A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid >> 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.

Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation.

We reported previously that the human T-cell lymphotrophic virus type I (HTLV-I)-associated adult T-cell leukemia line HuT-102 produces a cytokine designated interleukin (IL) T that requires interleukin (IL) 2 receptor beta-subunit expression for its action. Using anti-cytokine antibodies, we demonstrated that IL-T is identical to the simultaneously described IL-15. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5'-untranslated region (UTR) of IL-15. Normally, by alternative splicing, this 118-nucleotide R element represents the most 5' region of several HTLV-I transcripts including tax, rex, and env. The introduction of the R element eliminated over 200 nucleotides of the IL-15 5'-UTR, including 8 of 10 upstream AUGs that are present in normal IL-15 messages. On analysis of the 5'-UTR of normal IL-15, we demonstrated that the presence of these 10 upstream AUGs interferes with IL-15 mRNA translation. Thus, IL-15 synthesis by the adult T-cell leukemia line HuT- 102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I R element fusion message that lacks many upstream AUGs.

Immunocytochemical localization of the endogenous neuroexcitotoxin quinolinate in human peripheral blood monocytes/macrophages and the effect of human T-cell lymphotropic virus type I infection.

Quinolinate (Quin), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that appears to act through the N-methyl-D-aspartate receptor system, was localized in cultured human peripheral blood monocytes/macrophages (PBMOs) by using a recently developed immunocytochemical method. Quin immunoreactivity (Quin-IR) was increased in gamma interferon (IFN-gamma)-stimulated monocytes/macrophages (MOs). In addition, the precursors, tryptophan and kynurenine, significantly increased Quin-IR. Infection of MOs by human T-cell lymphotropic virus type I (HTLV-I) in vitro substantially increased both the number of Quin-IR cells and the intensity of Quin-IR. At the peak of the Quin-IR response, about 40% of the cells were Quin-IR positive. In contrast, only about 2-5% of the cells were positive for HTLV-I, as detected by both immunofluorescence for the HTLV-I antigens and PCR techniques for the HTLV-I Tax gene. These results suggest that HTLV-I-induced Quin production in MOs occurs by an indirect mechanism, perhaps via cytokines produced by the infection but not directly by the virus infection per se. The significance of these findings to the neuropathology of HTLV-I infection is discussed.

A two-subunit type I DNA topoisomerase (reverse gyrase) from an extreme hyperthermophile.

A recently described reverse gyrase from the hyperthermophilic methanogen Methanopyrus kandleri is the only known example of a heterodimeric type I topoisomerase. The enzyme is made up of a 42-kDa subunit which covalently interacts with DNA (RgyA) and a 138-kDa subunit which binds ATP (RgyB). We have now cloned and sequenced the genes for both subunits of this enzyme. Surprisingly, the universally conserved type I topoisomerase domain [Lima, C. D., Wang, J. C. & Mondragon, A. (1994) Nature (London) 367, 138-146] which has been found as a contiguous polypeptide in the prokaryotes and eukaryotes is shared between the protomers. The subdomain with the active-site tyrosine is entirely within RgyA, whereas the subdomain implicated in noncovalent binding of the cleaved DNA strand is contained entirely in RgyB. The appearance of this unique structure in a highly conserved enzyme family supports the hypothesis that the methanogens branched from other prokaryotes and eukaryotes very early in evolution.

Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway.

The WAF1/CIP1 protein has been identified as a downstream mediator of the tumor suppressor p53 in regulating cell cycle progression through a G1-phase check-point. Recent work has implicated the functional status of p53 as a critical determinant in the apoptotic response of certain cell lines to DNA damaging agents. By using human T-cell leukemia virus type I-transformed lymphoid cell lines that differ in their level and function of wild-type p53, we investigated the induction of WAF1/CIP1 and apoptosis after exposure to Adriamycin, a genotoxic agent. We found that regardless of the p53 status in these cell lines, WAF1/CIP1 RNA was rapidly induced in response to Adriamycin treatment. An elevated level of WAF1/CIP1 protein was observed as well. Additionally, we demonstrated that apoptosis was induced in all cell lines analyzed despite some having functionally inactive p53 protein. Our data suggest that a p53-independent pathway may play a role in the apoptotic response observed in some cell lines after exposure to DNA damaging agents.

Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndrome, rd5, whose linkage on mouse chromosome 7 to Hbb and tub has homology to human Usher I reported on human chromosome 11p15. The electroretinogram in homozygous rd5/rd5 mouse is never normal with reduced amplitudes that extinguish by 6 months. Auditory-evoked response testing demonstrates increased hearing thresholds more than control at 3 weeks of about 30 decibels (dB) that worsen to about 45 dB by 6 months.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.

Genetic heterogeneity in cystinuria: the SLC3A1 gene is linked to type I but not to type III cystinuria.

Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described (I, II, and III). An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. To assess whether mutations in SLC3A1 are involved in different cystinuria phenotypes, linkage with this gene and its nearest marker (D2S119) was analyzed in 22 families with type I and/or type III cystinuria. Linkage with heterogeneity was proved (alpha = 0.45; P < 0.008). Type I/I families showed homogeneous linkage to SLC3A1 (Zmax > 3.0 at theta = 0.00; alpha = 1), whereas types I/III and III/III were not linked. Our data suggest that type I cystinuria is due to mutations in the SLC3A1 gene, whereas another locus is responsible for type III. This result establishes genetic heterogeneity for cystinuria, classically considered as a multiallelic monogenic disease.

Fungal metabolic model for human type I hereditary tyrosinaemia.

Type I hereditary tyrosinaemia (HT1) is a severe human inborn disease resulting from loss of fumaryl-acetoacetate hydrolase (Fah). Homozygous disruption of the gene encoding Fah in mice causes neonatal lethality, seriously limiting use of this animal as a model. We report here that fahA, the gene encoding Fah in the fungus Aspergillus nidulans, encodes a polypeptide showing 47.1% identity to its human homologue, fahA disruption results in secretion of succinylacetone (a diagnostic compound for human type I tyrosinaemia) and phenylalanine toxicity. We have isolated spontaneous suppressor mutations preventing this toxicity, presumably representing loss-of-function mutations in genes acting upstream of fahA in the phenylalanine catabolic pathway. Analysis of a class of these mutations demonstrates that loss of homogentisate dioxygenase (leading to alkaptonuria in humans) prevents the effects of a Fah deficiency. Our results strongly suggest human homogentisate dioxygenase as a target for HT1 therapy and illustrate the usefulness of this fungus as an alternative to animal models for certain aspects of human metabolic diseases.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.