The involvement of nuclear factor-kappa B in cyclooxygenase-2 overexpression in murine colon cancer cells transduced with herpes simplex virus thymidine kinase gene.

We have previously reported that transduction of murine colon cancer cells (MC38) with herpes simplex virus thymidine kinase (HSV-tk) gene results in a significant enhancement of tumor growth rate in vivo and overexpression of cyclooxygenase-2 (COX-2). Our current study aimed to investigate the involvement of nuclear factor-kappa B (NF-kappaB), a pivotal transcriptional regulator of COX-2, in the upregulation of COX-2 expression by HSV-tk. It was found that HSV-tk gene transduction of MC38 cells results in significantly enhanced NF-kappaB activity, increased phosphorylation and degradation of inhibitor-kappa Balpha (IkappaBalpha) and enhanced translocation of NF-kappaB to the nucleus. Treatment of HSV-tk-transduced MC38 cells with sulfasalazine, a potent NF-kappaB inhibitor, led to dose-dependent inhibition of NF-kappaB activity, IkappaB phosphorylation and nuclear translocation of NF-kappaB, accompanied by significantly decreased COX-2 expression and reduced release of prostaglandin E2. Transient transfection experiments with COX-2 promoter constructs fused to luciferase reporter gene revealed that mutation in NF-kappaB-responsive element of COX-2 promoter significantly reduced promoter activity in HSV-tk-transduced MC38 and COS-7 cells, whereas it had no effect on promoter activity in the respective wild-type cells. At last, it was found that HSV-tk gene transduction causes significant enhancement of NF-kappaB activity and COX-2 expression in two additional tumor cell lines, 9L and T24. These findings suggest that HSV-tk gene transduction results in NF-kappaB pathway activation, which is essential for COX-2 overexpression by HSV-tk.

Plasmid vectors designed for the analysis of transcription termination signals.

We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage lambda oLpL operator and promoter. Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes. In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene. Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation. We used these plasmids to select a lambda DNA fragment which includes the N-unresponsive tJ transcriptional terminator. This DNA fragment was inserted between the cat and galK genes. Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region. Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII-. As a control, we showed a complete antitermination of the lambda t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids.

A four-amino-acid insertion in the ligand-binding domain inactivates hRXRbeta and renders dominant negative activity.

Retinoid X receptors (RXRs) are members of the steroid and thyroid hormone receptor superfamily of hormone-dependent transcription factors that mediate the pleiotropic effect of retinoids. Here, we report the initial characterization of an isoform of hRXR beta, termed hRXR beta3, which was previously identified as an H-2RIIBP isoform (Epplen and Epplen, 1992). The hRXR beta3 isoform cotains an in-frame insertion of four amino acids (SLSR) in the ligand binding domain at codon 419. The isoform is generated by alternate use of a 3' splice acceptor site and was detectable by reverse transcription polymerase chain reaction (RT-PCR) in all human tumor cell lines and mouse tissues examined. Chimeric receptors, in which the ligand-binding domain of hRXR alpha was substituted by the corresponding domain from hRXR beta3, were used to investigate the consequences of the SLSR insertion on the transactivation and DNA-binding functions of the chimeric receptor. Co-transfection assays revealed that a chimera RXR alpha/beta3 receptor failed to transactivate the RXR-specific CRBPII promoter, whereas the identical chimera lacking the SLSR insertion was active. The RXR alpha/beta3 receptor exhibited dominant negative activity against active retinoid X and retinoic acid receptors on retinoid-responsive promoters. Moreover, the RXR alpha/beta3 protein failed to interact physically with the retinoic acid receptor (RAR) to form heterodimers as detected by physical association assays, and failed to bind DNA containing an RAR-responsive element. Therefore, this suggests that the SLSR insertion in the ligand-binding domain of the RXR alpha/beta3 receptor is responsible for the altered behavior of the chimera. Our findings raise the possibility that RXR alpha/beta3, and perhaps hRXR beta3 isoform, function by titrating a limiting adaptor molecule that is involved in mediating retinoid function.

Autoregulation of cloned human protein kinase C beta and gamma gene promoters in U937 cells.

Protein kinase C (PKC) serine/threonine kinases transduce cellular signals initiated by phospholipase C activation and diacylglycerol production. Human gene sequences from the beta and gamma isoforms were cloned and sequenced, and transcriptional regulation was studied. The major PKC beta transcription initiation site was identified by primer extension and S1 nuclease protection. Additional transcription initiation sites were located within a CpG-rich region proximal to the ATG. The transcription initiation site of the PKC gamma gene was identified by primed cDNA synthesis. In transfection experiments, the PKC gamma promoter was expressed at high level in U937 and HL60 cells but not in COS-1 cells. A sequence motif (AnAGATTCanAGAGnCa), reiterated over at least 1 kb, was located approximately 1.5 kb 5' of the PKC gamma translation initiation codon. This repetitive motif is abundant in run-on RNA in the hematopoietic and epithelial cell lines tested. Analysis of promoter deletion constructs by transient transcription assays in U937, IM9, and COS-1 cells showed negative regulation of the PKC beta promoter by sequences located between -3,000 and -690. although no homology between PKC beta and PKC-gamma 5'-flanking sequences was observed, both PKC beta and PKC gamma promoters were potently induced by 12-phorbol 13-myristate in transfected U937 cells.

Ethnobotanical survey in the Palestinian area: a classification of the healing potential of medicinal plants.

An ethnobotanical survey was carried out in the West Bank to evaluate the relative efficacy of the plants used to treat skin diseases and prostate cancer. A total number of 102 informants, 30 years and older and either native born or had been living in the West Bank for more than 30 years, were interviewed using a previously prepared questionnaire. Of about 165 plant species mentioned by the informants, 63 (38.1%) were mentioned by three or more informants. On the basis of their primary uses, 21 of these plants were reported to relieve skin disorders, 17 for urinary system disorders, 16 for gastric disorders, nine for cancer and prostate disorders, eight for arthritis, five for respiratory problems, and five for other ailments. Indices on fidelity levels (FLs), relative popularity level (RPL), and rank-order priority (ROP) were calculated. Plants were classified in two groups: 'popular' (RPL=1) or 'unpopular' (RPL<1). The following plant species were classified as popular in this study: Teucrium polium, Matricaria aurea, Urtica pilulifera, Paronychia argentea, Petroselinum sativum, and Salvia fruticosa. The remaining 57 species were classified as 'unpopular'. Fifty-nine plants were claimed to be effective against cancer and prostate disorders, which include Arum dioscorides, U. pilulifera, Allium sativum, Viscum cruciatum, and Allium cepa.

Translation initiation of bacteriophage lambda gene cII requires integration host factor.

Escherichia coli integration host factor (IHF), a DNA-binding protein, positively regulates expression of the lambda cII gene. Purified IHF stimulates cII protein synthesis in vitro, suggesting a direct role for host factor in cII expression. Further evidence for a direct role for IHF was obtained with operon and gene fusions between cII and lacZ or cII and galE. Analysis of these fusions in vivo demonstrated that IHF is essential for the initiation of cII translation. Replacement of the entire cII coding sequence with lacZ yielded a gene fusion which was still IHF dependent. However, a cII-galE fusion carrying a hybrid ribosome binding region expressed galE in IHF mutants. These results indicate that sequences which make cII translation IHF dependent lie between the ribosome binding region and the initiating codon of cII. Failure to translate cII activates a transcription terminator located within cII and results in polar effects on downstream transcription. This polarity is suppressed by the lambda N antitermination function. When cloned into another context, the terminator is active in both wild-type and IHF mutant strains. The amino terminus of cII is located near an IHF binding site in a region with considerable dyad symmetry. The role of IHF in cII translation may be to prevent formation of an RNA-RNA duplex that sequesters the ribosome binding site of cII. The binding of IHF might influence RNA structure by altering the rate of the dissociation of RNA from the DNA template.

Identification and characterization of baxepsilon, a novel bax variant missing the BH2 and the transmembrane domains.

The Bax gene is a member of the Bcl2 family that functions to regulate the programmed cell death process. A number of Bax isoforms have been previously identified: alpha, beta, gamma, delta, and omega. Here we report the identification and characterization of an additional Bax variant, termed Baxepsilon. The newly identified Bax variant contains a 97-base insertion generated by alternative splicing which includes a previously unidentified exon between exons 4 and 5. The insertion causes the production of a truncated Bax protein, termed Baxepsilon, which encodes a protein of 164 residues with a calculated molecular weight of 18 kDa. The last 69 amino acids of Baxalpha that encompass the BH2 and the TM domains are missing in Baxepsilon. The Baxepsilon protein, when expressed as a GST fusion protein, associated efficiently with Baxalpha, Baxepsilon, Bcl2, and Bcl-xL. In addition, Baxepsilon was active in inducing apoptosis when tested in a transient transfection assay. Furthermore, the presence of antiapoptotic genes including Bcl2, Bcl-xL, and baculovirus p35 abrogated Baxepsilon and Baxalpha function. Although the newly identified Bax variant was detectable by RT-PCR in several normal mouse tissues, the role of this variant in controlling programmed cell death is currently unknown.