Transformation nonresponsive cells owe their resistance to lack of p65/nuclear factor-kappaB activation

Clonal variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to tumor promoter-induced neoplastic transformation exhibit differential activator protein-1 (AP-1) response. Transactivation of AP-1 appears to be necessary but not sufficient to promote transformation in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibition of nuclear factor-kappaB (NF-kappaB) when transformation is suppressed, suggesting that NF-kappaB may also play a role in neoplastic transformation. We report here that transactivation of NF-kappaB is inducible by tumor promoters in P+ but not in P- JB6 cells. Inhibition of NF-kappaB using a nondegradable mutant of IkappaBalpha suppressed inducible anchorage-independent transformation of P+ JB6 cells, suggesting that NF-kappaB activation is required for tumor promotion. Induced degradation of IkappaBalpha occurred in both P+ and P- JB6 cells, indicating that failure to activate NF-kappaB in P- JB6 cells cannot be attributed to failure to degrade IkappaBalpha. Slightly higher levels of nuclear p65 were seen in P+ than in P- JB6 cells. The p65-specific DNA binding activity was also higher in P+ cells upon induction by tumor necrosis factor-alpha, suggesting that differential NF-kappaB activation may be attributable to changes in p65 activity. Transactivation of p65 protein was substantially higher in P+ than in P- JB6 cells, as determined by assay of Gal4-p65 fusion constructs. Thus activated, p65 may be a limiting factor for NF-kappaB activation and transformation responses. Stable expression of p65 in P- JB6 cells conferred not only inducible NF-kappaB and AP-1 activation but also transformation response to tumor promoters. Therefore, p65/NF-kappaB appears to be not only necessary for but also sufficient to confer tumor promotion response. Although stable expression of p65 in P- cells produced p65 increases in whole cell extracts, only the transfectants exhibiting increased nuclear p65 showed transformation response. Thus, elevation of nuclear p65 appears to be a necessary step for a transformation response. The P-/p65 transfectants showing acquired transformation response also showed elevated p65-specific transactivation response, thus recapitulating the NF-kappaB phenotypes seen in P+ cells. Expression of a transactivation-deficient mutant of Jun or dominant-negative extracellular signal-regulated kinase suppressed both AP-1 activation and p65-specific transactivation in JB6 cells, suggesting that AP-1 activity is needed for p65 transactivation and consequently for NF-kappaB activation. Thus, the transformation nonresponsive P- JB6 cells owe their resistance to lack of NF-kappaB activation and p65 transactivation that appears in turn to be attributable to insufficient AP-1 activation.     

Two novel glycosides from the fruits of Morinda citrifolia (noni) inhibit AP-1 transactivation and cell transformation in the mouse epidermal JB6 cell line

The fruit juice of Morinda citrifolia (noni), a plant originally grown in the Hawaiian and Tahitian islands, has long been used by islanders to treat diseases, including cancer. Two novel glycosides, 6-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-glucopyranose and asperulosidic acid, extracted from the juice of noni fruits, were used to examine their effects on 12-O-tedtradecanoylphorbol-13-acetate (TPA)- and epidermal growth factor (EGF)-induced AP-1 transactivation and cell transformation in mouse epidermal JB6 cells. The results indicated that both compounds were effective in suppressing TPA- or EGF-induced cell transformation and associated AP-1 activity. TPA- or EGF-induced phosphorylation of c-Jun, but not extracellular signal-regulated kinases or p38 kinases, was also blocked by the compounds, indicating that c-Jun N-terminal kinases were critical in mediating TPA- or EGF-induced AP-1 activity and subsequent cell transformation in JB6 cells.     

Involvement of the Akt/mTOR pathway on EGF-induced cell transformation

Our previous study demonstrated that phosphatidylinositol 3-kinase (PI3K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells.     

Release of fibronectin is linked to tumor promotion: response of promotable and non-promotable clones of a mouse epidermal cell line

By use of indirect immunofluorescence technique and enzyme-linkedimmunosorbent assay we show that JB 6 mouse epidermal cellshave cell surface fibronectin (FN) and release FN into the culturemedium. The addition of 10^–8 M 12-O-tetradecanoylphorbol-13-acetate(TPA) to promotable clones caused a 2-fold enhancement of theFN release over solvent control. On the other hand, in non-promotableclones, TPA in concentrations of 10^–8 M or 10^–7M did not cause increased FN release. Mezerein, a non-phorbolditerpene and second-stage tumor promoter was also found tobe active in causing enhanced FN release in promotable but notin non-promotable clones. The vitamin A derivative retinoicacid (RA) antagonized the TPA-caused FN-release in promotableclones. RA had, however, no effect on the basic release patterns,when given alone or given to nonpromotable clones together withTPA. These results suggest that the increased release of FNmay be a required event for promotion to transformation. Ourview is derived from the observation that promotable clonesof the JB 6 cell line release increased amounts of FN into theirmedium upon promoter exposure while non-promotable clones areunaffected.     

Retinoic acid and beta-carotene inhibit fibronectin synthesis and release by fibroblasts; antagonism to phorbol ester

Previous work from our and other laboratories has shown thattumor promoters stimulated the loss of fibronectin (FN) fromthe cell surface of fibroblasts in culture; retinoic acid (RA)appeared to counteract this loss. We have now studied the actionof RA and carotenoids on FN synthesis and release. Using mousefibroblasts (C3H/1OT cells), we found that RA inhibited releaseof FN into the medium in a time- and concentration-dependentmanner (e.g. 90% inhibition in 48 h with 1 x 10-⁶ M RA). RAcaused inhibition of synthesis, as well as a time- and concentration-dependentdecrease in FN mRNA. A second phenomenon we observed was thegreatly increased binding of FN to the surface of the cells,both in dimeric and multimeric forms, caused by RA treatment.RA produced a striking inhibition of the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated FN release from the cellsurface usually associated with tumor promotion. We postulatethat the combined action of RA in causing decreased FN synthesisand increased FN binding to the cell surface is the reason forthe apparent antagonism of RA to the TPA stimulated releaseof FN. Surprisingly, beta-carotene (BC) and canthaxanthin (anon-provitamin A carotenoid) also inhibited the release of FNfrom these cells. The action of BC was specific, in that anantioxidant carotenold (trans-methyl bixin) and lycopene wereinactive. BC also inhibited FN synthesis and thus inhibitedthe TPA-stlmulated release of FN, similar to RA, but to a lesserextent. BC had no effect on the binding of FN to the cell surface.     

c-jun and multistage carcinogenesis: Association of overexpression of introduced c-jun with progression toward a neoplastic endpoint in mouse JB6 cells sensitive to tumor promoter-induced transformation

Tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) induce neoplastic transformation, elevated c-jun protein expression, and activator protein-1 (AP-1)-dependent gene expression in JB6 mouse epidermal cells sensitive to tumor promoters (clone 415a P+ cells). In contrast, JB6 cells resistant to tumor promoter-induced transformation (clone 307b P- cells) exhibit a greatly reduced TPA or EGF inducible c-jun expression and AP-1 activity. We have recently shown that induced AP-1 is necessary for tumor promoter-induced transformation of P+ cells because introduction of a dominant negative c-jun mutant into P+ cells inhibits both AP-1 dependent transactivation and the transformation response to tumor promoter. The intent of the investigation presented here was to test the hypothesis that elevation of AP-1 activity is sufficient to cause progression to the P+ phenotype in P-cells or to the transformed phenotype in P+ cells. Clonally derived P+ and P- recipient cells transfected with a human c-jun expression construct and overexpressing c-jun protein were tested for progression by assaying for constitutive or inducible anchorage independent phenotype and nude-mouse tumorigenicity. Overexpression of c-jun did not produce progression in P- cells but did increase the probability of progression in P+ cells (two of five transfectant cell lines progressed to the tumor phenotype). In addition, c-jun overexpression did not increase AP-1 activity in any of the P-/c-jun transfectants or in the two of five P+/c-jun transfectants that acquired the transformed phenotype. The P+/c-jun transfectants that showed elevated AP-1 activity did not progress to the tumor phenotype, demonstrating that an increase in AP-1 activity is insufficient for this progression. Since P+-to-tumor phenotype progression occurred in cells overexpressing c-jun but not AP-1, we propose that P+-to-transformed phenotype progression is c-jun dependent and AP-1 independent. © 1995 Wiley-Liss, Inc.     

Inhibitory effects of ascorbic acid on AP-1 activity and transformation of JB6 cells

Ascorbic acid (vitamin C) has been reported as an anti-cancer agent. Previous in vitro studies using either primary cell cultures from cancer patients or tumor cell lines have indicated that different kinds of tumor cells may have different sensitivities to ascorbic acid for the inhibition of tumorigeneity or growth. Because the JB6 mouse epidermal cell system has been used extensively as an in vitro model for the study of tumor promotion and progression, we assessed the effects of ascorbic acid on transformation in JB6 cell variants. The results show that ascorbic acid could inhibit 5.5% to 97.1% of transformation of JB6 P+ cell Cl 41-19 induced by TPA, EGF or EGF + insulin, but has no effect on anchorage-independent growth of JB6 transformed cell A33. Since our previous results indicated that induced AP-1 activity is required for tumor promoter induced-transformation, we tested whether inhibition of tumor promoter-induced transformation by ascorbic acid is through an AP-1 inhibition mechanism. Our results indicated that ascorbic acid inhibited AP-I activity at the same dose range for inhibition of transformation. These results demonstrated that ascorbic acid has inhibitory effects on JB6 cell tumor promoter-induced transformation, but no influence on tumor cell phenotype expression and provided the basic knowledge for understanding of vitamin C action on tumor prevention.     

Synergistic inhibition of phorbol ester-induced transformation of JB6 cells by transforming growth factor-beta and retinoic acid.

Transforming growth factor-beta (TGF-beta) plays a complex role as a regulator of proliferation and differentiation of many cell types, including cells of epithelial origin. In this study, we examined whether TGF-beta, alone or in combination with retinoic acid, was able to inhibit the transformation of the murine epidermal cell line JB6. When treated with phorbol myristate acetate (PMA) and other tumor promoters, the nontumorigenic and anchorage-dependent JB6 cells acquired a tumor phenotype, as shown by the acquisition of tumorigenicity and anchorage independence. We found that TGB-beta inhibited the PMA-induced transformation of a subclone of JB6 cells. The effect of TGF-beta was due to an anti-transformation promoting activity, rather than to generalized growth inhibition, since TGF-beta neither inhibited the growth of monolayer cultures of JB6 cells, nor affected the colony-forming efficiency in agar of the JB6-derived permanently transformed RT101 cell line. TGF-beta was synergistic with retinoic acid, a known anti-tumor promoter, in inhibiting the PMA-induced transformation of JB6 cells. Examination of TGF-beta receptor expression on JB6 cells, by both binding and affinity labeling, showed that treatment with PMA significantly decreased TGF-beta receptor expression while retinoic acid counteracted this effect of PMA, thus suggesting that the synergy between retinoic acid and TGF-beta may be due, at least in part, to modulation of TGF-beta receptor expression. TGF-beta, therefore, appears to function as an incomplete antipromoter whose action can be permitted and/or complemented by retinoic acid. Our data demonstrating that TGF-beta has anti-transformation promoting activity suggest that TGF-beta plays a role in maintaining homeostasis of epithelial cells, not only by regulating cell proliferation and differentiation, but also by counteracting events that lead to malignant transformation.     

Inhibitory effects of caffeine analogues on neoplastic transformation: structure-activity relationship

Some xanthine analogues, including 1,3,7-trimethylxanthine (caffeine) and 1,3-dimethylxanthine (theophylline), have been shown to exert anticancer activities in both cell culture and animal models. The present study focused on the relationship of structure and activity of 50 different caffeine analogues in preventing epidermal growth factor (EGF)-induced malignant transformation of mouse epidermal JB6 promotion-sensitive (P+) Cl41 (JB6 P+) cells. Results indicated that the inhibition of cell transformation by the 1,3,7-trialkylxanthines depends on the number of carbons at the alkyl groups R1 and R3, but not R7. Notably, 1-ethyl-3-hexylxanthine (xanthine 70) was the most effective compound for inhibiting EGF-induced neoplastic transformation among the 50 xanthine analogues tested. The 50% inhibition of cell transformation (ICT(50)) value for xanthine 70 was 48- or 75-fold less than the ICT(50) value of caffeine or theophylline, respectively. Further study revealed that xanthine 70 (5-40 muM) dose dependently inhibited EGF-induced transactivation of activator protein 1 (AP-1), whereas theophylline or caffeine (up to 500 muM) had no effect on AP-1 activity. In addition, xanthine 70 (10 muM) inhibited 12-O-tetradecanoylphorbol-13-acetate- or H-Ras-induced neoplastic transformation in JB6 P+ cells by 78.2 or 62.0%, respectively. Collectively, these results indicated that the number of carbons at R1 and R3 is important for the antitumor-promoting activity of the trialkylxanthines and xanthine 70 might be a promising anticancer agent.     

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

Evidence has been obtained that implicates the generation ofreactive oxygen species as an early and critical event in thepromotion of neoplastic transformation in mouse JB6 cells. Thetime courses for specific inhibition by CuZn-superoxide dismutase(CuZn-SOD) of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducedpromotion of neoplastic transformation in JB6 cells and forchanges in antioxidant enzyme activities associated with TPA-exposurewere examined. The antipromoting effect of CuZn-SOD was foundto be critically dependent on the time of addition of CuZn-SODrelative to the start of a 14-day exposure of cells to TPA.Treatment of JB6 P⁺ Clone 22 and Clone 41 cells with CuZn-SODfor 18 h before, simultaneously with or up to 1 h after exposureto TPA, all inhibited promotion of transformation maximally.Delay of addition of CuZn-SOD by 2 h or more after the startof TPA treatment resulted in a marked decrease in the promotioninhibitory effect. CuZn-SOD added 24 or 48 h after TPA had noeffect on promotion of transformation. Exposure of JB6 cellsto O₂^– (superoxide anion radical) generated exogenouslyby the aerobic xanthine oxidase reaction resulted in promotionof neoplastic transformation that was prevented by concurrentaddition of CuZn-SOD. Taken together these studies provide evidencethat increased superoxide anion generation within the first2 h following TPA exposure is an essential event in promotionof transformation in JB6 cells. Upon TPA exposure, JB6 Clone41 cells exhibited time-specific activity changes in the cellularSOD, glutathione peroxidase (GSH-Px), and catalase. SOD andGSH-Px activities were reduced to 54% and 26% respectively ofbasal levels within 2 h of TPA treatment. GSH-Px activity recoveredto basal levels within 4 h and CuZn-SOD within 48 h. Catalaseactivity was maximally reduced to 50% of basal within 1 h afterTPA treatment and rebounded to greater than basal levels within4 h. It is postulated that a c-kinase-dependent event inducesrapid elevation of superoxide anion following TPA exposure andthat this leads to reduced activity of antioxidant enzymes.Since antipromotion by exogenous CuZn-SOD is effective onlyduring the first 2 h following TPA exposure, this suggests thatthe promotion-relevant O₂^– elevation is transient.