RETROVIRUS MEDIATED TRANSFER OF ANTISENSE HUMAN C-MYC GENE INTO HUMAN ESOPHAGEAL CANCER-CELLS SUPPRESSED CELL-PROLIFERATION AND MALIGNANCY

A retroviral vector, called pDAM3, containing the neomycin resistant gene and the antisense human c-myc gene fragment (the third exon and 3'flanking sequence) was constructed. pDAM3 was introduced into amphotropic packaging cells PA317 by the calcium phosphate precipitation method. Several G418-resistant PA317 clones were isolated. The virus titer of these cell lines was determined by infectivity of their culture fluid to NIH/3T3 cells. The highest titer obtained was 8 x 10(5) G418-resistant colony forming units/ml. Clonal and pooled G418-resistant PA317 colonies with high titers were expanded and analyzed by Southern blot for the presence of intact viral sequences. All cell lines were found to harbor the internal sequences of the pDAM3 vector without any rearrangement. Recombinant virus DAM3 infected human esophageal cancer cell line EC8712 efficiently. The DAM3-infected EC8712 (called EC-DAM3) was found to contain the full DAM3 sequence (4.8 kb) by Southern blot analysis. Antisense myc RNA expressed in the EC-DAM3 cell was detected by RNA hybridization. Further studies indicated that [H-3]-thymidine incorporation in EC-DAM3 cells was reduced by 45% in average compared to that in untreated EC8712 cells. Growth rate of EC-DAM3 cells also decreased about 50%. DAM3-infected EC8712 cells lost their ability of forming tumor in nude mice. It thus appeared that the antisense myc gene introduced into EC8712 cells via retrovirus vector was capable of inhibiting cell proliferation and malignancy.