Biomedical subjects

Alexander N Lukashev

Publications and source records attributed to Alexander N Lukashev.

7 recordsLinked to original sources

PubMed2006

Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells.

BACKGROUND: The Wnt signaling pathway is activated by mutations in the APC and beta-catenin genes in many types of human cancer. beta-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. METHODS: Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. RESULTS: The E1A-HD2 fusion protein binds to beta-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of beta-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE) in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. CONCLUSION: Despite an increase in the nuclear pool of beta-catenin, the effects on viral activity in colon cancer cells were small, suggesting that factors acting downstream of beta-catenin are limiting for viral replication and toxicity in these cells. The approach of fusing E1A to a protein domain implicated in oncogenic signaling could be used to selectively increase the activity of oncolytic viruses targeting several other pathways defective in cancer.

Adenovirus E1A Proteins↗

PubMed2005

Late expression of nitroreductase in an oncolytic adenovirus sensitizes colon cancer cells to the prodrug CB1954.

We have constructed an oncolytic adenovirus expressing the Escherichia coli nitroreductase gene nfsB from an internal ribosome entry site (IRES) in the adenovirus L5 major late transcript. The virus (Tcf-NTR) has Tcf transcription factor-binding sites in the E1A, E1B, and E4 promoters, which restrict viral replication to cells that have activation of the Wnt signaling pathway. This virus was compared with an E1B-55K-deleted virus expressing nitroreductase (NTR) from a cytomegalovirus (CMV) promoter in the E1B-55K region [CRAd-NTR(PS1217H6)]. Both viruses express NTR in colorectal cancer cell lines and show increased cytopathic effect in the presence of the prodrug CB1954. Unlike the Tcf-NTR virus, the CMV-NTR virus expresses NTR in human lung fibroblasts and sensitizes these normal cells to CB1954. The in vivo activity of the viruses was tested in SW620 xenografts in nude mice by intravenous injection of 1,011 particles of virus followed 1 week later by intraperitoneal injections of CB1954. The CMV-NTR virus produced minimal effects in this model. The median time to form 1,000-mm(3) tumors in mice treated with the Tcf-NTR virus plus CB1954 was increased from 14 to 26 days (p=0.003), but this was due mainly to the direct oncolytic effect of the virus. Combination therapy with 3 x 10(11) particles of Tcf-NTR virus (given intravenously) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) (given orally) significantly improved survival (median, >50 days), and addition of CB1954 to this regimen further delayed tumor growth. These results show that the Tcf-NTR virus is more tumor selective and active than the CMV-NTR virus. At the level of transduction that can be achieved currently with oncolytic viruses given intravenously, drugs such as RAD001, which do not require activation by the virus, produce greater increases in efficacy than prodrugs such as CB1954.

Adenoviridae↗

PubMed2005

Evidence for recombination in Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) virus has attracted considerable attention recently and a number of phylogenetic studies have been published, based mostly on partial sequences of S and M RNA segments. In this study, available full-length S, M and L segment sequences of CCHF virus were checked for recombination. Similarity plots and bootscan analysis of the S segment suggested multiple recombination events between southern European, Asian and African CCHF virus strains, with additional evidence provided by phylogenetic trees, the hidden Markov model and probabilistic divergence measures methods. No unambiguous signs of recombination were observed for M and L segments; however, the results did not exclude the possibility of this. These findings, coupled with a recent report on reassortment in CCHF virus, suggest caution when assessing CCHF virus phylogeny based on short sequence fragments.

Animals↗

PubMed2005

Recombination in circulating Human enterovirus B: independent evolution of structural and non-structural genome regions.

The complete nucleotide sequences of eight Human enterovirus B (HEV-B) strains were determined, representing five serotypes, E6, E7, E11, CVB3 and CVB5, which were isolated in the former Soviet Union between 1998 and 2002. All strains were mosaic recombinants and only the VP2-VP3-VP1 genome region was similar to that of the corresponding prototype HEV-B strains. In seven of the eight strains studied, the 2C-3D genome region was most similar to the prototype E30, EV74 and EV75 strains, whilst the remaining strain was most similar to the prototype E1 and E9 strains in the non-structural protein genome region. Most viruses also bore marks of additional recombination events in this part of the genome. In the 5' non-translated region, all strains were more similar to the prototype E9 than to other enteroviruses. In most cases, recombination mapped to the VP4 and 2ABC genome regions. This, together with the star-like topology of the phylogenetic trees for these genome regions, identified these genome parts as recombination hot spots. These findings further support the concept of independent evolution of enterovirus genome fragments and indicate a requirement for more advanced typing approaches. A range of available phylogenetic methods was also compared for efficient detection of recombination in enteroviruses.

5' Untranslated Regions↗

PubMed2003

Recombination in circulating enteroviruses.

Recombination is a well-known phenomenon for enteroviruses. However, the actual extent of recombination in circulating nonpoliovirus enteroviruses is not known. We have analyzed the phylogenetic relationships in four genome regions, VP1, 2A, 3D, and the 5' nontranslated region (NTR), of 40 enterovirus B strains (coxsackie B viruses and echoviruses) representing 11 serotypes and isolated in 1981 to 2002 in the former Soviet Union states. In the VP1 region, strains of the same serotype expectedly grouped with their prototype strain. However, as early as the 2A region, phylogenetic grouping differed significantly from that in the VP1 region and indicated recombination within the 2A region. Moreover, in the 5' NTR and 3D region, only 1 strain of 40 grouped with its prototype strain. Instead, we observed a major group in both the 5' NTR and the 3D region that united most (in the 5' NTR) or all (in the 3D region) of the strains studied, regardless of the serotype. Subdivision within that major group in the 3D region correlated with the time of virus isolation but not with the serotype. Therefore, we conclude that a majority, if not all, circulating enterovirus B strains are recombinants relative to the prototype strains, isolated mostly in the 1950s. Moreover, the ubiquitous recombination has allowed different regions of the enterovirus genome to evolve independently. Thus, a novel model of enterovirus genetics is proposed: the enterovirus genome is a stable symbiosis of genes, and enterovirus species consist of a finite set of capsid genes responsible for different serotypes and a continuum of nonstructural protein genes that seem to evolve in a relatively independent manner.

5' Untranslated Regions↗

PubMedDate not supplied

Enterovirus uveitis.

Enterovirus uveitis (EU) is a new infant eye disease that was first observed in 1980. Three distinct subtypes of human echoviruses, EV19/K, EV11/A and EV11/B, caused five hospital outbreaks of EU in different Siberian cities in 1980-1989, affecting approximately 750 children, predominantly below 1 year of age. Sporadic EU cases were also retrospectively diagnosed in other regions of Russia and in different countries of the Former Soviet Union. The illness was characterised by rapid iris destruction and severe complications, including cataract and glaucoma. The disease has been a subject of intensive studies and was reproduced in lower primates after intraocular inoculation of isolated enterovirus strains. Importantly, prototype EV11 and EV19 strains did not induce notable disease in experimental monkeys. Some of the EU-causing strains were shown to be similar phylogenetically and in their pathogenetic properties to the enterovirus strains associated with multisystem hemorrhagic disease of newborns. In this review we present a summary of the vast epidemiological, virological, clinical and experimental data on this new form of ophthalmic infection.

Adult↗

PubMedDate not supplied

Role of recombination in evolution of enteroviruses.

Enteroviruses, members of the Picornaviridae family, comprise a large (over 70 serotypes) group of viruses that are ubiquitous in nature, infect different species and cause a wide range of diseases. Human enteroviruses were recently classified into five species, human enterovirus A-D and poliovirus. Recombination has long been known to be an important property of poliovirus genetics. Recently, several publications demonstrated that recombination is extremely frequent also in non-polio enteroviruses, and allows independent evolution of enterovirus genome fragments even on a microevolutionary scale. Prototype enterovirus strains were shown to have complex phylogenetic relations, and almost all modern enterovirus isolates turned out to be recombinants compared with the prototype strains. Recombination takes place strictly between members of the same species, and usually spares the capsid-encoding genome region. Therefore, it can be concluded that the enterovirus species exist as a worldwide reservoir of genetic material comprising a limited quantity of capsid gene sets defining a finite number of serotypes and a range of non-structural genes that recombine frequently to produce new virus variants. This new model of enterovirus genetics helps to explain the failure of previous attempts to connect serotype and disease profile in non-polio enteroviruses, and seriously questions existing typing approaches that are based solely on the capsid-encoding genome region. It remains to be determined what role recombination plays in the emergence of new enterovirus variants and in the macroevolution of animal enteroviruses and viruses of the picorna-like supergroup.

Enterovirus↗