Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.     

Dual activity of pyrrolidine dithiocarbamate on kappaB-dependent gene expression in U937 cells: II. Regulation by tumour necrosis factor-alpha.

In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-kappaB (NF-kappaB) signalling pathway induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-alpha (TNF-alpha)-induced NF-kappaB DNA binding activity but potentiated the effect of TNF-alpha on kappaB-dependent gene expression. The stimulatory effect of PDTC with TNF-alpha was not observed with an HIV-1 LTR reporter construct containing two mutated kappaB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two kappaB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-alpha, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on kappaB-dependent gene expression. Evidence that PDTC induced AP-1 DNA binding and AP-1 reporter gene activity, raised the hypothesis that the effect of PDTC was mediated by an interaction between the AP-1 pathway and p65(RelA). Co-transfection with expression vectors for p65(RelA) and the AP-1 subunits c-Fos and c-Jun resulted in a decrease in the stimulatory effect of PDTC on HIV-1 LTR activity. Co-transfection of p65(RelA) with Tam67, a dominant negative mutant of c-Jun defective in transactivation, stimulated the effect of PDTC on HIV-1 LTR activity. Evidence that the stimulatory effect of Tam67 with PDTC was reduced with c-Jun is consistent with the hypothesis.     

Activator protein-1 mediates induced but not basal epidermal growth factor receptor gene expression

BACKGROUND: The epidermal growth factor receptor (EGFR) is expressed at different levels in many cell types and found overexpressed in many cancers. EGFR expression is increased or decreased in response to extracellular stimuli. We examined the effect of increased c-Jun expression on EGFR promoter activity. MATERIALS AND METHODS: We used DNAse I foot-printing analysis to determine the binding of activator protein 1 (AP-1) to the promoter region. We also used cotransfection experiments and western blotting analysis to determine the effect of AP-1 family members on EGFR expression. RESULTS: AP-1 was able to bind to at least seven sites in the EGFR promoter region. Cotransfection of MCF-7 cells with a c-Jun expression vector and the EGFR promoter reporter resulted in a 7-fold increase in promoter activity. JunB, but not c-fos, also enhanced the EGFR promoter activity. An A-Fos-dominant negative shown to inhibit Jun-dependent transactivation was able to prevent c-Jun induction of the promoter activity, but only slightly decreased the basal activity of the promoter. Furthermore, the A-Fos dominant negative was able to inhibit phorbol ester induction of the EGFR promoter. Examination of EGFR expression of MCF-7 stable cell lines that overexpress c-Jun revealed an increase in EGFR expression. Additionally, a cisplatin-resistant cell line, A2780/CP70, which has an increase in AP-1 activity compared with the parental cell line, A2780, was found to have an increase in EGFR level. CONCLUSIONS: These results indicate that AP-1 can act to increase the expression of EGFR and may play a role in upregulation of EGFR in cancer cells.     

CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.     

SAG/ROC2 E3 ligase regulates skin carcinogenesis by stage-dependent targeting of c-Jun/AP1 and IkappaB-alpha/NF-kappaB

Sensitive to apoptosis gene (SAG)/regulator of cullins-2-Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase regulates cellular functions through ubiquitination and degradation of protein substrates. We report that, when expressed in mouse epidermis driven by the K14 promoter, SAG inhibited TPA-induced c-Jun levels and activator protein-1 (AP-1) activity in both in vitro primary culture, in vivo transgenic mice, and an AP-1- luciferase reporter mouse model. After AP-1 inactivation, epidermal proliferation induced by 7,12-dimethylbenz(a)-anthracene/12-O-tetradecanoylphorbol-13-acetate at the early stage of carcinogenesis was substantially inhibited. Later stage tumor formation was also substantially inhibited with prolonged latency and reduced frequency of tumor formation. Interestingly, SAG expression increased tumor size, not because of accelerated proliferation, but caused by reduced apoptosis resulting, at least in part, from nuclear factor kappaB (NF-kappaB) activation. Thus, SAG, in a manner depending on the availability of F-box proteins, demonstrated early-stage suppression of tumor formation by promoting c-Jun degradation, thereby inhibiting AP-1, and later-stage enhancement of tumor growth, by promoting inhibitor of kappaBalpha degradation to activate NF-kappaB and inhibit apoptosis.