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Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor
L P Li1, P M Schlag, T Blankenstein
1Max-Delbrück-Center for Molecular Medicine, Berlin, FRG.
Abstract:
A 'bottle-neck' for construction of autologous genetically engineered tumor vaccines and characterization of tumor antigens consists in the difficulty of establishing cell lines from human tumor material. We have constructed two retroviruses allowing transient expression of Simian virus 40 large T as an immortalizing agent. The first vector contains the genes for hygromycin and Herpes Simplex Virus thymidinkinase (TK), for positive and negative selection and the gene encoding large T. They are flanked by LoxP sites, the substrate of the bacteriophage recombinase Cre. The second retrovirus contains the genes for the Cre recombinase and puromycin as selection marker. By sequential infection of NIH3T3 cells with the two viruses, we have shown that the newly expressed large T gene can be deleted in a large proportion (> or =90%) of cells by site-specific recombination. Because the deletion included the TK gene, selection with gancyclovir against cells not having undergone recombination was possible. By infection with the large T retrovirus, cell lines could be easily established from mouse primary kidney cells, human fibroblasts, and cells derived from different surgical specimens of breast or colon cancer patients. One breast carcinoma cell line was further analyzed and shown to be of epithelial origin by characteristic markers (cytokeratins, mucin). This cell line grew continuously in culture for more than a year without any indication of a cell crisis. Infection with the cre-puro retrovirus and GCV selection resulted in complete excision of the large T gene as judged from antibody staining. Remarkably, these cells changed morphology and stopped proliferation comparable to the cells obtained from biopsy demonstrating the requirement of large T for growth. Therefore, this approach may facilitate molecular and cellular characterization of human tumors and other cell types where cell culturing is the limiting step, and gene therapy approaches involving autologous tumor cells.
Insights
Establishing human tumor cell lines for cancer vaccines is difficult. Researchers developed a novel retroviral system for transiently expressing Simian virus 40 large T antigen, enabling cell line creation and subsequent removal for tumor characterization.
Area of Science:
- Molecular Biology
- Oncology
- Virology
Background:
- Establishing primary human tumor cell lines for research is challenging.
- This limitation hinders the development of autologous tumor vaccines and antigen characterization.
- Efficient cell line generation is crucial for advancing cancer research and therapy.
Purpose of the Study:
- To develop a novel retroviral system for efficient immortalization and subsequent de-immortalization of human tumor cells.
- To overcome the bottleneck in establishing and characterizing primary human tumor cell lines.
- To facilitate the construction of autologous genetically engineered tumor vaccines.
Main Methods:
- Construction of two retroviral vectors for transient expression of Simian virus 40 large T antigen and Cre recombinase.
- Sequential infection of various cell types, including human tumor samples, with the retroviruses.
- Utilized site-specific recombination (LoxP/Cre system) for selective deletion of the large T antigen gene.
- Employed positive (hygromycin) and negative (gancyclovir) selection strategies.
- Characterized established cell lines using epithelial markers (cytokeratins, mucin).
Main Results:
- Successfully established continuous cell lines from mouse kidney, human fibroblasts, breast, and colon cancer specimens.
- Demonstrated efficient (>90%) deletion of the large T antigen gene via site-specific recombination.
- Showcased that removal of large T antigen leads to cessation of proliferation, mimicking primary tumor cells.
- Confirmed epithelial origin of a breast carcinoma cell line through marker analysis.
Conclusions:
- The developed retroviral system effectively overcomes limitations in establishing and culturing human tumor cell lines.
- This approach facilitates the molecular and cellular characterization of tumors.
- Enables advancements in gene therapy and the development of autologous tumor vaccines.