CAT Assay Research Articles

Specific expression of the pancreatic‐secretory‐trypsin‐inhibitor (PSTI) gene in hepatocellular carcinoma

Twenty hepatocellular carcinomas (HCC) were analyzed by Northern blotting to test the expression of pancreatic secretory trypsin inhibitor (PSTI). This gene was expressed in all HCCs, but not in other tumors, including mammary, thyroid, pulmonary and ovarian cancers. Some gastric and colonic cancers weakly expressed PSTI. Among cell lines examined in a similar manner, PSTI was expressed in all of 4 derived from hepatoma. On the other hand, among 15 cell lines derived from cancers other than hepatoma, only 3, derived from pancreatic, colonic and gastric cancers, weakly expressed PSTI. A CAT assay using a deletion set of the 5' region from the cloned PSTI gene has shown that in hepatoma cell lines, the expression of this gene is dependent on the presence of 2 regulatory regions that include an IL-6 responsive elements and an AP-I-binding site. However, in non-hepatoma cell lines, the 2 regulatory regions are not necessary for expression. The blood level of PSTI in 27 patients with HCC was significantly increased, and it was positively correlated with tumor size, suggesting that specific expression of PSTI in HCC causes this effect and that elevated blood level of PSTI without inflammation indicates the presence of HCC.

Characterization of the promoter region of the human c-kit proto-oncogene.

The c‐kit proto‐oncogene encodes a tyrosine kinase receptor for stem cell factor and plays a critical role in the growth and differentiation of various types of cells including hematopoietic stem cells. To investigate the mechanisms of its transcriptional regulation, we isolated the 5’flanking region of the human c‐kit gene and characterized its promoter activity in hematopoietic cells. Nucleotide sequence analysis revealed that the 1.2 kb 5’flanking region lacked a typical “TATA box,” but had a relatively high G + C content and four potential Sp1‐binding sites. Putative binding sites for AP‐2, basic helix‐loop‐helix proteins, Ets‐domain proteins, Myb and GATA‐1 were also found. Primer extension and S1 nuclease protection analyses of hematopoietic cells indicated that the major transcription start sites are 62 bp and 58 bp upstream of the translation start site. Essentially the same start sites were detected in non‐hematopoietic cells such as small cell lung carcinoma and glioblastoma: this single promoter in c‐kit is different from the multiple promoter system of c‐fms, a c‐kit‐related gene, in which at least two promoters are differently used in hematopoietic and non‐hematopoietic cells. An analysis of the c‐kit 5’flanking region using the bacterial chloramphenicol acetyltransferase gene (CAT assay) in human erythroleukemia HEL cells, which express the endogenous c‐kit mRNA at high levels, showed that a region from ‐180 to ‐22 is important for the expression of the c‐kit gene. In addition, a negative regulatory element(s) is suggested to be involved in the regulation of the c‐kit gene expression in mammals.

Presence of particular transcription regulatory elements in the 5'-intergenic region shared by the chicken H2A-III and H2B-V pair

The two chicken histone gene families, H2A and H2B, contain nine and eight members, respectively, within two major histone gene clusters. Six genes each from families H2A and H2B have been found to be closely associated in inverted directions as H2A/H2B gene pairs. Two previously sequenced H2A members ( H2A-I and H2A-II) encode the same amino acid (aa) sequence (class I), whereas seven sequenced H2B genes encode three different variants (classes I, II and III). In this study, we first sequenced H2A-III, a member of the H2A family, which is located in inverted orientation and 350 bp upstream from H2B-V, encoding the class-III H2B protein. The protein encoded by H2A-III differs from the class-I H2A protein in a single aa (Ala 70 → Pro; class II). As a step toward elucidation of the transcriptional regulation of the H2A and H2B families, we fused this 5'-intergenic region to the cat gene in inverted orientations to generate two chimeric plasmids, pH2A-III-350 and pH2B-V-350. Transient CAT assays using these constructs indicated that the promoter of H2B-V is more active than that of H2A-III. CAT assays with 5'-deletion mutants of H2A-III and H2B-V showed that they each possess particular transcriptional motifs which are located relatively close to, or apart from, their own coding regions. These findings, together with those reported previously on the H2A-V/H2B-II pair, suggest distinct manners of transcription regulation of different members of the chicken histone gene families, H2A and H2B.

Enhanced Activity of Human G6PD Promoter Transfected in HeLa Cells Producing High Levels of HIV-1 Tat

Glucose 6-phoshate dehydrogenase (G6PD) enzyme activity has an important role in the cell defence against oxidative damage produced by hydrogen peroxide, a compound known to activate HIV-1 expression through the intermediate of NF-κB. By means of CAT assays, in this paper we show that the Tat protein increases the rate of transcription from the human G6PD promoter in HeLa cells; furthermore, we report a similar effect of Tat on transcription driven from the viral RSV promoter. We did not observe Tat stimulation with the human CMV, SV40 or the basic RSV promoters. Dose-response curves indicate that Tat activates G6PD and RSV through a mechanism different from the major one operating on the HIV-1 LTR promoter. TAR-like structures are not involved, instead a short sequence close to the G6PD transcription start site and the RSV LTR enhancer is a good candidate for mediating the phenomena described in this paper. This sequence has some features in common with the NF-κB motif.

Identification in the sera from patients with advanced cancer of a factor which stimulates gene expression from human immunodeficiency virus type 1

From the sera of patients with advanced cancer, a novel factor called SDF (serum-derived factor) was partially purified. SDF was shown to stimulate transcription from the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) by transient CAT assay. It did not stimulate gene expression of various control promoters including Rous sarcoma virus, human c-fos, c-myc, c-H-ras and chicken β-actin genes. The SDF preparation did not contain any detectable TNF-α or TNF-β, and differed in its physicochemical properties from TNFs. We concluded that SDF might be a novel factor associated with the clinical features of advanced cancer. It is speculated that SDF might have some role in disease progression of AIDS as well as in the development of the cachectic conditions in AIDS associated with malignancies.

Transcriptional regulation of the gene encoding cholesterol 7α-hydroxylase in the rat

The cytochrome P450 enzyme, cholesterol 7a-hydroxylase (CYP7A), catalyses the first and rate-limiting step in the conversion of cholesterol to bile acids. Expression of the CYP7A gene is under complex physiological control, encompassing amongst others a feedback down-regulation by bile acids. Using the CYP7A cDNA of the rat as a probe, we isolated a rat genomic clone containing the 5' part of the gene, including approximately 3.6 kb of upstream sequences. Sequence analysis revealed the presence of several putative regulatory elements. Transient expression analyses of transfected primary hepatocytes demonstrated that the major transcription-activating region is located in the proximal 145 nucleotide (nt). Upon addition of taurocholate to the culture, a significant reduction of the transcriptional activity was observed, suggesting the presence of a bile acid-responsive element in the proximal region of the CYP7A promoter. In addition, evidence was obtained for the presence of a thyroxine-responsive site further upstream. After addition of taurocholate, steady-state CYP7A mRNA levels, as judged by Northern analysis of hepatocyte RNA, are eightfold reduced. On the other hand, the transcriptional activity of CYP7A, as shown both in CAT assays and run-on experiments, revealed only a threefold decrease. These experiments suggest that both transcriptional control and regulation of CYP7A mRNA stability play an important part in the feedback regulation of CYP7A activity in the rat.

Altered dexamethasone responsiveness and loss of growth control in tumorigenic mouse lung cell lines.

Glucocorticoid hormones induce differentiation, inhibit proliferation, and, in mice, reduce carcinogen-induced tumorigenesis of lung epithelial cells. Therefore we examined dexamethasone effects on tumorigenic and non-tumorigenic mouse lung epithelial-derived cell lines. Non-tumorigenic cells were growth inhibited and exhibited CAT activity in pMMTV-CAT transfectants in response to dexamethasone. Tumorigenic cell lines exhibited a range of responses to dexamethasone. While one tumorigenic line was growth-inhibited and responsive in CAT assays, 2 other tumorigenic cell lines were unresponsive both in CAT and in growth assays. A fourth tumorigenic cell line exhibited intermediate sensitivity in CAT assays and was actually growth-enhanced by dexamethasone. Although no difference between cell lines was observed in the abundance of glucocorticoid receptor protein on Western blots, the least dexamethasone-responsive tumorigenic lines exhibited very little binding of 3H-dexamethasone. Clones of tumorigenic lines stably transfected with the rat glucocorticoid receptor gene were more dexamethasone-sensitive in CAT assays and were growth-inhibited by dexamethasone. These data suggest that the neoplastic progression of cell lines derived from mouse lung frequently involves the acquisition of diminished glucocorticoid responsiveness.

Human hnRNP Protein A1 Gene Expression: Structural and Functional Characterization of the Promoter

hnRNP protein A1 (34 kDA, pl 9·5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues.A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

The equine herpesvirus 1 (EHV-1) UL3 gene, an ICP27 homolog, is necessary for full activation of gene expression directed by an EHV-1 late promoter.

We have previously reported that the equine herpesvirus 1 (EHV-1) XbaI G restriction fragment (nucleotides 1436 to 7943 relative to the left terminus of the EHV-1 genome [Kentucky A strain]) is required in combination with the EHV-1 immediate-early (IE) gene to achieve significant activation of two representative EHV-1 late promoter-chloramphenicol acetyltransferase (CAT) recombinants in transient expression assays. In this report, we demonstrate that the XbaI G-encoded UL3 gene (an ICP27 homolog) provides a trans-acting factor which acts (in combination with the EHV-1 IE gene product) to increase reporter gene expression directed by an EHV-1 late promoter-CAT recombinant plasmid. We show that cloned copies of UL3 can successfully substitute for the XbaI G fragment in CAT assays and that stop codon insertion within the UL3 open reading frame inhibits the ability of UL3 to activate reporter gene expression in trans.

Genomic organization and promoter function of the murine tumor necrosis factor receptor β gene

Using the tumor necrosis factor receptor β (TNFRβ) cDNA as a probe, overlapping clones from a genomic phage library were isolated which encompass the murine TNF receptor β gene. Analysis of the gene led to the identification of 10 exons, most of which were concentrated in two clusters. The boundaries of the exons do not match protein domains or characteristic motifs of the extracellular region of the TNFRβ. The 5'-flanking region of the gene shows a high density of G and C nucleotides with a strong overrepresentation of CpG dinucleotides. Most of the analyzed CpG were found to be nonmethylated, suggesting that this region is an HTF island. We revealed at least three transcriptional start sites which is likely due to the absence of classical TATA and CAAT sequences from the putative promoter region. CAT assays confirmed promoter activity of the 5'-flanking sequences. Surprisingly, some successively shortened promoter constructs displayed higher relative promoter activity than a full length clone. Preliminary experiments indicate that the promoter region of the TNFRβ gene does not respond to a variety of cytokines. In summary, the structural and functional analysis suggest that the TNFRβ expression is directed by a non-inducible housekeeping-type promoter.

Role of protein kinase system in the signal transduction of stretch-mediated protooncogene expression and hypertrophy of cardiac myocytes.

To examine the molecular mechanisms by which mechanical stimuli induce protooncogene expression and hypertrophy of cardiac myocytes directly, we cultured rat neonatal cardiac myocytes in deformable dishes and imposed mechanical load on adherent cultured cardiac myocytes by stretching the dishes. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin followed by the amino acid incorporation into cardiac proteins. CAT assay analysis indicated that the sequences containing serum response element were required for the efficient transcription of c-fos gene by stretching. This accumulation of c-fos mRNA by myocyte stretching was inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels. These findings suggests that mechanical stimuli might directly induce protooncogene expression possibly via protein kinase C activation. Furthermore, we observed the activation of MAP kinase by myocytes stretching. This result suggests that MAP kinase activation induced by mechanical stimuli might increase the efficiency of protein synthesis on ribosomes induced by mechanical stimuli.

Identification of a binding protein to the X gene promoter region of hepatitis B virus

The X protein of hepatitis B virus (HBV) is a transactivator to homologous and heterologous viral and cellular transcriptional regulatory elements. One sequence-specific binding protein, whose binding site located from nt 1102 to nt 1117 of HBV DNA, was identified by mobility shift assay and DNase I foot-printing analysis. A CAT assay experiment demonstrated this 16-bp binding site to have a promoter activity in the X gene transcription. The 58-bp DNA fragment (nt 1085 to nt 1142), which contains the above binding site, could be enhanced by the HBV enhancer. Mobility shift assay using the mutated 58-bp DNA fragments as probes, showed that the mutation, which damaged the palindrome structure between nt 1105 and nt 1112, resulted in loss of the binding activity. This mutation also remarkably reduced the promoter activity. The binding site differed from the target sequences of known transcriptional factors. This factor was thus concluded to be a binding protein to the X gene promoter (X-PBP) of HBV. A homology search demonstrated the binding site to be highly homologous to the promoter elements of human laminin receptor (2H5epitope) and lipoprotein receptor-related protein (LRP) genes.

DETERMINATION OF TRANSREPRESSION DOMAINS OF THE HUMAN N-MYC PROTEIN

The myc gene family, including c- and N-myc, is believed to play a critical role in the regulation of cell cycle and proliferation. The function of N-myc, in contrast to c-myc, is not clearly understood. Here we report that, using a N-myc expression vector in CAT and S1 nuclease protection assays, the N-myc protein trans-represses expression of the human N-myc, the mouse N-myc, and the human MHC class 1 antigen genes. The analysis of N-myc deletion mutants showed that the N-terminal region where there are amino acid sequences highly conserved in the myc family and the central region where the acidic amino acids are concentrated, are necessary for trans-repression activity. Also, the region near the C-terminus is relevant for DNA-protein and protein-protein interaction; in particular, the basic region, helix-loop-helix, and leucine zipper are important for activity.

Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist

We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothiglucose, which has disease-modifying activity in treated patients, ats as an IL-1 functional antagonist in this system.

Optimization of the CAT assay procedure by determining the initial rate of the enzymatic reaction

Optimization of the CAT assay procedure by determining the initial rate of the enzymatic reaction

Tissue-specific expression of the human neuropeptide Y gene in transgenic mice

Neuropeptide Y (NPY) is the most abundant neuropeptide detected in the mammalian brain, and is found throughout the central and peripheral nervous systems. This peptide is a proposed regulator of appetite, blood pressure, and pituitary hormone release. Previous experiments have demonstrated the ability of 5′ sequences within the human NPY gene to promote transcription in cultured neuronal cells. To identify sequences in this gene that regulate tissue-specific expression, a NPY/CAT fusion gene, containing approximately 850 bp of NPY sequences, was microinjected into fertilized mouse ova. Five lines of transgenic mice were derived from these ova and several tissues from mice of each line were tested for transgene expression using the CAT assay. One line demonstrated X-chromosome-linked transmission of the transgene while the other lines demonstrated autosomally-linked transmission. Three lines demonstrated transgene expression with significant levels of CAT activity detectable only in tissues which have been shown to express endogenous NPY. One autosomally-linked line did not demonstrate significant levels of transgene activity because the transgene appeared to have undergone structural alteration during genomic integration. No transgene activity was detected in either male of female mice from the X-linked line, suggesting a positional regulation of the transgene locus other than X-inactivation in this line. The present research demonstrated the NPY regulatory sequences included i in pCATNPYΔ796 sufficiently directed tissue-appropriate gene expression in transgenic mice.

Characterization of a specific erythromegakaryocytic enhancer within the glycoprotein IIb promoter.

The gene coding for glycoprotein IIb (GPIIb), the alpha subunit of platelet integrin GPIIb/IIIa is an early and specific marker of the megakaryocytic lineage. Thus, studies on the regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation in this lineage. The promoter region of this gene was isolated and analyzed to understand its tissue-specific transcriptional activity. A region extending from nucleotides -414 to -554 was found to be extremely important for the promoter function. Deletion of this region results in a 70% decrease of the promoter activity, as measured in CAT assays. This region has the properties of an enhancer. It is able to activate a heterologous promoter, in a distance- and orientation-independent manner, in both megakaryocytic and erythroid cells. This enhancer contains binding sites for nuclear factors and mutation of these sites, individually or together, abolish the enhancer activity. These nuclear factors are present in megakaryocytic and erythroid cell lineages, but they are absent in the other tested cells. One of the sites, named domain D, contains a TTATC motif that may interact with the transcription factor GATA1, active in erythroid and megakaryocytic cells. These results indicate that the promoter of a megakaryocytic gene contains a tissue specific enhancer, active in both the erythroid and the megakaryocytic lineages, and may implicate the erythroid factor GATA1.

Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene ( MDR1) expression caused by arsenite

Expression of the MDR1 gene, which encodes P-glycoprotein, is increased under some stress conditions. We have reported that quercetin, a bioflavonoid, inhibits the expression of heat-shock proteins. We have identified the effects of quercetin on the MDR1 gene expression in the human hepatocarcinoma cells line, HepG2. The increase of P-glycoprotein synthesis and MDR1 mRNA accumulation caused by exposure to arsenite were inhibited by quercetin. The CAT assay suggested that quercetin suppressed the transcriptional activation of the MDR1 gene after exposure to arsenite. Although many drugs that prevent the P-glycoprotein function have been reported, this is the first report to describe the inhibition of MDR1 expression by a reagent.

Host cell reactivation of cisplatin-damaged plasmid DNA in human non-T leukocyte cell lines

Host-cell reactivation of cisplatin-damaged pRSVcat was assessed in three B cell lines (SKW 6.4, WIL2-NS, RPMI 1788), the monocytic cell line THP-1, and promyelocytic HL-60 cells. IC 50 values following a 3-day exposure of the five leukocyte cell lines to cisplatin ranged from 0.45 to 1.92 μM. Transfer of pRSVcat into all cell lines was effected by electroporation and the resultant CAT activity was measured 24 h later by a rapid single vial CAT assay. CAT activity corresponding to an average of 0.06 units of purified CAT enzyme was expressed by WIL2-NS cells. Very low to no expression of the CAT vector was observed in all other cell lines studied, despite the presence of intracellular levels of 3H-labelled pRSVcat comparable to WIL2-NS. Epstein-Barr virus transformed B cells (SKW 6.4 and RPMI 1788) did not successfully perform host cell reactivation. In WIL2-NS cells, platination of pRSVcat to defined levels of 5–40 platinum molecules per plasmid led to a graded reduction in CAT activity expressed following transfection. Platination levels of 20 and 40 platinum molecules per plasmid did not alter the efficiency of transfer of pRSVcat into these cells by electroporation. Data obtained in this study suggests that EBV transformation may possibly be a negative influence on host cell-reactivation assays for cisplatin-DNA damaged plasmid in non-T human leukocytes.

Structural and functional organization of the HF.10 human zinc finger gene (ZNF35) located on chromosome 3p21–p22

We report the structural and functional characterization of the HF.10 zinc finger gene (ZNF35) in normal human cells, as well as a processed pseudogene. The HF.10 gene spans about 13 kb and it is interrupted by three introns. All 11 zinc finger DNA-binding domains are contiguously encoded within the last 3′ exon. The genomic region surrounding HF.10 exon 1 contains a CpG island and acts as a promoter in vitro. Using transient CAT assay in cotransfection experiments in cultured cells, we have determined that the HF.10 finger protein is a transcriptional transactivator. Restriction enzyme mapping and partial nucleotide sequencing of the HF.10 pseudogene indicated that is has arisen by retroposition of spliced HF.10 mRNA. In situ hybridization experiments revealed that both the functional locus and the pseudogene map to chromosome 3p21p22, a region that is frequently deleted in small cell lung and renal carcinomas. Hybridization of the HF.10 gene and the HF.10 pseudogene DNA probes to metaphases from a small cell lung carcinoma cell line with the 3p deletion revealed that both loci are part of the deleted chromosome region.