Ultraviolet-inactivated Sendai Virus Research Articles

Anticancer effect of inactivated Sendai virus strain Tianjin on human osteosarcoma HOS cells.

Ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) has been proved to have antitumor effects in many kinds of tumor cells. Here, we investigated the anticancer properties of UV-Tianjin on human osteosarcoma HOS cells and the underlying molecular mechanism. Apoptosis, intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential were determinedbyflow cytometryanalysis. The expression levels of apoptosis-related proteins weretestedby western blotting. The results showed that UV-Tianjin concentration-dependently induced apoptosis in HOS cells. UV-Tianjin-induced apoptosis was mediated by the mitochondrial pathway, which was confirmed by mitochondrial dysfunction, downregulation of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-xL (Bcl-xL) and myeloid cell leukemia-1 (Mcl-1), upregulation of B-cell lymphoma 2 associated X protein (Bax) and Bcl-2 Homologous Antagonist/Killer (Bak), as well as the cleavage of caspase-9 and -3. Further analysis showed that UV-Tianjin augmented the phosphorylation of c-Jun N-terminal kinase, the extracellular-regulated kinase and p38, the major components of mitogen-activated protein kinase (MAPK) pathways, as well as the generation of ROS. Moreover, UV-Tianjin-induced apoptosis was remarkably attenuated by MAPK inhibitors and ROS inhibitor. Taken together, our results indicated that UV-Tianjin exerts antitumor effects by inducing mitochondria-dependent apoptosis involving ROS generation and MAPK pathway in human osteosarcoma HOS cells.

Inactivated Tianjin strain, a novel genotype of Sendai virus, induces apoptosis in HeLa, NCI-H446 and Hep3B cells.

The Sendai virus strain Tianjin is a novel genotype of the Sendai virus. In previous studies, ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) demonstrated antitumor effects on human breast cancer cells. The aim of the present study was to investigate the in vitro antitumor effects of UV-Tianjin on the human cervical carcinoma HeLa, human small cell lung cancer NCI-H446 and human hepatocellular carcinoma Hep 3B cell lines, and the possible underlying mechanisms of these antitumor effects. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that UV-Tianjin treatment inhibited the proliferation of HeLa, NCI-H446 and Hep 3B cells in a dose- and time-dependent manner. Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide double staining indicated that UV-Tianjin induced dose-dependent apoptosis in all three cell lines with the most significant effect observed in the HeLa cell line. In the HeLa cell line, UV-Tianjin-induced apoptosis was further confirmed by the disruption of the mitochondria membrane potential and the activation of caspases, as demonstrated by fluorescent cationic dye and colorimetric assays, respectively. In addition, western blot analysis revealed that UV-Tianjin treatment resulted in significant upregulation of cytochrome c, apoptosis protease activating factor-1, Fas, Fas ligand and Fas-associated protein with death domain, and activated caspase-9, −8 and −3 in HeLa cells. Based on these results, it is hypothesized that UV-Tianjin exhibits anticancer activity in HeLa, NCI-H446 and Hep 3B cell lines via the induction of apoptosis. In conclusion, the results of the present study indicate that in the HeLa cell line, intrinsic and extrinsic apoptotic pathways may be involved in UV-Tianjin-induced apoptosis.

Inactivated Sendai virus strain Tianjin induces apoptosis in breast cancer MCF-7 cells by promoting caspase activation and Fas/FasL expression.

Virotherapy represents a promising new approach for treating cancer. Here the authors have analyzed the effect of ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human breast cancer MCF-7 cells in vitro and in vivo. In vitro, UV-Tianjin inhibited the proliferation of MCF-7, MDA-MB-231, and T47D breast cancer cell lines, although MCF-7 cells were most susceptible to UV-Tianjin treatment. Hoechst staining and flow cytometric analysis of UV-Tianjin-treated MCF-7 cells revealed that UV-Tianjin induced apoptosis in a dose-dependent manner. Moreover, UV-Tianjin treatment resulted in reductions in the mitochondria membrane potential of MCF-7 cells and regulated the levels and activities of Bcl-2, Bax, cyt c, caspases, Fas, and Fas ligand (FasL). In vivo, UV-Tianjin inhibited the growth of MCF-7 tumors in nude mice and increased tumor cell apoptosis compared with saline-treated controls. In addition, the percentage of tumor cells positive for cleaved versions of caspase-7, caspase-8, and caspase-9 was higher in UV-Tianjin-treated tumors than in saline-treated controls. In summary, UV-Tianjin exhibited the antitumor activity in human breast cancer MCF-7 cells both in vitro and in vivo. The UV-Tianjin treatment seemed to induce apoptosis by activating both the mitochondrial and death receptor apoptotic pathways.

Inactivated Sendai virus strain Tianjin induces apoptosis in human breast cancer MDA-MB-231 cells.

Sendai virus strain Tianjin is a novel genotype. Here, we investigate the antitumor and proapoptotic effects of ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human breast cancer MDA-MB-231 cells in vitro, as well as the involvement of the apoptotic pathway in the mechanism of UV-Tianjin-induced antitumor effects. MTT assays showed that treatment with UV-Tianjin dose-dependently inhibited the proliferation of MDA- MB-231 cells but not normal MCF 10A breast epithelium cells. Hoechst staining and flow cytometric analysis revealed that UV-Tianjin induced apoptosis of MDA-MB-231 cells in a dose-dependent manner. Moreover, UV-Tianjin treatment resulted in reduction in the mitochondria membrane potential (MMP) and release of cytochrome complex (cyt c) via regulation of Bax and Bcl-2, as well as activation of caspase-9, caspase-3, Fas, FasL and caspase-8 in MDA-MB-231 cells. In summary, our study suggests that UV-Tianjin exhibits anticancer activity in human breast cancer MDA-MB-231 cells through inducing apoptosis, which may involve both the endogenous mitochondrial and exogenous death receptor pathways.

Fusogenic liposomes and their suitability for gene delivery

A novel carrier system to deliver large exogenous molecules into the cytosol has advantages for efficient delivery compared with traditional transfection techniques. We previously prepared a fusogenic liposome (FL) system by fusing phospholipid-based conventional liposomes with ultraviolet inactivated Sendai virus, which possess membrane fusion activity derived from the Sendai virus accessory protein. The FLs deliver their contents rapidly and directly into the cytoplasm rather than by endocytosis via membrane fusion without cytotoxicity. This FL-mediated cytoplasmic delivery technique is a powerful tool with the potential for wide use in the field of gene regulation and for the development of gene therapy. This review focuses on the suitability of FLs as a versatile and effective gene delivery system.

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

Target-cell specificity of fusogenic liposomes: Membrane fusion-mediated macromolecule delivery into human blood mononuclear cells

Fusogenic liposome, a unique vector prepared by fusing ultraviolet-inactivated Sendai virus and liposome, is known to efficiently deliver content into various animal cells through membrane fusion. In this study, we examined the target-cell specificity of fusogenic liposome (FL)-mediated macromolecule delivery into human blood cells using diphtheria toxin fragment A (DTA) as a probe. Among the peripheral blood mononuclear cells (PBMC), FL was able to deliver its encapsulates into CD14 + monocytes and CD4 −/CD8 − T-cells, but not into CD19 + B-lymphocytes, CD4 + T-cells or CD8 + T-cells. The susceptibility of human leukemia cell lines to FL was similar to that of PBMC; the order of the reactivity was U937 (monoblastic leukemia)>MOLT4, Jurkat (T-lymphoma)>Daudi, BALL1 (B-lymphoma)>K562 (erythroblastic leukemia). Interestingly, FL showed similar binding activity to all of these leukemia cell lines. These findings indicate that, among blood cells, monocytes, monoblastic leukemia cells, CD4 −/CD8 − T-cells and T-lymphoma cells are preferable targets for FL-mediated macromolecule delivery. This is the first demonstration of the existence of non-permissive cells against FL. Our results also suggest that some molecules on target-cells other than the binding targets of SV-derived protein may participate in fusion between FL and cells.

Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F.

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.

Inhibition by interferon of Sendai virus cytolysis.

Treatment of HeLa cells with human interferons inhibited 51Cr release from cells induced by ultraviolet-inactivated Sendai virus. The inhibitory effect became apparent about 6 h after interferon treatment and persisted for 24 to 48 h. In the interferon-treated cells, the cytolysis was inhibited within 10 min after adding virus and the inhibitory action was suppressed by the treatment of the cells with cycloheximide. Mock interferon and mouse interferon did not inhibit the cytolysis and antiinterferon serum neutralized the effect of interferon. All these findings indicate that Sendai virus-induced cytolysis is inhibited by interferon per se. However, interferon did not have any influence on Sendai virus hemolysis.

Role of sendai virus fusion-glycoprotein in target cell susceptibility to cytotoxic T cells

IN mice, cytotoxic T lymphocytes (CTLs) are generated in vivo or in vitro against virally-infected syngeneic cells1–4. The specificity of killing requires that the target cells possess both viral gene product(s) and H–2K and/or D gene products which are identical to those on the effector cells5. Target cells coated with non-infectious (ultraviolet-inactivated) Sendai virus are also specifically lysed by syngeneic virus-specific CTLs6. We show here that it is primarily the fusion glycoprotein of the Sendai virus envelope which is essential for the formation of the target antigen on cells coated with ultraviolet-inactivated Sendai virus.

Helper factor(s) for growth of adeno-associated virus in cells transformed by adenovirus 12.

Evidence is presented that a helper factor(s) for growth of adeno-associated virus (AAV) is present in cells transformed by adenovirus type 12 (Ad12). The growth of AAV was observed in heterokaryons formed by fusion of human KB and Ad12-transformed rodent cells by using ultraviolet-inactivated Sendai virus without coinfection of cells with adenovirus. The presence of the helper factor(s) for AAV growth in rat cells transformed by the EcoRI-C fragment or the HindIII-G fragment of Ad12DNA suggests that the helper factor(s) induced by infection with adenovirus is the Ad12-specific T antigen.

Enhancement of potassium influx, in baby hamster kidney cells and chicken erythrocytes, during adsorption of parainfluenza 1 (Sendai) virus

Adsorption of ultraviolet-inactivated Sendai virus causes a marked but transient inhibition of cellular deoxyribonucleic acid synthesis in HeLa [ 13, baby hamster kidney (BHK 21), FL, l-929 and Chinese hamster cells [2]. Sendai virus adsorption also affects the chemical properties of cell membrane (Click, Fuchs and Kohn, unpublished data). In view of reports showing the inhibitory effect of high potassium concentration on cell division and deoxyribonucleic acid synthesis, in animal cells [3, 41, it was desirable to see whether virus adsorption had any effect on potassium influx in animal cells.

Electron Microscopic Observations on the Rescue of SV40 Virus From Transformed Cells

Several laboratories have reported that simian virus 40 (SV40) was rescued from transformed cells when the nonproducing cells were cocultivated or fused in the presence of ultraviolet inactivated Sendai virus (UV-SV), to potentially susceptible cells. Evidence obtained from studies in which nuclei from heterokaryons were isolated and separated on density gradients, indicated that rescued virus was first detected in the transformed nuclei of the heterokaryons formed during cell fusion. The present study was performed to determine how long after fusion SV40 virus particles could be found in the nuclei of the heterokaryons and to investigate the site of rescue by electron microscopy.

Induction of prophase in interphase nuclei by fusion with metaphase cells.

Fusion of an interphase cell with a metaphase cell results in profound changes in the interphase chromatin that have been called "chromosome pulverization" or "premature chromosome condensation" In addition to the usual light microscopy, the nature of the changes has been investigated in the present study with electron microscopy and biochemical techniques Metaphase and interphase cells were mixed and fused at 37 degrees C by means of ultraviolet-inactivated Sendai virus. After cell fusion, morphological changes in interphase nuclei occurred only in binucleate cells which contained one intact set of metaphase chromosomes Irrespective of the nuclear stage at the time of cell fusion, the morphologic changes that occurred 5-20 min later simulated very closely a sequence of events that characterizes the normal G(2)-prophase transition. Radioautography revealed that, late in the process, substantial amounts of RNA and probably protein were transferred from the interphase nucleus into the cytoplasm of fused cells. Thus, the findings indicate the existence in metaphase cells of factor(s) which are capable of initiating biochemical and morphological events in interphase nuclei intrinsic to the normal mitotic process.

Nature of transient inhibition of deoxyribonucleic acid synthesis in HeLa cells by parainfluenza virus 1 (Sendai).

Adsorption of ultraviolet-inactivated Sendai virus, at high or low multiplicity, to HeLa cells caused a transient increased incorporation of (3)H-thymidine into the cellular deoxyribonucleic acid (DNA). In HeLa cells synchronized by a double-thymidine block, this increased incorporation of thymidine during the S phase lasted from about 30 to 90 min after virus adsorption. The observations that the kinetics of accumulation of radioactive thymidine in the nucleotide pool did not differ in control and in the virus-treated cells and that the (32)P incorporation into the DNA of the virus-treated cells was inhibited at the same time indicate that the augmented incorporation of (3)H-thymidine into DNA results from a transient block in the endogenous pathway of thymidine synthesis. Chromatographic analysis of the nucleotide pool of the virus-treated cells labeled with (14)C-formate indicates that methylation of deoxyuridine monophosphate to thymidine monophosphate is inhibited. It is suggested that the inhibition is caused by a block of either the thymidilate synthetase or some step in the tetrahydrofolate cycle.

Human-mosquito somatic cell hybrids induced by ultraviolet-inactivated Sendai virus.

WE have recently achieved interphylum heterokaryon and hybrid formation from human (HeLa, clone 5 of S3, courtesy Dr Grace Leidy)1 and mosquito (Aedes aegyptae L., courtesy of the late Dr E. C. Suitor, Naval Medical Research Institute, Bethesda, Maryland)2 cell lines, using ultraviolet-inactivated Sendai virus to induce fusion3.

The influence of ultraviolet-inactivated Sendai virus on Marek's disease virus infection in tissue culture.

SUMMARY The presence of ultraviolet-inactivated Sendai virus increased the transfer of Marek’s disease virus from infected to uninfected cultured chick kidney cells. This effect was seen after incubation of infected and uninfected chick kidney cells in the presence of ultraviolet-inactivated Sendai virus at 4°. There was only a slight further increase in transfer of infection with subsequent incubation at 37°. The close apposition of infected and uninfected cells occurring during the agglutination produced by treatment with ultraviolet-inactivated Sendai virus in the cold, rather than complete cell fusion may have been the main means by which treatment with ultraviolet-inactivated Sendai virus increased the transfer of infection.

A quantitative comparison of formation of spontaneous and virus-produced viable hybrids.

Ultraviolet-inactivated Sendai virus used in conjunction with a selective system reproducibly gives high yields of viable hybrid cell lines. With two different crosses, the frequency of hybrid colonies obtained after virus treatment was found to be 100 times greater than the frequency of spontaneous hybrids, and this increase was found to show little variation between 33 and 1000 hemagglutinating units of virus. No differences have been found between the properties of hybrids obtained after Sendai treatment and those obtained from untreated mixed cultures of parental cells.

Activation of infectious SV40 synthesis in transformed cells.

The entire SV40 genome can be recovered from at least some SV40-transformed cell lines which do not sponltaneously produce this virus. Even after repeated cloning in the presence of SV40-antisera, these cell lines can be activated to produce SV40 if they are cocultivated with susceptible monkey kidney cells. Rescue of SV40 is greatly facilitated when mixtures of susceptible and transformed cells are treated with ultraviolet-inactivated Sendai virus (UV-Sendai), a procedure known to produce heterokaryons.1-9 Despite the presence of the entire SV40 genome in transformed cells, Sabin and Koch' failed to detect infectious SV40 DNA. The present study, using an infectious DNA assay of greatly increased sensitivity, also shows that superhelical (closed-circular) SV40 DNA molecules are absent from SV40-transformed cells. Infectious SV40 DNA can, however, be detected after susceptible cells and transformed cells are fused. The time required for emergence of infectious SV40 DNA is significant. T'he data to be presented show that after either fusion of transformed with susceptible cells or productive infection of CV-1 cells, there is about a 16to 19-hour delay before infectious SV40 DNA is made. Also described are attempts to rescue infectious SV40 DNA from transformed cell lines that have so far never yielded virus and from cell lines that are poor virus yielders.9 Materials and Methods. -Cell cultures: CV-1 is an established line of green monkey kidney cells. Transformed mouse kidney lines were obtained by inoculating primary mouse kidney cultures with norrmal SV40 (mKS-A or mKS-BUI00 lines)6 or with SV40 irradiated with UV light to l0oto 10-5 survival (mKS-U lines).' The TSV-5 and the H-50and 2X-10-transformed hamster cells have been described by Tournier et al.5 and by Melnick et al.,10 respectively. All cell lines were grown with R5a medium containing 0.5% lactalbumin hydrolysate and 10% calf serum (Hylands Laboratory, Los Angeles, Calif.). Cell fusion: Approximately 1-1.7 X 107 CV-1 cells were mixed at 4?C for 15 min with 107 transformed cells and 8,000--16,000 hemagglutination units (HAU) of UV-Sendai in a volume of 2 ml. The mixes were shaken for 20 min at 38?C in a water bath, 10 ml of growth medium was added, and the cells were centrifuged and resuspended in 10 ml of growth medium. Two-ml aliquots of cell mixes were seeded in 8-oz prescription bottles, 18 ml of growth medium added, and the cultures were incubated at 37?C for various periods of time. UV inactivation of Sendai virus: Sendai virus was grown in the allantoic cavity of 10to 11-day-old eggs for 48-72 hr at 37?C. After the eggs were chilled at 4?C overnight, the allantoic fluid was har vested arnd clarified by low-speed centrifugation. Sendai virus was pelleted by cenfti,ifugatioti: for 30 n-tin at 30,000 rpim (Spinco model L-2, no. 30 rotor), and the pellets wer e resusp)eided ill 1-)hosphabe-1)lffe le(I saline (1P138) solution'1 at 1/10 the original volume and stored i lthe fro2,en cond(lition. Aliquots were thawed immediately before use and dilulted with 4 vol of PIBS to a titer of 5000-8000 HAU per ml, and 1.6-ml aliquots in 60-mm Petri dishes were inactivated by UV iiradiation for 5 min at a distance of 25 em from the light source (G30T8 Sylvania germicidal lamp).