Note for NATS 101 with Professor Hirschboeck at UA
Note for NATS 101 with Professor Hirschboeck at UA
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Date Created: 02/06/15
UVA and UVB genetic damage in the p53 gene occur at different depths within solar keratosis Agar N 1Halliday Gl 1 Bametson Rls 1 and Jones Als z Faculty of Medicine Dermatology 1 Melanoma and Skin Cancer Research Institute of the University of Sydney at Royal Prince Alfred Hospital NSW2 Introduction Australia has the highest incidence of skin cancer in the world In this country skin cancer incidence signi cantly exceeds that of all other malignancies combined It is accepted that ultraviolet radiation is the main causative factor in skin cancer formation Till now most research has attributed these effects to the UVB fraction of sunlight We hypothesised that UVA in addition to UVB may be playing an important role in skin cancer formation In order to examine this we have mapped the pattern of mutational damage within the premalignant lesion solar keratosis in our target gene of interest the tumour suppressor gene p53 Methods Frozen sections were prepared from 8 Solar Keratosis excised from human volunteers attending the Dermatology Outpatients Clinic at RPAH Utilising the Pixcell II Laser Capture Microdissection Apparatus several areas were dissected from within each skin Histologically 2 epithelial rete pegs representative of each lesion were chosen Superficial and deep cells from each vertical peg were sampled with the removal of 20 cells per microdissection DNA was extracted from microdissected cells using protocols previously established 1 2ul of crude cell lysate was then directly used as a DNA template Exons 59 were ampli ed in all samples using primers designed by Schifter 2 under the following conditions Exons 59 Denatured at 950 C for 15min then 940C for 3min followed by Annealing over 50 cycles at 940C for 20 sec and 640C for 20 sec A final extension step of 720C for 10 minutes was then performed for Exon 5 only after which the specimens were kept on hold at 40C PCR product was purified using a Qiaquik Gel extraction kit prior to automated sequencing Applied Biosystems Model 373A Each amplicon was successfully sequenced completely in forward and reverse directions and the results repeated to con rm mutations Negative controls without DNA and positive controls K562 cell line DNAknown to contain the wild type p53 genomic sequence were also included in each PCR run RESULTS Utilising the methods described above we were able to reliably sequence 100 of microdissected keratinocytes Both UVA and UVB fingerprint mutations were detectable in Solar Keratosis and showed a striking spatial distribution See Fig l The majority of UVB fingerprint mutations 875 were found in the spinous layer or above in contrast to the basal distribution of UVA fingerprint mutations 88 in SK When the distribution of UV induced mutations was calculated as depth per mm from the skin surface and a Chi square analysis performed it was found that 100 of UVA mutations had a depth 5 04mm Whereas only 453 of UVB mutations were 5 04mm deep This difference was statistically signi cant p lt 0001 39 UVB Fingerprint mutation UVA Fingerprint mutation Fig 1 c ema c represen on o mrcro rssec ron results in SK DISCUSSION Utilising laser capture microdissection we report for the rst time a marked spatial distribution of UVB and UVA induced genetic damage in the premalignant skin lesion the solar keratosis The deeper localisation of UVA ngerprint mutations in the basal gerrninative layer of the epidermis suggests that UVA rather than UVB may be the dominant carcinogen in this formative region A lack of sensitivity in previous whole tissue sampling and failure to sample the basal layers where UVA is possibly exerting its maximal effects may explain why UVA induced DNA lesions have been difficult to identify in the past REFERENCES 1wwwARCTURcom 2Schifter M etal Epithelial P53 gene expression and mutational analysis combined with growth fraction assessment in oral lichen planus J Oral Pathol Med 27318 1998 This study implicates a much earlier and more significant genotoxic role for UVA in skin cancer formation than previously described Our results may help explain why the use of principally UVB blocking sunscreen has not altered the growing skin cancer incidence both here in Australia and worldwide
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