Efficacy of iclaprim against wild-type and thymidine kinase-deficient methicillin-resistant Staphylococcus aureus isolates in an in vitro fibrin clot model.

Iclaprim is a novel diaminopyrimidine antibiotic that is active against methicillin-resistant Staphylococcus aureus (MRSA). However, it is known that the activity of diaminopyrimidines against S. aureus is antagonized by thymidine through uptake and conversion to thymidylate by thymidine kinase. Unlike with humans, for whom thymidine levels are low, thymidine levels in rodents are high, thus precluding the accurate evaluation of iclaprim efficacy in animal models. We have studied the bactericidal activity of iclaprim against an isogenic pair of MRSA isolates, the wild-type parent AW6 and its thymidine kinase-deficient mutant AH1252, in an in vitro fibrin clot model. Clots, which were aimed at mimicking vegetation structure, were made from human or rat plasma containing either the parent AW6 or the mutant AH1252, and they were exposed to homologous serum supplemented with iclaprim (3.5 microg/ml), trimethoprim-sulfamethoxazole (TMP-SMX; 8/40 microg/ml), vancomycin (40 microg/ml), or saline, each of which was added one time for 48 h. In rat clots, iclaprim and TMP-SMX were bacteriostatic against the parent, AW6. In contrast, they were bactericidal (> or = 3 log10 CFU/clot killing of the original inoculum) against the mutant AH1252. Vancomycin was the most active drug against AW6 (P < 0.05), but it showed an activity similar those of iclaprim and TMP-SMX against AH1252. In human clots, iclaprim was bactericidal against both AW6 and AH1252 strains and was as effective as TMP-SMX and vancomycin (P > 0.05). Future studies of animals using simulated human kinetics of iclaprim and thymidine kinase-deficient MRSA, which eliminate the thymidine-induced confounding effect, are warranted to support the use of iclaprim in the treatment of severe MRSA infections in humans.

Charge dependent substrate activity of C3' and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

cAMP prevents the glucose-mediated stimulation of GLUT2 gene transcription in hepatocytes.

Glucose homoeostasis necessitates the presence in the liver of the high Km glucose transporter GLUT2. In hepatocytes, we and others have demonstrated that glucose stimulates GLUT2 gene expression in vivo and in vitro. This effect is transcriptionally regulated and requires glucose metabolism within the hepatocytes. In this report, we further characterized the cis-elements of the murine GLUT2 promoter, which confers glucose responsiveness on a reporter gene coding the chloramphenicol acetyl transferase (CAT) gene. 5'-Deletions of the murine GLUT2 promoter linked to the CAT reporter gene were transfected into a GLUT2 expressing hepatoma cell line (mhAT3F) and into primary cultured rat hepatocytes, and subsequently incubated at low and high glucose concentrations. Glucose stimulates gene transcription in a manner similar to that observed for the endogenous GLUT2 mRNA in both cell types; the -1308 to -212 bp region of the promoter contains the glucose-responsive elements. Furthermore, the -1308 to -338 bp region of the promoter contains repressor elements when tested in an heterologous thymidine kinase promoter. The glucose-induced GLUT2 mRNA accumulation was decreased by dibutyryl-cAMP both in mhAT3F cells and in primary hepatocytes. A putative cAMP-responsive element (CRE) is localized at the -1074/-1068 bp region of the promoter. The inhibitory effect of cAMP on GLUT2 gene expression was observed in hepatocytes transfected with constructs containing this CRE (-1308/+49 bp fragment), as well as with constructs not containing the consensus CRE (-312/+49 bp fragment). This suggests that the inhibitory effect of cAMP is not mediated by the putative binding site located in the repressor fragment of the GLUT2 promoter. Taken together, these data demonstrate that the elements conferring glucose and cAMP responsiveness on the GLUT2 gene are located within the -312/+49 region of the promoter.

Effect of Platinum-Based Chemoradiotherapy on Cellular Proliferation in Bone Marrow and Spleen, Estimated by 18F-FLT PET/CT in Patients with Locally Advanced Non-Small Cell Lung Cancer.

Historically, it has been difficult to monitor the acute impact of anticancer therapies on hematopoietic organs on a whole-body scale. Deeper understanding of the effect of treatments on bone marrow would be of great potential value in the rational design of intensive treatment regimens. 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a functional radiotracer used to study cellular proliferation. It is trapped in cells in proportion to thymidine-kinase 1 enzyme expression, which is upregulated during DNA synthesis. This study investigates the potential of (18)F-FLT to monitor acute effects of chemotherapy on cellular proliferation and its recovery in bone marrow, spleen, and liver during treatment with 2 different chemotherapy regimens.

Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

The Xenopus vitellogenin (vit) gene B1 estrogen-inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen-responsive elements (EREs), i.e. ERE-2 and ERE-1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor- and hormone-dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen-responsive unit (vitB1 ERU). Using in vitro protein-DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE-2 increases approximately 4- to 8-fold the binding affinity of the receptor to the adjacent less conserved ERE-1. Thus, we suggest that the observed synergistic estrogen-dependent transcription activation conferred by the pair of hormone-responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Functional analysis of the 5' flanking sequence of a vaccinia virus late gene.

A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity.

One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription.

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK- phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.

Development of novel immunotherapies against bladder cancer

Le cancer de la vessie est le deuxième cancer urologique le plus fréquent dans le monde. La plupart des patients (75%) sont initialement diagnostiqués avec un cancer non musculo- invasif. Après résection trans-urétrale, ie traitement standard pour ce type de lésion chez les patients présentant un risque important de récidive/progression consiste en une série d'instillations intravésicales du Bacille de Calmette-Guerin (i.e. le vaccin BCG). Cependant cette "BCG thérapie" est associée à des effets secondaires non négligeables et s'avère inefficace dans 30% des cas, des limitations donc importantes qui soulignent la nécessité de développer des stratégies thérapeutiques alternatives. L'utilisation d'antigènes associés aux tumeurs (TAA) comme vaccin, combinée à une application locale d'immunostimulants sur le site tumoral, est une approche prometteuse en vue de maximiser les réponses immunitaires anti-tumorales localement. Nous montrons que la bactérie vivante atténuée Ty21a, issue du vaccin Vivotif® contre la fièvre typhoïde, peut être utilisée comme immunostimulant intravésical (IVES), mais ce uniquement dans le cas où la bactérie est en phase exponentielle de croissance (Vivotif exp). En effet, l'instillation IVES de Vivotif exp à la suite d'une vaccination par un TAA, un antigène mineur d'histocompatibilité mâle H-Y (Uty), permet d'augmenter de 15 fois le nombre de cellules T CD8 totales et spécifiques de l'antigène dans la vessie. Le recrutement des cellules T est TLR4-dépendent, ce qui suggère un rôle des lipopolysaccharides du Vivotif exp. Par ailleurs, en comparaison avec le contenu bactérien de la capsule de Vivotif, les bactéries en phase exponentielle de croissance permettent également une augmentation préférentielle des chemokines C5/C5a, CXCL1, CXCL2 et CXCL5 dans la vessie, mais pas du nombre de cellules T exprimant les récepteurs apparentés (C5aR et CXCR2). De plus, combiner la vaccination Uty avec le Vivotif exp en IVES permet d'améliorer la survie des souris présentant une tumeur orthotopique de la vessie exprimant l'antigène Uty (lignée tumorale murine MB49). Puisque pour certains cancers, aucun TAA - du moins exprimé à tous les stades tumoraux - n'est identifié, il est nécessaire de développer d'autres approches non vaccinales. Dans une deuxième partie de ce travail de thèse, nous avons donc investigué deux stratégies permettant d'induire une destruction des cellules tumorales, la thérapie génique par gène de suicide, d'une part, et la thérapie photodynamique dans le proche infrarouge (NIR-PDT), d'autre part. Pour appliquer ces thérapies, nous avons utilisé comme vecteur sûr et non toxique une forme non réplicative du virus du « Human Papillomavirus » (HPV) capable de "pseudo-infecter" préférentiellement les souris présentant des tumeurs vésicales (MB49). L'utilisation de pseudovirions (PsV) HPV portant comme gène suicide la thymidine kinase, une enzyme du virus de l'herpès simplex, suivi d'un traitement par la prodrogue Ganciclovir, permet de tuer 90% des cellules MB49 in-vitro ainsi que de ralentir significativement le développement des tumeurs vésicales in-vivo. Par ailleurs, l'emploi de particules pseudo- virales HPV couplées à la phtalocyanine IR700, un pigment photosensible présentant un pouvoir cytotoxique une fois activé, permet de tuer, après application d'une lumière dans le proche infrarouge, quasi 100% des cellules MB49 in-vitro et, plus important, de régresser des tumeurs in-vivo. De façon générale, ce travail de thèse présente des approches thérapeutiques innovantes et prometteuses pour le traitement des patients avec un cancer non musculo-invasif de la vessie. -- Bladder cancer is the second most common urological malignancy in the world. At initial diagnosis, non-muscle invasive bladder cancer (NMIBC) accounts for 75% of bladder cancer. The standard of care of NMIBC consists of intravesical (IVES) treatments with Bacillus- Calmette-Guerin (BCG) following transurethral resections of the lesions. However, repeated BCG treatments are associated with significant side effects and treatment failure may occur in 30% of the cases, underlying the necessity of alternative therapeutic strategies. The use of tumor-associated antigens (TAA) as vaccines followed by the local application of immunostimulants where the tumor resides is a promising approach to increase anti-tumor immune responses locally. We show that live attenuated Ty21a bacteria used from the vivotif® vaccine against typhoid fever can efficiently be used as IVES immunostimulant, only if bacteria are grown to exponential phase (Vivotif exp). In this condition, IVES immunostimulation after TAA vaccination with a minor histocompatibility male antigen HY (Uty) resulted in more than 15-fold increase of both vaccine-specific and total CD8-T cells in the bladder. T cell recruitment was mediated by TLR-4 suggesting that it was mainly mediated by lipopolysaccharides of Vivotif exp. In addition, these bacteria, as compared to the bacterial content of the vivotif capsule preferentially increased C5/C5a, CXCL1, CXCL2 and CXCL5 chemokines, but not the numbers of T cells expressing the cognate receptors (C5aR and CXCR2). Combination of IVES Vivotif exp with Uty vaccination improved survival of mice with pre-established orthotopic Uty-expressing MB49 murine bladder tumors, as compared to vaccination alone. As known TAA are not identified in all cancers, or not expressed in all stages of the tumor, we further investigated two potent approaches able of initiating tumor-cell destruction, suicide-gene therapy and near-infrared (NIR) photodynamic therapy (PDT). Towards a safe and non-toxic application of these therapies, we used Human Papillomavirus (HPV) replication-defective vectors that were able to preferentially pseudo-infect MB49-tumor bearing mice. HPV pseudovirions (PsV) carrying the Herpex-Simplex virus thymidine kinase suicide-gene followed by treatment with the prodrug Ganciclovir resulted in 90% of MB49 cell-death in-vitro and was able to significantly reduce bladder tumor growth in-vivo. Furthermore, HPV virus-like particles coupled to a NIR phtalocyanine dye, IR700 in combination with specific NIR light led to almost 100% of MB49 cell-death in-vitro and more interestingly, to bladder tumors shrinkage in-vivo. Overall, in this thesis, we offer promising therapeutic approaches for application in NMIBC patients.

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.