A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells.

We have examined the regulation of the proliferating cell nuclear antigen gene (PCNA) in a hamster fibroblast cell line (tk-ts13) which is temperature sensitive for growth. These tk-ts13 cells, at the restrictive temperature, are growth arrested in the G1 phase of the cell cycle. The cells were stably transfected with a full length human PCNA gene, and the resulting cell lines (K525 cells) were analyzed. We find that, in growth arrested K525 cells, a cryptopromoter is activated in the transfected human PCNA gene. The cryptopromoter resides in intron 4 which is necessary for proper regulation of the PCNA gene. Removal of this intron leads to increased expression of PCNA in cells which have entered the G0 state. An Alu sequence residing in intron 4 is implicated as the promoter element which is active during growth arrest.     

Suppression of bcl-2 gene by RNA interference increases chemosensitivity to cisplatin in nasopharyngeal carcinoma cell line CNE1

To explore the effect of suppressing BCL-2 expression using RNA interference (RNAi) technique in nasopharyngeal carcinoma cell line CNE1. CNE1 cell lines stably expressing shRNAs targeted bcl-2 and GL3 gene were established and gene expression inhibition was assessed by Western blotting analysis. The effect of suppressing bcl-2 by RNAi on cell growth was studied, the apoptosis induction and the sensitization of CNE1 cells to cisplatin were quantified by MTT assay and flow cytometry. The results showed that: stable transfection of CNE1     

mRTVP-1, a novel p53 target gene with proapoptotic activities

We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.     

小麦几丁质酶基因Wch2的克隆与表达分析

利用小麦几丁质酶基因PCR特异片段为探针,分离克隆了一个小麦Chidl几丁质酶基因Wch2。该基因编码311个氨基酸,不含内含子,具有一个信号肽、一个富含半胱氨酸的几丁质结合区域、两个变异区、两个酶活性区域。Southern分析表明,在小麦基因组中Wch2有多个拷贝。秆锈菌接种诱导Wch2在一对小麦近等基因系中差异表达;在抗病系中国春Srll中,接种3d后Wch2开始表达,6d后表达量更高;而在感病等基因系中国春srll中,在所有取样分析的时间内均未检测到Wch2表达。将Wch2克隆到细菌表达载体pET22b,在细菌中表达的重组Wch2具有几丁质酶活性。这些结果说明,分离的Wch2基因在小麦秆锈菌诱导的抗性反应中具有重要作用。     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

Transient transfection of the human myeloid cell line Mono Mac 6 using electroporation

Leukemic cell lines such as Mono Mac 6 provide an excellent model for studying changes in gene expression during induction of cell differentiation. Mono Mac 6 cells can be induced to differentiate from their immature state to cells resembling morphologically and functionally mature monocytes and macrophages by various stimuli such as calcitriol and transforming growth factor-beta. During differentiation, the expression of differentiation markers such as the cell surface antigen CD14 or other differentiation-related genes such as 5-lipoxygenase are strongly increased. Thus, this cell line constitutes an excellent model system to study the regulation of gene expression by inducers of cell differentiation. However, myeloid cell lines are often refractory to transfection by calcium phosphate or DEAE dextran so that reporter gene assays are difficult to perform. We have established a transient transfection protocol for Mono Mac 6 cells using electroporation, a 5-lipoxygenase promoter luciferase reporter gene construct, and the secreted alkaline phosphatase as an internal standard.     

BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in a cell type-dependent manner

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.     

BECN1 and BIM interactions with MCL-1 determine fludarabine resistance in leukemic B cells

The purine analog fludarabine (Fd) is an essential therapeutic for chronic lymphocytic leukemia (CLL). Innate or acquired resistance to Fd is a significant clinical problem and is largely mediated by increased expression of BCL-2 family members. The antiapoptotic BCL-2 family proteins inhibit both apoptosis and autophagy, therefore, downregulation of antiapoptotic BCL-2 family proteins and enhanced autophagy must coexist in cells dying in response to an apoptosis inducing therapeutic. However, in the drug-resistant cells that have an increased dependence on antiapoptotic proteins, whether autophagy is also inhibited remains unclear. Here, we examined the role of the BCL-2 family in regulating cell death and autophagy in leukemic cell lines and their derivative isogenic Fd-resistant (FdR) cells. MCL-1 degradation following Fd treatment freed the proapoptotic effectors BIM and BECN1, thus leading to cell death-associated autophagy in Fd-sensitive cells. However, in FdR cells, low BIM expression and BECN1 sequestration by MCL-1 prevented cell death. Consistently, in sensitive cells inhibition of apoptosis using siBIM and of both the early-phase autophagy nucleation steps by siBECN1, shATG7 or 3-methyladenine and the late-phase autophagy by shLAMP2, significantly reduced Fd-induced cell death. Paradoxically, FdR cells were addicted to basal autophagy, which was dependent on AMP-activated protein kinase (AMPK) but not BECN1. Moreover, in FdR cells, inhibition of autophagy by shLAMP2, but not siBECN1, enhanced cell death. The BH3-mimetic obatoclax released BIM and BECN1 from MCL-1 in Fd-sensitive and BECN1 from MCL-1 in FdR cells, and was effective at killing both Fd-sensitive and - resistant leukemic cells, including primary CLL cells. Therefore, a differential regulation of autophagy through BECN1 and AMPK signaling in Fd-sensitive and - resistant cells determines the different possible outcomes of autophagy inhibition. These findings suggest effective means to overcome Fd resistance by induction of BIM-dependent apoptosis and activation of BECN1-dependent autophagy.     

T cell-specific expression of the human TNF-alpha gene involves a functional and highly conserved chromatin signature in intron 3

Using a phylogenetic approach, we identified highly conserved sequences within intron 3 of the human TNF-alpha gene. These sequences form cell type-specific DNase I hypersensitivity sites and display cell type-specific DNA-protein contacts in in vivo genomic footprints. Consistent with these results, intron 3 confers specific activity upon a TNF-alpha reporter gene in Jurkat T cells, but not THP-1 monocytic cells. Thus, using a combinatorial approach of phylogenetic analysis, DNase I hypersensitivity analysis, in vivo footprinting, and transfection analysis, we demonstrate that intronic regulatory elements are involved in the cell type-specific regulation of TNF-alpha gene expression.     

Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms

Downy mildew (Plasmopara halstedii (Farl.) Berlese et de Toni) is a serious foliar pathogen of cultivated sunflower (Helianthus annuus L.). Genetic resistance is conditioned by several linked downy mildew resistance gene specificities in the HaRGC1 cluster of TIR-NBS-LRR resistance gene candidates (RGCs) on linkage group 8. The complexity and diversity of the HaRGC1 cluster was assessed by multilocus intron fragment length polymorphism (IFLP) genotyping using a single pair of primers flanking a hypervariable intron located between the TIR and NBS domains. Two to 23 bands were amplified per germplasm accession. The size of the included intron ranged from 89 to 858 nucleotides. Forty-eight unique markers were distinguished among 24 elite inbred lines, six partially isogenic inbred lines, nine open-pollinated populations, four Native American land races, and 20 wild H. annuus populations. Nine haplotypes (based on 24 RGCs) were identified among elite inbred lines and were correlated with known downy mildew resistance specificities. Sixteen out of 39 RGCs identified in wild H. annuus populations were not observed in elite germplasm. Five partially isogenic downy mildew resistant lines developed from wild H. annuus and H. praecox donors carried eight RGCs not found in other elite inbred lines. Twenty-four HaRGC1 loci were mapped to a 2-4 cM segment of linkage group 8. The multilocus IFLP marker and duplicated, hypervariable microsatellite markers tightly linked to the HaRGC1 cluster are powerful tools for distinguishing downy mildew resistance gene specificities and identifying and introgressing new downy mildew resistance gene specificities from wild sunflowers.     

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.     

Cell-type specific and differential regulation of the human metallothionein genes. Correlation with DNA methylation and chromatin structure.

The expression of three human metallothionein genes, MT-IIA, MT-IF, and MT-IG was studied in the human hepatoblastoma (HepG2), the hepatocarcinoma (Hep3B2), the embryonic kidney (Hek 293), and the lymphoblastoid-derived (Wi-L2) cell lines. The pattern of expression of each specific MT gene in response to various heavy metals was different among the four cell lines studied indicating differential regulation of MT gene expression. The MT-IF or MT-IG and the MT-IIA genes were regulated in a cell-type specific manner in response to heavy metals and dexamethasone, respectively. DNA methylation was shown to be correlated to cell-type specific regulation of MT gene expression since 5-azacytidine treatment resulted in the expression of the MT-IF and MT-IG genes in response to cadmium and zinc in Wi-L2 cells, of the MT-IIA gene in response to dexamethasone in Wi-L2 cells, and of the MT-IG in response to zinc and copper in Hek 293 cells. Furthermore, transfection studies indicated that all the trans-acting factors necessary for the expression of these genes were present and functional in Wi-L2 and Hek 293 cells. The differential level of expression of the MT-IF and MT-IG genes in response to heavy metals in the Hek 293 cell line was shown to be correlated to their chromatin structure.