TLR3 Activation of Intratumoral CD103+ Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity.

An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103+ cDC1 and, to a much lesser extent CD11b+ cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8+ Granzyme B+ T cells, (lower Treg/CD8+ ratio) and an important expansion of tumor-antigen specific CD8+ T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field.

Effects of the fluorine substituent positions of the intercalating ligands on the binding behavior and third-strand stabilization of two Ru(II) complexes toward poly(U)•poly(A)*poly(U) triplex RNA.

Two new Ru(II) polypyridyl complexes containing fluorine substituents, [Ru(bpy)2(o-fpip)]2+ (Ru1, bpy=2,2'-bipyridine, o-fpip=2-(2-fluorophenyl)imidazo[4,5-f] [1,10]phenanthroline) and [Ru(bpy)2(p-fpip)]2+ (Ru2, p-fpip=2-(4-fluorophenyl)imidazo[4,5-f] [1,10]phenanthroline) have been synthesized as binders for poly(U)•poly(A)∗poly(U) triplex RNA. The binding of the two complexes with the triplex RNA has been investigated by spectroscopic methods and viscosity measurements. Analysis of the electronic absorption spectra indicates that the association of intercalating Ru2 with the triplex RNA is greater than that of Ru1, which is also supported by spectroscopic titrations and viscosity measurements. Thermal denaturation studies reflect that third-strand stabilization depend on the nature of the two complexes and Ru2 is more effective for stabilization of the triplex RNA. Circular dichroism spectra of the triplex RNA in the presence of metal complexes indicate that the binding-induced CD perturbation of the triplex structure is more obvious by Ru2. The main results obtained here suggest that the positions of fluorine substituent in the intercalating ligands have a significant effect on the two complexes stabilizing the third strand.

Stimulation of the toll-like receptor 3 promotes metabolic reprogramming in head and neck carcinoma cells.

In this study, a possible link between the innate immune recognition receptor TLR3 and metabolic reprogramming in Head and Neck carcinoma (HNC) cells was investigated. The effects of TLR3 stimulation/knock-down were assessed under several culture conditions in 4 HNC cell-lines by cell growth assays, targeted metabolomics, and glycolysis assays based on time-resolved analysis of proton release (Seahorse analyzer). The stimulation of TLR3 by its synthetic agonist Poly(A:U) resulted in a faster growth of HNC cells under low foetal calf serum conditions. Targeted analysis of glucose metabolism pathways demonstrated a tendency towards a shift from tricarboxylic acid cycle (Krebs cycle) to glycolysis and anabolic reactions in cells treated with Poly(A:U). Glycolysis assays confirmed that TLR3 stimulation enhanced the capacity of malignant cells to switch from oxidative phosphorylation to extra-mitochondrial glycolysis. We found evidence that HIF-1α is involved in this process: addition of the TLR3 agonist resulted in a higher cell concentration of the HIF-1α protein, even in normoxia, whereas knocking-down TLR3 resulted in a lower concentration, even in hypoxia. Finally, we assessed TLR3 expression by immunohistochemistry in a series of 7 HNSCC specimens and found that TLR3 was detected at higher levels in tumors displaying a hypoxic staining pattern. Overall, our results demonstrate that TLR3 stimulation induces the Warburg effect in HNC cells in vitro, and suggest that TLR3 may play a role in tumor adaptation to hypoxia.

In Vivo Visualizing the IFN-ß Response Required for Tumor Growth Control in a Therapeutic Model of Polyadenylic-Polyuridylic Acid Administration.

The crucial role that endogenously produced IFN-ß plays in eliciting an immune response against cancer has recently started to be elucidated. Endogenous IFN-ß has an important role in immune surveillance and control of tumor development. Accordingly, the role of TLR agonists as cancer therapeutic agents is being revisited via the strategy of intra/peritumoral injection with the idea of stimulating the production of endogenous type I IFN inside the tumor. Polyadenylic-polyuridylic acid (poly A:U) is a dsRNA mimetic explored empirically in cancer immunotherapy a long time ago with little knowledge regarding its mechanisms of action. In this work, we have in vivo visualized the IFN-ß required for the antitumor immune response elicited in a therapeutic model of poly A:U administration. In this study, we have identified the role of host type I IFNs, cell populations that are sources of IFN-ß in the tumor microenvironment, and other host requirements for tumor control in this model. One single peritumoral dose of poly A:U was sufficient to induce IFN-ß, readily visualized in vivo. IFN-ß production relied mainly on the activation of the transcription factor IFN regulatory factor 3 and the molecule UNC93B1, indicating that TLR3 is required for recognizing poly A:U. CD11c(+) cells were an important, but not the only source of IFN-ß. Host type I IFN signaling was absolutely required for the reduced tumor growth, prolonged mice survival, and the strong antitumor-specific immune response elicited upon poly A:U administration. These findings add new perspectives to the use of IFN-ß-inducing compounds in tumor therapy.

Effect of toll-like receptor 3 agonists on the functionality and metastatic properties of breast cancer cell model.

There exists compelling evidence that Toll-like receptor 3 (TLR3) agonists can directly affect human cancer cells. The aim of this study was to investigate anti-cancer effects of TLR3 agonist in human breast cell line. We assessed potential effects of poly (A:U) on human breast cell line (MDA-MB-231) on a dose-response and time-course basis. Human breast cell line MDA-MB-231 was treated with different concentrations of poly (A:U) and lipopolysaccharide (LPS). Then, the following assays were performed on the treated cells: dose-response and time-course cytotoxicity using colorimetric method; matrix metalloproteinase-2 (MMP-2) activity using gelatin zymography method; apoptosis using annexin-v flowcytometry method; and relative expression of TLR3 and MMP-2 mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Following treatments, dose- response and time-course cytotoxicity using a colorimetric method, (MMP-2) activity (using gelatin zymography), apoptosis (using annexin-v flowcytometry method) assays and expression of TLR3 and MMP-2 genes (using PCR method) were performed. Cytotoxicity and flowcytometry analysis of poly (A:U) showed that poly (A:U) do not have any cytotoxic and apoptotic effects in different concentrations used. MMP-2 activity analysis showed significant decrease in higher concentrations (50 and 100 µg/ ml) between treated and untreated cells. Moreover, poly A:U treated cells demonstrated decreased expression of MMP-2 gene in higher concentrations. Collectively, our data indicated that human breast cancer cell line (MDA-MB-231) was highly responsive to poly (A:U). The antimetastatic effect of direct poly (A:U) and TLR3 interactions in MDA-MB-231 cells could provide new approaches in malignant tumor therapeutic strategy.

Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN-ß production capable of improving dendritic cell function.

Viral double-stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN-ß autocrine loop, dsRNA-elicited IFN-ß production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN-ß at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte-derived DCs. IFN-ß produced by poly I:C-activated human cancer cells increased the capacity of monocyte-derived DCs to stimulate IFN-γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3(-/-) mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1(-/-) mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN-ß production and contribute to the antitumoral response.

Double domain swapping in bovine seminal RNase: formation of distinct N- and C-swapped tetramers and multimers with increasing biological activities.

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.

Probing the binding of two sugar bearing anticancer agents aristololactam-ß-(D)-glucoside and daunomycin to double stranded RNA polynucleotides: a combined spectroscopic and calorimetric study.

The plant alkaloid aristololactam-ß-d-glucoside and the anticancer chemotherapy drug daunomycin are two sugar bearing DNA binding antibiotics. The binding of these molecules to three double stranded ribonucleic acids, poly(A)·poly(U), poly(I)·poly(C) and poly(C)·poly(G), was studied using various biophysical techniques. Absorbance and fluorescence studies revealed that these molecules bound non-cooperatively to these ds RNAs with the binding affinities of the order 10(6) for daunomycin and 10(5) M(-1) for aristololactam-ß-d-glucoside. Fluorescence quenching and viscosity studies gave evidence for intercalative binding. The binding enhanced the melting temperature of poly(A)·poly(U) and poly(I)·poly(C) and the binding affinity values evaluated from the melting data were in agreement with that obtained from other techniques. Circular dichroism results suggested minor conformational perturbations of the RNA structures. The binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived from calorimetry were in agreement with that obtained from spectroscopic data. Daunomycin bound all the three RNAs stronger than aristololactam-ß-d-glucoside and the binding affinity varied as poly(A)·poly(U) > poly(I)·poly(C) > poly(C)·poly(G). The temperature dependence of the enthalpy changes yielded negative values of heat capacity changes for the complexation suggesting substantial hydrophobic contribution to the binding process. Furthermore, an enthalpy-entropy compensation behavior was also seen in all systems. These results provide new insights into binding of these small molecule drugs to double stranded RNA sequences.

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34(+) cells.

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-ß induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Binding of the anticancer alkaloid sanguinarine to double stranded RNAs: insights into the structural and energetics aspects.

Elucidation of the molecular aspects of small molecule-RNA complexation is of prime importance for rational RNA targeted drug design strategies. Towards this, the interaction of the cytotoxic plant alkaloid sanguinarine to three double stranded ribonucleic acids, poly (A).poly(U), poly(I).poly(C) and poly(C).poly(G) was studied using various biophysical and thermodynamic techniques. Absorbance and fluorescence studies showed that the alkaloid bound cooperatively to these RNAs with binding affinities of the order 10(4) M(-1). Fluorescence quenching and hydrodynamic studies gave evidence for intercalation of sanguinarine to these RNA duplexes. Isothermal titration calorimetric studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived were in agreement with the overall binding affinity values obtained from spectroscopic data. The binding of sanguinarine stabilized the melting of poly(A). poly(U) and poly(I).poly(C) and the binding data evaluated from the melting data were in agreement with that obtained from other techniques. The overall binding affinity of sanguinarine to these double stranded RNAs varied in the order, poly(A).poly(U) > poly(I).poly(C) >> poly(C).poly(G). The temperature dependence of the enthalpy changes afforded negative values of heat capacity changes for the binding of sanguinarine to poly(A).poly(U) and poly(I).poly(C), suggesting substantial hydrophobic contribution in the binding process. Further, enthalpy-entropy compensation phenomena was also seen in poly(A).poly(U) and poly(I).poly(C) systems that correlated to the strong binding involving a multiplicity of weak noncovalent interactions compared to the weak binding with poly(C).poly(G). These results further advance our understanding on the binding of small molecules that are specific binders to double stranded RNA sequences.

Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.

Many cancer cells express Toll-like receptors (TLR) that offer possible therapeutic targets. Polyadenylic-polyuridylic acid [poly(A:U)] is an agonist of the Toll-like receptor TLR3 that displays anticancer properties. In this study, we illustrate how the immunostimulatory and immunosuppressive effects of this agent can be uncoupled to therapeutic advantage. We took advantage of two TLR3-expressing tumor models that produced large amounts of CCL5 (a CCR5 ligand) and CXCL10 (a CXCR3 ligand) in response to type I IFN and poly(A:U), both in vitro and in vivo. Conventional chemotherapy or in vivo injection of poly(A:U), alone or in combination, failed to reduce tumor growth unless an immunochemotherapeutic regimen of vaccination against tumor antigens was included. CCL5 blockade improved the efficacy of immunochemotherapy, whereas CXCR3 blockade abolished its beneficial effects. These findings show how poly(A:U) can elicit production of a range of chemokines by tumor cells that reinforce immunostimulatory or immunosuppressive effects. Optimizing the anticancer effects of TLR3 agonists may require manipulating these chemokines or their receptors.

Phase III trial of adjuvant 5-fluorouracil and adriamycin versus 5-fluorouracil, adriamycin, and polyadenylic-polyuridylic acid (poly A:U) for locally advanced gastric cancer after curative surgery: final results of 15-year follow-up.

BACKGROUND: This phase III trial was to compare 5-fluorouracil (5-FU), adriamycin, and polyadenylic-polyuridylic acid (poly A:U) against 5-fluorouracil plus adriamycin (FA) for operable gastric cancer. PATIENTS AND METHODS: From 1984 to 1989, patients who had D(2-3) curative resection were randomly assigned to receive chemotherapy or chemoimmunotherapy. Chemotherapy consisted of 12 mg/kg 5-FU every week for 18 months and 40 mg/m2 adriamycin every 3 weeks for 12 cycles. Chemoimmunotherapy consisted of FA plus 100 mg of poly A:U weekly for six cycles and was followed 6 months later by six weekly 50-mg booster injections. RESULTS: A total of 292 patients were enrolled. After excluding 12 ineligible patients, 142 and 138 patients were allocated to each treatment. Patients were balanced with prognostic variables: age, sex, tumor location, differentiation, degree of tumor invasion (T2-T4a), and lymph node status (N0-N2). During the 15-year follow-up, chemoimmunotherapy significantly prolonged overall (P = 0.013) and recurrence-free (P = 0.005) survivals compared with chemotherapy alone. The survival benefits were prominent in the subset of patients with T3/T4a, N2, or stage III. Treatments were generally well tolerated in both arms. CONCLUSIONS: These results indicate a survival advantage of chemoimmunotherapy with a regimen of FA and poly A:U in curatively resected gastric adenocarcinoma.

Dramatic selectivity differences in the association of DNA and RNA models with new ethylene- and propylene diamine derivatives and their copper complexes.

The affinities of polyamines consisting of ethylenediamine units equipped with either one or two terminal naphthyl-, anthryl-, or acridyl units towards PolyA.PolyU as an RNA model, and Poly(dA).Poly(dT) as a DNA model are screened by measuring the melting point changes (DeltaT(m)) of the double strands, and also partially by a fluorimetric binding assay using ethidium bromide. The larger aromatic moieties with long spacers between them allow bisintercalation; this leads to an increased preference for DNA in comparison to RNA, where ion pairing of the ammonium centers with the major RNA groove phosphates dominates. Allosteric affinity control by metalation is achieved e.g. with Cu(2+) ions, which induce conformational distortions within the chains. With anthryl- in contrast to naphthyl derivatives intercalation can be so strong that distortion of the ethylenediamine chain by metalation is not powerful enough. A particularly high concentration of positive charges is accessible with tripodal derivatives built up from ethylenediamine and propylenediamine units; in the absence of aryl parts, which interfere with the RNA groove preference, one observes the highest affinity difference known until today, reflected in a melting point ratio of DeltaT(m(RNA))/DeltaT(m(DNA)) = 40, whereas other synthetic ligands reach only a DeltaT(m(RNA))/DeltaT(m(DNA)) ratio of about 3.

Copper insertion facilitates water-soluble porphyrin binding to rA.rU and rA.dT base pairs in duplex RNA and RNA.DNA hybrids.

The binding of the copper(II) complex of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to double-helical polynucleotides has been studied by optical absorption, circular dichroism (CD), and resonance Raman spectroscopic methods. The target polymers were RNA and RNA.DNA hybrids consisting of rA.rU, rI.rC, rA.dT, and rI.dC base pairs. Relative to the metal-free H(2)TMPyP [Uno, T., Hamasaki, K., Tanigawa, M., and Shimabayashi, S. (1997) Inorg. Chem. 36, 1676-1683], CuTMPyP binds to poly(rA).poly(dT) and poly(rA).poly(rU) with a greatly increased binding constant. The external self-stacking of the porphyrin on the surface of the polymers was evident from the strong conservative-type induced CD signals. The signal intensity correlated almost linearly with the number of stacking sites on the polymer except for poly(rA).poly(dT), which showed extraordinarily strong CD signals. Thus, the bound porphyrin may impose an ordered architecture on the polymer surface, the stacking being facilitated by the more planar nature of the CuTMPyP than the nonmetal counterpart. Resonance Raman spectra of the stacked CuTMPyP were indistinguishable from those of the intercalated one with positive delta(Cbeta-H) and negative delta(Cm-Py) bending shifts, and hence the stacked porphyrins are suggested to adopt a similar structure to that of intercalated ones. Porphyrin flattening by copper insertion opens a new avenue for medical applications of porphyrins, blocking biological events related to RNA and hybrids in malignant cells.

Polyadenylic-polyuridylic acid plus locoregional radiotherapy versus chemotherapy with CMF in operable breast cancer: a 14 year follow-up analysis of a randomized trial of the Fédération Nationale des Centres de Lutte contre le Cancer (FNCLCC).

With a median follow-up of 14 years, the combination of polyadenylic-polyuridylic acid plus locoregional radiotherapy (257 patients) has significantly improved disease-free survival (p = 0.03) and significantly reduced the incidence of metastases (p = 0.04) when compared to CMF alone (260 patients), in women with operable breast cancer. The trial does not, however, permit an appreciation of the respective role of radiotherapy and PolyAU in these results.

Peri-operative acute phase response and cytokine releasein women with breast cancer: modulation bypolyadenylic-polyuridylic acid.

AIMS: Acute phase reactants (APRs) are believed to play an important biological role in trauma, sepsis and malignant disease. We have investigated the induction of the APR, C-reactive protein (CRP), by the biological response modifier, polyadenylic-polyuridylic acid (PAPU) during the peri-operative period. METHODS: Twenty post-menopausal women with breast cancer undergoing mastectomy were randomized into a double blind, placebo-controlled, parallel group pilot study. PAPU (150 mg) or placebo was given intravenously the day prior to surgery (D -1), the day of surgery (D 0) and post-operatively on days (D 1, 3, 5, 7 and 14). Blood samples were collected on eight different days (D -2, -1, 0, 1, 2, 4, 6 and 18). CRP was significantly elevated in the PAPU group (P<0.05) on days 2 and 4, when compared with patients receiving placebo. The serum levels of cytokines believed to induce hepatic APRs, were also measured. RESULTS: The serum concentration of IL-6 was elevated on days 1, 2, 4 and 6 (P<0.05), TNF- alpha and IL-1 beta levels were increased on days 1 and 2 (P<0.05), respectively, while the serum level of soluble interleukin-2 receptor (sIL-2R) was elevated above the baseline on days 0, 2, 4, 6 and 18 in the PAPU group, when compared with the baseline. CONCLUSIONS: This modulation of acute phase response may have important implications for patients with cancer undergoing surgery.

Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species.

The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3'-untranslated region of the hamster beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for beta ARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.

Ultrastructural nucleolar alterations induced by an ametantrone--poly r(A-U) complex.

The current study has documented changes in the ultrastructure as well as in the intranucleolar distribution of rDNA and rRNA in RT4 (human transitional cell bladder carcinoma) cell nucleoli following a 3-h exposure to toxic doses of 50 microM ametantrone (AMT), 200 microM poly (adenylate-uridylate) (poly r(A-U) or an AMT/poly r(A-U) combination with an AMT/polyribonucleotide ratio of 1:4 and a poly r(A-U) concentration of 200 microM. While the main nucleolar components (fibrillar center (F), dense fibrillar component (D), granular component (G) and interstices (I) can be discerned following all treatments, the nucleoli exhibit: compaction, segregation, a decrease in the number of F, an increase in the size of remaining F, margination of intranucleolar chromatin and retention of intranucleolar pre-rRNA and rRNA. The relative abilities of the test agents to induce nucleolar compaction are AMT/poly r(A-U) > poly r(A-U) > AMT > sham-treated, while the abilities of the test agents to induce the remaining nucleolar changes are AMT/poly r(A-U) > or = AMT > poly r(A-U) > sham-treated cells. Poly r(A-U) and the induced interferon induce nucleolar compaction, while AMT produces nucleolar segregation. These results are consistent with a model in which the poly r(A-U) and/or the AMT inhibit DNA transcription and rRNA processing as well as the release of nascent preribosomes from the nucleolus.