Inactivation of the PTCH tumor suppressor gene occurs in a subset of sporadic medulloblastomas, suggesting that alterations in the PTCH pathway may be important in the development of this tumor. In order to address the frequency of genetic alterations affecting genes in this pathway, we used a combination of loss of heterozygosity (LOH) analysis, single‐stranded conformational polymorphism (SSCP) analysis, and direct sequencing of DNA samples from sporadic primitive neuroectodermal tumors (PNETs). To identify alterations in the PTCH gene, we performed LOH analysis on 37 tumor DNA samples. Of those with matched constitutional DNA samples, one demonstrated LOH. Of those without matched constitutional DNA, six were homozygous with all markers. All exons of the PTCH gene were sequenced in these seven tumors, and three mutations were found. To identify alterations in the SHH and SMO genes, we analyzed all exons of both genes in 24 tumors with SSCP and sequenced any exons that showed aberrant band patterns. No mutations were found in either SHH or SMO in any tumor. We also identified the following genes as candidate tumor suppressors based on their roles in controlling hh/ptc signaling in Drosophila: EN‐1 and EN‐2, deletion of which results in a lack of cerebellar development in mice; SMAD family members 1–7, and protein kinase A subunits RIα, RIβ, RIIβ, Cα, and Cβ. Each of these genes was investigated in a panel of 24 matched constitutional and tumor DNA samples. Our search revealed no mutations in any of these genes. Thus, PTCH is the only gene in this complex pathway that is mutated with notable frequency in PNET. Genes Chromosomes Cancer 27:44–51, 2000. © 2000 Wiley‐Liss, Inc.

SummaryThe cleavage of C3 is a critical step for complement (C) activation in the classical and alternative pathways. This reaction is controUed by the regulators of C activation protein family. Membrane cofactor protein (MCP) is a cofactor for the factor I-mediated inactivation of C3b and C4b. As a widely distributed membrane protein, MCP may protect host cells from inadvertent C activation. Human MCP has recently been shown to protect transfected rodent cells from human C-mediated lysis. In this report the relationship of MCP expression to C3b deposition and cytoprotection was examined using NIH/3T3 cells transfected with human MCP and exposed to human serum as a source of C and naturally occurring anti-mouse antibody. MCP inhibited C3b deposition in a dose-dependent fashion and inhibited lysis of the mouse cells expressing it. MCP did not inhibit lysis on bystander cells. These results demonstrate the protective role of MCP, at the cellular level, by an intrinsic mechanism.T he human C system is an independent immune system that can separate self from nonself and function as a potent effector system (1). C components are spontaneously and continuously deposited on self tissue in small amounts (2), and in larger quantities during inflammatory reactions. Membrane cofactor protein (MCP; 1 CD46) and decay-accderating factor (DAF; CD55) are membrane glycoproteins that protect host cells from damage by the C system (reviewed in references 3 and 4). MCP is a cofactor for the factor I-mediated cleavage of C3b and C4b, while DAF accelerates decay dissociation of the C3 and C5 convertases. Both proteins are widely distributed on hematopoietic, endothelial, and epithdial cells. They are also highly expressed on certain malignant cell lines (3-7) and reproductive tissues (8).DAF is a glycolipid-anchored protein and functions intrinsically; that is, it protects only the cell on which it is expressed (9). The same mechanism has been postulated for MCP (3,10,11). Although experiments to date are consistent with this interpretation (11-15), the question has not been directly tested.Oglesby et al. (14), Atkinson et al. (15), and Lublin and Abbreviations used in this paper: DAF, decay-accderating factor; MCP, membrane cofactor protein; NHS, normal human serum.Coyne (13) have used nonhuman cell lines transfected with the human MCP or DAF gene to demonstrate the capacity of these proteins to protect cells from C-mediated damage. Seya et al. (12) have also shown that human T cell lines expressing MCP but no other C regulatory protein became susceptible to lysis by the alternative pathway if the activity of MCP was blocked by a mAb. To further assess the mechanism of the inhibitory effect of MCP on C activation, mouse fibroblasts were transfected with human MCP and then exposed to human serum. Only those cells on which MCP was expressed were protected from lysis by human C. This intrinsic mechanism of cytoprotection by MCP and the quantitative aspects of its activity could be of interest to tumor, transplant, and reproductive imm...

Among the best-characterized genetic alterations in gliomas is the amplification of the epidermal growth factor receptor (EGFR) gene, present in f40% of glioblastoma multiforme, and frequently associated with the EGFRvIII gene rearrangement. We have previously shown that attenuated vaccine strains of measles virus have potent antitumor activity against gliomas, and identified H protein mutations, which ablate recognition of the natural measles virus receptors CD46 and SLAM. Retargeted recombinant viruses were generated from the measles Edmonston-NSe vaccine strain displaying a singlechain antibody against EGFRvIII at the COOH terminus of H and containing the marker green fluorescent protein (GFP) gene in position 1. Two different H mutants were employed: H SNS (V451S, Y481N, and A527S)-CD46 blind, and H AA (Y481A and R533A)-CD46 and SLAM blind. MV-GFP virus was used as a positive control. Both EGFRvIII-retargeted viruses had significant antitumor activity against EGFRvIII-expressing glioblastoma multiforme but no cytopathic effect against normal cells. In an orthotopic model of EGFRvIII-expressing GBM39 xenografts, there was comparable therapeutic efficacy between retargeted strains and unmodified MV-GFP and statistically significant prolongation of survival in treated animals compared with the control group (P = 0.001). Formation of syncytia was observed in tumors treated with retargeted viruses, with a surrounding infiltrate consisting of macrophages and natural killer cells. In summary, EGFRvIIIretargeted oncolytic measles virus strains have comparable therapeutic efficacy with the unmodified MV-GFP strain against EGFRvIII-expressing glioma lines and xenografts with improved therapeutic index, a finding with potential translational implications in glioma virotherapy. (Cancer Res 2006; 66(24): 11840-50)

A retargeted measles virus strain MV-GFP-H(AA)-scEGFR was generated by engineering the MV-NSe Edmonston vaccine strain to incorporate both CD46 (Y481A) and signaling lymphocyte activation molecule (SLAM) (R533A) ablating mutations in the hemagglutinin protein in combination with the display of a single-chain antibody against epidermal growth factor receptor (EGFR) at the C terminus of hemagglutinin. The unmodified MV-GFP virus was used as a positive control. Specificity of the EGFR retargeted virus was demonstrated in non-permissive Chinese hamster ovary (CHO) cells stably transfected to express either the natural receptors CD46 or SLAM or the target receptors EGFR and EGFRvIII. In vitro, the retargeted virus had potent antitumor activity against EGFR- or EGFRvIII-overexpressing primary glioblastoma multi-forme (GBM) cell lines that was comparable to the activity of the unmodified MV-GFP virus. Intratumoral administration of MV-GFP-H(AA)-scEGFRvIII in orthotopic GBM12 xenografts resulted in tumor regression, as demonstrated by bioluminescence imaging and significant prolongation of survival, that was comparable to the effect of the unmodified strain. In contrast to MV-GFP, central nervous system administration of the targeted MV-GFP-H(AA)-scEGFR virus in measles replication-permissive Ifnar(ko) CD46 transgenic mice resulted in no neurotoxicity. In conclusion, EGFR-retargeted measles virus strains have comparable therapeutic efficacy to the unmodified virus in glioma cells overexpressing EGFR or EGFRvIII in vivo and in vitro, and improved therapeutic index, a finding with potential translational implications in glioma virotherapy.

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 (125I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of 123I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 × 106 TCID50) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of 131I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by 131I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.