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Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts
Abstract:
The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.
Insights
Normal human fibroblasts and xeroderma pigmentosum variant (XP4BE) cells show different responses to ultraviolet light (UV) induced mutations. XP4BE cells exhibit higher mutation frequencies after UV exposure, indicating impaired DNA repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Ultraviolet (UV) radiation induces DNA damage, leading to mutations.
- Xeroderma pigmentosum variant (XP4BE) cells are known to have defects in postreplication DNA repair.
- Understanding differential cellular responses to UV damage is crucial for cancer research and photobiology.
Purpose of the Study:
- To compare the UV-induced mutability of normal human fibroblasts and XP4BE fibroblasts.
- To investigate the role of postreplication repair in UV mutagenesis.
- To quantify the frequency of 6-thioguanine resistance (TGr) mutations after UV exposure.
Main Methods:
- Human fibroblasts (normal and XP4BE) were exposed to varying doses of UV radiation.
- Cells were cultured to allow for the expression of the 6-thioguanine resistance (TGr) phenotype.
- Mutation frequencies were determined by selecting for TGr colonies at a concentration of 20 micrograms TG/ml.
Main Results:
- XP4BE cells showed a significantly higher frequency of UV-induced TGr mutations compared to normal fibroblasts at equivalent UV doses.
- The induced mutation frequency in normal cells was linear with UV dose, extrapolating to zero mutants at a low UV dose.
- XP4BE cells exhibited a non-linear relationship between induced mutation frequency and the logarithm of cell survival.
Conclusions:
- Normal postreplication repair is largely error-free until a threshold UV dose is exceeded.
- XP4BE cells, deficient in postreplication repair, are more susceptible to UV-induced mutations.
- These findings highlight the critical role of efficient DNA repair in preventing UV mutagenesis.