Four-day duration of tumor promoter exposure required to transform JB6 promotion-sensitive cells to anchorage independence.

The JB6 mouse epidermal cell lines have been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 1 to 100 ng/ml results in the irreversible acquisition of tumorigenicity in nude mice and anchorage-independent growth. Among the biochemical responses which occur during TPA treatment is a decrease in procollagen synthesis.

Early superoxide dismutase-sensitive event promotes neoplastic transformation in mouse epidermal JB6 cells.

Evidence has been obtained that implicates the generation of reactive oxygen species as an early and critical event in the promotion of neoplastic transformation in mouse JB6 cells. The time courses for specific inhibition by CuZn-superoxide dismutase (CuZn-SOD) of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of neoplastic transformation in JB6 cells and for changes in antioxidant enzyme activities associated with TPA-exposure were examined. The antipromoting effect of CuZn-SOD was found to be critically dependent on the time of addition of CuZn-SOD relative to the start of a 14-day exposure of cells to TPA.

Lifespan extension of basal cell nevus syndrome fibroblasts by transfection with mouse pro or v-myc genes.

Dermal fibroblasts from patients with the autosomal dominant cancer-prone disease Basal Cell Nevus Syndrome (BCNS) exhibit a serum dependence, anchorage dependence and in vitro lifespan (about 20 population doublings or less) similar to those of fibroblasts from normal age-, race- and sex-matched controls. Transfection with v-myc or with an activated mouse pro-I gene (which specifies sensitivity to promotion of neoplastic transformation in JB6 mouse epidermal cells) specifically conferred partial immortality on the BCNS fibroblasts by substantially extending their population doubling levels by more than 19 population doublings. This suggests that either v-myc or pro-I gene can cooperate with BCNS gene(s) to produce an extension of lifespan or partial immortality.

Possible involvement of a lanthanide-sensitive protein kinase C substrate in lanthanide promotion of neoplastic transformation.

The rare earth elements lanthanum and terbium (0.1-1.0 mM), pharmacological analogs of calcium, induced neoplastic transformation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive (P+) and to a lesser extent TPA-resistant (P-) preneoplastic mouse JB6 epidermal cells.

Effect of hyperthermia on combination interferon treatment: enhancement of the antiproliferative activity against murine B-16 melanoma.

Previous studies have evaluated the effects of hyperthermia on the antiproliferative activity of interferon. The activities of all three types of interferon have been shown to be synergistically enhanced by hyperthermic conditions. Further, the antiproliferative activity of interferon has been shown to be synergistically enhanced by combinations of gamma-plus alpha- or beta-interferon.

Extracellular calcium requirement for promotion of transformation in JB6 cells.

Extracellular calcium is required in the induction of neoplastic transformation of preneoplastic mouse JB6 epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Depleting extra-cellular calcium by chelation or by use of commercial calcium-depleted medium inhibited TPA-promoted transformation of promotion-sensitive JB6 cells with a half-maximal inhibition at 1.2 mM calcium.

Effect of hyperthermia on the antiproliferative activities of murine alpha-, beta-, and gamma-interferon: differential enhancement of murine gamma-interferon.

Fever is frequently an important side effect of interferon (IFN) therapy. Studies have shown that culturing interferon-treated cells at elevated temperature heightens the antiproliferative activity of IFN-alpha and IFN-beta. Since IFN-gamma has also been shown to be a potent antiproliferative agent, the effect of elevated temperature on IFN-gamma activity was compared to its effect on IFN-alpha and IFN-beta.

A transforming activity not detected by DNA transfer to NIH 3T3 cells is detected by JB6 mouse epidermal cells.

Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity.

Benzoyl peroxide promotion of transformation of JB6 mouse epidermal cells: inhibition by ganglioside GT but not retinoic acid.

Benzoyl peroxide (BzPo), a free radical generator with tumor promoting activity on mouse skin, is shown to promote neoplastic transformation of JB6 mouse epidermal cells in vitro. Repeated exposures to BzPo are required to readily detect promotion of transformation of JB6 cells. Markedly reduced net synthesis of the major epidermal ganglioside, trisialoganglioside GT1b, (GT) occurs with BzPo treatment as with other tumor promoters active in this system.

Role of reactive oxygen in tumor promotion: implication of superoxide anion in promotion of neoplastic transformation in JB-6 cells by TPA.

The role of reactive oxygen (RO) in the promotion of neoplastic transformation of JB6 mouse epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated using inhibitors of RO itself or RO generating systems of seven different types. Bovine erythrocyte CuZn superoxide dismutase (SOD) maximally decreased anchorage-independent (AI) colony induction by TPA in semi-solid agar in a dose-dependent manner to 10% of TPA control level. The inhibitory effect was specifically on induction of transformation, not expression of transformation.

Transformation and tumor promoter sensitive phosphoproteins in JB-6 mouse epidermal cells: one is also sensitive to heat stress.

JB-6 mouse epidermal cells undergo irreversible transformation when exposed to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Phosphoprotein changes related to transformation were sought in four tumor cell lines related to JB-6 cells. Two dimensional polyacrylamide gel electrophoresis showed altered abundances of five phosphoproteins in the tumor cell lines compared with five untransformed clones.

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.

Transfer of sensitivity to tumor promoters by transfection of DNA from sensitive into insensitive mouse JB6 epidermal cells.

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P(+)) and nonpromotable (P(-)) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Mitigation of intestinal cytotoxicity of cisplatin by EDTA in rats.

Cisplatin (cis-dichlorodiammineplatinum-II), an antitumor agent containing platinum, produced acute necrotizing enteritis in rats. This toxic side effect was significantly reduced by oral treatment with CaNa2EDTA. The protective effect of CaNa2EDTA against intestinal cytotoxicity of cisplatin was dose-related.

Hexose uptake as an indicator of JB6 mouse epidermal cell resistance to the mitogenic activity of TPA.

JB6 mouse epidermal cells have been selected for resistance to the tumor-promoting phorbol diester TPA for (1) the plateau density mitogenic (M) response, and (2) the promotion of tumor cell phenotype (P) response. The purpose of this study was to determine the relationship of hexose uptake to the two TPA-dependent processes. Monolayers of JB6 mouse epidermal cells showing one of four different phenotypes (M+P+, M+P-, M-P+, M-P-) were exposed to 60 nM [3H(G)]2-deoxy-D-glucose (2DG) with or without TPA (10 ng/ml) stimulation.

Membrane and genetic events in tumor promotion: studies with promoter resistant variants of JB6 cells.

The derivation and properties of JB6 mouse epidermal clonal cell lines are reviewed and the conclusions that can be drawn from studies with the JB6 mouse epidermal system are summarized. Promoter induced mitogenic stimulation, epidermal growth factor (EGF) 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.

Tumor-promoter-resistant cells lack trisialoganglioside response.

JB6 mouse epidermal cells shift irreversibly to tumor cell phenotype (anchorage independence and tumorigenicity) on treatment with phorbol esters and other tumor promoters. Exposure to phorbol 12-myristate 13-acetate (PMA) decreased the de novo synthesis of trisialoganglioside (GT) in these "promotable" JB6 cells to 5-10% of that of untreated cells. The GT decrease occurred consistently in promotion-sensitive cells and not in promotion-resistant variants.

Phorbol diester and epidermal growth factor receptors in 12-O-tetradecanoylphorbol-13-acetate-resistant and -sensitive mouse epidermal cells.

Several cell variants have been isolated from promotable mouse JB6 epidermal cells which are resistant either to mitogenic stimulation at quiescence or to promotion of anchorage independence by 12-O-tetradecanoylphorbol-13-acetate (TPA). Such resistant variants would be expected to lack one or more steps in the TPA response pathway leading to mitogenesis or promotion of tumor cell phenotype. This report is concerned with determining whether resistance is attributable to lack of receptors for phorbol diesters or epidermal growth factor (EGF, a potential mediator) or to absence of receptor down modulation following ligand binding.

Suppression of natural killing in vitro by monocytes and polymorphonuclear leukocytes: requirement for reactive metabolites of oxygen.

Natural killer cells spontaneously lyse certain tumor cells and may defend against malignancy. We have previously shown that natural killing (NK) by human peripheral blood mononuclear cells (PBMC) is suppressed in vitro by phorbol diester tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We here demonstrate that suppression of NK is mediated by monocytes or polymorphonuclear leukocytes (PMN) and that suppression is dependent on the generation of reactive forms of molecular oxygen (RO), particularly hydrogen peroxide (H2O2).

Natural killing of tumor cells by human peripheral blood cells. Suppression of killing in vitro by tumor-promoting phorbol diesters.

Tumor-promoting phorbol diesters were shown to suppress natural killing in vitro by human peripheral blood mononuclear cells. The inhibitory effect of different phorbol diesters and their analogues correlated with their potency as tumor promoters, the most effective agent being 12-O-tetradecanoylphorbol-13-acetate (TPA). Both peripheral blood cells and targets specifically bound TPA, and natural killing could be inhibited by pretreatment of either cell population with TPA, though this was less effective than direct addition of TPA to the assay.