hygromycin-a and Colonic-Neoplasms

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.

Topics: 3T3 Cells; Animals; Antigens, Polyomavirus Transforming; Breast Neoplasms; Carcinoma; Cell Division; Cell Line, Transformed; Cinnamates; Colonic Neoplasms; Gene Deletion; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Humans; Hygromycin B; Integrases; Mice; Neoplasms; Puromycin; Recombination, Genetic; Retroviridae; Simian virus 40; Simplexvirus; Thymidine Kinase; Tumor Cells, Cultured; Viral Proteins

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Topics: beta-Galactosidase; Cinnamates; Colonic Neoplasms; DNA Damage; DNA, Neoplasm; DNA, Viral; Genes, Reporter; Humans; Hygromycin B; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Plasmids; Repressor Proteins; Transfection; Tumor Cells, Cultured; Virus Integration