess an active excision repair pathway that recognizes and removes transplatin-induced DNA-protein crosslinks (DPC).
- Xeroderma pigmentosum group A cells, deficient in excision repair, are unable to efficiently remove DPC, leading to increased sensitivity to transplatin.
- These findings highlight the importance of DPC repair in cellular resistance to platinum-based chemotherapeutics.