Douglas E. Brash

Sunlight and the onset of skin cancer

The photons of sunlight precipitate a series of genetic events in skin leading to cancer. These events involve somatic mutations as well as inherited alleles. Competition between cell populations ensues, as a single mutated cell expands into a clone. Thus cancer involves both a single-cell problem and a many-cell problem; in skin cancer, sunlight appears to drive both.

UV MUTAGENIC PHOTOPRODUCTS IN Escherichia coli AND HUMAN CELLS: A MOLECULAR GENETICS PERSPECTIVE ON HUMAN SKIN CANCER†

Abstract— The relevance of photoproducts produced by 254 nm irradiation to human skin cancer is first critically evaluated. Experiments identifying the mutagenic photoproducts at 254 nm are then described. Mutations are primarily due to the(6–4) photoproduct and the cyclobutane pyrimidine dimer, both in E. coli and in human cells. The(6–4) photoproduct may be more important in E. coli and the cyclobutane dimer more important in mammalian cells. In human cells, mutations occur at the C of a TC, CT, or CC cyclobutane dimer, but not at TT cyclobutane dimers, and also appear to occur, less frequently, at the C of TC and CC(6–4) photoproducts. The local structure of DNA is more important in determining the frequency of mutation at a site than is the photoproduct frequency at that site. The effect of DNA structure appears to be due to site‐specific lethality.

Transgenic expression of survivin in keratinocytes counteracts UVB-induced apoptosis and cooperates with loss of p53

The inhibitor of apoptosis protein survivin has been implicated in both cell cycle control and apoptosis resistance. To discriminate between these different roles, we used transgenic expression of survivin in the skin as a model for cell proliferation, differentiation, and apoptosis. Transgenic mice expressing survivin under the control of a keratin-14 promoter developed normally, without histologic abnormalities of the skin or hair, epidermal hyperplasia, or developmental abnormalities of basal or suprabasal epidermis. Keratinocyte proliferation assessed under basal conditions, or after ultraviolet-B (UVB) irradiation, or phorbol ester stimulation was unchanged in survivin transgenic mice. In contrast, survivin expression inhibited UVB-induced apoptosis in vitro and in vivo (i.e., sunburn cell formation), whereas it did not affect Fas-induced cell death. When crossed with p53 knockout mice, transgenic expression of survivin in a p53(+/-) background substituted for the loss of a second p53 allele and further inhibited UVB-induced apoptosis. These data provide the first in vivo evidence that survivin inhibits apoptosis and suggest that this pathway may oppose the elimination of cancerous cells by p53.

Escaping the stem cell compartment: Sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations

Once mutated, a single cell must expand into a clone before becoming significant for carcinogenesis. The forces driving clonal expansion and the obstacles that must be overcome are poorly understood. In a genetic mechanism, acquiring a second mutation conferring a proliferative advantage would enable the cell to expand autonomously. If carcinogen exposure instead induced a physiological change, clonal expansion would require the carcinogens continued presence. To determine which is the case, we studied microscopic clones of keratinocytes mutated in the p53 tumor suppressor gene. Carcinogen exposure was controlled by irradiating mice with 280–320 nm UV radiation (UVB), sunlights principal carcinogenic component; expansion of mutant clones was observed in epidermal sheets. p53-mutant clones grew only during chronic UVB exposure. Therefore, clonal expansion was not triggered by a proliferative mutation but was instead continually driven by UVB. Unexpectedly, the clone size distribution showed periodicity with maxima at estimated intervals of 16 ± 6 cells, the size of the epidermal proliferating unit in murine dorsal skin. In the absence of UVB, rare “imprisoned clones” increased in cell number without increasing in area. We conclude that: stem cell compartments act as physical barriers to clonal expansion of a p53-mutant keratinocyte; a rate-limiting step in clonal expansion is the colonization of an adjacent compartment; and sustained UVB enables the p53-mutant keratinocyte to colonize without incurring an additional mutation.

A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen.

Antibodies against a conserved RNA-binding protein, the Ro 60-kDa autoantigen, occur in 24–60% of all patients with systemic lupus erythematosus. Anti-Ro antibodies are correlated with photosensitivity and cutaneous lesions in these patients and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with complete congenital heart block and photosensitive skin lesions. In higher eukaryotes, the Ro protein binds small RNAs of unknown function known as Y RNAs. Because the Ro protein also binds misfolded 5S rRNA precursors, it is proposed to function in a quality-control pathway for ribosome biogenesis. Consistent with a role in the recognition or repair of intracellular damage, an orthologue of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival of this bacterium after UV irradiation. Here, we show that mice lacking the Ro protein develop an autoimmune syndrome characterized by anti-ribosome antibodies, anti-chromatin antibodies, and glomerulonephritis. Moreover, in one strain background, Ro–/– mice display increased sensitivity to irradiation with UV light. Thus, one function of this major human autoantigen may be to protect against autoantibody development, possibly by sequestering defective ribonucleoproteins from immune surveillance. Furthermore, the finding that mice lacking the Ro protein are photosensitive suggests that loss of Ro function could contribute to the photosensitivity associated with anti-Ro antibodies in humans.

UV Signature Mutations

Sequencing complete tumor genomes and exomes has sparked the cancer fields interest in mutation signatures for identifying the tumors carcinogen. This review and meta‐analysis discusses signatures and their proper use. We first distinguish between a mutagens canonical mutations—deviations from a random distribution of base changes to create a pattern typical of that mutagen—and the subset of signature mutations, which are unique to that mutagen and permit inference backward from mutations to mutagen. To verify UV signature mutations, we assembled literature datasets on cells exposed to UVC, UVB, UVA, or solar simulator light (SSL) and tested canonical UV mutation features as criteria for clustering datasets. A confirmed UV signature was: ≥60% of mutations are C→T at a dipyrimidine site, with ≥5% CC→TT. Other canonical features such as a bias for mutations on the nontranscribed strand or at the 3′ pyrimidine had limited application. The most robust classifier combined these features with criteria for the rarity of non‐UV canonical mutations. In addition, several signatures proposed for specific UV wavelengths were limited to specific genes or species; UVs nonsignature mutations may cause melanoma BRAF mutations; and the mutagen for sunlight‐related skin neoplasms may vary between continents.

New careers for antioxidants.

It is a remarkable fact that antioxidants are nontoxic compounds that reduce the incidence of cancer. Antioxidant nutrients such as vitamin E, β-carotene, lycopene, and selenium are regularly found to reduce the risk of lung, prostate, stomach, or total cancers, as well as oral precancers, in epidemiologic studies (1). Foods containing these nutrients are similarly effective, as are nondietary antioxidants such as green tea phenols and various Oriental herbal medicines (2). In general, the level of risk reduction is on the order of 0.6, which might encouragingly be extrapolated to (0.6) n for those of us with n organs at risk. The risk reduction from an antioxidant can be 3-fold in the elderly, in smokers, and in subpopulations deficient in a second antioxidant (1). This result suggests that functional redundancy of antioxidant systems is hiding the importance of their biological roles until two actors have been removed, as often happens with gene knockouts. These nutrients, as well as nonnutrient antioxidants such as N -acetyl cysteine, also slow the appearance of tumors in mice (2–4). Why do antioxidants have these effects? Biochemically, attention has traditionally centered on the obvious suspect, the ability of these compounds to scavenge free radicals, especially reactive oxygen species. Lipid-soluble antioxidants such as vitamin E act as chain-breakers to stop the propagation of sequential free radical reactions, as can water-soluble antioxidants such as vitamin C. Eradicating radicals will reduce damage to DNA and membranes (Fig. 1). But antioxidants have other molecular consequences, including inhibiting generation of reactive oxygen species, inhibiting metabolic activation of carcinogens, and altering the intracellular redox potential (5). The latter occurs with those water-soluble antioxidants having a high reducing potential, such as vitamin C and N -acetyl cysteine, which change the cells redox state. Redox state, in turn, regulates the activity of many transcription …

Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells.

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).

Common Contaminants in Next-Generation Sequencing That Hinder Discovery of Low-Abundance Microbes

Unbiased high-throughput sequencing of whole metagenome shotgun DNA libraries is a promising new approach to identifying microbes in clinical specimens, which, unlike other techniques, is not limited to known sequences. Unlike most sequencing applications, it is highly sensitive to laboratory contaminants as these will appear to originate from the clinical specimens. To assess the extent and diversity of sequence contaminants, we aligned 57 “1000 Genomes Project” sequencing runs from six centers against the four largest NCBI BLAST databases, detecting reads of diverse contaminant species in all runs and identifying the most common of these contaminant genera (Bradyrhizobium) in assembled genomes from the NCBI Genome database. Many of these microorganisms have been reported as contaminants of ultrapure water systems. Studies aiming to identify novel microbes in clinical specimens will greatly benefit from not only preventive measures such as extensive UV irradiation of water and cross-validation using independent techniques, but also a concerted effort to sequence the complete genomes of common contaminants so that they may be subtracted computationally.

Local recurrence versus new primary: Clinical analysis of 82 breast relapses and potential applications for genetic fingerprinting☆

PURPOSE The purpose of this study was to perform a detailed clinical pathological analysis of breast relapses in patients treated with conservative surgery and radiation therapy in an effort to classify those relapses as true local recurrences or second primary tumors, and to assess the prognostic and therapeutic implications of such a classification system. METHODS AND MATERIALS Of 990 patients treated with conservative surgery and radiation therapy at our facilities prior to December 1987, 82 patients have experienced a relapse in the conservatively treated breast as the primary site of failure. Patients were classified as having new primary tumors if they fulfilled any one of the following criteria: a) breast relapse occurring at a site distinctly removed from the original tumor; b) histology of the breast relapse compared with the original tumor consistent with a new primary; or c) DNA flow cytometry converting from an aneuploid primary to a diploid relapse. RESULTS As of 2/92, with a median follow-up of 5.4 years from the time of breast relapse, the overall 5-year survival rate following breast relapse was 55%. Forty-seven patients were classified as true recurrences and 33 patients were classified as new primaries. Patients classified as true recurrences had a shorter median time to breast relapse than patients classified as new primaries (3.16 years vs. 5.42 years, p < .05) and an inferior post breast recurrence survival rate compared to patients classified as new primaries (36% vs. 89%, p < .05). Residual disease outside of the recurrent tumor bed was also noted to be more frequent in patients classified as true recurrences compared to patients classified as new primaries (48% vs. 16%, p < .05). CONCLUSION Based on the clinical and pathological criteria outlined, it appears that a significant portion of patients experiencing a relapse in the conservatively treated breast may have new primary tumors as opposed to true local relapses. Distinction between a true recurrence and a new primary tumor may have significant prognostic implications. Uncertainties associated with the clinical and pathological criteria are presented and further investigations with genetic fingerprinting techniques to establish the clonality of breast relapses are presented and discussed.