Riikka Pellinen

Oncolytic Semliki Forest Virus Vector as a Novel Candidate against Unresectable Osteosarcoma

Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Delta24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP-treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system.

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models.

Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication‐competent SFV vector VA7‐EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7‐EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection). This was at least partially due to a strong type I interferon (IFN) response in the tumors. The animals did not display any signs of abnormal behavior or encephalitis, even though SFV‐positive foci were detected in the brain after the initial blood viremia. Intracranial rat tumors were injected directly with SFV A7(74) virus and monitored with magnetic resonance imaging. Tumor growth was significantly reduced (p < 0.05) with one virus injection, but the tumor size continued to increase after a lag period and none of the treated animals survived. Three virus injections or T‐cell suppression with dexamethasone did not significantly improve treatment efficacy. It appeared that the local virotherapy induced extensive production of neutralizing anti‐SFV antibodies that most likely contributed to the insufficient treatment efficacy. In conclusion, we show here that SFV A7(74) is a potential oncolytic agent for cancer virotherapy, but major immunological hurdles may need to be overcome before the virus can be clinically tested.

The involvement of mitochondria and the caspase-9 activation pathway in rituximab-induced apoptosis in FL cells

Despite the wide use of anti-CD20 antibody rituximab in the cancer treatment of B cell malignancies, the signalling pathways of CD20-induced apoptosis are still not understood. By using dominant negative (DN)-caspase-9 overexpressing follicular lymphoma cells we demonstrated that the activation of caspase-9 was essential for rituximab-mediated apoptosis. The death receptor pathway mediated by caspase-8 activation was not involved in rituximab-mediated apoptosis since overexpression of FLIPshort or FLIPlong proteins, inhibitors of caspase-8 activation, could not inhibit rituximab-induced apoptosis. However, the death receptor pathway activation by anti-Fas antibodies showed an additive effect on rituximab-induced apoptosis. The stabilisation of the mitochondrial outer membrane by Bcl-xL overexpression inhibited cell death, showing the important role of mitochondria in rituximab-induced apoptosis. Interestingly, the rituximab-induced release of cytochrome c and collapse of mitochondrial membrane potential were regulated by caspase-9. We suggest that caspase-9 and downstream caspases may feed back to mitochondria to amplify mitochondrial disruption during intrinsic apoptosis.

Replication competent Semliki Forest virus prolongs survival in experimental lung cancer

We evaluated the therapeutic potential of the replication competent vector VA7‐EGFP, which is based on the avirulent Semliki Forest virus (SFV) strain A7 (74) carrying the EGFP marker gene in an orthotopic lung cancer tumor model in nude mice. We have previously shown that this oncolytic vector destroys tumor cells efficiently in vitro and in vivo (in subcutaneous tumor model). Tumor growth in animals with orthotopically implanted adenocarcinoma cells (A549) were monitored during the study with small animal CT. We show that locally administered virotherapy with VA7‐EGFP increased survival rate in experimental lung cancer significantly (p < 0.001) comparable to results obtained with the second generation conditionally replicating adenoviral vector Ad5‐Δ24TK‐GFP, used for comparison. The limited efficacy in systemically administered oncolytic viruses is the essential problem in oncolytic virotherapy and also in this study we were not able to elicit significant response with systemic administration route. Despite the fact that tumor microenvironment in orthotopic lung cancer is more optimal, viruses failed to home to the tumors and were unable to initiate efficient intratumoral replication. Clearly, the efficacy of virotherapy is influenced by many factors such as the route of virus administration, immunological and physiological barriers and cancer cell–specific features (IFN‐responsiveness).

Dexamethasone-induced apoptosis and up-regulation of Bim is dependent on glycogen synthase kinase-3.

Glucocorticoids are commonly used in the treatment of lymphoid malignancies. In this study, we show that apoptosis induced by dexamethasone (Dex), a synthetic glucocorticoid, was dependent on mitochondria, since overexpression of Bcl-X(L) prevented Dex-induced apoptotic changes. Dominant negative (DN) caspase-9 also prevented Dex-induced apoptotic changes including the loss of mitochondrial membrane potential indicating that caspase-9 controls mitochondrial changes. In addition, we evaluated the role of glycogen synthase kinase (GSK3) in Dex-induced apoptosis. Inhibition of GSK3 attenuated Dex-induced up-regulation of Bim, loss of mitochondrial membrane potential, release of cyt c and DNA fragmentation. These results indicate that GSK3 contributes to Dex-induced apoptosis by controlling up-regulation of Bim.

Type I interferon response against viral and non-viral gene transfer in human tumor and primary cell lines

Type I interferon (IFN‐α/β) response is one of the major host defence mechanisms against viruses. Some recent reports suggest that IFNs may interfere with the efficacy of both non‐viral and virus‐vector‐mediated therapeutic gene transfer.

PDTC enables type I TRAIL signaling in type II follicular lymphoma cells.

Based on Bcl-X(L) overexpression studies we identified type I and type II follicular lymphoma cell lines in response to TRAIL. We demonstrate here that either amount of caspase-8 activation or Bid cleavage could not define the dependence on mitochondria. Furthermore, an inhibitor of NF-kappaB, PDTC, enabled TRAIL to activate type I apoptotic pathway in type II cells. However, an inhibitor of IKK did not switch apoptosis to type I pathway in type II cells, indicating that NF-kappaB might not be responsible for the switch.

HOX-GFP and WOX-GFP lentivirus vectors for inner ear gene transfer.

Conclusion. GFP transgene was expressed in the lining cells of the perilymphatic space. Lentivirus vectors are safe and cause only minimal inflammatory reaction. Transgene products can be delivered into the perilymph by utilizing lentivirus vectors. Objectives. To analyze the efficiency and safety of lentiviral vectors HOX-GFP and WOX-GFP in intracochlear gene transfer. Materials and methods. Lentivirus vectors were tested for their transduction efficiency in vivo in CD-1 mice. Half of the animals were pretreated with kanamycin. Lentivirus vector or saline (1 µl) was injected into the inner ear. All the animals were sacrificed 14 days after the surgery and the cochleae and selected organs were analyzed immunohistochemically. Results. HOX-GFP and WOX-GFP expression was restricted to the lining cells of the scala tympani and scala vestibuli. No GFP expression was seen in the organ of Corti or the spiral ganglion. Aminoglycoside treatment had no effect on the expression of these vectors. The distant spread of lentivirus vectors was minimal; only the liver of one animal showed some GFP expression. Inflammatory reaction caused by these vectors was mild. Few inflammatory cells were found in the perilymphatic space of the cochlea and in the vestibular organ.

Inducible expression of antizyme 1 in prostate cancer cell lines after lentivirus mediated gene transfer

The prostate has the highest level of polyamines among all tissues, and it is the only tissue in which polyamines are purposely synthesized for export. It has been suggested that the high local polyamine concentrations suppress cell growth of primary prostatic carcinomas and that this growth control is lost when cancer cells metastasize. It has also been shown that the sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cell lines. In this study, we evaluated the sensitivity of poorly metastatic (LNCaP) and highly metastatic (DU145) prostate cancer cell lines to conditional antizyme 1 over-expression. Antizyme 1 induction resulted in a marked loss of ODC activity and polyamine uptake in both cell lines. However, the proliferation of LNCaP cells was repressed by antizyme 1 induction, whereas the proliferation of DU 145 cells was not affected. Neither cell line showed any reduction in polyamine pools after manipulation nor did polyamine addition into the medium save the LNCaP cells from the growth retardation. The growth inhibition of LNCaP cells was accompanied by accumulation of cells in the G1 phase and depletion of cyclin E1 protein. These results confirm that different prostate cancer cell lines show diverse sensitivities to antizyme 1 which may not be directly polyamine related. The high gene transfer capacity of the used lentiviral vector makes the present approach a useful tool to study the therapeutic potential of antizyme 1 both in cell cultures and experimental animals.

Recombination of replicon and helper RNAs and the emergence of propagation-competent vectors upon Sindbis virus vector production

Sindbis vectors have shown remarkable antitumor efficacy and tumor-targeting capacity in animal models and hold promise for cancer therapy. Different packaging systems are used to produce propagation-incompetent Sindbis vectors. However, the vectors produced using either DH-BB single helper RNA or split helper RNA can spread in permissive cell cultures. We investigated the mechanisms of vector spreading and show, here, that recombination occurs between the replicon and DH-BB helper RNA, leading to formation of the full-length virus genome. Split helper RNA may not completely prevent wild-type reversion, although the frequency is greatly reduced. Contrary to propagation of Sindbis DH-BB vectors, Sindbis split helper vectors were frequently able to spread without cytopathic effect (CPE), a feature that was linked to wild-type reversion. Our results support the hypothesis that the non-cytopathic local spreading constantly observed with Sindbis split helper vector results from unspecific packaging of helper RNAs into vector particles and co-infection with particles containing replicon and helper RNAs. Several malignant cell lines with defective interferon responses were found to be permissive for non-cytopathic spreading of the Sindbis split helper vector. Interferon-α suppressed the spreading providing a possible option to control the vector.