Modulation of Reoviral Cytolysis (I): Combination Therapeutics.

Patients with stage IV gastric cancer suffer from dismal outcomes, a challenge especially in many Asian populations and for which new therapeutic options are needed. To explore this issue, we used oncolytic reovirus in combination with currently used chemotherapeutic drugs (irinotecan, paclitaxel, and docetaxel) for the treatment of gastric and other gastrointestinal cancer cells in vitro and in a mouse model. Cell viability in vitro was quantified by WST-1 assays in human cancer cell lines treated with reovirus and/or chemotherapeutic agents. The expression of reovirus protein and caspase activity was determined by flow cytometry. For in vivo studies, athymic mice received intratumoral injections of reovirus in combination with irinotecan or paclitaxel, after which tumor size was monitored. In contrast to expectations, we found that reoviral oncolysis was only poorly correlated with Ras pathway activation. Even so, the combination of reovirus with chemotherapeutic agents showed synergistic cytopathic effects in vitro, plus enhanced reovirus replication and apoptosis. In vivo experiments showed that reovirus alone can reduce tumor size and that the combination of reovirus with chemotherapeutic agents enhances this effect. Thus, we find that oncolytic reovirus therapy is effective against gastric cancer. Moreover, the combination of reovirus and chemotherapeutic agents synergistically enhanced cytotoxicity in human gastric cancer cell lines in vitro and in vivo. Our data support the use of reovirus in combination with chemotherapy in further clinical trials, and highlight the need for better biomarkers for reoviral oncolytic responsiveness.

Post-Passage rock inhibition induces cytoskeletal aberrations and apoptosis in Human embryonic stem cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are prone to anoikis after single cell dissociation. The small molecule, Y-27632 is known to increase survival of hESCs and hiPSCs by inhibiting the Rho-associated protein kinase (ROCK). However, the underlying mechanisms are still unclear. Here, we thoroughly screened small molecules to investigate the adhesion and survival of hESCs in adherent culture. Y-27632 provided higher adhesion and survival of hESCs by increased cell migration and preventing cell blebbing in single dissociated cells. The combination of Matrigel with poly-d-lysine increased the attachment and survival of dissociated cells via actin filament and microtubule spreading in Y-27632-treated cells. Although Y-27632 prevented apoptosis by suppressing actin filament contraction, microtubule bundling, and blebbing, prolonged Y-27632 treatment presented a different morphology in the attached growing hESC colony. It induced apoptosis of cells by promoting cytoplasmic spread, E-cadherin structural change, and increased detachment. It also induced actin cytoskeleton disruption, combined with microtubule and intermediate filament elongation. For optimal hPSC culture, our research suggests that Y-27632 should be removed shortly after passaging.

Virological characteristics of occult hepatitis B virus in a North American cohort of human immunodeficiency virus type 1-positive patients on dual active anti-HBV/HIV therapy.

BACKGROUND: Occult hepatitis B virus infection (OBI) is defined as low-level HBV DNA presence in serum, liver and/or peripheral blood mononuclear cells (PBMC) in individuals that lack serum hepatitis B virus surface antigen (HBsAg). HIV+ patients with OBI may be at risk for HBV reactivation, and often receive dual active anti-HBV/HIV therapy, such as lamivudine (LMV). OBJECTIVES: To determine the presence of OBI in a North American cohort of HIV-1-positive patients. STUDY DESIGN/METHODS: 45 HIV-1-positive, serum HBsAg-negative patients, reactive for antibodies to HBV core antigen (anti-HBc), were tested for HBV DNA in plasma and for HBV DNA and covalently closed circular DNA (cccDNA) in PBMC. Ten patients were re-tested after ∼5-10 years, including genotyping and clonal sequence analysis of the HBV polymerase (P) gene and overlapping HBV surface (S) gene from 8 PBMC samples. RESULTS: Overall, 42% (19/45) tested HBV DNA positive, especially in PBMC (18/45), including 3/18 that were reactive for HBV cccDNA, compared to 17% (8/45) that were HBV DNA reactive in plasma. In 8 patients on LMV, sequence analysis in PBMC showed that all were HBV genotype C or D. Several carried HBV P region variants at residues associated with anti-HBV drug resistance and overlapping S gene region within the major HBsAg "a determinant". CONCLUSION: OBI is common in HIV-positive, anti-HBc reactive patients on anti-HBV/HIV therapy, particularly in PBMC. HBV sequence analysis revealed that all had HBV genotype C or D and often had P/overlapping S gene variants possibly associated with dual-active anti-HIV/HBV therapy.

Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors.

Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

NO-ß-catenin crosstalk modulates primitive streak formation prior to embryonic stem cell osteogenic differentiation.

Nitric oxide (NO) has been shown to play a crucial role in bone formation in vivo. We sought to determine the temporal effect of NO on murine embryonic stem cells (ESCs) under culture conditions that promote osteogenesis. Expression profiles of NO pathway members and osteoblast-specific markers were analyzed using appropriate assays. We found that NO was supportive of osteogenesis specifically during an early phase of in vitro development (days 3-5). Furthermore, ESCs stably overexpressing the inducible NO synthase showed accelerated and enhanced osteogenesis in vitro and in bone explant cultures. To determine the role of NO in early lineage commitment, a stage in ESC differentiation equivalent to primitive streak formation in vivo, ESCs were transfected with a T-brachyury-GFP reporter. Expression levels of T-brachyury and one of its upstream regulators, ß-catenin, the major effector in the canonical Wnt pathway, were responsive to NO levels in differentiating primitive streak-like cells. Our results indicate that NO may be involved in early differentiation through regulation of ß-catenin and T-brachyury, controlling the specification of primitive-streak-like cells, which may continue through differentiation to later become osteoblasts.

Peptide vaccination is superior to genetic vaccination using a recombineered bacteriophage λ subunit vaccine.

Genetic immunization holds promise as a vaccination method, but has so far proven ineffective in large primate and human trials. Herein, we examined the relative merits of genetic immunization and peptide immunization using bacteriophage λ. Bacteriophage λ has proven effective in immune challenge models using both immunization methods, but there has never been a direct comparison of efficacy and of the quality of immune response. In the current study, this vector was produced using a combination of cis and trans phage display. When antibody titers were measured from immunized animals together with IL-2, IL-4 and IFNγ production from splenocytes in vitro, we found that proteins displayed on λ were superior at eliciting an immune response in comparison to genetic immunization with λ. We also found that the antibodies produced in response to immunization with λ displayed proteins bound more epitopes than those produced in response to genetic immunization. Finally, the general immune response to λ inoculation, whether peptide or genetic, was dominated by a Th1 response, as determined by IFNγ and IL-4 concentration, or by a higher concentration of IgG2a antibodies.

Implantation serine proteinase 1 exhibits mixed substrate specificity that silences signaling via proteinase-activated receptors.

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.

Oncolytic viral therapy for prostate cancer: efficacy of reovirus as a biological therapeutic.

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.

PURPOSE: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. EXPERIMENTAL DESIGN: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. RESULTS: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. CONCLUSIONS: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

Systemic reovirus therapy of metastatic cancer in immune-competent mice.

The human reovirus is an oncolytic virus that specifically targets cancer cells with an activated Ras pathway. Because it is replication competent and highly specific for cancer cells, this virus has the potential to be an effective antimetastatic cancer agent through remote site delivery. In this study, we exploited the ability of reovirus to replicate in murine cells to test the efficacy of this virus in eliminating distal and/or metastatic tumors in immune-competent mice. We found that i.v. therapy with reovirus not only inhibited metastatic tumor growth but also led to a significant improvement in animal survival. Combining i.v. reovirus treatment with immune suppression (cyclosporine A or anti-CD4/anti-CD8 antibodies) resulted in further reduction in tumor size and a considerable prolongation in survival, compared with viral therapy alone. Combined therapy was also effective in overcoming a preexisting immunity to reovirus (a common occurrence in humans and thus a potential impediment to oncolytic effectiveness) to induce metastatic tumor regression. This is the first study to use systemic delivery of an oncolytic agent in conjunction with immune-suppressive drugs to effectively prolong animal survival. Altogether, our results suggest that i.v. reovirus therapy may present a feasible, novel alternative in the treatment of metastatic cancer in humans.

Reovirus therapy of lymphoid malignancies.

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.

Oncolytic reovirus against ovarian and colon cancer.

Reovirus selectively replicates in and destroys cancer cells with an activated Ras signaling pathway. In this study, we evaluated the feasibility of using reovirus (serotype 3, strain Dearing) as an antihuman colon and ovarian cancer agent. In in vitro studies, reovirus infection in human colon and ovarian cell lines was assessed by cytopathic effect as detected by light microscopy, [(35)S]Methionine labeling of infected cells for viral protein synthesis and progeny virus production by plaque assay. We observed that reovirus efficiently infected all five human colon cancer cell lines (Caco-2, DLD-1, HCT-116, HT-29, and SW48) and four human ovarian cancer cell lines (MDAH2774, PA-1, SKOV3, and SW626) which were tested, but not a normal colon cell line (CCD-18Co) or a normal ovarian cell line (NOV-31). We also observed that the Ras activity in the human colon and ovarian cancer cell lines was elevated compared with that in normal colon and ovarian cell lines. In animal models, intraneoplastic as well as i.v. inoculation of reovirus resulted in significant regression of established s.c. human colon and ovarian tumors implanted at the hind flank. Histological studies revealed that reovirus infection in vivo was restricted to tumor cells, whereas the surrounding normal tissue remained uninfected. Additionally, in an i.p. human ovarian cancer xenograft model, inhibition of ascites tumor formation and the survival of animals treated with live reovirus was significantly greater than of control mice treated with UV-inactivated reovirus. Reovirus infection in ex vivo primary human ovarian tumor surgical samples was also confirmed, further demonstrating the potential of reovirus therapy. These results suggest that reovirus holds promise as a novel agent for human colon and ovarian cancer therapy.

Reovirus oncolysis of human breast cancer.

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.