ZEBRAFISH MODEL FOR MELANOMA

Our lab recently transitioned to using zebrafish as a model for the investigating the early events leading to melanoma. I learned the advantages of using zebrafish while I was a US Fulbright Research Fellow in Ireland (Jan-July 2008) at the Dublin Institute of Technology’s Facility for Optical Characterization and Spectroscopy (FOCAS). During this time I worked with the Radiation and Environmental Science group on the cellular and subcellular responses to UVA radiation. While there I became acquainted with several labs in Ireland who were using zebrafish for research. I was very much impressed with how zebrafish could help me answer important questions about how melanocyte stem cells might be involved in UV-induced melanoma.

INSTALLATION OF ZEBRAFISH HABITAT

After returning to campus, I received a Faculty Merit Award to purchase a continuously cycling Aquaneering System to start a zebrafish research colony. This is now located in the Animal Care Facility in the Science Building at Ferris State University. Each tank is a baffle/tank system that ebbs water in a circular motion to ensure flushing and water turnover. Ultraviolet (UV) sterilizers (110,000 microwatt-s cm⁻²) are used to disinfect the water and prevent the spread of disease in the re-circulating system. The water temperature is maintained at 28±0.5°C. The system continuously circulates water from the tanks through Siporax™ strainers, through a fiber mechanical filtration system, and finally into a chamber containing foam filters and activated carbon inserts. Zebrafish are maintained with a 14:10 h light/dark cycle and fed living brine shrimp twice per day.

Ryan Freye (standing left) is now in pharmacy school. He was instrumental in beginning stages of our research program to document all the procedures for zebrafish breeding and rearing, and took a major role in performing and documenting the fish radiation protocols. Richard Marble (standing right), Director of the Animal Care Facility, has overall responsibility for the management of the zebrafish colony. Jim Hoerter (seated) is P.I. for the project.

Shortly after returning to Ferris State University, O’Reilly-Pol and Johnson’s paper was published demonstrating how neocuproine can be used to control the differentiation of melanocytes from melanocyte adult stem cells. This technique provides a powerful method to determine if UVA in sunlight can damage melanocyte stem cells and contribute to the development of melanoma. Although human epidermal melanocyte stem cells have not been definitely isolated, they are likely to exist based on the presence of stem cell markers on cells located on the basal layer of the epidermis. Zebrafish is the only animal model thus far that permits the elimination of adult melanocytes without affecting their ability to regenerate them from adult melanocyte stem.

I recently received an NIH Academic Research Enhancement Award ($347,000) to investigate the effects of UVA/UVB irradiation on melanocyte stem cells. My research will help to determine if the cellular origin of melanoma is the melanocyte stem cell.

UVA/UVB RADIATION AND MELANOMA

The role that UVA (320-400 nm) and UVB (290-320 nm) radiation plays in the development of melanoma is not clear. UVA radiation, which is more penetrating than UVB, may cause the accumulation of mutations in melanocyte stem cells, contributing to the development of melanoma when these stem cells divide to replace damaged adult melanocytes in the basal layer of the epidermis. Hopefully, our studies will provide insights as to how sunlight UVA and UVB might be contributing to the development of melanoma.

Exposure to ultraviolet light is an important causative factor in melanoma, although the relationship between risk and exposure is complex. Intermittent sun exposure and sunburn history are important variables in the development of melanoma (1). It is controversial as to whether the UVB (290-320 nm) or the UVA (320-400 nm) wavelengths of solar radiation is more important in melanoma development (2,3). But epidemiological evidence suggests that both UVA and UVB are involved in melanoma causation (4,5). It is still unclear how a normal melanocyte becomes a melanoma cell, and how the properties of the normal melanocyte and/or its progenitors are utilized in its transformation. Determining the etiology of melanoma and the mechanisms involved in melanoma genesis are required for the development of more effective early detection and prevention and treatment strategies.

UVA (320-400 nm) plays a much more important role in melanoma than previous thought (6). More than 20% of UVA reaches the basal layers of the skin compared to less than 10% for UVB (7). Thus, much more UVA is able to penetrate down to the basal layer of the epidermis where the stem cells reside and may play an important role in the early stages of transformation (7,8). Absorption of UVA leads to the production of reactive oxygen and nitrogen species that can damage major biomolecules including DNA and membrane lipids. Various types of damage induced in these molecules lead to significant biological effects including cytotoxicity, mutations and alternations in cell signaling pathways (9). Longer-term effects such as persistent genomic instability and bystander effects have also been observed following UVA treatment of mammalian cells (10-12).

RESEARCH OBJECTIVES

Melanoma is the most deadly form of human skin cancer. Over the past several decades, the incidence of melanoma has steadily risen in the United States with about 60,000 cases and 8,000 deaths reported each year. Although solar UVB radiation plays a leading role in melanoma, it is still not clear where the damage actually originates. The long-standing hypothesis is that mature melanocytes accumulate mutations over time until critical genes are damaged and the melanocyte is transformed. My research plan will investigate an alternative hypothesis that solar radiation damages melanocyte stem cells, recently discovered in the myelin sheath of nerves innervating the skin.

The long-range goal of my undergraduate research lab is to identify the early molecular events responsible for the development of melanoma. The primary objective of this research project is to test the hypothesis that melanoma is initiated in the melanocyte stem cell after exposure to solar UVB/UVA irradiation. This research plan has the following specific aims: (1) determine if UVB/UVA irradiation of melanocyte stem cells with activated human BRAF and NRAS oncogenes will increase the incidence of melanocytic tumors (2) determine if additional UVB/UVA irradiation of the regenerated adult melanocytes derived from these previously irradiated stem cells will increase the number of melanocytic tumors, (3) determine if a melanocyte in early stages of differentiation is more susceptible to UVB/UVA-induced transformation than one in later stages of differentiation, (4) determine if the tumor suppressor p53 affects UVB/UVA damage/repair of melanocyte stem cells, and (5) determine if cellular senescence, known to be a powerful tumor-suppressive process that prevents excessive proliferation, is suppressed by UVB/UVA irradiation of either melanocyte stem cells or adult melanocytes. We will take advantage of the zebrafish melanoma model. Zebrafish genes controlling melanocyte development and differentiation are highly conserved and very similar to mammals. The unique advantage of the zebrafish model system is that it permits synchronization and control of melanocyte regeneration, permitting us to selectively irradiate the entire melanocyte stem population at once and/or the entire adult melanocyte population at the same stage of differentiation. This synchronization cannot be achieved in mammalian systems.

Our working hypothesis is that melanocyte stem cells accumulate UVB/UVA-induced mutations over time due to defects in repair pathways. Later in life when adult melanocytes are regenerated from these stem cells they will carry a mutational load and will be more susceptible to UVB/UVA-induced transformation. Melanocytes exposed to UVB/UVA at different stages differentiation will give forth different melanocytic lesions ranging from invasive malignant melanoma to isolated atypical epidermal melanocytes or nevi. The experimental plan will utilize a recently reported method for ablating adult melanocytes but not melanocyte stem cells, permitting us to irradiate only the melanocyte stem cell population and then observing how this affects regenerated melanocytes derived from these stem cells. The entire melanocyte population is regenerated simultaneously within 4 weeks after drug washout, permitting us to irradiate at different stages of differentiation by simply changing the time of irradiation. We will utilize transgenic human BRAF and NRAS activated oncogenes, as well as a p53 mutant under control of a melanocyte promoter to accelerate the development of tumors.

We expose zebrafish to UVA and UVB using a circular radiation chamber. Zebrafish are kept in the dark to prevent following treatment to photoreactivation repair. All zebrafish experiments are performed in accordance to regulatory standards as accepted by the Institutional Animal Care and Use Committee (IACUC) at Ferris State University. This animal care facility is accredited by AAALAC which validates the institution's commitment to the highest standard of animal care and use recognized in this country.

RESEARCH ASSISTANTS

Patrick Bradley, Lab Manager.

Alexandria Casillas, NIH Post-Baccalaureate Fellow.

Rebecca Finder, Zebrafish Habitat Director

Danielle Chambers, Research Assistant

Kimberly Johnson, Research Assistant

Carli Denholm, Research Assistant

Brandon Weiswasser, Research Assistant

Lauren Clements, Research Assistant

Sarah Gilbert, Research Assistant

Katey Williams and Albert Jiao, Math/Science Center High School

REFERENCES

1. Gandini, S., F. Sera, M. S. Cattaruzza, P. Pasquini, O. Picconi, P. Boyle, and C. F. Melchi (2005) Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure. Eur. J. Cancer 41, 45-60.

2. Wang, S. Q., R. Setlow, M. Berwick, D. Polsky, A. A. Marghoob, A. W. Kopf, and R. S. Bart (2001) Ultraviolet A and melanoma: a review. J. Am. Acad. Dermatol. 44, 837-846.

3. De Fabo, E. C., F. P. Noonan, T. Fears, and G. Merlino (2004) Ultraviolet B but not ultraviolet A radiation initiates melanoma. Cancer Res. 64, 6372-6376.

4. Oliveria, S., S. Dusza, and M. Berwick (2001) Issues in the epidemiology of melanoma. Expert. Rev. Anticancer Ther. 1, 453-459.

5. Garland, C. F., F. C. Garland, and E. D. Gorham (2003) Epidemiologic evidence for different roles of ultraviolet A and B radiation in melanoma mortality rates. Ann. Epidemiol. 13, 395-404.

6. Wang, L. E., P. Xiong, S. S. Strom, L. H. Goldberg, J. E. Lee, M. I. Ross, P. F. Mansfield, J. E. Gershenwald, V. G. Prieto, J. N. Cormier, M. Duvic, G. L. Clayman, R. S. Weber, S. M. Lippman, C. I. Amos, M. R. Spitz, and Q. Wei (2005) In vitro sensitivity to ultraviolet B light and skin cancer risk: a case-control analysis. J. Natl. Cancer Inst. 97, 1822-1831.

7. Bruls, W. A., H. Slaper, J. C. van der Leun, and L. Berrens (1984) Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem. Photobiol. 40, 485-494.

8. Bruls, W. A., W. H. van, and J. C. van der Leun (1984) Transmission of UV-radiation through human epidermal layers as a factor influencing the minimal erythema dose. Photochem. Photobiol. 39, 63-67.

9. McMillan, T. J., E. Leatherman, A. Ridley, J. Shorrocks, S. E. Tobi, and J. R. Whiteside (2008) Cellular effects of long wavelength UV light (UVA) in mammalian cells. J. Pharm. Pharmacol. 60, 969-976.

10. Dahle, J., E. ngell-Petersen, H. B. Steen, and J. Moan (2001) Bystander effects in cell death induced by photodynamic treatment UVA radiation and inhibitors of ATP synthesis. Photochem. Photobiol. 73, 378-387.

11. Dahle, J. and E. Kvam (2003) Induction of delayed mutations and chromosomal instability in fibroblasts after UVA-, UVB-, and X-radiation. Cancer Res. 63, 1464-1469.

12. Dahle, J., E. Kvam, and T. Stokke (2005) Bystander effects in UV-induced genomic instability: antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation. J. Carcinog. 4, 11.