Transfer of the E6 and E7 genes of human papillomavirus type 18 into human epithelial cells via recombinant retrovirus infection.
A retroviral vector carrying the E6 and E7 genes of HPV type 18 was transfected into a packaging cell line, Ampho psi 2. Thirteen recombinant viruses carrying the E6 and E7 genes were obtained. The titers of these recombinant viruses were estimated by infecting BALB/c3T3 cells and then counting the number of G418r colonies. Presence of HPV E6/E7 genes was confirmed by the PCR method and sequence-specific primers. The expression of E7 gene was examined by RT-PCR method. Results showed that the titers were ranged between 0.2 and 1.2 x 10(3) CFU/ml and the E7 transcripts were detected in all 13 cell clones. These E6 and E7-containing cell clones were able to grow in soft agar, indicating the E6/E7 delivered by the recombinant retroviruses retained their transformation function. These recombinant viruses were then used to infect human NPC cell lines, NPC-TW076 and -TW039 and cell clones resistant to G418 were obtained. Using Western blot analysis and HPV type 18 E6-specific monoclonal antibody, HPV-CIP5, these cells were shown to contain a protein with a molecular mass of 18 kDa. Our data indicated that the HPV E6/E7-containing recombinant retroviruses were capable of infecting human cell lines. The potential of using these recombinant retroviruses to immortalize human primary epithelial cells was discussed.
Wu
C J
CJ
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
Chang
K S
KS
Chang
Y S
YS
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000821:virology; D006801:Humans; D051379:Mice; D009303:Nasopharyngeal Neoplasms / Q000821:virology*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral