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|Title:||UV Enhanced Mutagenesis of Adenovirus in Human Fibroblasts|
|Abstract:||<p>UV-induced cellular "error-prone" repair was examined by the comparative determination of the enhanced reactivation (UVER) and mutagenesis (UVEM) among viral progeny resulting from singIe cycle lytic infections of intact or UV-irradiated Ad5ts36 and Ad5ts125 in unirradiated or UV-irradiated confluent human fibroblasts. The induction of phenotypic wild type revertants among the viral progeny was determined by plaquing at 33°C (permissive) and 39°C (nonpermissive) on HaLa or KB indicator monoIayers. UV-irradiation of Ad5ts36 and Ad5ts125 resulted in a dose dependent increase in the UV-Induced reversion frequency (RF) of viral progeny and a dose dependent exponential decrease in progeny survival in unirradiated normal fibroblasts. Kinetic analysis of UV-induced reversion suggests that 2.5 ± 0.3 (Ad5ts36) and 2.4 ± 0.5 (Ad5ts125) "hits" were required to produce a targeted reversion event among the viral progeny derived from normal human fibrolasts. The expression of UVER and UVEM was examined as a function of Increasing time delay between cellular UV-irradiation and viral infection for various time periods. UV-irradiation of normal human fibroblasts (10 J/m/2), carried out 24 hours prior to viral infection, resulted in an increased RF for both unirradiated (untargeted increase) or UV-irradiated (targeted increase) virus. A maximal UVEM of 1.4 ± 0.2 was observed concomitantly with the maximal UVER (3.4 ± 0.8) and targeted increase (1.9 ± 0.3) when viral infection was delayed 24 hours following cellular UV-irradiation.</p> <p>The maximal untargeted increase (2.0 ± 0.5) was observed when viral infection was immediately following cellular irradiation and declined as viral infections were delayed for longer time intervals. Different time courses for the targeted and untargeted increases suggested that these processes may be separateIy reguIated in human fibroblast cells.</p> <p>DNA repair-deficient human fibroblast strains from patients with ataxia telanglectasia (AT), xeroderma plgmentosum (XP) and Cockyane's syndrome (CS) were also examined for the expression of UVER and/or UVEM of UV-irradiated adenovirus. AT fibroblasts expressed a significantly reduced UVEM (0.31 ± 0.12) of UV-irradiated adenovirus as compared to normals when viral infections were delayed 24 hours following celluIar UV-irradiation. A fibroblasts also displayed abnormally reduced UVER of UV-irradiated adenovirus when viral infections immediately followed cellular UV-irradiation. Aberrant expression of both UVER and UVEM of UV-irradiated adenovirus in AT suggests that these cells may be deficient in an error-prone DNA repair process or express enhanced error-free repair. Similarity, the XP variant strain studied displayed some aberration of UVEM when compared to normals indicated by a reduced UVEM (0.52 ± 0.2) of UV-irradiated adenovirus for viral infections delayed 24 hours after cellular irradiation. UV-induced reversion of adenovirus progeny derived from unirradiated XP 25RO (complementation group A) was hypermutable on a per unit dose basis but when corrected for survival, reversion was similar to that seen for normals. This suggests excision repair is an error-free process. UVER and UVEM of UV-irradiated adenovirus in CS fibroblasts (a non cancer prone condition) were similar to that observed in normals when viral infections were 24 hours after cellular irradiation. These results suggest that fibroblasts from cancer-prone individuals (AT, XPA, XP var) all express abnormal parameters of UV-induced mutagenesis. The underIying mechanism(s) responsible for this abnormal mutagenesis may ultimately contribute to the cancer-prone nature of these syndromes.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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