Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells

The involvement of H2O2 in cataract development has been established inboth human patients and animal models. At the molecular level H2O2 has beenobserved to cause damage to DNA, protein and lipid. To explore the oxidativestress response of the lens system at the gene expression level, we haveexamined the effects of H2O2 on the mRNA change of the proto-oncogenes,c-jun, c-fos and c-myc in a rabbit lens cell line, N/N1003A. H2O2 treatmentof the rabbit lens epithelial cells for 60 min induces quick up-regulationof both c-jun and c-fos mRNAs. The maximal induction is 38 fold for c-jun at150 µM and 72 fold for c-fos at 250 µM H2O2. Treatment ofN/N1003A cells with 50-250 µM H2O2 for 60 min leads to a 2-5 foldincrease of the c-myc mRNA level. H2O2 also induces an up-regulation intransactivity of the activating protein-1 (AP-1) as shown with a reportergene driven by a prolactin gene promoter with 4 copies of AP-1 binding sitesinserted in the upstream of the promoter. Maximal induction occurs with 150µM H2O2. In the same system, the antioxidants, N-acetyl-cysteine (NAC)and pyrrolidine dithiocarbamate (PDTC) at concentrations shown toup-regulate the mRNAs of both c-jun and c-fos, also enhance thetransactivity of AP-1. NAC and PDTC have different effects in modulating theinduction of AP-1 activity by H2O2 and TPA. These results reveal thatoxidative stress regulates expression of various regulatory genes in lenssystems, which likely affects cell proliferation, differentiation andviability and thus affect normal lens functions.     

The proto-oncogenes c-fos and c-jun modulate thyroid hormone inhibition of human thyrotropin beta subunit gene expression in opposite directions.

The sequence from -1 to +6 bp in the hTSH beta gene contains overlapping putative thyroid hormone and AP-1 response elements. We demonstrate interaction between the AP-1 constituents c-fos and c-jun and thyroid hormone receptor in this region by transient transfection experiments using a -125 to +37 bp hTSH beta fragment. T3 inhibition was completely abolished by c-jun, but increased threefold by c-fos. A single transversion mutation at +2 bp restored T3 inhibition in the presence of c-jun and markedly reduced binding of purified c-jun by gel mobility shift assay. Thus, c-fos and c-jun influence T3 inhibition of hTSH beta expression in opposite directions acting through a response element shared with thyroid hormone receptor. Control of the relative cellular levels of these two proto-oncogenes may play a major role in modulating thyroid hormone inhibitory responses.     

Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice

Previous studies indicated that a DNA fragment comprising nucleotides (nt) ?164 to +45 of the RTBV promoter is sufficient to drive phloem-specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt ?3 to +5) and Box II (nt ?53 to ?39) were identified by DNA-protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem-specific gene expression, among which are a direct repeat (ASL Box, nt ?98 to ?79) and a GATA motif (nt ?143 to ?135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss-of-competition in electrophoretic mobility shift assays. A DNA fragment comprising nt ?164 to ?32, which contains Box II, the ASL Box and the GATA motif, conferred phloem-specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the ?103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue-specific gene expression. Similar studies in the ?164 to +45 promoter fragment indicated that the ?164 to ?103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the ?103 to ?32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem-specific gene expression.     

Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3′ enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3′ enhancer was active only in CD34⁺ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34⁺ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3′ enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160 kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

High expression of long non-coding RNA H19 is required for efficient tumorigenesis induced by Bcr-Abl oncogene

Dysregulation of non-coding RNA H19 has been observed in various tumors. However, it remains unknown whether H19 is involved in Bcr-Abl-induced leukemia. Here, we demonstrate a critical requirement for H19 in Bcr-Abl-mediated tumorigenesis. H19 was highly expressed in Bcr-Abl-transformed cell lines and primary cells derived from patients in a Bcr-Abl kinase-dependent manner. Silencing H19 expression sensitized leukemic cells to undergo imatinib-induced apoptosis and inhibited Bcr-Abl-induced tumor growth. Furthermore, H19 was shown to be regulated by c-Myc in Bcr-Abl-expressing cells. These results reveal an important role H19 plays in Bcr-Abl-mediated transformation and provide novel insights into complex mechanisms underlying Bcr-Abl-induced cancers.     

Retinoic acid repressed the expression of c-fos and c-jun and induced apoptosis in regenerating rat liver after partial hepatectomy.

Retinoic acid (RA), which was injected within 4 h after partial hepatectomy (PH), inhibited DNA synthesis in regenerating liver. The inhibition was accompanied by apoptosis, evidenced by in situ end labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and reached a maximum at 8 h after injection. Northern blot analysis revealed that RA repressed the expression of c-fos and c-jun at 15 and 30 min with the up-regulation of retinoic acid receptor gamma (RARgamma) and RARbeta at 2 h after PH. The transglutaminase II mRNA level and activity were increased by RA injection at 4 h and 8 h after PH, respectively. The mRNA levels of thymidylate synthase and thymidine kinase, which are rate determining enzymes of DNA synthesis, decreased in RA injected rats. No change was seen in the expression of p53 and p21WAF1/CIP1 which have been suggested to participate in the apoptosis process. These results suggest that RA exerts the antiproliferative activity only on the early stage of liver regeneration accompanied by the repression of c-fos and c-jun expression and induction of apoptosis.