以转录因子AP-1为靶的decoy核酸体内外抗肿瘤研究

合成了双链寡聚核苷酸——decoy核酸,其与靶转录因子AP-1有高亲和性,可进入细胞作为decoy顺式元件,通过抑制特异的转录因子和调控区域的结合,调控基因转录而改变基因的表达.在体内外抗肿瘤试验中, decoy核酸有显著抑制肿瘤细胞增殖的作用,可以成为潜在性的肿瘤基因治疗药物.     

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.     

Induction of plasminogen activator inhibitor-2 is associated with suppression of invasive activity in TPA-mediated differentiation of human prostate cancer cells

We previously reported that 12-O-tetra-decanoylphorbol-13-acetate (TPA) induces microglia-like differentiation and decreases malignancy in human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying differentiation and decrease of malignancy in TSU-Pr1 cells treated with TPA, we attempted to identify genes expressed differentially during the differentiation using differential display. We successfully detected plasminogen activator inhibitor type-2 (PAI-2) as one gene up-regulated by TPA treatment. The change in expression of PAI-2 by TPA was blocked by treatment with protein kinase C or mitogen-activated protein kinase inhibitors. We also found that secretion of PAI-2 protein was increased by TPA treatment. Moreover, we demonstrated that suppression of invasive activity of TSU-Pr1 cells by TPA treatment was blocked by co-treatment with anti-PAI-2 antibody. These results suggest that induction of PAI-2 is associated with suppression of invasive activity in TSU-Pr1 cells treated with TPA.     

Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells

Emerging evidence has suggested a critical role for activator protein-1 (AP)-1 in regulating various cellular functions. The goal of this study was to investigate the effects of Helicobacter pylori and mitogen-activated protein kinases (MAPK) on AP-1 subcomponents expression and AP-1 DNA-binding activity in gastric epithelial cells. We found that H. pylori infection resulted in a time- and dose-dependent increase in the expression of the proteins c-Jun, JunB, JunD, Fra-1, and c-Fos, which make up the major AP-1 DNA-binding proteins in AGS and MKN45 cells, while the expression levels of Fra-2 and FosB remained unchanged. Helicobacter pylori infection and MAPK inhibition altered AP-1 subcomponent protein expression and AP-1 DNA-binding activity, but did not change the overall subcomponent composition. Different clinical isolates of H. pylori showed various abilities to induce AP-1 DNA binding. Mutation of cagA, cagPAI, or vacA, and the nonphosphorylateable CagA mutant (cagA(EPISA)) resulted in less H. pylori-induced AP-1 DNA-binding activity, while mutation of the H. pylori flagella had no effect. extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) each selectively regulated AP-1 subcomponent expression and DNA-binding activity. These results provide more insight into how H. pylori and MAPK modulate AP-1 subcomponents in gastric epithelial cells to alter the expression of downstream target genes and affect cellular functions.     

Load application induces changes in the expression levels of Sox-9, FGFR-3 and VEGF in condylar chondrocytes

Experimental and clinical observations have proven the modulatory effects of mechanical loading on the development and maintenance of cartilage architecture. Here we examined the involvement of Sox-9, FGFR-3 and VEGF (pivotal factors controlling cartilage development and growth) in the mechano-transduction pathway of mandibular condylar cartilage by changing the dynamics of the transmitted load via changes in food hardness. To this end, condyle cartilage tissue of rats fed with hard or soft food was analyzed immunohistochemically at various time points. Our findings demonstrate that different mechanical loading conditions in condylar chondrocytes trigger differentiation-/maturation-related processes by affecting the expression levels of these factors, ultimately influencing condylar cartilage growth.     

Jun DNA-binding is modulated by mutations between the leucines or by direct interaction of fos with the TGACTCA sequence

Mutations between the leucines of the "leucine zipper" domain of Jun D can either decrease (Asn 301 to Ala) or increase (Thr 307, Ala 308, to Glu, Val) homodimer formation and specific binding to DNA even though such changes do not modify the predicted alpha-helical structure of this region. As shown previously, addition of Fos strongly increases the affinity of Jun for DNA by forming a heterodimer. The jun down mutation (Asn 301 to Ala) also diminishes DNA binding by the Fos-Jun D heterodimer. These data strongly support the coiled coil conformation of this region where residues adjacent to the leucines are also important for dimer formation. Ultraviolet cross-linking experiments have shown that both Fos and Jun directly contact the TGACTCA palindromic sequence defined as a TPA (12-O-tetradecanoyl phorbol-13-acetate) response element or TRE. Both Jun homodimers and Jun-Fos heterodimers bind this TRE as well as the cAMP responsive element (CRE or TGACGTCA) with comparable affinities. While strong c-Jun or Jun D binding requires a perfect palindrome, Jun-Fos complexes can also efficiently recognize sequences where the right half of the palindrome is less conserved (TGACTAA or TGACGCA).