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- Backvall, H., et al.
(författare)
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Mutation spectra of epidermal p53 clones adjacent to basal cell carcinoma and squamous cell carcinoma
- 2004
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Ingår i: Experimental dermatology. - : Wiley. - 0906-6705 .- 1600-0625. ; 13:10, s. 643-650
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Tidskriftsartikel (refereegranskat)abstract
- Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.
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| 3. |
- Sivertsson, Åsa, 1969-
(författare)
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Detection and analysis of genetic alterations in normal skin and skin tumours
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin.Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis.After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique.Keywords:single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras
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