Jari E. Heikkilä

Oncolytic viruses in cancer therapy

Abstract Oncolytic virotherapy is a promising form of gene therapy for cancer, employing nature’s own agents to find and destroy malignant cells. The purpose of this review is to provide an introduction to this very topical field of research and to point out some of the current observations, insights and ideas circulating in the literature. We have strived to acknowledge as many different oncolytic viruses as possible to give a broader picture of targeting cancer using viruses. Some of the newest additions to the panel of oncolytic viruses include the avian adenovirus, foamy virus, myxoma virus, yaba-like disease virus, echovirus type 1, bovine herpesvirus 4, Saimiri virus, feline panleukopenia virus, Sendai virus and the non-human coronaviruses. Although promising, virotherapy still faces many obstacles that need to be addressed, including the emergence of virus-resistant tumor cells.

Ionic Dependence of Membrane Potential and Glutamate Receptor‐Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS‐C2‐(5)

Abstract: Membrane potentials of particles present in a sub‐cellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absor‐bance of a cyanine dye, DiS‐C2‐5. The membrane potential of the particles seems to be dependent on both Cl− and K+ diffusion potentials, as judged from dependence of the ab‐sorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl− was replaced with gluconate. The apparent high Cl− permeability of the membrane preparation was reduced in the presence of pic‐rotoxin, a finding suggesting endogenous activation of receptor‐linked Cl− channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4‐aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N‐methyl‐d‐aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl− permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmit‐ter receptor‐linked membrane potential changes with optical probes of membrane potential.

Targeted inhibition of human collagenase-3 (MMP-13) expression inhibits squamous cell carcinoma growth in vivo.

Squamous cell carcinomas (SCCs) of the head and neck are characterized by a high tendency for local invasion and metastasis to lymph nodes. Collagenase-3 (MMP-13) is specifically expressed by tumor cells in SCCs of the head and neck and its expression correlates with their invasion capacity. To specifically examine the role of MMP-13 in the growth and invasion of SCC, we constructed a hammerhead ribozyme targeted against human MMP-13 mRNA. The anti-MMP-13 ribozyme effectively cleaved MMP-13 transcripts in vitro. Adenoviral delivery of the anti-MMP-13 ribozyme to cutaneous metastatic SCC cells in culture resulted in potent and specific inhibition of the production of proMMP-13 and markedly suppressed invasion of SCC cells through Matrigel. In addition, adenoviral delivery of anti-MMP-13 ribozyme promoted apoptosis in SCC cells within 72 h. Intratumoral injection of anti-MMP-13 ribozyme coding adenovirus into human SCC xenografts established in SCID mice potently suppressed tumor growth, inhibited MMP-13 expression and gelatinolytic activity and reduced the number of proliferating cells within the tumors. These results provide evidence for an important role for MMP-13 in SCC growth and invasion and identify MMP-13 as a promising target for ribozyme-based therapy of SCC in vivo.

Oncolytic Capacity of Attenuated Replicative Semliki Forest Virus in Human Melanoma Xenografts in Severe Combined Immunodeficient Mice

Oncolytic viruses have gained attention as a novel form of cancer treatment. Many viral vectors in use today have been rendered safe by deletion of genes encoding viral structural proteins, thus making them unable to spread beyond the first infected cells. Hence, such replication-deficient constructs may lack efficacy. Here, we analyzed the oncolytic potential of the replication-competent vector VA7-EGFP, based on the avirulent Semliki Forest virus (SFV) strain A7(74), to kill cancer cells in culture as well as to target s.c. human melanoma xenografts in severe combined immunodeficient (SCID) mice. VA7-EGFP was able to infect most cancer cell lines studied, leading to complete lysis of the cells within 72 hours after infection. In SCID mice grafted with A2058 human melanoma, marked regression of the xenografts was observed following a single injection of 10(6) plaque-forming units of virus given either i.p., i.v., or intratumorally. Histologic analysis revealed the presence of virus not only in all treated tumors but also in the brains of the treated mice, causing progressing neuropathology beginning at day 16 after infection. Following initial oncolysis, clusters of viable tumor cells were observed embedded in connective tissue, and at later stages, encapsulated tumor nodules had formed. Infection of melanoma cells from explant cultures of these nodules revealed that a portion of the cells were resistant to virus. To be eligible for use in virotherapy, the ability of avirulent SFV to spread within tumor tissue may have to be improved and the biological safety of the virus may have to be addressed thoroughly in higher animals.

Staurosporine induces a neuronal phenotype in SH-SY5Y human neuroblastoma cells that resembles that induced by the phorbol ester 12-O-tetradecanoyl phorbol-13 acetate TPA)

Treatment of SH‐SY5Y human neuroblastoma cells with the protein kinase inhibitor staurosporine, induced both morphological and functional differentiation in these cells. The effects of staurosporine were comparable to those induced by the protein kinase C (PKC) activator. 12‐O tetradecanoyl phorbol 13‐acetate (TPA), with respect to induction of neuronal differentiation, i.e. neurite outgrowth, inhibition of DNA synthesis, induction and down‐regulation of c‐myc protein expression, induction of mRNA for both neuropeptide Y (NPY) and growth associated protein 43 (GAP‐43) and stimulation of tyrosine hydroxylase expression. Staurosporine failed to translocate PKC to the membrane fraction or to stimulate phosphorylation of the endogenous PKC substrate M r 80,000 (p8O). Instead, staurosporine inhibited TPA‐induced phosphorylation of p80.

Sphingosine Kinase as an Oncogene: Autocrine Sphingosine 1-Phoshate Modulates ML-1 Thyroid Carcinoma Cell Migration by a Mechanism Dependent on Protein Kinase C-α and ERK1/2

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

Human embryonic stem cell-derived mesenchymal stromal cell transplantation in a rat hind limb injury model

BACKGROUND AIMS Mesenchymal stromal cells (MSC) have been used in a wide variety of pre-clinical experiments and in an increasing number of human clinical trials. Although many of these studies have shown different levels of engraftment, the exact fate of MSC after transplantation and the tissue response to their engraftment have not been investigated in detail. In the present work we studied the distribution of human MSC in a rat hind limb ischemic injury model immediately after transplantation and also analyzed the recipient tissue response to transplanted cells. METHODS We tracked the in vivo fate of the transplanted MSC utilizing bioluminescence imaging, fluorescence microscopy and gene/protein expression analysis in a rat hind limb ischemia model. We also monitored the viability of transplanted cells by graft versus recipient expression analysis and determined the angiogenic and proliferative effect of transplantation by histologic staining. RESULTS According to imaging analysis only a small portion of cells persisted for an extended period of time at the site of injury. Interestingly, recipient versus graft expression studies showed increased synthesis of rat-origin angiogenic factors and no human-origin mRNA or protein synthesis in transplanted tissues. More importantly, despite the lack of robust engraftment or growth factor secretion the transplantation procedure exerted a significant pro-angiogenic and pro-proliferative effect, which was mediated by angiogenic and mitogenic signaling pathways. CONCLUSIONS Our results show an immediate temporal tissue effect in response to MSC transplantation that may represent a novel indirect paracrine mechanism for the beneficial effects of cell transplantation observed in injured tissues.

Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice

Background VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma. Methodology/Principal Findings In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point. Conclusion VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus.

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.

Differential Roles of Fibroblast Growth Factor Receptors (FGFR) 1, 2 and 3 in the Regulation of S115 Breast Cancer Cell Growth

Fibroblast growth factors (FGFs) regulate the growth and progression of breast cancer. FGF signaling is transduced through FGF receptors 1–4, which have oncogenic or anti-oncogenic roles depending on the ligand and the cellular context. Our aim was to clarify the roles of FGFR1–3 in breast cancer cell growth in vitro and in vivo. Pools of S115 mouse breast cancer cells expressing shRNA against FGFR1, 2 and 3 were created by lentiviral gene transfer, resulting in cells with downregulated expression of FGFR1, FGFR2 or FGFR3 (shR1, shR2 and shR3 cells, respectively) and shLacZ controls. FGFR1-silenced shR1 cells formed small, poorly vascularized tumors in nude mice. Silencing of FGFR2 in shR2 cells was associated with strong upregulation of FGFR1 expression and the formation of large, highly vascularized tumors compared to the control tumors. Silencing FGFR3 did not affect cell survival or tumor growth. Overexpressing FGFR2 in control cells did not affect FGFR1 expression, suggesting that high FGFR1 expression in shR2 cells and tumors was associated with FGFR2 silencing by indirect mechanisms. The expression of FGFR1 was, however, increased by the addition of FGF-8 to starved shLacZ or MCF-7 cells and decreased by the FGFR inhibitor PD173074 in shR2 cells with an elevated FGFR1 level. In conclusion, our results demonstrate that FGFR1 is crucial for S115 breast cancer cell proliferation and tumor growth and angiogenesis, whereas FGFR2 and FGFR3 are less critical for the growth of these cells. The results also suggest that the expression of FGFR1 itself is regulated by FGF-8 and FGF signaling, which may be of importance in breast tumors expressing FGFs at a high level.