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At least 811 records · Page 45Linked to original sources
PubMed2005

Expression of human TFF3 in relation to growth of HT-29 cell subpopulations: involvement of PI3-K but not STAT6.

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1-4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.

Cell Differentiation↗
PubMed2000

Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha.

The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor-alpha TNF-alpha, however, the number of strands that form the tight junctions decreases, and apoptosis is induced in intestinal epithelial cells. These morphological changes lead to a rise of transepithelial ion permeability, because the paracellular ion permeability increases and leaks associated with sites of apoptosis increase by number and magnitude. Thus apoptosis and degradation of tight junctions contribute to the increased permeability observed after exposure to TNF-alpha. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokine-secreting macrophages--for example, leak flux diarrhea and invasion of bacterial enterotoxins.

Apoptosis↗
PubMed2000

Apoptosis and intestinal barrier function.

The signal transduction pathways of the induction of apoptosis in the gastrointestinal tract have in part been discovered. However, almost nothing is known about the functional influence of apoptotic signals on intestinal barrier function. In this study the effect of camptothecin-induced apoptosis in HT-29/B6 monolayers and the influence of apoptosis on epithelial barrier function were characterized. We demonstrated that camptothecin causes a decrease of transepithelial resistance and an increase in fluxes of the paracellular marker [3H]mannitol. Camptothecin increased the apoptotic rate and the conductance of single-cell apoptosis as measured by the conductance scanning technique. We conclude that in our model of HT-29/B6 cells camptothecin is a potent inductor of apoptosis that causes significant barrier defects measured by the Ussing chamber technique and the conductance scanning technique. Based on these results we are able to investigate the effect of other cytokines--TGF-beta, for instance, and its role in apoptotic conditions.

Apoptosis↗
PubMed2002

Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.

Restitution of single-cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time-lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single-cell loss from an HT-29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 +/- 0.05 microS 2 min post lesion to 0.17 +/- 0.02 microS 8 min post lesion, n = 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO-1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin-ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML-7. The Rho-associated protein kinase inhibitor Y-27632 did not affect restitution. After loosening of intercellular contacts in low Ca(2+) Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12-fold higher in low Ca(2+) Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single-cell defects comprises rapid closure by an actinomyosin 'purse-string' mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.

Actins↗
PubMed2006

Induction of AKR1C2 by phase II inducers: identification of a distal consensus antioxidant response element regulated by NRF2.

AKR1C2, also referred to as the human bile acid binder and 3alpha-hydroxysteroid dehydrogenase type III, is a multifunctional oxidoreductase able to stereoselectively reduce steroids as well as oxidize or reduce polyaromatic hydrocarbons. Previously, this same protein was also identified by its robust induction by phase II inducers in HT29 cells. In HepG2 cells, both AKR1C2 and AKR1C1 (97% sequence homology) were induced by phase II inducers but not the highly related AKR1C3 and AKR1C4 family members (84% sequence homology). We now report the initial characterization of the proximal promoter of AKR1C2 in HepG2 cell line and the identification of a potent enhancer-like element responsive to phase II inducers located approximately 5.5 kilobases upstream from the transcription start site. DNA sequence analysis of this enhancer element revealed that it contained a consensus antioxidant response element (ARE), which was confirmed by mutation analysis. Treatment with phase II inducers leads to increased accumulation of nuclear factor-erythroid 2 p45-related factor (NRF) 2 in the nucleus, which was associated with increased binding to this ARE as determined by electrophoretic mobility shift assay. Transient transfection with Nrf2 increased the transcriptional activity of the ARE of AKR1C2 comparable with that observed with phase II inducers. Chromatin immunoprecipitation (ChIP) analysis also confirmed increased NRF2 binding to the ARE after induction by a phase II inducer. The AKR1C1 promoter also harbored this same ARE element in a highly homologous region, which was also bound by NRF2 in a ChiP analysis. No induction of the ARE of AKR1C2 was detected in Nrf2-/- fibroblasts. The regulation of AKR1C2 by this distal ARE suggests that AKR1C2 detoxifies products of reactive oxidant injury, which has important implications for both hormone and xenobiotic metabolism.

Base Sequence↗
PubMed1998

Cytochalasin-induced actin disruption of polarized enterocytes can augment internalization of bacteria.

Cytochalasin-induced actin disruption has often been associated with decreased bacterial internalization by cultured epithelial cells, although polarized enterocytes have not been systematically studied. In assays using confluent polarized HT-29 enterocytes, cytochalasin D appeared to increase internalization of wild-type Salmonella typhimurium, Proteus mirabilis, and Escherichia coli. HeLa and HEp-2 epithelial cells, as well as HT-29 and Caco-2 enterocytes, were used to clarify this unexpected observation. Resulting data showed that cytochalasin D was associated with increased internalization of S. typhimurium and P. mirabilis by both HT-29 and Caco-2 enterocytes and with increased internalization of E. coli by HT-29 enterocytes; with either HeLa or HEp-2 cells, cytochalasin was associated with no change or a decrease in internalization of these same bacterial strains. Cytochalasin caused decreased internalization of Listeria monocytogenes by HT-29, Caco-2, HeLa, and HEp-2 cells, indicating that cytochalasin did not consistently augment bacterial internalization by polarized enterocytes. Fluorescein-labeled phalloidin confirmed marked disruption of filamentous actin in cytochalasin-treated HT-29, Caco-2, HeLa, and HEp-2 cells. Cytochalasin had no noticeable effect on epithelial viability but caused distorted apical microvilli, cell rounding, and separation of adjacent enterocytes in confluent cultures (with a corresponding decrease in transepithelial electrical resistance). Scanning electron microscopy showed that cytochalasin-induced enhanced bacterial internalization was associated with preferential bacterial adherence on the exposed enterocyte lateral surface. Colchicine, used to disrupt microtubules, had no noticeable effect on bacterial internalization by HT-29 or Caco-2 enterocytes. These data indicated that for HT-29 and Caco-2 enterocytes, cytochalasin-induced disruption of filamentous actin might augment internalization of some bacterial species by a mechanism that appeared to involve exposure of the enterocyte lateral surface.

Actins↗
PubMed1999

Mycobacterium avium infection of epithelial cells results in inhibition or delay in the release of interleukin-8 and RANTES.

Mycobacterium avium is an opportunistic pathogen in AIDS patients, who acquire the infection mainly through the gastrointestinal tract. Previous studies in vitro have shown that M. avium invades epithelial cells of both intestinal and laryngeal origin. In addition, M. avium enters the intestinal mucosa of healthy mice. Because M. avium invasion of the intestinal mucosa in vivo initially is not accompanied by significant influx of inflammatory cells, we sought to determine whether M. avium would trigger chemokine release upon entry into epithelial cells by using HT-29 intestinal and HEp-2 laryngeal epithelial cell lines. Chemokine synthesis was measured both by the presence of specific mRNA and protein secretion in the cell culture supernatant as determined by enzyme-linked immunosorbent assay. Infection of HT-29 intestinal cells with M. avium did not induce the release of interleukin-8 (IL-8) or RANTES for up to 7 days postinfection. However, infection of HEp-2 cells resulted in the release of IL-8 and RANTES at 72 h. Similar findings were observed with other AIDS M. avium isolates belonging to different serovars. Secretion of IL-8 by HEp-2 cells was dependent upon bacterial uptake. In addition, prior infection with M. avium suppressed IL-8 production by HT-29 cells infected with Salmonella typhimurium. Our results suggest that M. avium infection of epithelial cells is associated with a delay in IL-8 and RANTES production which, in the case of HT-29, is prolonged up to 1 week. These findings may explain the weak inflammatory response after intestinal mucosa invasion in mice and are probably related with the ability of the bacterium to evade the host's immune response.

Chemokine CCL5↗
PubMed1999

Consequences of reduction of Klebsiella pneumoniae capsule expression on interactions of this bacterium with epithelial cells.

Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.

Bacterial Adhesion↗
PubMed1999

Characterization of candidate live oral Salmonella typhi vaccine strains harboring defined mutations in aroA, aroC, and htrA.

The properties of two candidate Salmonella typhi-based live oral typhoid vaccine strains, BRD691 (S. typhi Ty2 harboring mutations in aroA and aroC) and BRD1116 (S. typhi Ty2 harboring mutations in aroA, aroC, and htrA), were compared in a number of in vitro and in vivo assays. BRD1116 exhibited an increased susceptibility to oxidative stress compared with BRD691, but both strains were equally resistant to heat shock. Both strains showed a similar ability to invade Caco-2 and HT-29 epithelial cells and U937 macrophage-like cells, but BRD1116 was less efficient at surviving in epithelial cells than BRD691. BRD1116 and BRD691 were equally susceptible to intracellular killing within U937 cells. Similar findings were demonstrated in vivo, with BRD1116 being less able to survive and translocate to secondary sites of infection when inoculated into the lumen of human intestinal xenografts in SCID mice. However, translocation of BRD1116 to spleens and livers in SCID mice occurred as efficiently as that of BRD691 when inoculated intraperitonally. The ability of BRD1116 to increase the secretion of interleukin-8 following infection of HT-29 epithelial cells was comparable to that of BRD691. Therefore, loss of the HtrA protease in S. typhi does not seem to alter its ability to invade epithelial cells or macrophages or to induce proinflammatory cytokines such as IL-8 but significantly reduces intracellular survival in human intestinal epithelial cells in vitro and in vivo.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1999

Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S.

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa 388 and the non-ExoS-producing strain 388DeltaS. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388DeltaS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388DeltaS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P. aeruginosa infectious process by rendering cells nonfunctional.

ADP Ribose Transferases↗
PubMed2004

Differential regulation of beta-defensin gene expression during Cryptosporidium parvum infection.

Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. beta-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial beta-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates beta-defensin gene expression. Downregulation of murine beta-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human beta-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.

Animals↗
PubMed2007

p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines.

While Newcastle disease virus (NDV) causes serious infections in birds, it is apparently nonpathogenic in mammalian species, including humans. Previous observations and small-scale clinical trials indicated that NDV exerts oncolytic effects. Isolates of NDV were found to have selective affinity to transformed cells. We previously showed that the attenuated NDV strain MTH-68/H causes apoptotic cell death in cultures of PC12 rat pheochromocytoma cells. The aim of the present study was to extend MTH-68/H cytotoxicity testing with human tumor cell lines and to analyze certain biochemical aspects of its oncolytic effect. MTH-68/H was found to be able to kill a wide range of transformed cells by apoptosis. While caspase-8 and caspase-9 are not involved in MTH-68/H-induced apoptosis, activation of caspase-3 and caspase-12 was detected in virus-infected PC12 cells. A human glioblastoma cell line with repressible expression of the p53 protein did not show any difference in MTH-68/H sensitivity in its p53-expressing and p53-depleted states, indicating that the apoptotic process induced by MTH-68/H does not depend on p53. Apoptosis was accompanied by virus replication in two tumor cell lines tested (PC12 cells and HeLa human cervical cells), and signs of endoplasmic reticulum stress (phosphorylation of protein kinase R-like endoplasmic reticulum kinase and eIF2alpha) were also detected in transformed cells. In contrast, proliferation of nontransformed mouse and rat fibroblast cell lines and human primary fibroblasts was not affected by MTH-68/H treatment. MTH-68/H thus selectively kills tumor cell cultures by inducing endoplasmic reticulum stress leading to p53-independent apoptotic cell death.

Animals↗
PubMed2006

A microRNA signature of hypoxia.

Recent research has identified critical roles for microRNAs in a large number of cellular processes, including tumorigenic transformation. While significant progress has been made towards understanding the mechanisms of gene regulation by microRNAs, much less is known about factors affecting the expression of these noncoding transcripts. Here, we demonstrate for the first time a functional link between hypoxia, a well-documented tumor microenvironment factor, and microRNA expression. Microarray-based expression profiles revealed that a specific spectrum of microRNAs (including miR-23, -24, -26, -27, -103, -107, -181, -210, and -213) is induced in response to low oxygen, at least some via a hypoxia-inducible-factor-dependent mechanism. Select members of this group (miR-26, -107, and -210) decrease proapoptotic signaling in a hypoxic environment, suggesting an impact of these transcripts on tumor formation. Interestingly, the vast majority of hypoxia-induced microRNAs are also overexpressed in a variety of human tumors.

Apoptosis↗
PubMed1999

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

5-Lipoxygenase-Activating Proteins↗
PubMed2000

Topography of genetic loci in tissue samples: towards new diagnostic tool using interphase FISH and high-resolution image analysis techniques.

Using single and dual colour fluorescence in situ hybridisation (FISH) combined with image analysis techniques the topographic characteristics of genes and centromeres in nuclei of human colon tissue cells were investigated. The distributions of distances from the centre-of-nucleus to genes (centromeres) and from genes to genes (centromeres to centromeres) were studied in normal colon tissue cells found in the neighbourhood of tumour samples, in tumour cell line HT-29 and in promyelocytic HL-60 cell line for comparison. Our results show that the topography of genetic loci determined in 3D-fixed cell tissue corresponds to that obtained for 2D-fixed cells separated from the tissue. The distributions of the centre-of-nucleus to gene (centromere) distances and gene to gene (centromere to centromere) distances and their average values are different for various genetic loci but similar for normal colon tissue cells, HT-29 colon tumour cell line and HL-60 promyelocytic cell line. It suggests that the arrangement of genetic loci in cell nucleus is conserved in different types of human cells. The investigations of trisomic loci in HT-29 cells revealed that the location of the third genetic element is not different from the location of two homologues in diploid cells. We have shown that the topographic parameters used in our experiments for different genetic elements are not tissue or tumour specific. In order to validate high-resolution cytometry for oncology, further investigations should include more precise parameters reflecting the state of chromatin in the neighbourhood of critical oncogenes or tumour suppresser genes.

Cell Nucleus↗
PubMed2004

Increased immunogenicity of colon cancer cells by selective depletion of cytochrome C.

We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo "necrotic" cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.

Animals↗
PubMed2004

Galectin-3, a novel binding partner of beta-catenin.

Galectin-3 (gal-3), a pleiotrophic protein, is an important regulator of tumor metastasis, which like beta-catenin shuttles between the nucleus and the cytosol in a phosphorylation-dependent manner. We report herein that beta-catenin stimulation of cyclin D1 and c-myc expression is gal-3 dependent. Gal-3 binds to beta-catenin/Tcf complex, colocalizes with beta-catenin in the nucleus, and induces the transcriptional activity of Tcf-4 as determined by the TOP/FOPFLASH reporter system. We have identified the beta-catenin-gal-3-binding sequences, which are in the NH2 and COOH termini of the proteins encompassing amino acid residues 1 to 131 and 143 to 250, respectively. These data indicate that gal-3 is a novel binding partner for beta-catenin involved in the regulation of Wnt/beta-catenin signaling pathway.

Breast Neoplasms↗
PubMed2005

RAD001 (everolimus) improves the efficacy of replicating adenoviruses that target colon cancer.

Selectively replicating adenoviruses have the potential to cure cancer but have shown little efficacy in clinical trials. We have tested the ability of the mTOR kinase inhibitor RAD001 (everolimus) to enhance the response of xenografts to an oncolytic adenovirus. The virus has Tcf sites inserted in the early viral promoters and replicates selectively in cells with activation of the Wnt signaling pathway. To enhance tumor cell infection, an integrin targeting peptide (CDCRGDCFC) was inserted into the fiber gene of the virus. RAD001 combines three useful properties: it inhibits tumor cell growth directly, blocks angiogenesis, and suppresses the immune response. RAD001 does not block viral protein expression, DNA replication, or cytopathic effect in tumor cells in vitro. After 6 weeks of daily RAD001 treatment, ongoing viral DNA replication could be detected in tumor xenografts, showing that RAD001 does not inhibit virus replication in vivo. I.v. injection of virus alone produced a small delay in xenograft growth, whereas combination therapy substantially prolonged the survival of the mice. We suggest that collapsing the tumor vasculature after the initial infection traps the virus and facilitates local spread within the tumor. Unlike conventional drugs, which require continued access to the tumor through the vascular system, oncolytic viruses are in principle less sensitive to late reductions in perfusion because they are produced locally within the tumor.

Adenoviridae↗