Lentiviral Vector Transfer of Either Tumstatin or Tum–4 Gene Fails to Inhibit Proliferation of Ocular Choroidal Melanoma and Retinoblastoma Cell Cultures

Abstract

Abstract: : Purpose: To evaluate the effect of a lentiviral vector containing either the full–length Tumstatin or the Tum–4 domain transduced to ocular choroidal melanoma (OCM1) and retinoblastoma (Weri–RB1) cell cultures. Methods: The 735 bp cDNA for full length Tumstatin was cloned into the pHR’CMV vector with a C–terminal six–histidine tag. The 57 bp Tum–4 domain was amplified by PCR using Tumstatin as the template and also cloned into the pHR’CMV vector with a C–terminal six–histidine tag. Lentivirus particles were produced by standard 3–plasmid co–transfection into 293T cells, collected and concentrated by ultracentrifugation. RT–PCR from transduced human microvascular endothelial cells (HMVEC) was used to test the ability of the virus to infect and express the Tumstatin and Tum–4 genes. OCM1 and Weri–RB1 cells were cultured using standard cell culture techniques. In 96–well plates, cell proliferation was determined by cell count and by cell proliferation reagent WST–1 on days 2, 4, 6, 9, and 11 after the second transduction. Western blotting with anti–his–tag antibody was performed to detect Tumstatin and Tum–4 proteins in the cell lysate and the media from transfected and transduced OCM1 cells. Results: RT–PCR from transduced HMVEC confirmed the mRNA transcription of Tumstatin and Tum–4 transgenes. The transduction with these two lentiviruses failed to inhibit OCM1 and Weri–RB1 cell proliferation by the WST–1 assays at every time point studied. Moreover, we were unable to detect Tumstatin and Tum–4 proteins in both the cell lysate and the media from transfected and transduced OCM1 cells by Western blot analysis. Conclusions: These findings suggest that lentivirus–mediated gene transfer of either Tumstatin or Tum–4 is insufficient to express functioning products able to inhibit OCM1 and Weri–RB1 cell proliferation.

Keywords: gene transfer/gene therapy • retinoblastoma • melanoma