Adherence of human monocytes and NK cells to human TNF-alpha-stimulated porcine endothelial cells

Discordant xenograft models undergoing delayed rejection response are characterized by xenograft infiltration with host monocytes and NK cells, associated with the release of large quantities of pro-inflammatory cytokines, such as TNF-alpha. In the present study, human monocytes (PBMo)/NK cells (PBNK) isolated from peripheral blood and cultured porcine aortic endothelial cells (PAEC) treated with recombinant human TNF-alpha (rhTNF-alpha) were used to investigate their adhesive interactions and mAbs against porcine E-selectin, human CD11a and CD49d were used to test their relative contributions to such intercellular adhesions. The PBMo exhibited significantly greater adherence to resting (unstimulated) PAEC than PBNK. The rhTNF-alpha upregulated E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression on PAEC and augmented the adhesiveness of PAEC for PBMo and PBNK in a time- and dose-dependent manner. In mAb blocking assays, anti-E-selectin, anti-CD11a and anti-CD49d mAbs did not inhibit PBMo adherence to rhTNF-alpha-stimulated PAEC when used singly, but resulted in a maximal inhibitory effect when used in combination. Regarding PBNK, anti-E-selectin mAb had no marked influence on PBNK adherence. The combined use of anti-CD11a and anti-CD49d mAbs produced additive reduction in the PBNK binding to rhTNF-alpha-stimulated PAEC, even to far below baseline (unstimulated) levels. Therefore, it is concluded that human TNF-alpha promotes the adhesiveness of PAEC for human monocytes and NK cells and that the mechanism underlying the increased adherence differs for PBMo and PBNK.     

The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion mutants of the two proteins to interact directly, suggesting that an interaction between the RS-domain of ASF/SF2 and a region between amino acid residues 208-735 on topoisomerase I accounts for the observed effect. Consistently, phosphorylation of the RS-domain with either topoisomerase I or a human cell extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors.     

Effects of antioxidant and nitric oxide on chemokine production in TNF-alpha-stimulated human dermal microvascular endothelial cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-kappaB) induced by TNF-alpha (10 ng/ml). Treatment with TNF-alpha for 8 h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h, but not by 1 mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-kappaB were induced by TNF-alpha for 2 h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-kappaB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

Effects of modification of the active site tyrosine of human DNA topoisomerase I

The human topoisomerase I-mediated DNA relaxation reaction was studied following modification of the enzyme at the active site tyrosine (position 723). A series of unnatural tyrosine analogues was incorporated into the active site of human topoisomerase I by utilizing misacylated suppressor tRNAs in an in vitro protein synthesizing system. The relaxation activities of the modified human topoisomerase I analogues having varied steric, electronic, and stereochemical features were all greatly diminished relative to that of the wild type. It was found that modifications involving replacement of the nucleophilic tyrosine OH group with NH2, SH, or I groups eliminated DNA relaxation activity, as did changing the orientation of the nucleophilic tyrosine OH group. Only tyrosine analogues having the phenolic OH group in the normal position with respect to the protein backbone were active; the relative activities could be rationalized in chemical terms on the basis of the H-bonding and the electronic effects of the substituents attached to the meta position of the aromatic ring. In addition, the poisoning of one of the modified human topoisomerase I analogues, as part of covalent binary complexes with DNA, by CPT and 20-thio CPT was evaluated.     

Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.     

Structure of the human type I DNA topoisomerase gene

We describe the molecular organization of the human gene coding for type I DNA topoisomerase. The coding sequence is split into 21 exons distributed over at least 85 kilobase pairs (kb) of human genomic DNA. The sizes of the 20 introns vary widely between 0.2 and at least 30 kb and all contain the sequence elements known to be required for pre-mRNA splicing. Several of the intron sequences separate exons encoding parts of the enzyme that are highly conserved between human and yeast suggesting that at least some of the exons may code for individual, structurally, or functionally important domains of the enzyme. We also describe the promoter sequence of the human topoisomerase I gene and show that it is composed of distinct functional elements.     

Characterization of a camptothecin-resistant human DNA topoisomerase I

Topoisomerase I purified from a camptothecin-resistant human leukemia cell line and from the parental, camptothecin-sensitive line were compared in vitro. Relaxation of supercoiled DNA by the wild type enzyme was inhibited in the presence of camptothecin, while the mutant enzyme was unimpaired. Camptothecin altered the cleavage pattern of the wild type but not of the mutant enzyme. The stability of cleavable complexes was studied at a preferred topoisomerase I-binding sequence recognized by both enzymes. Camptothecin greatly enhanced the kinetic stability of the cleavable complex formed by the wild type enzyme, whereas that of the mutant enzyme was only marginally affected. In the absence of camptothecin, the cleavable complex formed by the mutant enzyme was stabilized relative to that of the wild type by several criteria. Thus, the mutant enzyme cleaved the topoisomerase I recognition sequence with 2-fold higher efficiency than the wild type enzyme. The mutant cleavable complex had a higher kinetic stability and was less sensitive to salt dissociation than the wild type complex. Furthermore, the mutant enzyme formed cleavable complexes in the absence of divalent cations, which were required for complex formation by the wild type enzyme.     

Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage

We have studied the effect of the antitumor drug, camptothecin, on the interaction of human topoisomerase I with DNA at the sequence level. At a low molar ratio of enzyme to DNA, cleavage is prominent and unique, located at a previously described hexadecameric recognition sequence, while a number of strong additional cleavage sites appear in the presence of the drug. Camptothecin stimulates cleavage at the recognition sequence less than twofold, whereas cleavage at the additional sites is stimulated up to 200-fold. Camptothecin greatly enhances the stability of the cleavable complexes formed at the additional sites, whereas the complex formed at the hexadecameric sequence is only marginally affected. Cleavage was eliminated at certain sites in the presence of camptothecin. Taken together these observations demonstrate that at least three types of potential eukaryotic topoisomerase I cleavage sites can be distinguished by the use of camptothecin. Comparison of the sequences at the additional cleavage sites in the presence of camptothecin reveals that the most frequently cleaved dinucleotide is TG with no consensus for the flanking nucleotides.     

DNA strand-breaks induced by the topoisomerase I inhibitor camptothecin in unstimulated human white blood cells

Camptothecin (CPT) and actinomicyn-induced strand-breaks, repair and apoptosis in unstimulated human blood cells were studied using the DNA comet assay, and electrophoresis of low molecular weight DNA extracts. On the one hand, incubation of G0 leukocytes for 1 h with CPT induced DNA strand-breaks that were observed using the single cell gel electrophoresis technique. On the other hand, internucleosomal DNA fragments were not observed, suggesting that apoptosis had not occurred. DNA-strand-breaks caused by CPT were repaired 24 h after treatment; the migration of DNA fragments was assessed by a reduction in the number of comets. These data strongly suggest that the unexpected clastogenic effect of this topoisomerase I inhibitor is not due to the collision of the cleavage complex with the replication fork, since replication does not occur in G0. In our opinion, this effect could be due instead to the topoisomerase I enzyme being able to bind DNA in the absence of replication, probably in a way that is not strictly related to the progression of the cell cycle. Thus, CPT does not provoke apoptosis in quiescent leukocytes.     

Rapid purification and structural study of DNA topoisomerase I from human Burkitt lymphoma Raji cells

We have developed a rapid purification method for DNA topoisomerase I from Raji cells, a human Burkitt lymphoma cell line, using ammonium sulfate fractionation followed by chromatography on a Mono S column (FPLC, Pharmacia). By this method, the enzyme could be purified to near homogeneity within one day. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel revealed that the final preparation is mainly composed of a 100-kDa protein. The major enzyme activity sedimented through a glycerol density gradient at 5.7S, accompanied with a minor peak at 8.7S. The former may correspond to the monomer of the 100-kDa polypeptide, and the latter, to its dimeric form. The gel filtration study of the crude extract revealed an active molecular species of 200 kDa, in addition to 100 kDa, and lower molecular weight forms. These results suggest that DNA topoisomerase I is largely in monomeric form, but also has a minor population of the dimeric form.     

DNA binding induces conformational transition within human DNA topoisomerase I in solution

We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.     

Increased DNA topoisomerase I activity in aging human cell chromatin

Chromatin-associated DNA topoisomerase I activity was measured in human diploid fibroblasts during in vitro aging. No diilerence was detected as a function of cell age in the nicking and the closing activities of the DNA-unwinding enzyme. The capacity of type-I topoisomerase to relax superhelical DNA molecules was, however, increased in aged cells. An age-related increase in nucleoprotein content was also observed.