Differential transformation efficiency but not AP-1 induction under anchorage-dependent and -independent conditions.

The JB6 mouse epidermal cell system has been extensively used as an in vitro model for the study of tumor promotion. The present study aimed to assess the relevance of monolayer measurements to the process of transformation, which is induced more efficiently under anchorage-independent (AI) conditions. Although it would be ideal to use identical conditions for studying tumor promoter-induced transformation and biochemical and molecular events that may cause the process, it is not feasible in the case of soft agar conditions because cells cannot be readily recovered. In the present report, we used liquid medium over agar as an AI condition that permitted efficient recovery of cells. Responses to tumor promoter have been compared with those in monolayer and semisolid agar. Results indicate that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced similar magnitude concentration-dependent transformation of JB6 cells under both of the AI conditions, namely soft agar and over-agar. Under anchorage-dependent (AD) conditions of exposure to TPA, the transformation efficiency was much lower than that seen under AI conditions. Mechanical detachment of monolayer cells after 5-10 days TPA exposure enriched the transformed phenotype. Activator protein 1 transcriptional activity measured at 12 h was induced equally under AD and AI conditions, and thus is not an early limiting event that could explain the lower transformation efficiency seen under AD conditions. To summarize, the over-agar and monolayer assays described in this study can be considered valid for the study of early biochemical and molecular events relevant to the promotion of transformation measured in soft agar.

Comparison of mouse pro-1 and pro-2 transfectants for responses to tumor promoters and antipromoters.

A previous report demonstrated that mouse JB6 cells transformed to promotion sensitive (P+) phenotype by transfection with an activated promotion sensitivity (pro) gene showed both evidence for the presence of the transfected gene and sensitivity to phorbol ester induced transformation similar to that observed in parental P+ cells. In addition, pro-1 and pro-2 transfectants were similar to each other in phorbol ester response. The current report extends these findings to ask whether pro-1 or pro-2 transfectants are also sensitive to promotion of transformation by other classes of tumor promoters such as epidermal growth factor (EGF), lanthanides, and phthalate esters and to inhibition of phorbol ester promoted transformation by several classes of antipromoters. The results showed that both pro-1 and pro-2 transfectants resembled parental P+ cells in sensitivity to promotion of anchorage independent transformation by lanthanides and by diethylhexylphthalate. In addition both pro-1 and pro-2 transfectants showed inhibition of phorbol ester induced transformation by antipromoters ganglioside GT1b, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and forskolin. Thus the pathways implicated by these inducers and inhibitors of transformation appear similar to those implicated for parental P+ cells and similar when controlled by pro-1 or pro-2. The single differential response was that of EGF-induced transformation. pro-2 transfectants but not pro-1 transfectants were sensitive to EGF-induced neoplastic transformation. The nonresponsiveness could not be attributed to lack of EGF receptors since 125I-EGF binding to pro-1 transfectants was similar to that for pro-2 transfectants and parental P+ cells. Thus pro genes transfer responsiveness to a C-kinase mediated promotion of transformation pathway and to putatively non-C kinase pathways triggered by lanthanides or phthalate esters, but not necessarily to an EGF receptor kinase mediated pathway.

Transfer by pro gene transfection of tumor promoter-sensitive phenotype to promotion-insensitive JB6 cells.

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 is 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.

Role of specific membrane and genetic changes in the mechanism of tumour promotion. Studies with promoter-resistant variants.

Several cell culture model systems in current use for studying tumour promotion mechanism are reviewed briefly. The conclusions that can be drawn from studies with the JB6 mouse epidermal system are summarized. Promoter-induced mitogenic stimulation, epidermal growth factor receptor binding and stimulated hexose transport are apparently not required for promotion of neoplastic transformation in JB6 cells by phorbol esters and other promoters. Phorbol ester receptor binding (or protein kinase C activation) and switched-off collagen synthesis may be required, but definitive proof is not available. Decreased cell surface trisialoganglioside synthesis and one or more genes that determine promotion sensitivity appear to distinguish sensitive from resistant cells and to be required for promotion of neoplastic transformation in JB6 cells.

Dissociation of mitogenesis and late-stage promotion of tumor cell phenotype by phorbol esters: mitogen-resistant variants are sensitive to promotion.

The JB-6 mouse epidermal cell line has been used as a model system for studying the mechanism of late-stage promoter-dependent preneoplastic progression. The studies reported here are concerned with determining whether there is a requirement for mitogenic stimulation in promotion of anchorage independence and tumorigenicity in JB-6 cells. Such a requirement would predict that variants selected for 12-O-tetradecanoylphorbol 13-acetate (TPA) mitogen resistance would also be promotion resistant. Promotion-responsive cell lines have been selected for resistance to TPA-induced mitogenic stimulation at plateau density by cotreatment with colchicine and removal of mitogen-responsive colchicine-detached cells. The selected TPA-resistant population of cells showed a mitogenic response that was diminished by a factor of four but showed no diminution in the promotion-of-anchorage-independence response to TPA. Mitogen-resistant clonal lines derived from the selected population fell into three phenotypic classes when assayed in soft agar: (i) anchorage-independent transformants; (ii) variants resistant to promotion of anchorage independence by TPA; and (iii) variants sensitive to promotion by TPA. The existence of the latter class (i.e., the mitogen-resistant promotable variants) indicates a lack of obligatory causal relationship between TPa-induced mitogenesis and late-stage promotion and, thereby suggests that the two events can be dissociated.