[The State of The Jak/Stat Pathway Affects the Sensitivity of TumorCells to Oncolytic Enteroviruses].

A test of the sensitivity of seven colon cancer cell lines to a panel of 12 nonpathogenic human enteroviruses revealed significant differences in the ability of tumor cells to become infected and replicate different viral strains. Among the factors that can affect the sensitivity of cells to viruses are differences in the state of the mechanisms of antiviral protection, associated with a reaction to type I interferons. Using the two colon cancer cell lines CaCo2 and LIM1215 as a model, significant differences were revealed in the ability of cells to defend themselves against virus infection after 16 hours of treatment with 1000 units/mL of interferon-alpha. To study the effect of the state of the interferon response system, represented by the Jak/STAT signaling pathway, on the sensitivity of cells to different strains of enteroviruses, HEK293T cell lines were used. These are capable of supporting replication of each of the tested enteroviruses, as well as maintaining the ability to protect against viral infection after the treatment with interferon. Using the CRISPR/Cas9 system, HEK293T sublines with knockouts of the IFNAR1 and STAT2 genes were obtained. The sensitivity of control and knockout cells to infection with five strains of enteroviruses and the vesicular stomatitis virus was analyzed. It was noted that knockout of the IFNAR1 and STAT2 genes resulted in an increased sensitivity to all tested viruses. In knockout cells, the levels of reproduction of the vaccine derived of poliovirus type 1, Echoviruses 7 and 30, and Coxsackie viruses B5 and A7 were also significantly increased in comparison with the control HEK293T cells. Thus, deficiencies in the Jak/STAT signaling pathway in tumor cells lead to an overall increase in the sensitivity to oncolytic viruses.

Oncolytic Activity of the Vaccine Strain of Type 3 Poliovirus on the Model of Rat Glioma C6 Cells.

Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/ß (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.

Relationship between Cell Receptors and Tumor Cell Sensitivity to Oncolytic Enteroviruses.

Replicative ability of 5 oncolytic enterovirus strains was evaluated on a panel of 18 human normal and tumor cells. The capacity of each cell line to support replication of enterovirus strains varied. Cell lines weakly replicating one virus could be highly sensitive to another viral strain. Differences in the expression of CXADR cell receptor did not correlate with susceptibility to infection and replication of Coxsackie B virus, but complete inactivation of CXADR gene and poliovirus receptor gene (PVR) led to loss of the sensitivity to Coxsackie B5 and poliovirus, respectively. Detection of additional expression markers will contribute to understanding the causes of different sensitivity of tumor cells to viruses.

[Oncolytic Paramyxoviruses: Mechanism of Action, Preclinical and Clinical Studies].

Preclinical studies demonstrate that a broad spectrum of human and animal malignant cells can be killed by oncolytic paramyxoviruses, which includes cells of ecto-, endo- and mesodermal origin. In clinical trials, significant reduction or even complete elimination of primary tumors and established metastases has been reported. Different routes of virus administration (intratumoral, intravenous, intradermal, intraperito-neal, or intrapleural) and single- vs. multiple-dose administration schemes have been explored. The reported side effects were grades 1 and 2, with the most common among them being mild fever. There are certain advantages in using paramyxoviruses as oncolytic agents compared to members of other virus families exist. Thanks to cytoplasmic replication, paramyxoviruses do not integrate the host genome or engage in recombination, which makes them safer and more attractive candidates for widely used therapeutic oncolysis than ret-roviruses or some DNA viruses. The list of oncolytic Paramyxoviridae members includes the attenuated measles virus, mumps virus, low pathogenic Newcastle disease, and Sendai viruses. Metastatic cancer cells frequently overexpress certain surface molecules that can serve as receptors for oncolytic paramyxoviruses. This promotes specific viral attachment to these malignant cells. Paramyxoviruses are capable of inducing efficient syncytium-mediated lysis of cancer cells and elicit strong immune stimulation, which dramatically enforces anticancer immune surveillance. In general, preclinical studies and phases I-III of clinical trials yield very encouraging results and warrant continued research of oncolytic paramyxoviruses as a particularly valuable addition to the existing panel of cancer-fighting approaches.

Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

[Oncolytic viruses for therapy of malignant glioma]. / Onkoliticheskie virusy v terapii zlokachestvennykh gliom.

Effective treatment of malignant brain tumors is still an open problem. Location of tumor in vital areas of the brain significantly limits capasities of surgical treatment. The presence of tumor stem cells resistant to radiation and anticancer drugs in brain tumor complicates use of chemoradiotherapy and causes a high rate of disease recurrence. A technological improvement in bioselection and production of recombinant resulted in creation of viruses with potent oncolytic properties against glial tumors. Recent studies, including clinical trials, showed, that majority of oncolytic viruses are safe. Despite the impressive results of the viral therapy in some patients, the treatment of other patients is not effective; therefore, further improvement of the methods of oncolytic virotherapy is necessary. High genetic heterogeneity of glial tumor cells even within a single tumor determines differences in individual sensitivity of tumor cells to oncolytic viruses. This review analyses the most successful oncolytic virus strains, including those which had reached clinical trials, and discusses the prospects for new approaches to virotherapy of gliomas.

[Oncotoxic proteins in cancer therapy: mechanisms of action].

Cancer therapeutics based on protein biomolecules that exhibit selective toxic of inhibiting effects towards tumor cells without affecting normal tissue, are gaining extensive attention in cancer research. This heterogenous group of proteins consists of several subgroups, among them, are engineered cancer antigen-specific antibodies that suppress tumor growth by blocking proliferation-inducing receptors, or by direct action of a covalently attached toxin. Another subgroup of anticancer proteins that also represents promising potential therapeutic agents is oncotoxic proteins that can selectively trigger proapoptotic signaling in cancer cells. The oncotoxic proteins target such commonly disturbed processes in tumor calls as enhanced cell proliferation, altered cell-cycle control, deficient apoptotic response, inhibited mitochondrial respiration and activated glycolysis. The introduction of oncotoxic proteins to the clinic might substantially widen and upgrade modern arsenal of anticancer therapeutics.

Mechanisms of Oncolysis by Paramyxovirus Sendai.

Some viral strains of the Paramyxoviridae family may be used as anti-tumor agents. Oncolytic paramyxoviruses include attenuated strains of the measles virus, Newcastle disease virus, and Sendai virus. These viral strains, and the Sendai virus in particular, can preferentially induce the death of malignant, rather than normal, cells. The death of cancer cells results from both direct killing by the virus and through virus-induced activation of anticancer immunity. Sialic-acid-containing glycoproteins that are overexpressed in cancer cells serve as receptors for some oncolytic paramyxoviruses and ensure preferential interaction of paramyxoviruses with malignant cells. Frequent genetic defects in interferon and apoptotic response systems that are common to cancer cells ensure better susceptibility of malignant cells to viruses. The Sendai virus as a Paramyxovirus is capable of inducing the formation of syncytia, multinuclear cell structures which promote viral infection spread within a tumor without virus exposure to host neutralizing antibodies. As a result, the Sendai virus can cause mass killing of malignant cells and tumor destruction. Oncolytic paramyxoviruses can also promote the immune-mediated elimination of malignant cells. In particular, they are powerful inducers of interferon and other cytokynes promoting antitumor activity of various cell components of the immune response, such as dendritic and natural killer cells, as well as cytotoxic T lymphocytes. Taken together these mechanisms explain the impressive oncolytic activity of paramyxoviruses that hold promise as future, efficient anticancer therapeutics.

A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4.

Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13-17 h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells.

Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.

The Role of Dihydroorotate Dehydrogenase in Apoptosis Induction in Response to Inhibition of the Mitochondrial Respiratory Chain Complex III.

A mechanism for the induction of programmed cell death (apoptosis) upon dysfunction of the mitochondrial respiratory chain has been studied. Previously, we had found that inhibition of mitochondrial cytochrome bc1, a component of the electron transport chain complex III, leads to activation of tumor suppressor p53, followed by apoptosis induction. The mitochondrial respiratory chain is coupled to the de novo pyrimidine biosynthesis pathway via the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). The p53 activation induced in response to the inhibition of the electron transport chain complex III has been shown to be triggered by the impairment of the de novo pyrimidine biosynthesis due to the suppression of DHODH. However, it remained unclear whether the suppression of the DHODH function is the main cause of the observed apoptotic cell death. Here, we show that apoptosis in human colon carcinoma cells induced by the mitochondrial respiratory chain complex III inhibition can be prevented by supplementation with uridine or orotate (products of the reaction catalyzed by DHODH) rather than with dihydroorotate (a DHODH substrate). We conclude that apoptosis is induced in response to the impairment of the de novo pyrimidine biosynthesis caused by the inhibition of DHODH. The conclusion is supported by the experiment showing that downregulation of DHODH by RNA interference leads to accumulation of the p53 tumor suppressor and to apoptotic cell death.

[Oncolytic properties of some orthopoxviruses, adenoviruses and parvoviruses in human glioma cells].

UNLABELLED: Currently one of the most promising approaches in development of cancer virotherapy is based on the ability of oncolytic viruses to selective infection and lysis of tumor cells. AIM: The goal of the study was to identify and evaluate perspective oncolytic viruses capable of selectively destroying human glioma cells. PATIENTS AND METHODS: Original GB2m, GA14m and GB22m glioma cell cultures derived from patients were used for evaluating in vitro oncolytic activity of some typical orthopoxviruses, adenoviruses and parvoviruses. RESULTS: The oncolytic activity in the human glioma cell models was confirmed for LIVP and WR strains of vaccinia virus, Adel2 and Ad2del strains with deletions within E1B/55K gene and derived from human adenoviruses type 2 and 5, respectively. CONCLUSIONS: We consider these oncolytic viruses as promising agents for the treatment of human malignant glioma.

[Apoptin enhances the oncolytic activity of vaccinia virus].

Chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain L-IVP) genome. The insertion has replaced major part of the viral C11R gene encoding viral growth factor (VGF), which is important for the virulence. The recombinant virus VVdGF-ApoS24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. The expression apoptin gene from a synthetic early-late promoter of vaccinia virus effectively provides accumulation of the protein in the cells infected with the VVdGF-ApoS24/2 virus. Despite the presence of virus growth factor signal peptide at apoptin N-terminal secretion of the recombinant protein into culture medium did not occur. The recombinant virus VVdGF-ApoS24/2 was found to have a significantly greater selective lyticactivity on human cancer cell lines (A549, A431, U87MG, RD and MCF7) as compared with the parent strain L-IVP and its variant VVdGF2/6 with the deletion of the C11R gene. The results suggest that the use of apoptin represents a promising approach for improving the natural anticancer activities of vaccinia virus.

[Oncolytic adenoviruses in anti-cancer therapy: current status and perspectives].

Lytic viral infection results in production of viral progeny, and lysis of the infected cells. Tumor cells are usually more sensitive to virus infection. Studies of viral oncolysis indicate that it could represent a promising alternative approach to cancer therapy. The ability of viruses to kill selectively cancer cells had been noticed for quite a long time ago. However, only in recent years, based on deeper understanding of molecular biology of viruses and the cell and due to the development of modern methods for directed modification of viruses, there emerged a real opportunity for development of virus variants with improved therapeutic potential. Adenoviruses represent one of the most studied models of oncolytic viruses. The DNA-containing viruses are very suitable for genetic manipulation and show minimal pathogenicity. The review summarizes data on directions and approaches aiming generation of highly efficient variants of oncolytic adenoviruses. The approaches include introduction of directed genetic modifications into viral genome, accelerated selection of oncolytic viral variants following treatment with mutagens, the use of adenoviruses as vectors for introduction of therapeutic gene products, optimization of viral delivery systems, minimalization of negative effects from the host immune system etc. The dynamic development of studies in the field holds promise for introduction into clinical practice of many variants of oncolytic adenoviruses in the very near future.

[Protein complementation as tool for studying protein-protein interactions in living cells].

Association and degradation of protein complexes play essential role in a majority of normal and pathologic processes, which take place in living cell. Studying the underlying mechanisms of those interactions would give deeper understanding of specific causes of disease progression and would allow developing new therapeutic strategies. The majority of technical approaches currently used for detecting protein association include in vitro protein extraction and purification, whereas more relevant results require methods that can be used in vivo. One of a few approaches for in vivo protein association detection is based on reporter protein fragment complementation. Reporter systems based on protein complementation rely on reconstitution of reporter protein fluorescent or enzymatic activity which occurs upon reassociation of protein fragments and could be measured by colorimetry, luminometry or fluorimetry. Protein complementation is widely used to develop reporter systems for analysis of protein interactions, for functional dissection of signal transduction pathways and for performing high-throughput screenings to discover new protein interaction partners. Currently developed approaches that utilize protein fragment complementation have possibilities that extend far beyond simple detection of interaction in a pair of proteins.

[Oncolytic enteroviruses].

Increasing information concerning molecular biology of viruses and virus-cell interactions makes it possible to use viruses as a tool in effort to treat cancer diseases. As a rule, tumor cells are highly sensitive to viruses that may be used in cancer therapy. Therewith, applications of viral oncolysis in treatment of cancer diseases assume maximum possible safety of used viruses for patient and environment. Human enteroviruses are one of the most convenient sources to generate oncolytic viruses. Many of enteroviruses are non-pathogenic for humans or cause mild disease. Progress in genetic engineering permits to develop attenuated enterovirus variants with high safety and selectivity. This review focuses on the main members of Enterovirus genus, such as Coxsackieviruses, and vaccine strains as promising source for development of oncolytic agents, applicable for cancer therapy. It reviews data concerning recently developed and tested oncolytic variants of enteroviruses and discusses perspectives of their application in cancer therapy and problems, concerning their improvement and practical use.

[Oncolytic poxviruses].

The latest data on selection and construction of poxviruses capable of specifically lysing tumor cells of different genesis, inducing antitumor immunity and apoptosis of malignant cells are discussed. The review concerns several directions: virus attenuation, insertion of immunomodulatory protein genes, and anti-tumor protein genes. Thymidine kinase and viral growth factor genes make the greatest contribution to the virus attenuation as their inactivation results in the virus inability to replicate in non-dividing cells, thereby contributing to increased selectivity with respect to tumor cells. Among the immunomodulatory proteins, interleukins 2, 12, and granulocyte-macrophage colony-stimulating factor proved to be most promising for oncolytic virotherapy. An attempt to use p53 protein gene expressed by vaccinia virus for addressed apoptosis of tumor cells was reported. The use of the double and triple viral recombinants carrying genes of multidirectional action seems to be most promising. Encouraging results were obtained using vaccinia virus in the oncotherapy with prodrugs and angiogenesis inhibitors. At present, two poxviral strains are undergoing Phase III clinical trials as anti-tumor preparations in the USA.

[P66shc action on resistance of colon carcinoma RKO cells to oxidative stress].

P66shc protein is an alternative transcript product of SHC1 gene. While two other isoforms (p52shc and p46shc) have adaptor function in RAS signaling pathway, p66shc regulates reactive oxygen species (ROS) level. P66shc genome knockout significantly extends lifespan in mice. Though p66shc was determined to translocate into mitochondria and led to increase in intracellular ROS, the mechanism by which the protein take part in signaling pathways that regulates resistance to cellular stresses remains poorly studied. P66shc has an important role in carcinogenesis and its increased expression correlates with poor prognosis in colon cancer. In this work we have applied RNA interference using lentiviral constructions that express short hairpin RNA (shRNA) against N-terminal CH2 domain of p66shc isoform. Using this approach p66 but not p52 and p46 SHC1 isoform expression was selectively suppressed in colon carcinoma RKO cells. RKO cells with p66shc knockdown have shown to be more resistant to oxidative stress induced by hydrogen peroxide or serum starvation. Fragmentation of mitochondria that depends on mitochondrial ROS accumulation during oxidative stress was significantly decreased in this cells. The data obtained are in agreement with hypothesis that p66shc participates in ROS accumulation in mitochondria and by this means promotes induction of apoptosis.

[Oncolytic parvoviruses. A new approaches for cancer therapy].

Parvoviruses such as parvovirus H-1 (H-1PV) may selectively infect and lysis cancer cells. The parvoviruses also induce an immune system to eliminate the tumor cells through the formation of anti-cancer immunity. One of the possible mechanisms of antitumor activity is associated with the direct induction of apoptosis by parvoviral proteins NS1 and 11 kDa. Parvovirus-based vectors are promising for gene therapy of oncological diseases and genetic disorders in humans. Parvoviruses were successfully used for the experimental treatment on animal models of human glioma, neuroblastomas, lymphomas, pancreatic carcinoma, carcinomas and breast tumors. ParvOryx is the first oncolytic preparation constructed on the base of H-1PV; its phase I/IIa clinical trials in patients with glioblastoma multiforme are in process.

[Oncolytic viruses in the therapy of gliomas].

Despite the advances of modern medicine, malignant glioblastoma cure remains an elusive goal. Both the invasive nature and location in vital areas of the brain make this type of tumors difficult for surgical treatment, while the current adjuvant therapy is not as successful as expected. Frequent recurrence and invasiveness of malignant gliomas is due to resistance of glioma stem cells to conventional radiation and chemotherapy. Technological advances in constructing recombinant viruses have allowed creating strains with high oncolytic activity toward glial tumors. Many of these strains have passed Phase I of clinical trials and demonstrated high safety. Despite the obvious potential of the approach, efficiency of the existing strains is still far from being sufficient for effectively curing the disease and require further improvement. The review summarizes results obtained with the most successful variants of oncolytic viruses that come down to the clinical trials and discusses the prospects for new approaches in virotherapy of malignant gliomas.