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Role of thymosin beta4 in tumor metastasis and angiogenesis.

BACKGROUND: Expression of the small peptide thymosin beta4 is associated with angiogenesis induction, accelerated wound healing, and the metastatic potential of tumor cells. However, little is known about the mechanism(s) by which thymosin beta4 promotes metastasis. METHODS: Northern blot analysis and immunohistochemistry were used to examine thymosin beta4 expression in mouse melanoma B16 cell lines and in B16-F10 cells derived from metastatic mouse lung tumors, respectively. B16-F10 cells infected with adenoviruses containing a thymosin beta4 expression vector or an empty vector were injected subcutaneously and intravenously into C57BL/6 mice to evaluate tumor growth and metastatic potential, respectively. In vitro assays were used to study cell migration, invasion, matrix metalloproteinase activity, cell proliferation, and angiogenic activity of adenovirus-infected B16-F10 cells. Statistical significance of all results was analyzed by two-tailed Student's t tests. RESULTS: Thymosin beta4 mRNA was expressed in primary cultured B16-F10 cells derived from lung metastases and in B16-F10 cells that had formed lung tumors after being injected into mice but not in the B16-F1, B16-F10, or B16-BL6 cell lines. The mean tumor sizes in mice 20 days after injection with B16-F10 cells infected with thymosin beta4-expressing adenovirus and with control adenovirus were 21.7 mm (95% confidence interval [CI] = 17.7 to 25.7 mm) and 13.3 mm (95% CI = 11.1 to 15.3 mm), respectively (difference = 8.4 mm; P =.036). The mean numbers of metastatic lung nodules in mice (n = 20) 2 weeks after intravenous injection with thymosin beta4-expressing adenovirus and with control adenovirus were 46.7 (95% CI = 35.0 to 57.7) and 10.9 (95% CI = 6.2 to 15.6), respectively (difference = 35.8 metastatic lung nodules, P<.001). Thymosin beta4 overexpression was associated with a mean 2.3-fold increase (95% CI = 1.9- to 2.7-fold increase; P<.001) in B16-F10 cell migration and a mean 4.4-fold increase (95% CI = 3.3- to 5.5-fold increase; P<.001) in the number of blood vessels in solid tumors derived from injected B16-F10 cells but had no effect on cell invasion, proliferation, or matrix metalloproteinase activity. This induction of angiogenesis by thymosin beta4 was associated with induction of vascular endothelial growth factor expression. CONCLUSION: Thymosin beta4 may stimulate tumor metastasis by activating cell migration and angiogenesis.

Adenoviridae↗

The histone deacetylase inhibitor FK228 preferentially enhances adenovirus transgene expression in malignant cells.

PURPOSE: Efficient adenovirus infection requires coxsackie-adenovirus receptor (CAR) and alpha(v) integrin. Whereas many malignant cells express these proteins poorly, normal tissues, especially liver, express high levels and are susceptible to adenovirus infection. Our previous studies showed that treatment of cancer cell lines with low concentrations of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in Phase II clinical trials, before infection was associated with an increase in adenovirus transgene expression. The purpose of these studies was to analyze the effects of FK228 on cultured normal human cells before initiating animal studies. EXPERIMENTAL DESIGN: Cancer and normal cells from the corresponding tissue were treated with FK228 and analyzed for the proteins needed for infection and the infection efficiency. RESULTS: Treatment of cancer cell lines with 1 ng/ml FK228 increased CAR RNA, alpha(v) integrin RNA, and histone H3 acetylation levels, and was associated with a 4-10-fold increase in the number of infected cells expressing the transgene. Similar treatment of normal human mammary epithelial cells, renal proximal tubule epithelial cells, and hepatocytes had little effect. The insensitivity of cultured normal cells may be explained, in part, by expression of the drug efflux pump P-glycoprotein, because addition of the P-glycoprotein inhibitor XR9576 (tariquidar) with FK228 resulted in increased histone acetylation and CAR expression. CONCLUSION: These studies suggest that low concentrations of FK228 preferentially increase the efficiency of adenoviral transgene expression in cancer cells compared with cultured normal cells from the corresponding tissue and may increase the efficiency of adenovirus therapies in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Papular and papulovesicular acrolocated eruptions and viral infections.

Twenty-six children with a symmetric papular or papulovesicular acrolocated eruption were studied to evaluate a possible link with viral infection. A history of a recent immunization was present in four patients, two of whom had a polio vaccine virus isolated from their stool samples. There was serologic evidence of a recent Epstein-Barr virus infection in one patient, adenovirus infection in another, a concurrent Epstein-Barr virus and adenovirus infection in a third, and cytomegalovirus infection in a fourth. Viral cultures were positive in two patients for adenovirus and in two others for rotavirus. Although some of the virologic findings could have been coincidental, this study confirms that multiple viral infections can be related to most papular and papulovesicular acrolocated eruptions of childhood. Virologic investigations in the earlier phases of the eruption are recommended to identify the etiologic factors of these lesions.

Adenoviridae Infections↗

Translation in vitro of total nuclear RNA from HeLa cell nuclei infected with adenovirus 2.

Total heterogeneous nuclear RNA from HeLa cells infected with adenovirus for 18-20 hr stimulates amino acid incorporation into protein in a cell-free system from Ehrlich ascites tumors. This stimulation of protein synthesis by the nuclear RNA requires intact nuclei. The role of nuclei in this system is unknown, but evidence is presented that the nuclei are involved in the conversion of high-molecular-weight RNA to low-molecular-weight species. Some of the newly synthesized polypeptides appear to resemble the virion polypeptides.

Adenoviridae↗

Respiratory adenovirus-like infection in a rose-ringed parakeet (Psittacula krameri).

Intranuclear inclusions were observed under light microscopy in the bronchial epithelial cells of a recently purchased female rose-ringed parakeet that died of chlamydiosis. Transmission electron microscopy revealed the presence of numerous particles of adenovirus morphology. A latent adenovirus infection may have become more severe following chlamydiosis and the stress of handling.

Adenoviridae Infections↗

Is persistent adenovirus 12 infection involved in coeliac disease? A search for viral DNA using the polymerase chain reaction.

It has been shown that partial amino acid sequence homology between alpha gliadin and an early region protein (E1B-58 kDa) of adenovirus 12 results in immunological cross reaction. This led to the proposal that prior infection by adenovirus 12 could be associated with the development of coeliac disease. To examine this hypothesis, evidence was sought of persistent adenovirus 12 infection in the small intestinal mucosa of patients with coeliac disease. DNA isolated from biopsy samples from 24 control and 18 coeliac disease patients was analysed by the polymerase chain reaction for adenovirus 12 DNA encoding the E1B-58 kDa protein. Four of 18 coeliac disease and two of 24 control patients were positive. There is thus a low prevalence of this infection on both groups of patients but certainly no significantly increased incidence in coeliac disease. These results suggest that persistent adenovirus 12 infection is not a major element in the pathogenesis of coeliac disease.

Adenoviridae↗

Increasing incidence of adenovirus disease in bone marrow transplant recipients.

Adenovirus infections in 201 bone marrow transplant (BMT) recipients over 4 years were retrospectively reviewed. Forty-two patients (20.9%) had positive adenovirus cultures after BMT. There was a higher incidence of adenovirus infections in pediatric patients than in adults (31.3% vs. 13.6%, P = .003). In addition, the time of onset of adenovirus infection after transplant was earlier in pediatric patients (mean, < 30 days) than in adults (> 90 days). Adenovirus type 35 was the most common serotype identified. One-third of adenovirus-positive patients had definite or probable adenovirus disease. Moderate to severe acute graft-versus-host disease and isolation of adenovirus from two or more sites were significant risk factors for adenovirus disease. This report documents a higher incidence of both adenovirus infection and disease than do previous studies. Adenovirus may emerge as a more frequent pathogen as more high-risk BMT transplants are done.

Adenovirus Infections, Human↗

Enhanced deoxyribonucleic acid polymerase activity in human embryonic kidney cultures infected with adenovirus 2 or 12.

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.

Adenoviridae↗

The effect of latent adenovirus 5 infection on cigarette smoke-induced lung inflammation.

The aim of this study was to test the hypothesis that latent adenovirus (Ad) 5 infection increases the lung inflammation that follows a single acute exposure to cigarette smoke. A recently developed model of latent adenoviral infection in guinea-pigs was used. Twelve animals were infected with Ad5 (10(8) plaque-forming units) and 12 animals were sham-infected. Thirty five days later six Ad5-infected and six sham-infected animals were exposed to the smoke from five cigarettes. The remaining animals were used as controls for both infection and smoking. As markers of inflammation, the volume fraction of macrophages, T-lymphocytes, neutrophils and eosinophils were measured by quantitative histology. We found that latent Ad5-infection alone, doubled the number of macrophages in the lung parenchyma and that smoking alone, doubled the volume fraction of neutrophils in the airway wall and the volume fraction of macrophages in the lung parenchyma. Neither viral infection nor smoking, alone, had an effect on T-lymphocytes or eosinophils. However, the combination of viral infection and smoking doubled the T-lymphocyte helper cells and quadrupled the volume fraction of macrophages in the lung parenchyma. We conclude that in guinea-pigs, latent adenovirus 5 infection increases the inflammation that follows a single acute exposure to cigarette smoke, by increasing the volume fraction of macrophages and T-lymphocyte helper cells.

Adenoviridae Infections↗

The behavior of the coiled body in cells infected with adenovirus in vitro.

The coiled body is a phylogenetically conserved nuclear organelle whose function is not known. Probes for detection of p80-coilin, an 80 kDa protein enriched in the coiled body, have made possible studies determining the behavior of the coiled body during the cell cycle, in proliferating cells, as well as reports suggesting some relationship of the coiled body to mRNA splicing and to the nucleolus. The objective of this study is to examine the distribution of p80-coilin and nucleolar proteins in cells infected with adenovirus in vitro. HeLa cells grown as monolayers were infected with successive dilutions of type 5 human adenovirus culture and fixed in methanol/acetone at different time points. Single and double indirect immunofluorescence was performed with human autoantibodies to p80-coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM-Scl, and To, as well as rabbit polyclonal serum to p80-coilin (R288) and mouse monoclonal antibody to adenovirus 72-kDa DNA-binding protein. Indirect immunofluorescence (IIF) with anti-p80-coilin antibodies showed that the usual bright dot-like coiled body staining pattern was replaced in infected cells by 1-5 clusters of tiny dots at the periphery of the nucleus. This phenomenon was first detected within 12 h of infection and affected more severely cells with increased length and load of infection. Cells subjected to heat shock presented no such alteration. Double IIF showed cells with abnormal coiled body appearance expressed the viral 72-kDa DNA-binding protein. Nucleolar proteins RNA polymerase I and NOR-90/hUBF became associated with the p80-coilin-enriched clusters and were no longer detected in the nucleolus. Other nucleolar proteins, like PM-Scl and To, remained associated to the nucleolus and were not detected in the newly formed clusters. Fibrillarin had a heterogeneous behavior, being restricted to the nucleolus in some infected cells while in some others it was associated with the p80-coilin-enriched clusters. Thus our results showed that in vitro adenovirus infection induced radical redistribution of nucleolar and coiled body constituents into newly formed structures characterized by clusters of tiny dots in the periphery of the nucleus. The fact that three major proteins involved in rRNA synthesis and processing colocalized with p80-coilin in these clusters may bring additional support to the idea that the coiled body and p80-coilin may be implicated in functions related to the nucleolus.

Adenoviridae Infections↗

T antigen synthesis and resistance to interferon in human adenovirus type 12 infected chick cells.

Tye specific T antigen formation has been demonstrated in primary and secondary chick embryo cells (CEC) infected with adenovirus type 12. The frequency of cells synthesizing T antigen was closely dependent on the multiplicity of infection (MOI). At a MOI of 2.85 TCD50 per cell, T antigen was formed in 50% of cells. Cycloheximide inhibited T antigen formation while cytosine arabinoside had no such effect. CEC infected with adenovirus 12 produced interferon and T antigen, both appearing early and at about the same time of infection Exogenous chick interferon had no inhibitory effect on the formation of T antigen. In adenovirus 12-infected CEC, virus specific structural antigen could not be detected.

Adenoviruses, Human↗

Cytokines in adenoviral disease in children: association of interleukin-6, interleukin-8, and tumor necrosis factor alpha levels with clinical outcome.

To explore the pathogenic mechanisms involved in adenovirus infection, we evaluated total levels of immunoglobulins, antiadenovirus antibodies, adenovirus-specific circulating immune complexes, and cytokines in serum samples obtained from 38 hospitalized children with adenovirus infection. According to their clinical findings and outcome, the infections were classified as follows: (1) moderate (group I, n = 10), (2) severe (group II, n = 12), and (3) fatal (group III, n = 16). About 60% of the children had elevated IgM levels. IgG-containing adenovirus-specific circulating immune complexes were initially detected in 7 of 16 group III patients, 4 of whom had low serum levels of the third component of complement. A decrease in initial antiadenovirus IgG antibodies was observed in 3 of 10 patients in group III. Serum interleukin-6 was not detected in group I (none of 10), but was present in group II (7 of 12, p = 0.016) and group III (13 of 16, p < 0.001). Interleukin-8 was detected in all groups; values in fatal cases were significantly higher than in surviving children. Tumor necrosis factor alpha was not observed in group I (none of 10) and was uncommon in group II (2 of 12) but was frequently detected in group III (9 of 15, p = 0.01). Interleukin-1 and interleukin-4 were rarely detected in serum samples. Increased concentrations of interleukin-6, interleukin-8, and tumor necrosis factor alpha were associated with hypoperfusion, febrile peaks, tonic-clonic seizures, and septic shock. In 5 of 10 patients in groups II and III, autoantibodies specific for smooth muscle were found. Our findings indicate that high serum values for interleukin-6, interleukin-8, and tumor necrosis factor alpha are associated with severity of adenovirus infection.

Adenovirus Infections, Human↗

Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells.

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours post-infection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

Adenoviruses, Human↗

The adenovirus E3-14.5K protein which is required for prevention of TNF cytolysis and for down-regulation of the EGF receptor contains phosphoserine.

The E3-14.5K and E3-10.4K proteins form a complex and function to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. Both 14.5K and 10.4K are cytoplasmic membrane proteins with a Ccyt orientation in the membrane. We show here that 14.5K is phosphorylated on serine residues in cells infected by adenoviruses that synthesize both 14.5K and 10.4K. 14.5K is phosphorylated on both serine and threonine in cells infected by a mutant that does not synthesize 10.4K; thus, the presence or absence of 10.4K affects the phosphorylation of 14.5K. Phosphotyrosine was not detected. 14.5K is also phosphorylated when translated in vitro in a rabbit reticulocyte extract. Both in vivo and in vitro, at least one of the phosphorylation sites is near the C-terminus, in the cytoplasmic domain of 14.5K. This C-terminal region of 14.5K is the most conserved among Ad5, Ad2, Ad3, and Ad7, and it is essential for 14.5K to prevent tumor necrosis factor cytolysis.

Adenovirus Early Proteins↗

Viral DNA synthesis in vitro with the inclusions isolated from adenovirus 12-infected cells.

A fraction defined as the inclusions was isolated by banding in CsC1 gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18S, 22S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.

Adenoviruses, Human↗