A biological marker of exposure, or biomarker, is defined as cellular, biochemical, or molecular alterations that are measurable in biological media such as human tissues cells or fluids (1). A biomarker may also be defined any parameter that can be used to measure an interaction between a biological system and an environment agent, which may be chemical, physical or biological (10). Biological markers can be further classified into categories representative of the spectrum from exposure to disease. These categories include: internal dose, biologically effective dose, early response, altered structure and function and disease. Moreover, a marker of biologically effective dose represents the actual amount of an absorbed chemical that interacts with critical subcellular targets measured either in the target tissue or surrogate tissue. DNA and protein adducts are examples of markers of biologically effective dose (1).
Some carcinogens alter genes in living cells, while others generate reactive chemical species that cause oxidative stress. UV radiation, however, affects a variety of molecules in living tissues, including DNA (9). Additionally, the use of biological markers of exposure may lead to the provision of better exposure estimates, the validation of other measures of exposure, and an increased knowledge of mechanisms of disease by providing evidence to intermediate steps in the pathway from exposure to disease(1).
Current and Ongoing Studies
1. Research Groups
Key Action 4 is a research group dedicated to identifying carcinogenic environmental factors, explaining how they exert their effects, detecting early signs of exposure and oxidative stress, and developing and evaluating strategies for countering carcinogenic processes before they lead to cancer. While their ultimate goal is to focus on preventative intervention while identifying UV radiation damage to the skin, individuals are currently developing biomarkers for measuring oxidative stress. These biomarkers are currently being developed in cultured cells and will be validated by normal and overexposed skin (9).
A. Title: Comparison of the expression of p53, p21, Bax and the induction of apoptosis between patients with basal cell carcinoma and normal controls in response to ultraviolet irradiation.
Aim: Ultraviolet light (UV) is known to cause DNA damage in the epidermis. The damaged DNA is repaired or deleted by apoptosis to prevent the generation of cancer. It has been suggested that a deficient apoptotic mechanism may predispose individuals to skin cancer. Therefore, the response of normal controls and patients with basal cell carcinoma (BCC) to UV irradiation was investigated.
Methods: The buttock skin from normal volunteers and patients with BCC was irradiated using solar simulated radiation (SSR). SSR mimics the effect of natural sunlight. Skin biopsies were excised and examined for p53, p21, and Bax protein expression and for the induction of apoptosis.
Results: At 33 hours after UV irradiation, the induction of apoptosis was significantly higher (p = 0.04) in patients with BCC than in normal volunteers (Mann Whitney test). A trend towards higher p21 expression was found at 33 hours in patients with BCC (mean, 18.69 positive cells/field) than in normal volunteers (mean, 9.89), although this difference was not significant (p = 0.05 positive cells/field).
Conclusion: These results may imply that patients with BCC have enhanced sensitivity to UV irradiation or that there is some defect in the cell arrest or repair pathways, which results in damaged cells been pushed into apoptosis rather than repair (11).
B. Title: Protein Kinases and UV induces Stress Response
Exposure of mammalian cells or skin to ultraviolet (UV) radiation in DNA damage and induction of a stress response called the UV response. This induction response is mediated by several transcription factors, including AP-1, NF-kB and p53. Only p53 is likely to be directly induced in response to UV-damaged DNA. AP-1 and NF-kB, on the other hand, are activated through signal transduction cascades that appear to be elicited by effects of short wavelength UV on the cell surface, independently of DNA damage. Both pathways rely on specific signal-responsive protein kinases. The protein kinases that stimulate AP-1 activity in response to UV irradiation are JNK and p38. The protein kinase involved in NF-kB activation has not been molecularly identified. As it phosphorylates the IkB inhibitors of NF-kB, we refer to it as the IkB kinase. We propose to further investigate the mechanism by which exposure to UV leads to activation of JNK, p38 and the IkB kinase by establishing a cell-free system in which these kinases can be activated by UV. We also plan to molecularly clone the UV responsive IkB kinase and study its activation mechanism. Most importantly, however, our studies will focus on the physiological roles of these protein kinases in the response of mammalian cells to UV radiation, especially their potential involvement in protection against UV damage. This aspect of the UV response is the least well understood. Activation of AP-1 and NF-kB have both been proposed to be involved in diverse and conflicting responses including apoptosis, tumor promotion, protection against radiation induced damage and aging. As UV light is a common carcinogen and genotoxic agent to which we are exposed, understanding the function of UV activated protein kinases is of great physiological and clinical importance. Using fibroblasts and T cells derived from knockout mice that are deficient in critical components of AP-1 and NF-kB activation we will investigate whether these pathways are protective or damaging. We also plan to identify genes that are specifically induced in response to JNK and c-Jun activation by UV radiation. These studies should shed new light on the mechanisms underlying cellular responses to UV radiation. These studies will also contribute to understanding how cells respond to other forms of radiation (18).
C. Title: p53-dependent DNA repair and apoptosis respond differently to high- and low-dose ultraviolet radiation p53 plays an essential part in the maintenance of the cellular genetic stability after a DNA-damaging event such as ultraviolet (UV) radiation. Following UV radiation, the amount of p53 protein is elevated. The increased p53 is believed to induce cell cycle arrest, promote nucleotide excision repair (NER) and apoptosis. To study if cells respond differently to high- and low-dose UV radiation, we examined the DNA repair efficiency and apoptosis rate of human and murine fibroblasts after UV radiation. Using a host cell reactivation assay, we found that NER was increased after low doses but not after high doses of UV radiation. In contrast, apoptosis occurred only after the cells received high doses (over 200 J/m2), but not low doses of UVB. The induction of both NER and apoptosis was observed only in p53+/+ murine fibroblasts, not in p53-/- cells, indicating that both stress response mechanisms are dependent on wild-type p53 function. UV radiation induced the expression of p53 protein in a dose-dependent manner up to 400 J/m2. In contrast, p21waf1/cip1 was induced only after low doses and bax only after high doses of UV radiation, supporting the roles of p21waf1/cip1 and bax in NER and apoptosis, respectively. Taken together, these results indicate that cellular stress response to UV radiation depends on UV dose, DNA repair after low doses and apoptosis after high doses, and that both mechanisms are dependent on wild-type p53 function (17).
D. Title: Risk Assessment of DNA repair Heterogeneity in Humans
RPROJ The ubiquitous environmental gentoxicant, ultraviolet light (UV), produces DNA damage, which if left unrepaired, can give rise to several biological effects including cell death, mutations and cancer. The human disease, xeroderma pigmentosum, exemplifies how reduced DNA repair capacity can lead to a higher incidence of cell lethality, higher mutation frequencies and a predisposition to cancer. The repair of UV-induced DNA damage is achieved by a complex interaction of >10 different gene products in a process called nucleotide excision repair. There is increasing evidence that DNA repair capacity, like any phenotypic trait, is heterogeneously distributed among the human population. Therefore certain individuals within the human population with low DNA repair capacity may be more prone to the adverse biological effects of environmental genotoxic agents, such as UV light. The goal of this study is to validate and apply within the human population several biomarkers of exposure and susceptibility to the environmental mutagen, ultraviolet light. In order to achieve this goal we will examine DNA repair capacity, cytotoxicity, and mutation frequency in peripheral lymphocytes of patients with skin cancer. More specifically the patient cohorts will be defined by the presence of one or more basal cell or squamous cell carcinomas and will be stratified into three age groups: l) less than 40 years old, 2) 40-50 years old, and 3) greater than 50 years old. Two major age-independent subgroups will be those patients with multiple skin cancers (MSC), (6 or greater) and patients with site clustering of skin cancers at specific body surfaces. All patients will be paired to age/sex-matched controls. We will test the hypothesis that low DNA repair capacity and high in vivo mutant frequency will correlate with increased risk of skin cancer using two DNA repair assays (SA1&2), a cytotoxicity assay (SA3) and an assay which measures the in vivo mutant frequency in a somatic gene (hypoxanthine phosphoribosyl transferase, HPRT) (SA4). Finally, in vitro studies of repair in isolated mutant clones will define the intra-individual interclonal heterogeneity of susceptibility to UV damage (SA6). The long-term goal of this study is to better understand the role of DNA repair in cancer susceptibility (19).
E. Proteomics in Uveal Melanoma
A new tool in the search for tumour markers is proteomic technology. Proteomics (or protein profiling) is the study of the proteome, the protein complement of the genome. The advantage of this technique in comparison with genomics is that the actual protein production can be measured. Gene microarrays determine levels of mRNA but do not necessarily predict the level of the corresponding proteins in a cell. In this study, we evaluated the use of proteomics in the aqueous humour of uveal melanoma patients compared with control patients using the surface-enhanced laser desorption ionization time-of-flight technique. The protein mass spectra of aqueous humours from 24 uveal melanoma eyes were compared with 24 control eyes using a strong anion exchange surface protein chip array. On the basis of two proteins (4543.43 and 6853.30 kDa), the aqueous humour of melanoma eyes and control eyes could be distinguished in 89% of cases. Therefore, proteomic evaluation might be helpful in finding diagnostic markers for uveal melanoma patients (20).
1. Talbott, E. O. and Craun, G.F. (1995). Introduction to Environmental Epidemiology. Boca Raton: Lewis Publishers.
9. Quality of Life and Management of Living Resources Programme implemented under the Fifth Framework Programme (1999). Europe Key Action 4 Environment and Health.Accessed 11/23/03
10. World Health Organization (1993). Environmental Health Information: Air Quality Guidelines. Accessed 11/23/03.
11. Murphy et al. Comparison of the expression of p53, p21, Bax and the induction of apoptosis between patients with basal cell carcinoma and normal controls in response to ultraviolet irradiation. Journal of Clinical Pathology.2002; 55:829-833.
17. Li, G and Ho, V C. p53-dependent DNA repair and apoptosis respond differently to high- and low-dose ultraviolet radiation. Br J Dermatol .1998 Jul; 139(1):3-10 [EMIC]
18. Karin et al.(2000) Protein Kinases and UV induces Stress Response.
19. National Institutes of Health TOXNET. Risk Assessment of DNA Repair Heterogeneity in Humans.
20. Missotten et al. Proteomics in Uveal Melanoma. Melanoma Research. 13(6):627-629, December 2003.
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