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γ₁34.5-deleted HSV-1-expressing human cytomegalovirus IRS1 gene kills human glioblastoma cells as efficiently as wild-type HSV-1 in normoxia or hypoxia.

Friedman GK, Nan L, Haas MC, Kelly VM, Moore BP, Langford CP, Xu H, Han X, Beierle EA, Markert JM, Cassady KA, Gillespie GY - Gene Ther. (2014)

Bottom Line: Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia.Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia.Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Research Program, Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA.

ABSTRACT
Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

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Percentage (mean + SD) of CD133+ GBM-X6 cells infected by ICP34.5- HSV-1, chimeric C154, and wild-type HSV-1 (M2001) at various MOI in normoxia or 1% hypoxia at 30 hours as measured by GFP and APC expression by FACS analysis.
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Figure 1: Percentage (mean + SD) of CD133+ GBM-X6 cells infected by ICP34.5- HSV-1, chimeric C154, and wild-type HSV-1 (M2001) at various MOI in normoxia or 1% hypoxia at 30 hours as measured by GFP and APC expression by FACS analysis.

Mentions: We next compared the abilities of the chimeric virus, the γ134.5-deleted virus and a wild type HSV-1 (M2001) to infect CD133+ GSCs from GBM-X6, the xenoline that was most resistant to the γ134.5-deleted virus, in normoxia and hypoxia. By 30 hours post-infection, the C154 virus infected a significantly greater number of CD133+ cells in both hypoxia and normoxia at each MOI (1, 3.3, and 10) compared to the ICP34.5- virus (Figure 1). At an MOI of 3.3, the chimeric virus infected 4-fold more CD133+ cells than the γ134.5-deleted virus in normoxia and 7-fold more CD133+ cells in hypoxia. Despite GBM-X6 being the most resistant xenoline, C154 was able to infect over half (55.9 ± 4.6%) of the CD133+ cells in hypoxia at 10 MOI within 30 hours compared to only 8.2 ± 0.9% for the γ134.5-deleted virus. At a low MOI of 1, the chimeric virus infected fewer cells than wild-type HSV-1; however at an MOI of 3.3 and 10, the chimeric virus was as effective at infecting cells as wild type virus. At 10 MOI, C154 infected 46.9 ± 4.6% of CD133+ cells in normoxia, and the wild-type virus infected 44.8 ± 5.1% (p=.62); in hypoxia, C154 infected 55.9 ± 4.6% of CD133+ cells versus 48. 9 ± 3.4% for the wild-type virus (p=.10). Importantly, in all three xenolines, the chimeric virus was able to infect as many CD133+ cells in hypoxia as in normoxia at various MOI (Figure 2). Taken together, these data show that the chimeric HCMV/HSV-1 infects CD133+ GSCs similar to wild-type HSV-1 except at low MOI. Furthermore, the chimeric virus is not limited to the same degree as the γ134.5-deleted virus in hypoxia, and its ability to infect GSCs is not limited by hypoxia like the γ134.5-deleted virus.


γ₁34.5-deleted HSV-1-expressing human cytomegalovirus IRS1 gene kills human glioblastoma cells as efficiently as wild-type HSV-1 in normoxia or hypoxia.

Friedman GK, Nan L, Haas MC, Kelly VM, Moore BP, Langford CP, Xu H, Han X, Beierle EA, Markert JM, Cassady KA, Gillespie GY - Gene Ther. (2014)

Percentage (mean + SD) of CD133+ GBM-X6 cells infected by ICP34.5- HSV-1, chimeric C154, and wild-type HSV-1 (M2001) at various MOI in normoxia or 1% hypoxia at 30 hours as measured by GFP and APC expression by FACS analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383690&req=5

Figure 1: Percentage (mean + SD) of CD133+ GBM-X6 cells infected by ICP34.5- HSV-1, chimeric C154, and wild-type HSV-1 (M2001) at various MOI in normoxia or 1% hypoxia at 30 hours as measured by GFP and APC expression by FACS analysis.
Mentions: We next compared the abilities of the chimeric virus, the γ134.5-deleted virus and a wild type HSV-1 (M2001) to infect CD133+ GSCs from GBM-X6, the xenoline that was most resistant to the γ134.5-deleted virus, in normoxia and hypoxia. By 30 hours post-infection, the C154 virus infected a significantly greater number of CD133+ cells in both hypoxia and normoxia at each MOI (1, 3.3, and 10) compared to the ICP34.5- virus (Figure 1). At an MOI of 3.3, the chimeric virus infected 4-fold more CD133+ cells than the γ134.5-deleted virus in normoxia and 7-fold more CD133+ cells in hypoxia. Despite GBM-X6 being the most resistant xenoline, C154 was able to infect over half (55.9 ± 4.6%) of the CD133+ cells in hypoxia at 10 MOI within 30 hours compared to only 8.2 ± 0.9% for the γ134.5-deleted virus. At a low MOI of 1, the chimeric virus infected fewer cells than wild-type HSV-1; however at an MOI of 3.3 and 10, the chimeric virus was as effective at infecting cells as wild type virus. At 10 MOI, C154 infected 46.9 ± 4.6% of CD133+ cells in normoxia, and the wild-type virus infected 44.8 ± 5.1% (p=.62); in hypoxia, C154 infected 55.9 ± 4.6% of CD133+ cells versus 48. 9 ± 3.4% for the wild-type virus (p=.10). Importantly, in all three xenolines, the chimeric virus was able to infect as many CD133+ cells in hypoxia as in normoxia at various MOI (Figure 2). Taken together, these data show that the chimeric HCMV/HSV-1 infects CD133+ GSCs similar to wild-type HSV-1 except at low MOI. Furthermore, the chimeric virus is not limited to the same degree as the γ134.5-deleted virus in hypoxia, and its ability to infect GSCs is not limited by hypoxia like the γ134.5-deleted virus.

Bottom Line: Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia.Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia.Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Research Program, Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA.

ABSTRACT
Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Show MeSH
Related in: MedlinePlus