Introduction

1. IL-8 and Gro-α factors have the ability to shift dormant MOLT-3 cells into an aggressive or active state Amanda Zych Review of Literature Introduction • Without angiogenesis tumors do not grow (Masuya 2001) • Vascular endothelial growth factor (VEGF) is known to induce angiogenesis (Watson 2000) • Cancer patients are known to have more VEGF within the white blood cells and platelets (Toi 2001) • VEGF is known to be associated with the following types of cancers: melanoma, ovarian carcinoma, prostate carcinoma, and colon carcinoma (Lurje 2008) • VEGF is also known to cause cells to become non-dormant (Montaldo 2000) • IL-8 and VEGF are factors that are known to assist each other • Another factor, Gro-α is an analog of IL-8 and therefore shows some of the same characteristics as IL-8 (Baggiolini 1994) • The structure of Gro-α is similar to IL-8 (Geiser 1993) • Kaposi sarcoma, or KS is a tumor that has the ability to overcome inhibition to different cells they come into contact with (Montaldo 2000) • KS-IMM cells, or ‘feeder’ cells are inducers of angiogenesis that allow cells to become angiogenic and therefore non-dormant (Indraccolo 2006) • KS-IMM has been found to produce IL-8 (Albini 1997) • MOLT-3 cells, or T lymphoblastic leukemia are resistant to apoptosis (Indraccolo 2006) • When KS-IMM and MOLT-3 cells were added together angiogenesis increased (Indraccolo 2006) • The RT-PCR analysis of the coinjection of MOLT-3 and KS-IMM showed that IL-8 and Gro-α were positive for KS-IMM but not MOLT-3 (Indraccolo 2006) • The coinjection of the two types of cells were needed; without KS-IMM, the MOLT-3 cells remained dormant (Indraccolo 2006) • A tumor cell is thought to stay in a state of dormancy because the tumor cell cannot perform angiogenesis:the formation of blood vessels from preexisting ones • A cell’s ability to leave a state of dormancy is thought to happen because of factors or inducers that play an active role in causing a cell to perform angiogenesis • An inducer such as Vascular Endothelial Growth Factor, or VEGF is known to cause this switch in angiogenesis • Short-term angiogenesis by an inducer is a reason there is an escape from tumor dormancy • VEGF factor is similar to KS-IMM cells by causing cells to become active and aggressive • KS or Kaposi sarcoma is a tumor that can overcome the inhibition to different cells it comes into contact with • Itactivates other cells by inducing angiogenesis and therefore become non-dormant • VEGF is found in KS-IMM and MOLT-3 cells, however, it is weakly present in MOLT-3 cells so the cells remain dormant. • It is uncertain whether the VEGF factor is the only inducer or if there are others, with which many papers have suggested, such as Interleukin-8 (IL-8) and (Gro-α). Predicted Results Methods Grow MOLT-3 and KS-IMM cells Flowchart Treatment with different gene factors After 6 days isolate 100ng/mL RNA from treatments: IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF, IL-8/Gro-α/VEGF, control no treatment (-), and control treatment VEGF (+) After 6 days (constant with paper) isolate 100mg/mL Protein from treatments: IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF, IL-8/Gro-α/VEGF, control no treatment (-), and control treatment VEGF (+) Conclusions Figure 1. shows the results of the RT-PCR with the genes (on left side): IL-8, Gro-α, MMP-1, MMP-2, and Ang-2 within the treatments (on top) of IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF, IL-8/Gro-α/VEGF, control no treatment (-), and control treatment VEGF (+) in an electrophoresis gel with 1.5% agarose gel . All the treatments increase with the genes except for the negative control with no treatment • IL-8 and Gro-α factors should be shown to change the dormancy of tumor cells. • These factors would therefore be angiogenic nature • They would show an increase expression of genes and protein levels. • As a results of their increase expression, Il-8 and Gro-α activate MOLT-3 cells into an active, aggressive state • With these cells in left in their dormant state by these factors then the relapse of cancer would not occur • Future Aim/Treatment: • A future study would be to take this a step further by using an antibody to keep the factors from turning on and aiding the IL-8 and Gro-α • Treat an animal with the IL-8 antibody, IL-33 to detect Il-8 and test if it is possible to stop or inactivate IL-8 • Do the same for Gro-α only use antibody Alpha-4 • The animals would then be dissected to see if the tumor cells remained inactive when IL-8 and Gro-α were inactive Extract out proteins from treatments using Western Blot method Run RT-PCR using mRNA from treatments against the genes IL-8, Gro-α, MMP-1, MMP-2, and Ang-2 • Figure 3. • his electrophoresis gel was taken from an RT-PCR (PNAS, Indraccolo, 2006) • MOLT-3 cells were coinjected with irradiated KS-IMM cells. • The MOLT-3 cells were shown to weakly express VEGF cells and were negative for all other factors. • However, when MOLT-3 and KS-IMM cells were coinjected together they showed a presence of VEGF, bFGF, Gro-α, IL-8, GM-CSF, SCF, IL-15, and β-actin (which is the control for both cells). Analyze bands and compare results to + (VEGF treatment) and – control (no treatment) Literature Cited Albini, Adriana, Paglieri, Isabella, Orengo, Giorgia, Carlone, Sebastiano, Aluigi, Maria Grazia, DeMarchi, Roberto, Matteucci, Cristian, Mantovani, Alberto, Carozzi, Franca, Donini, Silivia, and Benelli, Roberto. 1997 May 11. The β-core fragment of human chorionic gonodotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line. AIDS. 11, 713-726. Baggiolini, Marco, Lotetscher, Pius, and Moser, Bernhard. 1994 November 23. Interleukin-8 and the Chemokine Family. International Journal of Immunopharmacology. 17, 103-108. Geiser, Thomas, Dewald, Beatrice, Ehrengruber, Markus U., Clark-Lewis, Ian, and Baggiolini, Marco. 1993. The Interleukin- 8-related Chemotactic Cytokines Gro-α, Gro-β, and Gro-γ Activate Human Neutrophil and Basophil Leukocytes. The Journal of Biological Chemistry. 268, 15419-15424. Indraccolo, Stefano, Stievano, Laura, Minuzzo, Sonia, Tosella, Valeria, Esposito, Giovanni, Piovan, Erich, Zamarchi, Rita, Chieco-Bianchi, Luigi, and Amadori, Alberto. 2006 March 14. Interruption of tumor dormancy by a transient angiogenic burst within the tumor microrenvironment. 103, 4216-4221. Available from: www.pnas.org/cgi/doi/10.1073/pnas.0506200103. Lurje, G., Zhang, W., Schultheis, A. M., Yang, D., Groshen, S., Hendifar, A. E., Husain, H., Gordon, M. A., Nagashima, F., Chang, H. M., and Lenz, H.-J. 2008 June 11. Polymorphisms in VEGF and IL-8 predict tumor recurrence in stage III colon cancer. Annuals of oncology. 7, 1734-1741. Masuya, Daiki , Huang, Cheng-long, Liu, Dage, Kameyama, Kotaro, Hayashi, Elichi, Yamauchi, Akira, Kobayashi, Shoji, Haba, Reiji, and Yokomise, Hiroyasu. 2001 August 17. The Intratumoral Expression of Vascular Endothelial Growth Factor and Interleukin-8 Associated with Angiogenesis in Nonsmall Cell Lung Carcinoma Patients. Cancer. 92, 2628-2638. Montaldo, Fabrizio, Maffe, Antonella, Morini, Monica, Noonan, Douglas, Giordano, Silvia, Albini, Adriana, and Prat, Maria. 2000 August. Expression of Functional Tyrosine Kinases on Immortalized Kaposi's Sarcoma Cells. Journal of Cellular Physiology. 18, 246-254. Toi, Masakazu, Matsumoto, Tomoe, and Bando, Hiroko. 2001 November. Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications. The Lancet Oncology. 2, 667-673. Watson, Carolyn J., Webb, Nicholas J. A., Bottomley, Martyn J., and Brenchley, Paul E. C. 2000 August 8. Identification of Polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production. Cytokine. 12, 1232-1235. Hypothesis IL-8 and Gro-α affect the ability to shift dormant MOLT-3 cells into an aggressive state. IL-8 and Gro-α were analyzed by gene expression through the following genes: MMP-1, MMP-2, Ang-2, IL-8, and Gro-α using the following treatments: IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF, IL-8/Gro-α/VEGF, control no treatment (-), and control treatment VEGF (+). Then they were analyzed by protein expression using the same genes and treatments with the addition of the antibody IL-33 for IL-8 and the antibody alpha-4 for Gro-α Aims: (1) Determine that gene expression of IL-8 and Gro-α are associated with the angiogenic process (2) Prove that protein expression of IL-8 and Gro-α are associated with the angiogenic process Figure 2. shows the results of the Western Blot with the genes (on left side): IL-8, Gro-α, MMP-1, MMP-2, and Ang-2 within the treatments (on top) of IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF, IL-8/Gro-α/VEGF, control no treatment (-), and control treatment VEGF (+) Acknowledgements I would like to thank Dr. Kaltreider for all his help in making this proposal the best that it can be.