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Transfer by pro Gene Transfection of Tumor Promoter-sensitive Phenotype to Promotion-insensitive JB6 Cells

Cell Biology Section, Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701-1013

Transfection of activated promotion sensitivity genes (pro genes) confers on insensitive (P^-) cells susceptibility to induction of anchorage-independent growth by tumor-promoting phorbol esters. Promotion-sensitive (P⁺) JB6 cell variants, from which activated pro-1 and pro-2 were cloned, respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and various nonphorbol tumor promoters with anchorage-independent transformation that is irreversible 60–80% of the time. Anchorage-independent (Tx) clonal lines derived from these TPA-induced agar colonies were also tumorigenic in nude mice. This report has addressed the question of whether the phenotypes associated with parental P⁺ cells are transferred by transfection of activated pro-1 and pro-2. Clonal lines were established after transfection of JB6 P^- cells with activated pro-1 or pro-2, induction of anchorage-independent colony formation by TPA, and growth of individual agar colonies to yield clonal transfectant lines. The lines so derived from transfected populations included Tx, P⁺, and P^- lines, reflecting irreversible neoplastic transformation and greater and lesser degrees of preneoplastic progression, respectively. The anchorage-independent transfectants were found to be tumorigenic. Since untransfected P^- cells subjected to the same single-cycle TPA treatment and cloning in agar yielded no anchorage-independent and few P⁺ transfectants, the appearance of P⁺ and Tx transfectants after pro-1 and pro-2 transfection in therefore likely to be due to the transfected pro genes. Indirect assay of pro gene uptake by quick-blot hybridization of transfectant cell DNA with the vectors into which pro genes had been cloned confirmed the association of transferred P⁺ and Tx phenotypes with the presence of the transfected DNA. Finally, assay of the sensitivity of P⁺ pro-1 and pro-2 transfectants to transformation by TPA at various concentrations showed that transfection with pro-1 or pro-2 conferred about equal responses that were somewhat lower than those observed with parental P⁺ controls. Taken together these data indicate that promotion-insensitive JB6 cells need only an activated pro gene and TPA exposure to become neoplastically transformed.

¹ To whom requests for reprints should be addressed.

² Present address: Bionetics Research Inc., Frederick Cancer Research Facility, Frederick, MD 21701.

³ Present address: Biology Research Faculty and Facility, 10075-20 Tyler Place, Ijamsville, MD 21754.

Received 4/13/87. Revised 9/28/87. Accepted 11/18/87.

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