Low-level transforming activity of an activated Ras gene under the control of a vaccinia virus p40 promoter is abrogated by truncation of the Ras cDNA.
Many human cancers have been shown to contain activated forms of the Ras proto-oncogene. Mutations comprising amino acid changes at codons 12, 13 and 61 therefore represent unique targets for cancer immunotherapy. Recombinant Vaccinia viruses encoding point mutated Ras oncogenes have raised issues concerning the safety and transforming ability of these recombinant vaccines. Vaccinia virus, a representative of the orthopox virus genus, is a large DNA virus that is cytopathogenic and that replicates in the cytoplasm of the infected cell. However, it remains unclear whether orthopox viruses are capable of genetic interactions with infected cells. Our studies show that DNA isolated from cells infected with a recombinant Vaccinia virus expressing mutated Ras constituted a poor reagent for transfection into NIH3T3 cells for transformation analysis. Stable integration of a recombinant Vaccinia virus expressing mutant Ras DNA was not detected in recipient cells. This study also demonstrates that the crossover plasmids used to generate the recombinant virus where the activated Ras gene is under the control of a Vaccinia virus early promoter had low but detectable transforming efficiency in the NIH3T3 transformation assay. Analysis of the transfected cells indicated that Ras transcription was initiated upstream of the Vaccinia virus promoter. The introduction of wobble mutations as well as the truncation of the Ras protein removed the transforming capabilities of the crossover vector. This study demonstrates the potential problems and solutions in the use of point mutated oncogenes in live vectors for cancer vaccine development.
Cutler
M L
ML
Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Shupert
W L
WL
Schlom
J
J
Kantor
J
J
D016475:3T3 Cells; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002472:Cell Transformation, Viral / Q000235:genetics; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*
D003062:Codon; D018076:DNA, Complementary; D004279:DNA, Viral; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)