Effects of melanoma cells on human umbilical vein endothelial cell gene expression and cytokine release
Monika Burness, Class of 2004
H. Richard Alexander, M.D.
Head, Surgical Metabolism Section
Surgery Branch, National Cancer Institute
National Institutes of Health
Danny R. Welch, Ph.D.
Associate Professor, Pathology
Jake Gittlen Cancer Research Institute
May 29- August 3, 2001
To characterize the effects of melanoma cells on human umbilical vein endothelial cell gene expression and cytokine release
Research suggests that endothelial cells, which comprise the neovasculature of tumors, are intimately involved in tumor growth and development. Folkman asserts that malignant cells and their associated endothelial cells are dynamically related (1); tumors secrete factors that may have effects on endothelial cells, such as cytokines that increase adhesion molecule expression, while endothelial cells influence tumors with cytokines such as chemoattractants (2).
One cytokine that has been extensively studied for its effects on tumors is tumor necrosis factor (TNF). TNF has notable effects on tumors, causing a hemorrhagic necrosis of the tumor neovasculature when administered, resulting in the formation of eschars within the tumor. Although it is highly toxic, and thus not administered systemically, TNF is used in isolated tissue perfusion in conjunction with chemotherapeutic agents (3, 4). The effects on tumors are thought to be mediated mostly by the procoagulant effects of TNF (mediated by tissue factor), and less so by direct cytopathic effects on the tumor (5).
TNF is secreted by activated mononuclear phagocytes, and also by many other cell types, including tumor (6). It can increase expression of a number of different proteins by endothelial cells; TNF alone or in combination with other cytokines has been shown to induce endothelial cell secretion of many cytokines, including IL-1, IL-5, IL-6, IL-8, and IL-11 (2, 6). In addition, tissue factor is more highly expressed after treatment of endothelial cells with TNF (7)
While TNF has direct actions on endothelial cells, other cytokines act by increasing the sensitivity of the cells to TNF. It has been shown that endothelial cell monocyte-activating polypeptide II (EMAP-II), a cytokine secreted by tumors, up-regulates the expression of the TNF receptor 1 (TNF-R1) on endothelial cells (8). (Two different receptors for TNF are expressed on various cells; TNF-R1 is thought to mediate most of the vascular effects of TNF). EMAP-II also induces dose-dependent apoptosis in endothelial cells (5).
This project seeks to characterize the effects, at both the levels of the gene and the cell, of tumors on endothelial cells. An in vitro model of tumor cells and their associated neovasculature will be established via coculture of either a TNF-sensitive or a TNF-resistant tumor line with human umbilical vein endothelial cells (hUVECs). Due to factors such as EMAP-II, it is expected that the endothelial cells will respond differently to the tumor lines. Microarray analysis will be used to evaluate endothelial cell gene expression after coculture with the tumor cells. A cytokine release assay will be performed on hUVECs after coculture and treatment with TNF to assess the affects of the tumors on endothelial cell cytokine production in response to TNF.
Two human melanoma cell lines (1286 and Pmel, primary cell lines from patients treated at the NIH) will be cocultured with human umbilical vein endothelial cells (hUVECs). Coculture will be performed in 6-well plates containing 1µm-pore cell culture inserts. After a 48-hour incubation, the hUVECs will be harvested and the total RNA extracted. mRNA will be amplified (9) and hybridized to a 6.5K microarray. Gene expression of tumor-cocultured endothelial cells will be analyzed versus a control of hUVECs cocultured with hUVECs and versus other tumor-cocultures.
For the cytokine release assay, hUVECs will be cocultured with tumor lines as described above (or, alternately, hUVECs will be bathed in a tumor-conditioned medium), then treated with TNF. The cells will be assayed for cytokine secretion at 0, 1, 4, and 8 hours after treatment. Enzyme linked immunosorbent assays (ELISAs) will be performed to assay for IL-8, IL-6, and TNF-R1 and coagulation analysis will be used to assay for tissue factor.
To verify that the cell lines used are indeed TNF sensitive or resistant (1286 and Pmel, respectively), TNF sensitivity will be assessed in vivo. Female athymic nude mice will be inoculated subcutaneously with 2x106 tumor cells suspended in HBSS (200µl). Mice will be treated with 10 µg TNF or saline via direct tail vein injection when tumors are >10 mm diameter in size. Subsequent tumor regression, which is expected to correlate with TNF sensitivity, will be assessed in the groups receiving TNF as compared to control groups.
- Review literature on effects of cytokines on tumor and endothelial cells, and effects of tumors on endothelial cells
- Carry out experiments as outlined in Methods section
- Prepare draft and final MSR paper
- Provide guidance in project development
- Provide access to necessary equipment and reagents
- Review draft and final MSR paper
1. Folkman, J. (1997) Tumor Angiogenesis. Cancer Medicine, 4th edition, volume 1:181.
2. Krishnaswamy G, Kelley J, Yerra L, Smith JK, Chi DS. (1999) Human Endothelium as a Source of Multifunctional Cytokines: Molecular Regulation and Possible Role in Human Disease. Journal of Interferon and Cytokine Research 19(2): 91-104.
3. Alexander HR Jr, Bartlett DL, Libutti SK. (1998) Isolated hepatic perfusion: a potentially effective treatment for patients with metastatic or primary cancers confined to the liver. Cancer J Sci Am 4(1):2-11.
4. Fraker DL, Alexander HR, Andrich M, Rosenberg SA. 1996. Treatment of patients with melanoma of the extremity using hyperthermic isolated limb perfusion with melphalan, tumor necrosis factor, and interferon gamma: results of a tumor necrosis factor dose-escalation study. J Clin Oncol 14(2):479-89.
5. Kayton M, Libutti S. (2001) Endothelial Monocyte Activating Polypeptide II (EMAP II) Enhances the Effect of TNF on Tumor-Associated Vasculature. Current Opinion in Investigational Drugs 2(1): 136-138.
6. Encyclopedia of Immunology, 2nd Edition. (1998) Academic Press.
7. Nawroth PP, Stern DM. (1986) Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 163(3):740-5.
8. Berger A, et. al. (2000) Tumour Necrosis Factor Receptor I (p55) is Upregulated on Endothelial Cells by Exposure to the Tumour-derived Cytokine Endothelial Monocyte-Activating Polypeptide II (EMAP-II). Cytokine 12 (7): 992-1000.
9. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM. (2000) High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 18(4):457-9.
- H. Richard Alexander, M.D.
- Danny R. Welch, Ph.D.
- Monika L. Burness
I do x or do not give permission for my proposal to possibly be published on the College of Medicine website.