Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells

The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (–123 to –108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.     

HL-60 cells isolated for resistance to vincristine are defective in 12-O-tetradecanoylphorbol-13-acetate induced differentiation and the formation of a functional AP-1 complex.

HL-60 cells isolated for resistance to vincristine are multidrug resistant and defective in the cellular accumulation of drug. Further studies demonstrate that these cells are also highly defective in 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation to macrophages. Analysis of this system demonstrates that certain protooncogenes which may contribute to differentiation are expressed at similar levels in sensitive and resistant cells. Thus, treatment of cells with TPA results in a reduction in the levels of c-myb and c-myc mRNA, while the expression of c-fos, c-jun, and junB is greatly enhanced. Immunoprecipitation experiments also demonstrate a TPA induced increase in the c-jun protein in both sensitive and resistant cells. Gel mobility shift assays show that TPA induces AP-1 formation in sensitive cells, whereas in parallel experiments with the HL-60/Vinc isolate, AP-1 is essentially absent. It has been found, however, that in resistant cells which have reverted to drug sensitivity, the levels of TPA inducible AP-1 is essentially identical to that of sensitive cells. Revertant and sensitive cells differentiate at similar levels in the presence of TPA. These studies therefore demonstrate that HL-60/Vinc cells are defective in the TPA induction of a functional AP-1 complex and that this may account for the inability of these cells to differentiate to macrophages. The molecular basis of the finding that AP-1 is not formed in resistant cells remains to be determined.     

Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism

Ultraviolet (UV) light-induced activation of activator protein-1 (AP-1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP-1 activation. We have, therefore, examined the effects of two well-known inducers of phase II enzymes, sulforaphane (SF) and tert-butylhydroquinone (tBHQ), on UVB-induced AP-1 activation, with an AP-1-luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase-1 (QR-1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1-10 microM SF or 11-45 microM tBHQ for 24 h resulted in up to 1.4-fold and 1.7-fold increase of QR-1 activity, respectively, and up to 1.5-fold and 1.6-fold increases in cellular GSH levels, respectively. AP-1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m(2) of UVB. While the above SF treatment dose-dependently reduced the UVB-induced AP-1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB-induced AP-1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB-induced AP-1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP-1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB-induced AP-1 activation. The inhibitory effect of SF on UVB-induced AP-1 activation appears to be at least partly due to the direct inhibition of AP-1 DNA binding activity. This direct effect of SF on AP-1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP-1 activation.     

Regulation of the transcription factor AP-1 in benign and malignant mouse keratinocyte cells

The mouse benign keratinocyte cell line 308 was previously shown to have less AP-1 DNA binding and transactivation ability than its malignant variant 10Gy5. Because elevated AP-1 activity in 10Gy5 appears to be critical for its malignant phenotype, we were interested in examining the molecular mechanisms that regulate activator protein 1 (AP-1) in this system. In both 308 and 10Gy5 cells, c-fos, fra-2, c-jun, jun B, and jun D were capable of binding to an AP-1 DNA binding site as determined by antibody clearance gel mobility shift assays. By western analysis, jun B steady-state nuclear and cytoplasmic protein levels were reduced in 10Gy5 cells as compared with 308 cells and jun B steady-state mRNA levels were similar in the two cell lines. The rate of jun B protein synthesis was decreased in 10Gy5 cells in comparison with 308 cells. Gel mobility shift experiments indicated that AP-1 inhibitory proteins were not present in the cytoplasm of 308 cells. Oxidation-reduction posttranslational modification was not a major mechanism of AP-1 regulation in these cells as shown by 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) gel mobility shift assay of nuclear protein treated with a reducing agent and by western analysis for ref-1 protein. Overall phosphorylation of AP-1 proteins in 308 and 10Gy5 cells was examined by ³²P orthophosphate labeling and immunoprecipitation. A difference in jun B protein overall phosphorylation was observed in the two cell lines. Our experiments suggest that decreased jun B protein levels may be a mechanism that results in elevated AP-1 activity in malignant 10Gy5 cells. Mol. Carcinog. 18:26–36, 1997. © 1997 Wiley-Liss, Inc.     

Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.     

Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 proto-oncogene, fra-1, and S-phase alterations are mediated via oxidative stress

Crystalline silica has been classified as a group 1 human carcinogen in the lung. However, its mechanisms of action on pulmonary epithelial cells which give rise to lung cancers are unclear. Using a nontransformed alveolar type II epithelial cell line (C10), we show that alpha-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P < or = 0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P < or = 0.05). Addition of tetramethylthiourea inhibited silica-associated increases infra-1 and proportions of cells in S-phase (P < or = .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, ie., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers.     

High-level expression of human c-jun gene causes cellular transformation of chicken embryo fibroblasts

To analyze the transforming activity of c-jun, a Rous sarcoma virus (RSV) variant that carries human c-jun instead of v-src (JH1) was constructed. After infection onto chicken embryo fibroblasts (CEF), JH1 formed foci with a titer comparable to that of wild-type RSV, and the infected cells grew in soft agar, indicating that the human c-jun gene has transforming potential, like the v-jun gene. The expression of Fra-2, one of the recently isolated Fos-related antigens, but not Fos was detected in both JH1-infected CEF and CEF infected with the control retrovirus vector (DS3). Gel shift analysis using nuclear extracts from DS3-infected CEF revealed that the Fra-2/Jun complex contributes to the basal level of AP-1 DNA binding activity. A similar activity was detected in JH1-infected CEF, but these cells have an additional AP-1 binding activity derived from Jun homodimers that seems to play important roles in the cellular transformation.     

Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.