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.     

Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175.

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.     

Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro.

When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene.     

Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12

We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.     

Expression of herpes simplex virus thymidine kinase gene in aquatic filamentous fungus Achlya ambisexualis

We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.     

PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.     

Factors Governing Expression of the Herpes Simplex Virus Gene for Thymidine Kinase in Clonal Derivatives of Transformed Mouse L Cells

The cells used in this study are sublines of a transformed mouse L cell line (designated H2) that carries the herpes simplex virus (HSV) gene for thymidine kinase (tk) as well as other viral genetic information acquired after exposure of the parental Ltk(-) cells to UV-irradiated HSV type 1. These sublines of the H2 cell line were isolated by cloning under nonselective conditions and were shown to express widely different levels of viral tk. Selective media were used to isolate phenotypically tk(-) and tk(+) variants in sequence from one of the clonal derivatives. As previously reported, superinfection of the tk(+) cell lines with tk(-) HSV type 1 resulted in enhancement of tk activity. A new finding was that viral tk activity could be induced by superinfection in at least 30% of cells from the phenotypically tk(-) sublines, indicating that a functional viral tk gene was retained in a significant proportion of the cells. Experiments were designed to test for the presence of regulatory factors that could influence tk expression in the nonsuperinfected sublines of H2. Absence of freely diffusible regulatory factors was indicated by the finding that the fusion of phenotypically tk(-) and tk(+) cells and untransformed cells in appropriate combinations did not affect the levels of tk detected. Moreover, there was no evidence for the presence in phenotypically tk(+) transformed cells of HSV-specific regulatory factors that could influence expression of tk from a superinfecting viral genome. Phenotypically tk(+) sublines of H2 were found to differ from the phenotypically tk(-) sublines and from untransformed cells in that the tk(+) cells synthesized viral proteins earlier and produced greater yields of infectious HSV progeny after superinfection with wild-type tk(+) virus. We can conclude that the absence of tk expression in the tk(-) H2 sublines cannot be accounted for by rearrangements or loss of DNA sequences encoding the enzyme itself or of sequences necessary for induction of the gene by superinfecting HSV. Moreover, it appears that the expression of tk in the tk(+) H2 sublines correlates with the presence of some factor that can enhance (or the absence of some factor that can depress) HSV replication and gene expression.