Some reovirus stuff...from the AACR 2000 abstracts...Best of Luck, Where'd He Go?
BTW...a link to the Oncolytics thread...
Subject 31728
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Publishing ID: 2224
Reovirus as a Potential Anti-Breast Cancer Therapeutic
Kara L Norman, Matt C Coffey, Kensuke Hirasawa, Douglas J Demetrick, James E Strong, Patrick W K Lee, Univ of Calgary, Calgary, AB, Canada; Oncolytics Biotech Inc, Calgary, AB, Canada.
Previously we have shown that the human reovirus that is restricted to replicate in those cells with an activated ras pathway could be used as an effective oncolytic agent against human glioblastoma xenographs in a SCID mouse model. Here we present evidence that reovirus could potentially be used as an oncolytic agent against mammary tumors. Although ras mutations are infrequent in the etiology of breast cancer, aberrant Ras/MAPK signaling via upstream signal elements is common. We examined five breast cancer cell lines; MDA-MB-468, MCF7, MDA-MB-435, T47D, and SK-BR-3 as well as a cell line derived from normal breast tissue, HBL-100 for in vitro reovirus replication. All five of the tumor derived cell lines were infectable by reovirus while the HBL-100 was unable to effectively replicate the virus. To determine if the Ras pathway was indeed activated in these cell lines the level of MAPK phosphorylation was assessed in the presence and absence of serum. Those cell lines that were infectable exhibited constitutive MAPK phosphorylation even in the absence of mitogen indicating ras pathway activation. To determine if reovirus could be used as an oncolytic agent in vivo against breast tumors, SCID mice were implanted with MDA-MB-468 human tumor xenographs. Following the establishment of palpable tumors the mice were treated with a single injection of reovirus and tumor size was monitored for a four-week period. The single injection resulted in dramatic regression of the tumor size. Finally the ability of reovirus to act against primary breast cancer tumors was determined.
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Publishing ID: 2223
STUDY OF REOVIRUS-INDUCED CYTOPATHIC EFFECT (CPE), RAS AND DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE (PKR) IN HUMAN TUMOR CELL LINES.
L-T Song, I Gelman, J F Holland, T Ohuma, Mount Sinai Sch of Medicine, New York, NY.
Reovirus was reported to selectively kill tumor cells with an activated ras pathway (Science 282: 1332, 1998). We studied the relationship between reovirus-induced CPE and the ras and PKR protein levels in 8 human tumor cell lines. "Normal" NIH/3T3 and LLC-MK2 cell lines served as controls. Reovirus infection with MOI of 10 pfu/cell caused CPE on day 3 in all tumor cell lines and LLC-MK2, in decreasing order, (4+) MCF-7 breast cancer = U-87MGdEGFR glioblastoma subline expressing mutated EGFR = U-87MGwtEGFR > U-87MG glioblastoma = LLC-MK2 > HEp-2 laryngeal carcinoma > DND-1A melanoma = 2780 ovarian carcinoma = JAR choriocarcinoma (1+). Presence of viral RNA in the infected cells on day 4 detected by PAGE in combination with silver staining confirmed that CPE was the result of viral infection. The degree of CPE was correlated with the ras protein levels assayed with pan-ras antibody; in decreasing order, (3+) MCF-7 = U-87dEGFR = LLC-MK2 > U-87wtEGFR = U-87 = HEp-2 > DND-1A = 2780 = JAR (1+). Phosphorylated PKR protein levels measured by Western blot using 71/10 anti-PKR antibody did not correlate with the degree of CPE or Ras protein levels, however. NIH/3T3 cells had weakly positive ras protein and low PKR levels but absent CPE; no viral RNA was detected. These results confirm that ras levels are important for reovirus-induced CPE. Pre-infection PKR levels are not a determinant for CPE. The mechanism of reovirus resistance in NIH/3T3 cells was not explainable by Ras or PKR levels.
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Publishing ID: 4654
The Efficacy of Using Reovirus as a Treatment for Malignant Gliomas
Mary E Wilcox, Zhong Shi, Barry N Rewcastle, Mathew Coffey, James E Strong, Patrick Wk Lee, Peter A Forsyth, Univ of Calgary, Calgary, AB, Canada.
Background: Other potential viral therapies in gliomas have included herpes virus TK susceptible vectors and adenoviruses which selectively infect p53 deficient tumors. We have found that reovirus, a double stranded RNA virus, selectively infects ras-activated but not normal cells. Ras-activated cells dephosphorylate PKR and release a translational block (normally seen in normal cells) and viral protein synthesis subsequently ensues. Malignant gliomas are ideal candidate tumors because they commonly have ras-activation. We determined the effect of reovirus infection on the growth of malignant glioma cell lines: U87 and 9L both in subcutaneous flank models and intracerebrally. Results: 24 cell lines were tested in vitro for ras pathway activation and viral infection. A strong positive correlation was found for reovirus infection and MAPK actovation. In vivo (subcutaneous flank model) a striking inhibition of tumor growth, including complete tumor regression was seen (p=0.0001 ANOVA); only microscopic foci of residual tumor were found in most animals. Further, remote administration of reovirus infected and killed contra-lateral tumors (distant from injection site). Immunohistochemistry showed viral infection in both un-injected and injected tumors whereas it was absent from all other tissues. In immune competent animal models, antibodies to reovirus had no effect on the reproduction of previous results. In an intracerebral model, reovirus was found to dramatically reduce tumor burden. Further studies however are required to determine appropriate dosing regimen. Conclusions: Reovirus produced an inhibition and regression of glioma growth and warrants further investigation to be present in an intracerebral location.
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Publishing ID: 777
Reovirus as a novel prostate cancer therapeutic
Michael Nodwell, Chandini Thirukkumaran, Joanne Leuder, Sandra Nishikawa, Peter A Forsyth, Randall Johnston, Patrick Wk Lee, Don Gordon Morris, Univ of Calgary, Calgary, AB, Canada.
Intro: Reovirus is a common isolate of human respiratory and gastro- intestinal tracts, that is not associated with significant human disease. We have recently reported that reovirus susceptibility parallels "ras" activation (or up/downstream elements) in a myriad of cell lines (Science 282,1332-1334, 1998). This study investigates reovirus as a potential prostate cancer therapeutic. Methods: The prostate carcinoma cell lines DU-145 and PC-3 were incubated with live and U.V. inactivated (control) reovirus (strain Dearing, serotype 3) at a multiplicity of infection of 40 for up to 72 hours. Apoptosis was measured via CPE, flow cytometry (annexin V abs), PARP cleavage products via western blotting and DNA laddering. Both cell lines were grown in SCID/NOD mice as a xenograft hind flank model by s.q. injection of 107 cells, letting the tumors grow to approx. 0.5 cm2 and then injecting reovirus (108 PFU) intralesionally. Results: Both DU-145 and PC-3 were found to be exquistly sensitive to reovirus with over 80% of the cells dead by 72 hrs. To confirm that apoptosis was a major mode of cell death, Annexin V staining increased to reach 48% and 47% of DU-145 and PC-3 cells, respectively over the same time period. Further, the caspase-dependent cleavage of poly(ADP-ribose) polymerase via western blots paralleled the annexin V results. After a single intratumoral injection of reovirus, both DU-145 (N=20) and PC-3 (N=12) tumor containing mice significantly regressed compared to U.V. inactivated reovirus controls which eventually needed to be euthanized. Conclusions: Unmanipulated reovirus is cytopathic to DU-145 and PC-3 prostate cancer cell lines in vitro and in vivo. CPE, flow cytometry and PARP caspase cleavage products confirm that apoptosis is the major mechanism of cell death.
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