The identification of thymidylate synthase peptide domains located in the interface region that bind thymidylate synthase mRNA

Thymidylate synthase (TS) is a critical chemotherapeutic target and intracellular levels of TS are an important determinant of sensitivity to TS inhibitors. Translational autoregulation represents one cellular mechanism for controlling the level of expression of TS. This mechanism involves the binding of TS protein to its own messenger RNA (mRNA), thus, repressing translational efficiency. The presence of excess substrate or inhibitors of TS leads to derepression of protein binding to mRNA, resulting in increased translational efficiency and ultimately increased levels of TS protein. TS protein has been shown to bind to two distinct areas on its mRNA. The goal of the present work is to define the TS domains responsible for this interaction. Using a separate series of overlapping 17-mer peptides spanning the length of both the human and Escherichia coli TS sequences, we have identified six potential domains located in the interface region of the TS protein that bind TS mRNA. The identified domains that bind TS mRNA include three concordant regions in both the human and E. coli peptide series. Five of the six binding peptides contain at least one invariant arginine residue, which has been shown to be critical in other well-defined protein-RNA interactions. These data suggest that the identified highly conserved protein domains, which occur at the homodimeric interface of TS, represent potential participating sites for binding of TS protein to its mRNA.     

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

Thymidylate synthase (TS) is an important target of several chemotherapeutic agents, including 5-FU and raltitrexed (Tomudex). During TS inhibition, TTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). Thus, BER initiated by uracil DNA glycosylase (UDG) activity has been hypothesized to influence the toxicity induced by TS inhibitors. In this study we created a human cell line expressing the Ugi protein inhibitor of UNG family of UDGs, which reduces cellular UDG activity by at least 45-fold. Genomic uracil incorporation was directly measured by mass spectrometry following treatment with TS inhibitors. Genomic uracil levels were increased over 4-fold following TS inhibition in the Ugi-expressing cells, but did not detectably increase in UNG proficient cells. Despite the difference in genomic uracil levels, there was no difference in toxicity between the UNG proficient and UNG-inhibited cells to folate or nucleotide-based inhibitors of TS. Cell cycle analysis showed that UNG proficient and UNG-inhibited cells arrested in early S-phase and resumed replication progression during recovery from RTX treatment almost identically. The induction of gamma-H2AX was measured following TS inhibition as a measure of whether uracil excision promoted DNA double strand break formation during S-phase arrest. Although gamma-H2AX was detectable following TS inhibition, there was no difference between UNG proficient and UNG-inhibited cells. We therefore conclude that uracil excision initiated by UNG does not adequately explain the toxicity caused by TS inhibition in this model.     

ODN 491, a novel antisense oligodeoxynucleotide that targets thymidylate synthase, exerts cell-specific effects in human tumor cell lines

Thymidylate synthase (TS) is essential for DNA replication and is a target for cancer chemotherapy. However, toxicity to normal cells and tumor cell drug resistance necessitate development of new therapeutic strategies. One such strategy is to use antisense (AS) technology to reduce TS mRNA and protein levels in treated cells. We have developed oligodeoxynucleotides (ODNs) that target different regions of TS mRNA, inhibit human tumor cell proliferation as single agents, and enhance cytotoxicity of clinically useful TS protein-targeting drugs. Here we describe ODN 491, a novel 20mer AS ODN complementary to a previously untargeted portion of the TS mRNA coding region. AS ODN 491 decreased TS mRNA levels to different degrees in a panel of human tumor-derived cell lines, and induced different physiological effects in a tumor cell line-dependent manner. ODN 491 (like AS TS ODN 83, previously shown to be effective) decreased TS protein levels in HeLa cells with a concomitant increase in sensitivity to TS-targeting chemotherapeutics. However (and contrary to HeLa cell response to an AS ODN 83), it did not, as a single agent, inhibit HeLa cell proliferation. In MCF-7 cells, ODN 491 treatment was less effective at reducing TS mRNA and did not reduce TS protein, nor did it enhance sensitivity to TS-targeting or other chemotherapeutics. Moreover, specifically in MCF-7 cells but not HeLa cells, ODN 491 as a single agent induced apoptosis. These data indicate that AS TS ODN 491 is an effective AS reagent targeting a novel TS mRNA region. However, treatment of tumor cell lines with AS TS ODNs targeting different TS mRNA regions results in a pattern of physiological effects that varies in a tumor cell line-specific fashion. In addition, the capacity of different AS TS ODNs to induce physiological effects does not correlate well with their capacity to reduce TS mRNA and/or protein and, further, depends on the region of TS mRNA selected for targeting. Recognition of tumor cell-specific and mRNA region-specific variability in response to AS TS ODNs will be important in designing AS TS ODNs for potential clinical use.     

Analysis of thymidylate synthase gene amplification and of mRNA levels in the cell cycle

We report that the gene for thymidylate synthase (TS) is amplified in the mouse cell line L1210:C15 that was selectively grown in increasing concentrations of the competitive inhibitor of thymidylate synthase, CB3717. The gene is amplified 50-fold compared to the parental cell line. Amplification has not been accompanied by any major rearrangements, and the increase in gene copy number is reflected in elevation of thymidylate synthase mRNA levels. The amplification is relatively stable as there was only a 2- to 3-fold decrease in the number of amplified TS genes when cells were grown in the absence of selection for 375 generations. We also observe a 30- to 40-fold increase in number of copies of the dihydrofolate reductase gene with 7-fold elevation of the RNA product, and we suggest that this may be due to cross-inhibition of dihydrofolate reductase by CB3717. Thymidylate synthase mRNA levels in L1210 and L1210:C15 show no variation within the different phases of the cell cycle but are significantly reduced during quiescence.     

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.     

Regulatory mechanism of Toona sinensis on mouse leydig cell steroidogenesis

Toona sinensis (TS), a kind of arbor, widely distributes nowadays in Asia. The leaves of TS have been used as an effective nutritious food in Chinese society for a long time. It was reported that Toona sinensis can induce apoptosis of cancer cells, reduce plasma glucose in diabetic rats, and improve lipolysis of differentiated 3T3-L1 adipocyte and its uptake of glucose. It has also been shown that TS may increase dynamic activity of human sperm. Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity. Primary mouse Leydig cells were purified to conduct the in vitro experiments. Different concentrations of crude Toona sinensis were added to primary mouse Leydig cells and the testosterone production was determined. The results showed that crude TS significantly inhibited both basal and human chorionic gonadotropin (hCG)-stimulated testosterone productions in dose dependent manner, respectively (P<0.05). Crude TS also reduced the forskolin- and dibutyryl-cAMP (dbcAMP)-stimulated testosterone production (P<0.05), which indicated that crude TS might affect protein kinase A (PKA) signal transduction pathway at the site after the formation of cyclic AMP. Moreover, TS inhibited Leydig cell steroidogenesis by suppressing the activity of steroidogenic enzymes including P450 side chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 20 alpha-hydroxylase and 17 beta-hydroxysteroid dehydrogenase (P<0.05). In summary, these results suggested that TS inhibited steroidogenesis by suppressing the cAMP-PKA signaling pathway and the activities of steroidogenic enzymes in normal mouse Leydig cells.     

FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells

Signaling by fibroblast growth factor (FGF) is essential is for trophoblast stem (TS) cells and preimplantation embryos. FGF4 provides essential signaling, but the expression of the complete set of 23 FGF family members has not been analyzed. Here, semi-quantitative RT-PCR and microarray analyses were used to define expression of all FGF ligand mRNA. RT-PCR was done for developmentally important FGF subfamilies, FGF10/FGF22 and FGF8/FGF17/FGF18 as well as FGF11. FGF4 and FGF18 are detected at highest levels by RT-PCR and microarrays. FGF10 was detected at low levels in both assays. FGF11 was detected at moderate levels by microarray, but not by RT-PCR. FGF17 was detected at low levels by array and moderate levels by RT-PCR. FGF8 and FGF22 were detected by RT-PCR, but not by microarrays during late cleavage divisions. FGF8, FGF5, and FGF9 were detected in the oocyte by microarray. FGF2, FGF3, and FGF7 were not detected by RT-PCR or microarrays and FGF13, FGF14, and FGF23 were not detected by microarray. Since a major role of FGF is to maintain TS cells, we tested human and mouse placental cell lines and early gestation human placenta for expression of FGF ligands. Expression in mouse TS cells was compared with preimplantation embryos, and human placental cell line expression was compared with human placenta, to infer which ligands are expressed in placental lineage vs. other cell lineages. The data suggest that human and mouse placenta share FGF18 and its high expression suggests preimplantation and early placental function.     

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase

Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.     

Human Cdc14A Reverses CDK1 Phosphorylation of Cdc25A on Serines 115 and 320

Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin-B1-dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle-regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation-mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability.     

Angiotensin-converting enzyme signalling in human preadipocytes and adipocytes

Angiotensin-converting enzyme (ACE, kininase II) is a plasma membrane zinc metallopeptidase that acts as a key enzyme for the extracellular conversion of vasoactive peptides. Recently, ACE outside-in signalling in endothelial cells has been described. The present study tested the hypothesis that ACE signalling is not restricted to endothelial cells and may act as an additional peptide receptor on human preadipocytes and adipocytes. ACE protein levels were not changed during adipose conversion of human primary preadipocytes. The enzyme was primarily localized to the non-detergent-resistant fraction of the membrane and phosphorylated in non-dividing cells. Antibody arrays of whole cell lysate detected putative ACE-interacting proteins, which all share important roles in cell cycle control and/or apoptosis. These findings suggest that ACE is a versatile molecule, involved both in the regulation of extracellular peptide concentrations and direct intracellular signalling. In human adipose cells ACE may potentially influence exit from the cell cycle, differentiation, and programmed cell death signalling.     

The properdin-like type I repeats of human thrombospondin contain a cell attachment site

Thrombospondin (TS) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to TS. These include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an RGDA sequence of TS. We have characterized a mAb against human TS, designated A4.1, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for A4.1 lies within the amino terminal half of the central stalklike region of TS which is distinct from the three known cell attachment sites. This region of TS is recovered in a 50-kD peptide after chymotryptic digestion of TS in EDTA. It contains the procollagen-like domain of TS as well as three type I repeats of a 60-residue segment homologous to two malarial proteins and the complement proteins properdin, and factors C6 through C9. The purified chymotryptic fragment is an effective attachment factor for G361 cells. A4.1 blocks adhesion to the 50-kD domain, as do some sulfated glycoconjugates. RGD (and RGE) peptides and mAbs against other domains of TS are not inhibitory. Peptides (19 mers) based on the core homology sequence of the three type I repeats of TS are potent attachment factors for these cells, and this adhesion is also inhibited by sulfated glycoconjugates. A polyclonal antibody raised against one of these peptides inhibits adhesion of G361 cells to the peptides, to the 50-kD fragment and to intact TS. Thus a new cell-adhesion site has been identified in TS whose sequence is very similar to the site identified in region II of the circumsporozoite protein of malaria parasites (Rich, K. A., F. W. George IV, J. L. Law, and W. J. Martin. 1990. Science (Wash. DC) 249:1574-1577. Thus there may be a common receptor which binds TS, malarial proteins, and properdin.     

Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucleotide induces protective mucosal and systemic trypanosoma cruzi immunity involving CD8+ CTL and B cell-mediated cross-priming

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.     

Selenium and asthma

Se is a potent nutritional antioxidant important for various aspects of human health. Because asthma has been demonstrated to involve increased oxidative stress, levels of Se intake have been hypothesized to play an important role in the pathogenesis of asthma. However, significant associations between Se status and prevalence or severity of asthma have not been consistently demonstrated in human studies. This highlights both the complex etiology of human asthma and the inherent problems with correlative nutritional studies. In this review, the different findings in human studies are discussed along with results from limited intervention studies. Mouse models of asthma have provided more definitive results suggesting that the benefits of Se supplementation may depend on an individual’s initial Se status. This likely involves T helper cell differentiation and the mechanistic studies that have provided important insight into the effects of Se levels on immune cell function are summarized. Importantly, the benefits and adverse effects of Se supplementation must both be considered in using this nutritional supplement for treating asthma. With this in mind new approaches are discussed that may provide more safe and effective means for using Se supplementation for asthma or other disorders involving inflammation or immunity.