GROWTH-INHIBITION OF HUMAN-PAPILLOMAVIRUS-16 DNA-POSITIVE MOUSE-TUMORBY ANTISENSE RNA TRANSCRIBED FROM U6 PROMOTER

Over 95% of cervical carcinomas are human papillomavirus (HPV) DNA pos itive, and the expression of the E6 and E7 genes is required for the m aintenance of malignant phenotype, Here, antisense sequences targeted against the HPV16 E6 and E7 genes were cloned as U6/antisense chimeric genes under the control of the U6 RNA gene promoter. Cationic liposom e-mediated transfection of these plasmids into human 293 cells and HPV 16 DNA-positive mouse C3 tumor cells were performed, and the chimeric U6/antisense transcripts mere detected. The U6 promoter could express RNA up to 360 nucleotides in length. In a cotransfection study, the E7 antisense plasmid (hut not the sense control) caused the down-regulat ion of E7 gene expression. Similarly, decreased E7 protein levels were observed in the C3 cells that were transfected with E7 antisense plas mid. In vitro growth inhibition was studied by delivering liposome-ant isense plasmid complex to C3 tumor cells. Up to 50% growth inhibition was achieved with antisense plasmids, whereas only about 15% growth in hibition was observed with sense plasmids. Transfection into human cer vical carcinoma C33A cells and mouse melanoma BL6 cells, which contain no HPV DNA, showed no growth inhibition. These results indicate that growth inhibition resulted from specific E6/E7 down-regulation of the tumors or cells. Furthermore, intratumor injection of DNA-liposome com plex containing either E6 or E7 antisense plasmid resulted in signific ant growth inhibition of C3 tumors but not BL6 tumors, grown in a syng eneic mouse model. This promising result indicates that E6/E7 antisens e sequences expressed in the contest of the U6 gene might be useful fo r gene therapy of cervical carcinoma.