Apigenin is a nonmutagenic chemopreventive agent found in fruits and green vegetables. In this study, we used two different epithelial cell lines (308 mouse keratinocytes and HCT116 colon carcinoma cells) to determine the effect of apigenin on the mitogen-activated protein kinase (MAPK) cascade. Apigenin induced a dose-dependent phosphorylation of both extracellular signal-regulated protein kinase (ERK) and p38 kinase but had little effect on the phosphorylation of c-jun amino terminal kinase (JNK). We used immunoprecipitation-coupled kinase assays to show that apigenin increased the kinase activity of ERK and p38 but not JNK. Consistent with these results, we found that apigenin induced a 7.4-fold induction in the phosphorylation of Elk, the downstream phosphorylation target of ERK kinase. Similarly, apigenin induced a 3.2-fold induction in the phosphorylation of activating transcription factor-2, the downstream phosphorylation target of p38 kinase. Little change was observed in the phosphorylation of c-jun, the phosphorylation target of JNK. These data suggest that part of the chemopreventive activity of apigenin may be mediated by its ability to modulate the MAPK cascade.
The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1^E4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2^E4 transcripts resulting from 2 splice donor sites in the 5' part of E2, while the splice acceptor site is the one used for E1^E4. Both E2^E4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2^E4-S and E2^E4-L. Whereas we could not differentiate E2^E4-S from E1^E4 in vivo, E2^E4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2^E4 products.
Exposure of mammalian cells to genotoxic stress results in activation of the c-jun amino-terminal kinase (JNK)-stress-activated protein kinase (SAPK) pathway and induction of DNA repair enzymes and cell cycle-regulatory proteins such as p53 and p21waf1. The p53 tumor suppressor protein transmits signals that activate p21waf1 gene expression. The p21waf1 protein then restricts cell-cycle progression, thereby allowing time for DNA repair to occur. In this study, we investigated the effects of modulation of the level of wild-type and mutant p53 protein on basal JNK1 activity in the A1-5 rat fibroblast cell line. This cell line contains a p53 gene coding for a temperature-sensitive p53 protein, which allows us to regulate the relative level of wild-type and mutant p53 protein produced in cells. Using the immune complex kinase assay to measure JNK1 activity, we demonstrated that cells expressing the wild-type-conformation p53 protein (when grown at 32.5 degrees C) exhibited a very low level of JNK1 activity. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal level of JNK1 activity was significantly higher than at 32.5 degrees C. We also demonstrated protein-protein interactions between the p53, p21waf1, and JNK1 proteins in this cell line. Both wild-type p53 protein (expressed at 32.5 degrees C) and mutant p53(val135) protein (expressed at 37 degrees C and 39 degrees C) were present in immunocomplexes of JNK1 protein. Under conditions where wild-type p53 protein was present to induce p21waf1 expression (at 32.5 degrees C), a higher level of p21waf1 protein was also detected in the JNK1 immunocomplexes than in those at 37 degrees C and 39 degrees C. We next investigated the effect that co-association of p53 protein and p21waf1 protein would have on JNK1 activity. We measured basal levels of JNK1 activity in cells expressing wild-type p53 and p21waf1, or in p21waf1-null cells, and demonstrated that cells expressing both p53 and p21waf1 proteins exhibited an approximately threefold lower basal level of JNK1 activity when compared with p21waf1-null cells. To confirm that p21waf1 protein expression in cells resulted in reduced JNK1 activity, we transfected p21waf1-/- cells with a p21waf1 expression vector. We observed that JNK1 activity was inhibited after exogenous p21waf1 protein was expressed in these cells. Our results provide evidence for modulation of the JNK1 pathway by p53 and p21waf1 proteins and support the hypothesis that modulation of JNK1 activity occurred through protein-protein interactions between JNK1, p53, and p21waf1 proteins.
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