Attenuated reovirus displays oncolysis with reduced host toxicity.

BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. METHODS: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. RESULTS: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated σ1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. CONCLUSION: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.

Competitive inhibition of hsp70 gene expression causes thermosensitivity.

A novel method has been developed for modulating the expression of an endogenous chromosomal gene in a higher eukaryote, by competitive inhibition at the level of gene transcription. The gene studied was the hsp70 gene, which encodes a 72-kilodalton (kD) heat shock protein that is synthesized after thermal stress. The 5' control region of the hsp70 gene was inserted on a plasmid containing the eukaryotic gene for dihydrofolate reductase. The hybrid plasmid was then introduced into a Chinese hamster ovary cell line and elevated in copy number approximately 20,000-fold by selection of cells with methotrexate. Heat-inducible expression from the intact hsp70 gene was reduced by at least 90% in the modified cells when compared with the induction in control cells, and the modified cells also displayed elevated thermosensitivity. The change in heat shock protein synthesis is presumably caused by competition among the increased number of binding sites for the heat-shock transcription factor, leading to altered expression from the native heat shock gene. These results support a role for heat shock protein in the recovery of mammalian cells from acute thermal stress.

Translational potentiation of messenger RNA with secondary structure in Xenopus.

Differential translation of messenger RNA (mRNA) with stable secondary structure in the 5' untranslated leader may contribute to the dramatic changes in protein synthetic patterns that occur during oogenesis and early development. Plasmids that contained the bacterial gene chloramphenicol acetyltransferase and which encoded mRNA with (hpCAT) or without (CAT) a stable hairpin secondary structure in the 5' noncoding region were transcribed in vitro, and the resulting mRNAs were injected into Xenopus oocytes, eggs, and early embryos. During early oogenesis, hpCAT mRNA was translated at less than 3 percent of the efficiency of CAT mRNA. The relative translational potential of hpCAT reached 100 percent in the newly fertilized egg and returned to approximately 3 percent after the midblastula transition.

Acquired resistance to reoviral oncolysis in Ras-transformed fibrosarcoma cells.

Reovirus shows considerable potential as an oncolytic agent for Ras-activated tumors and is currently in clinical trials. Here we ask whether such tumor cell lines can acquire resistance to reoviral oncolysis. We challenged human HT1080 fibrosarcoma cells that carry a Ras mutation by prolonged exposure to reovirus, thereby yielding highly virus-resistant HTR1 cells. These cells are persistently infected with reovirus, exhibit high Ras activity and retain the original Ras gene mutation, showing that resistance to reovirus can be displayed in cells with active Ras. The HTR1 cells also exhibit reduced cellular cathepsin B activity, which normally contributes to viral entry and activation. Persistently infected HTR1 cells were not tumorigenic in vivo, whereas immunologically cured virus-free HTR1 cells were highly tumorigenic. Thus, acquisition of resistance to reovirus may constrain therapeutic strategies. To determine whether reoviral resistance is associated with a general reduction in apoptotic potential, we challenged the HTR1 cells with apoptotic inducers and E1B-defective adenovirus, resulting in significant apoptosis and cell death following both approaches. Therefore, even if resistance to reoviral oncolysis should arise in tumor cells in vivo, other therapeutic strategies may nevertheless remain effective.

Reovirus decreases azoxymethane-induced aberrant crypt foci and colon cancer in a rodent model.

Reovirus type 3 Dearing has demonstrated oncolytic efficacy in vitro and in vivo against a variety of cancer cell lines, tumor xenografts and syngeneic cancer models. In this study, we investigated the effectiveness of reovirus against aberrant crypt foci (ACF) and colon cancer induced by the carcinogen azoxymethane (AOM) in an immunocompetent rat model. Sprague-Dawley rats received 15 mg/kg AOM intraperitoneally once per week for 4 weeks and reovirus was administered rectally once a week for 5 weeks starting 20 weeks after the last dose of AOM. Two weeks after completion of reovirus therapy, animals were examined for tumor burden in the colon and other tissues. Reovirus-treated animals showed a decrease in total ACF numbers (P=0.014), in large ACFs (P=0.0069) and in tumor number (P=0.03) compared to vehicle-treated animals. Fewer obstructing tumors in the colon (P=0.07) and duodenum (P=0.03) and reduced hepatic metastases were also noted. In addition, a tumor cell line derived from hepatic metastases was found to be susceptible to reovirus in vitro. Our results show that repeated rectal reovirus administration had some efficacy in the treatment and prevention of AOM-induced ACFs, colon cancers and metastases.

Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

Enzastaurin: A lesson in drug development.

Enzastaurin is an orally administered drug that was intended for the treatment of solid and haematological cancers. It was initially developed as an isozyme specific inhibitor of protein kinase Cß (PKCß), which is involved in both the AKT and MAPK signalling pathways that are active in many cancers. Enzastaurin had shown encouraging preclinical results for the prevention of angiogenesis, inhibition of proliferation and induction of apoptosis as well as showing limited cytotoxicity within phase I clinical trials. However, during its assessment in phase II and III clinical trials the efficacy of enzastaurin was poor both in combination with other drugs and as a single agent. In this review, we will discuss the development of enzastaurin from drug design to clinical testing, exploring target identification, validation and preclinical assessment. Finally, we will consider the clinical evaluation of enzastaurin as an example of the challenges associated with drug development. In particular, we discuss the poor translation of drug efficacy from preclinical animal models, inappropriate end point analysis, limited standards in phase I clinical trials, insufficient use of biomarker analysis and also patient stratification, all of which contributed to the failure to achieve approval of enzastaurin as an anticancer therapeutic.

UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences.

The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12-48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.

Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India--a pilot study.

Expression of the oncofoetal glycoprotein, carcinoembryonic antigen (CEA), has been observed in a number of malignancies and is also being pursued as a target for anti-cancer therapy. This study explored the status of this biochemical entity in the oral squamous cell carcinoma (SCC) in South India caused by extensive chewing habits. Squamous cell carcinoma in the study belonged to grade I and grade II. Tumour staging of the patients recruited in the study ranged from T2N1M0 to T4N3M0. Of the grade II cases studied, 88% (7 out of 8) showed expression of CEA. The 2 cases of grade I SCC of buccal mucosa also showed positive anti-CEA staining. If the results from this pilot study can be validated with a larger sample size, a role can be attributed to this tumour marker in oral neoplasia, thereby opening up avenues for using CEA as an additional diagnostic marker in oral SCC in this population and as a possible target for anti-cancer therapy.

Reovirus as an oncolytic agent against experimental human malignant gliomas.

BACKGROUND: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. METHODS: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. RESULTS: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P =.0002 for both U251N and U87) and in two intracerebral (P =.0004 for U251N and P =.0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P =.0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. CONCLUSIONS: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers.

A novel candidate tumor suppressor, ING1, is involved in the regulation of apoptosis.

We have recently cloned a novel growth inhibitor and candidate tumor suppressor called p33ING1 (I. Garkavtsev et al., Nature Genet., 14: 415-420, 1996). Because some tumor suppressors participate in the regulation of apoptosis, we hypothesized that the ING1 gene may also play a role in this process. Our results show that p33ING1 levels increase upon the induction of apoptosis in P19 teratocarcinoma cells by serum deprivation. Elevated expression of ING1 in P19 and rodent fibroblast cells containing a tetracycline-controlled human c-myc gene enhanced the extent of serum starvation-induced apoptosis. This suggests that the pathway by which ING1 modulates cell death is synergistic with Myc-dependent apoptosis. Conversely, constitutive expression of an antisense construct of INGI conferred protection against apoptosis in these cells. These data support the idea that loss of proper ING1 function may facilitate tumorigenesis, in part, by reducing the cell's sensitivity to apoptosis.

A possible role for c-Myc oncoproteins in post-transcriptional regulation of ribosomal RNA.

Overexpression of members of the myc family of oncogenes contributes to the development of many vertebrate malignancies. Although several functions for myc gene products have been proposed, the targets for Myc activity during oncogenesis or normal development remain to be identified. Oocytes of Xenopus laevis, which are non-dividing cells that accumulate abundant c-Myc protein, provide an unusual opportunity to examine c-Myc function. We have investigated whether the accumulation of massive amounts of ribosomal RNA (rRNA) by Xenopus laevis oocytes may be related to their elevated expression of myc proto-oncogenes. Our data show that anti-Myc antibodies and some (but not all) c-Myc peptides from conserved regions of the c-Myc protein enhance the turnover of rRNA synthesized in homogenates of oocyte nuclei. These results suggest that one or more members of the Myc protein family may be involved in post-transcriptional regulation of rRNA metabolism. The regulation by c-Myc of rRNA could account, in part, for the generalized stimulation of cells during tumorigenesis.

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle.

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.

Caveolin-1 is regulated by c-myc and suppresses c-myc-induced apoptosis.

Recent data indicating that overexpression of caveolin-1 as well as c-myc are relatively common features of advanced prostate cancer prompted us to test for potential cooperative interactions between caveolin-1 and c-myc that would be consistent with malignant progression. We used the well-characterized Rat1AmycERT cells to show that the caveolin-1 gene is down-regulated at the level of transcription by c-myc. By maintaining relatively high levels of caveolin-1 with an adenoviral vector or in stably transfected clones we show that caveolin-1 can suppress c-myc-induced apoptosis. Further we established human prostate cancer cell lines with the mycER construct and show that clones with increased caveolin-1 are more resistant to myc-induced apoptosis and have increased capacity for growth in soft agar when c-myc is activated.

Marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340.

Synthetic matrix metalloproteinase (MMP) inhibitors have activity against a variety of tumors in preclinical models but have not been studied in gliomas. We determined the effect of AG3340, a novel synthetic MMP inhibitor with Ki values against gelatinases in the low picomolar range, on the growth of a human malignant glioma cell line (U87) in SCID-NOD mice. Mice were injected s.c. with U87 cells. Tumors were allowed to grow to a size of approximately 0.5 x 0.5 cm (after about 3 weeks), and the mice were randomized to receive either: (a) 100 mg/kg AG3340 in vehicle; or (b) vehicle control (0.5% carboxymethyl cellulose, 0.1% pluronic F68), both given daily i.p. Tumor area was measured twice weekly, and animals were sacrificed when moribund, or earlier if premorbid histology was examined. In vivo inhibition of tumor growth was profound, with AG3340 decreasing tumor size by 78% compared with controls after 31 days (when controls were sacrificed; P < 0.01, Wilcoxon test). Control animals survived 31 days after the i.p. injections began, and AG3340 mice survived 71 days, representing a >2-fold increase in survival associated with tumor growth delay. Histological examination found that AG3340-treated tumors were smaller, had lower rates of proliferation, and significantly less invasion than control-treated tumors. Hepatic or pulmonary metastases were not seen in either group. In a separate experiment, the tumors were smaller and sampled after a shorter duration of treatment; the changes in proliferation were more marked and occurred earlier than differences in tumor invasion between the two groups. Furthermore, in vitro cell growth was not inhibited at AG3340 concentrations of <1 mM. AG3340 plasma concentrations in vivo, 1 h after administration, ranged from 67 to 365 nM. Thus, AG3340 produced a profound inhibition of glioma tumor growth and invasion. AG3340 markedly increased survival in this in vivo glioma model. Treatment with AG3340 may be potentially useful in patients with malignant gliomas.

Localisation, quantitation, and characterisation of neuropeptide F- and FMRFamide-immunoreactive peptides in turbellarians and a monogenean: a comparative study.

Over the past decade it has become clear that the nervous systems of platyhelminths are both complex and highly developed, particularly in peptidergic elements. The central position of an ancestral flatworm in the evolution of the Bilateria has placed a greater importance on the study of modern flatworms. Using antisera generated to the C-terminal region of platyhelminth neuropeptide F and the molluscan neuropeptide, FMRFamide, in immunocytochemistry at both light and ultrastructural levels, immunoreactivities have been localised within the nervous systems of three species of triclad turbellarians, Dugesia lugubris, Dendrocoelum lacteum, and Polycelis nigra, and one species of monogenean trematode, Diclidophora merlangi. Extensive immunostaining was obtained with both antisera throughout the central and peripheral nervous systems of all species studied, but intensity and abundance was significantly greater in the turbellarians. Indirect electron-immunogold labeling demonstrated that immunoreactivity to both neuropeptides was often colocalised in neurosecretory vesicles, although discrete populations of vesicles were also observed. Radioimmunoassay of extracts of all species confirmed that neuropeptide F immunoreactivity was consistently more abundant than FMRFamide immunoreactivity, and that the levels of both in the three turbellarians were several orders of magnitude greater than those found in the monogenean. Chromatographic analyses of turbellarian extracts revealed that neuropeptide F and FMRFamide immunoreactivities were attributable to different peptides. These data imply that the neuropeptidergic systems systems of turbellarians are considerably more extensive than those of monogeneans, and would suggest that a regression has occurred in the latter as a consequence of the adoption of a mere sedentary parasitic lifestyle.

Widespread distribution of histamine in the nervous system of a trematode flatworm.

In general, most flatworms contain very little histamine (HA) and their nervous systems often lack, or contain very few, histaminergic elements. However, preliminary studies in our laboratory have revealed that the frog lung parasite, Haplometra cylindracea (Trematoda: Digenea), contains HA in a very high concentration. For this reason, the present study was undertaken to study the localization and synthesis of HA in this worm by using immunocytochemistry and high-pressure liquid chromatography (HPLC). Essentially all parts of the nervous system of H. cylindracea showed HA-like immunoreactivity. The paired cerebral ganglia and nerves emanating from these, including the longitudinal nerve cords, were intensely immunoreactive. The musculature of the pharynx, oral and ventral suckers, and those of the reproductive organs were all innervated by HA-immunoreactive fibers. Fiber plexuses beneath the tegument and throughout the parenchyma also showed HA-like immunoreactivity. HPLC studies revealed one of the highest HA concentrations in the animal kingdom, 6.49 +/- 1.36 nmole/mg protein, in the worm. The frog lung and blood contained very low concentrations of HA and could be excluded as sources for HA, while an enzyme assay revealed that the worm produces HA by decarboxylation of histidine. Thus, it is likely that H. cylindracea uses HA as a neurotransmitter or modulator.

The peptidergic nervous system of the triclad turbellarian, Bdelloura candida (Maricola, Bdellouridae): an immunocytochemical study using an antiserum raised to an endogenous neuropeptide, GYIRFamide.

The organisation of the nervous system of Bdelloura candida (Tricladida, Maricola, and Bdellouridae) was studied by immunocytochemistry, by using an antiserum raised to the authentic B. candida FMRFamide-related peptide (FaRP), GYIRFamide. Immunostaining was intense and abundant throughout both the central and peripheral nervous systems, being localised to the brain, the longitudinal nerve cords and their transverse and lateral connections, the pharyngeal plexus, the extensive sub-epidermal and sub-muscular plexuses, and elements of the reproductive apparatus. Compared to an earlier anatomical investigation of this species, and also to the neuroanatomy of other triclad turbellarians, the pattern of GYIRFamide-immunoreactivity reveals differences in the following aspects: the shape and structure of the brain, the distribution of longitudinal nerve cords and their relationships with the peripheral nervous system, the structure and distribution of the lateral nerves and the transverse connectives between the longitudinal nerve cords, organisation of the pharyngeal nervous system, and innervation of the eyespots and epidermal sensory structures. Although this study focuses on a descriptive account of the neuroanatomy of Bdelloura candida, by using anti-GYIRFamide as a neuronal marker, the possible functions of the native peptide are also discussed. The quality and reproducibility of the immunostaining obtained during this work highlights the effectiveness of the GYIRFamide antiserum in the neuroanatomical study of flatworms, and also the suitability of B. candida as a model species in studies of the turbellarian nervous system.

Immunocytochemical evidence for the involvement of an FMRFamide-related peptide in egg production in the flatworm parasite Polystoma nearcticum.

The monogenean flatworm Polystoma nearcticum exhibits reproductive synchrony with its treefrog host, Hyla versicolor, and becomes reproductively active only during the short period of host sexual activity at spawning. In this way, it provides a useful model system for exploring factors that may influence egg production in flatworm parasites. One such factor is the peptidergic innervation of the egg chamber or ootype. By using immunocytochemical techniques, the occurrence and distribution of GYIRFamide-like immunoreactivity, an authentic flatworm FMRFamide-related peptide (FaRP), have been monitored in the cells and fibres innervating the reproductive apparatus of worms collected at different stages of host sexual activity. Serotonin (5-HT) immunoreactivity in the worm was mapped for comparison. Extensive immunostaining for the FaRP and 5-HT was obtained throughout both the central and the peripheral nervous systems of worms, which were recovered from reproductively active frogs. In contrast, the innervation of the ootype of worms that were determined to be sexually inactive, including those recovered from frogs postspawning, showed little or no immunoreactivity for the FaRP; immunostaining for 5-HT in the ootype was unaffected by the reproductive state of the worm. These results indicate that FaRP expression in the neurons of the ootype innervation of P. nearcticum coincides with the parasite's brief period of egg production and, thus, provides evidence that regulatory peptides may be involved in the egg-assembly mechanism in flatworm parasites.

Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas.

Malignant gliomas maintain a poor prognosis and survival rate due to their marked local invasive growth and neovascularization. Matrix metalloproteinases (MMPs) have been implicated in glioma invasion and angiogenesis, but it is unknown whether they are produced by the tumor cells or surrounding stroma. Using in situ hybridization and immunohistochemistry, we found expression of mRNA for both gelatinase-A (MMP2) and gelatinase-B (MMP9) localized to tumor cells and vascular structures in glioma sections. Gelatinase-A protein expression was detected most prominently in tumor cells, with very little signal seen in vasculature. Gelatinase-B protein expression was prominent in vascular structures but was also expressed in tumor cells. Our data show that these proteases are produced by glioma cells and vascular structures and suggest that synthetic MMP inhibitors might be useful in this disease.