DT-diaphorase activity in NSCLC and SCLC cell lines: a role for fos/jun regulation

To assess the potential differential lung tumour expression of NAD(P)H:quinone reductase (NQO1), the human (h) NQO1 promoter was characterized in gene transfer studies. A deletion panel of 5' flanking hNQO1 promoter constructs was made and tested in transient transfection assays in NSCLC and SCLC cell lines. The largest hNQO1 construct (-1539/+115) containing the antioxidant response element (ARE), exhibited robust levels of reporter activity in the NSCLC (H460, H520, and A549) cell lines and expression was over 12 to 77-fold higher than the minimal (-259/+115) promoter construct. In contrast, there was little difference in promoter activity between the largest and minimal promoter construct in the SCLC (H146, H82 and H187) cell lines. Deletion of the sites for NFkappaB and AP-2 and the XRE did not significantly affect hNQO1 promoter activity in either the NSCLC or SCLC cell lines. Robust promoter activity in NSCLC lines was mediated by a 359 bp segment of the proximal promoter that contained a canonical AP-1 binding site, TGACTCAG, within the ARE. Gel supershift assays with various specific Fos/Jun antibodies identified Fra1, Fra2 and Jun B binding activity in NSCLC cells to a promoter fragment (-477 to -438) spanning the AP-1 site, whereas SCLC do not appear to express functional Fra or Jun B. These results suggest a possible role for AP-1 activity in the differential expression of hNQO1 in NSCLC.     

Identification of a novel SP3 binding site in the promoter of human IGFBP4 gene: role of SP3 and AP-1 in regulating promoter activity in CaCo2 cells

Insulin-like growth factor binding protein 4 (IGFBP4/BP4) gene expression plays an important role in the transition from proliferation to differentiation of a human colon cancer cell line, CaCo2. We recently cloned and identified multiple cis elements (including putative binding sites for activator protein 1 (AP-1) and specificity proteins (Sps) ) in the promoter of human BP4 gene, and measured a significant upregulation of the promoter activity in response to c-Jun. We therefore examined the role of the single AP-1 site (-869/-863) and other cis elements, in regulating the expression of hBP4 gene, in the current studies. Deletion of a 25 bp sequence from -872 to -848, which contains the AP-1 site, significantly reduced BP4 promoter activity by approximately 50%. Surprisingly, mutation of the AP-1 site did not produce significant alteration in the activity of the BP4 promoter. However, mutation of 7 bp (5'-TGCTGCA) at the 3' end of the AP-1 site resulted in significantly decreasing the promoter activity by >50%. Proteins bound to the 25 bp probe (-872/-848) could be supershifted by antibodies specific for JunD and Sp3 in an EMSA. JunD binding was abolished on mutation of the AP-1 site and Sp3 binding was abolished on mutation of the 7 bp at -861/-855; binding of the purified Sp3 protein to the 25 bp probe was similarly abolished on mutation of the newly discovered Sp3 binding site (TGCTGCA). BP4 promoter activity was upregulated in insect cells in response to Sp3 expression, confirming a functional importance of the novel Sp3 binding site. These studies suggest that the Sp3 binding site, rather than the AP-1 site, may be playing a significant role in regulating the expression of IGFBP4 gene in CaCo2 cells.     

Characterization of the human ABL promoter regions

Gel retardation and DNAase 1 footprinting experiments have been performed to characterize the promoter sequences of exon 1a and 1b of the human ABL gene. Several Sp1 motifs and CCAAT boxes are found to be protected by nuclear proteins in the 1b promoter but none of the 7 reported Sp1 sites in 1a were found to bind protein. Multiple sets of initiation sites seem to exist in the 1b promoter region which may represent individual initiation sites, distributed over a DNA region of up to 700 bp. Starting with the most distal initiation site, 1a and 1b ABL promoter sequences show a high degree of homology, suggesting that one is derived from the other. However, multiple evolutionary changes in the 1a promoter sequence indicate that type 1a ABL expression may be differently regulated than 1b.     

Activation and suppression of a cryptic promoter in the intron of the human melanoma-associated ME491 antigen gene.

A deletion mutant of the human melanoma-associated ME491 antigen gene starting at the first intron (lambda R31) differentially mediates the antigen expression depending on the cell type. Cryptic promoter activity residing in a 270-base-pair (bp) fragment of the first intron was examined by primer extension analysis and recombinant chloramphenicol acetyltransferase (CAT) assay. The cryptic promoter, further localized within a 153-bp fragment (fr153BN), exerted its effect in Ltk- and H-ras-transformed NIH3T3 (3T3-Hras) but not in parental NIH3T3 cells. The results suggested that the cryptic promoter was associated with a novel ras-responsive positive regulatory element, since fr153BN did not contain an AP-1-binding sequence motif, known as the ras-responsive enhancer element. The cryptic promoter activity of fr153BN was suppressed by an upstream 121-bp fragment (fr121SB) which contained a consensus sequence motif for binding of a repressor protein, GC factor, and regions showing sequence similarity with putative cis-acting repressor elements found in the vimentin gene. The degree of the suppression was greater in 3T3-Hras than in Ltk- cells. These positive and negative regulatory elements may be differentially involved in the regulation of ME491 antigen expression depending on the cell type.     

Activation and Suppression of a Cryptic Promoter in the Intron of the Human Melanoma-associated ME491 Antigen Gene

A deletion mutant of the human melanoma-associated ME491 antigen gene starting at the first intron (λR31) differentially mediates the antigen expression depending on the cell type. Cryptic promoter activity residing in a 270-base-pair (bp) fragment of the first intron was examined by primer extension analysis and recombinant chloramphenicol acetyltransferase (CAT) assay. The cryptic promoter, further localized within a 153-bp fragment (fr153BN), exerted its effect in Ltk^- and H- ras- transformed NIH3T3 (3T3-Hras) but not in parental NIH3T3 cells. The results suggested that the cryptic promoter was associated with a novel ras-responsive positive regulatory element, since fr153BN did not contain an AP-1-binding sequence motif, known as the rew-responsive enhancer element. The cryptic promoter activity of fr153BN was suppressed by an upstream 121-bp fragment (fr121SB) which contained a consensus sequence motif for binding of a repressor protein, GC factor, and regions showing sequence similarity with putative cis -acting repressor elements found in the vimentin gene. The degree of the suppression was greater in 3T3-Hras than in Ltk^- cells. These positive and negative regulatory elements may be differentially involved in the regulation of ME491 antigen expression depending on the cell type.     

PC-1基因在肿瘤组织中的表达及其启动子区的克隆和活性分析

背景与目的：PC－1是在骨转移和雄激素非依赖的前列腺癌细胞株C4－2中高表达的新基因。本文拟研究PC－1基因在多种人肿瘤组织和正常组织中的表达水平,并对该基因的启动子区进行克隆及活性分析。方法：以PC－1基因特异性DNA序列为探针与10对肿瘤和正常组织的总RNA进行杂交;以C4－2细胞基因组DNA为模板,PCR扩增PC－1基因翻译起始位点上游DNA序列。PCR产物定向克隆到含荧光素酶报告基因的载体pGL3-Basic上,将重组质粒瞬时转染C4－2细胞,荧光素酶定量分析检测启动子活性。结果：多肿瘤组织中PC－1基因的表达水平明显高于相应的正常组织中的表达;翻译起始位点上游340bp的片段没有启动子活性,而ATG前1099bp、1337bp,1579bp,1831bp和4939bp长度的片段都表现出启动子的功能活性。结论：PC－1基因在人前列腺癌以及多种肿瘤组织中被特异激活,表明该基因的功能可能和肿瘤的发生有关;研究的初步结果表明4939bp长度的启动子活性最强,以1831bp和4939bp之间可能含有转录增强子元件。     

Characterization of human and mouse c-fes cDNA clones and identification of the 5' end of the gene

cDNA clones of the human c-fes mRNA were isolated. Nucleotide analysis showed that c-fes mRNA contains a 2514 nucleotide open reading frame, which could encode for a 93 kDa protein, and both 5' and 3' v-fes nonhomologous sequences. Primer extension experiments confirmed that the longest isolated cDNAs are about the same length as the entire human c-fes mRNA. Sequence comparison between human c-fes cDNA and the corresponding genomic regions identified a 5' viral-non homologous exon (exon 1) located 491 bp upstream from the first v-fes homologous exon. The genomic region surrounding c-fes exon 1 contains a CpG island and acts as a promoter in vitro. Analysis of the 5' end of mouse c-fes cDNA suggested that the 5' human and mouse gene structure are similar.