1. The Effects of Glutamine Concentration on the Development of B16 Melanoma Yi Wang Advisor: Dr. Spilatro Introduction Melanoma, widely referred to as the “black cancer”, is commonly found on skin but occasionally on eyes. Both the incidence and the risk relating to death are relatively high. Every year, about 60,000 new cases of melanoma are diagnosed in the United States, and around 48,000 melanoma relating deaths are reported worldwide. The characteristics of melanoma include massive cell growth and abnormal melanin production. Melanin is a pigment with a dark brown to black color and is synthesized from tyrosine within cells. The scheme of melanin synthesis is shown in Scheme 1. One of the major enzymes that catalyzes the series of conversions is tyrosinase. B16 is a murine melanoma cell line and is widely used as a model to study human melanoma. The research of Aubert et al. indicates that the presence of glutamine in medium induces the B16 cell proliferation. 1 This project aimed to investigate the effect of the glutamine concentration in medium on the two major characteristics of B16 melanoma cells – cell proliferation and cell differentiation. The increase in glutamine concentration was expected to induce cell growth. Since cell differentiation is believed to inversely relate to cell proliferation, the higher glutamine concentration was expected to inhibit cell differentiation. Melanin production and tyrosinase activity were used to measure cell differentiation. Results What was the effect of glutamine concentration on growth rate? Cells were cultured in media with glutamine concentrations of 0, 2, 4, and 8mM. In the control group, medium but no cells were added into wells. The growth curves were constructed by plotting the protein concentration vs. time (Figure 1A). In glutamine-free medium, cells detached from the plate. In media with higher glutamine concentrations, there was no significant difference observed in the growth curves. Thus, glutamine concentrations from 0.25 – 1.5mM were then tested (Figure 1B). Within this range, as the glutamine concentration increased, the growth rates were correspondingly higher. A linear correlation was obtained between the growth rate and the glutamine concentration (Figure 1C). Tyrosine L-Dopa Dopachrome Dopaquinone Melanin 1 * 2 Scheme 1. The mechanism for the synthesis of melanin from tyrosine The reactions catalyzed by tyrosinase 1. Hydroxylation 2. Oxidation Materials and Methods Cell Growth Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with different concentrations of glutamine. After seeding the cells on 12- well plates, medium was replaced daily. Cells were lysed in Tris buffer. After being incubated on ice for 10 minutes and centrifuged at 4°C, the supernatants of lysates were used to measure protein concentrations through BCA assays. Melanin Production Cells were cultured in DMEM with different glutamine concentrations for 6 days. Cells were lysed in 1N NaOH solution and incubated at 60°C for 1 hour. 2 After being centrifuged, the melanin concentrations of supernatants were calculated by measuring absorbance at 470nm. 2 The protein concentrations of supernatants were measured by BCA assay. Tyrosinase Activity After 6 days of culture in DMEM with different glutamine concentrations, cell were lysed with RIPA buffer (10mM Tris-HCl, pH 7.5, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl, 1mM EDTA, and 1mM PMSF) and incubated at 4°C for 30 minutes. 3 After being centrifuged, tyrosinase activity of the supernatants was measured using the L-DOPA assay. The L-DOPA solution (50mM phosphate buffer pH 6.8, 0.05% L-DOPA) was mixed with the supernatants and the change in absorbance at 475nm over time was monitored. 2 The supernatants were heat inactivated at 95°C as a control. Figure 1. The effect of glutamine concentration in medium on the growth rate of B16 melanoma cells. In the control group, medium but no cells were added to wells. Protein was extracted from cells and protein concentration was measured by BCA assay. (A,B) Growth Curves. (C) The slopes of the linear portions in growth curves were used as growth rate. Figure 2. The effect of glutamine concentration on melanin production of B16 melanoma cells. Cells were lysed in 1N NaOH. The absorbance of the lysate supernatant at 470nm was used to calculate melanin concentration, and the protein concentration of supernatant was measured by BCA assay. Melanin production was defined as melanin concentration divided by protein concentration. Figure 3. The effect of glutamine concentration on tyrosinase activity of B16 melanoma cells. Cells were lysed in RIPA buffer. The reaction mixture, which contained L-DOPA solution and lysate supernatant, was monitored for absorbance at 475nm. The protein concentration of supernatant was measured using the BCA assay. Specific activity was defined as unit of activity divided by protein concentration. Discussion In B16 melanoma cells, cell growth, melanin production and tyrosinase increased with glutamine concentration, but more slowly above 2 mM glutamine. The similar patterns in Figure 3 and 4 implied that tyrosinase activity was correlated with melanin production in these B16 melanoma cells. The experimental results partially agree with the hypothesis. The increase in the glutamine concentration showed a positive effect on cell growth. However, an increase in glutamine concentration also resulted in a positive effect on melanin production and tyrosinase activity. These results indicate that melanin production and tyrosinase activity might not reflect cell differentiation and might not be a good indicator of metastatic characteristics. Acknowledge I want to thank Dr. Spilatro for all his patient help and advice throughout the project. I also want to thank Dr. Brown for his advice and the capstone classmates for comments on the presentations. Reference 1.Aubert C, Rouge F, Voulot C. 1990. New Variants of the B16 Melanoma: Tumorigenicity and Metasatic Properties Under Different Culture Conditions. Journal of the National Cancer Institute. 82(11): 952-958. 2.Hirata N, Naruto S, Ohguchi K, Akao Y, Nozawa Y, Iinuma M, Matsuda H. 2007. Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells. Bioorganic and Medicinal Chemistry. 15: 4897-4902. What was the effect of glutamine concentration on melanin production? Melanin production was plotted against the glutamine concentration (Figure 2). Melanin production was observed to increase along with the glutamine concentration. Above 2mM, melanin production slowed and leveled off. What was the effect of glutamine concentration on tyrosinase activity? No activity was observed in the control with RIPA buffer only nor in heat inactivated lysate supernatants. The specific activity was plotted against the glutamine concentration inmedium (Figure 4). As the glutamine concentration increased, the tyrosinase activity was also elevated. Beyond 2mM, the tyrosinase activity tended to level off. *