Assessment of Lentiviral Delivered Tumstatin and Tum–4, novel Anti–Angiogenic and Anti–Tumor proteins, on the Inhibition of Melanoma and Retinoblastoma Cell Proliferation

Abstract

Abstract: : Purpose: Tumstatin is a 245 amino acid cleaved product of the carboxy terminal non–collagenous end (NC1) of the α3 chain of type IV collagen, and contains separate anti–angiogenic (Tum–5) and anti–tumor (Tum–4) domains. Since intraocular growth of tumors is dependent upon both tumor cell proliferation and tumor vascularization, tumstatin is likely to be bifunctional in its ability to inhibit melanoma or retinoblastoma growth. We aim to examine the effect on proliferation of melanoma and retinoblastoma cells transduced with a lentiviral vector containing either the full–length tumstatin or the Tum–4 domain. Methods: The cDNA for full length Tumstatin was cloned into the pHR’CMV–IRES–eGFP vector. The 57bp Tum–4 domain was amplified by PCR using Tumstatin as the template and also cloned into the pHR’CMV–IRES–eGFP vector. Lentivirus particles were produced by standard 3 plasmid co–transfection into 293T cells, collected and concentrated by ultracentrifugation. RT–PCR from transduced microvascular endothelial cells was used to test the ability of the virus to infect and express the Tumstatin and Tum–4 genes. Melanoma and retinoblastoma cells (ATCC) were cultured using standard cell culture techniques. Cell proliferation was determined by cell count and by the MTT method. Results: Full length Tumstatin and the Tum–4 domain were successfully cloned into the viral backbone. Viral particles capable of expressing the mRNA for the Tumstatin and Tum–4 transgenes were confirmed by RT–PCR from transduced microvascular endothelial cells. Calculation of differences in cell proliferation between transduced and non–transduced cells is underway. Conclusions: Tumstatin is a unique molecule with bifunctional properties. This molecule not only induces endothelial cell apoptosis but can also inhibit tumor cells. A lentiviral reagent containing this gene may prove useful in the treatment of intraocular tumors as well as ocular neovascular disease.

Keywords: gene transfer/gene therapy • melanoma • retinoblastoma