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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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(a) Activated p38 MAPK (p-p38 MAPK) and Hsp27 (p-Hsp27) in GBM-XD456 cells (control) infected for 24 hours with ICP34.5- virus at 10 MOI. At time point 0 hours (virus infection only), a single dose of p38 MAPK inhibitor was added at 10μM. At 2, 4, 6 and 24hrs after the inhibitor was added, cells were infected with ICP34.5- virus for 24 hrs. (b) Virus recovery (mean + SD) in GBM-XD456 cells 24 and 48 hours post-infection with ICP34.5- or chimeric C154 virus in normoxia with (Inhibitor) and without (Control) a single 10μM dose of p38 MAPK inhibitor 24 hours prior to infection. (c) Activated Hsp27 (p-Hsp27) in GBM-XD456 cells 48 hour post-transfection with Hsp27 SiRNA or scrambled siRNA (control).
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Figure 7: (a) Activated p38 MAPK (p-p38 MAPK) and Hsp27 (p-Hsp27) in GBM-XD456 cells (control) infected for 24 hours with ICP34.5- virus at 10 MOI. At time point 0 hours (virus infection only), a single dose of p38 MAPK inhibitor was added at 10μM. At 2, 4, 6 and 24hrs after the inhibitor was added, cells were infected with ICP34.5- virus for 24 hrs. (b) Virus recovery (mean + SD) in GBM-XD456 cells 24 and 48 hours post-infection with ICP34.5- or chimeric C154 virus in normoxia with (Inhibitor) and without (Control) a single 10μM dose of p38 MAPK inhibitor 24 hours prior to infection. (c) Activated Hsp27 (p-Hsp27) in GBM-XD456 cells 48 hour post-transfection with Hsp27 SiRNA or scrambled siRNA (control).

Mentions: Since p38 MAPK signaling was greater in normoxia, we next sought to determine if inhibiting p38 MAPK activation in normoxia could decrease virus yields. GBM-XD456 cells were treated with a single 10μM dose of p38 MAPK inhibitor (SB 203580). At 2, 4, 6 or 24 hours after the inhibitor was added, cells were infected with ICP34.5- virus for 24 hours and p-p38 MAPK and p-Hsp27 were assessed by western blotting. Adding inhibitor 2, 4 or 6 hours prior to infection resulted in decreased p-p38 MAPK signal with the greatest decrease at 6 hours (61% of the control (total p38 MAPK); Figure 7a). By 24 hours the signal intensity of p-p38 MAPK was back to 81% of the control. Hsp27 was inhibited at all time points with greatest inhibition at 24 hours (52% of the control). The results demonstrate that activation of p38 MAPK and Hsp27 was inhibited by a single dose of SB 203580.


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)

(a) Activated p38 MAPK (p-p38 MAPK) and Hsp27 (p-Hsp27) in GBM-XD456 cells (control) infected for 24 hours with ICP34.5- virus at 10 MOI. At time point 0 hours (virus infection only), a single dose of p38 MAPK inhibitor was added at 10μM. At 2, 4, 6 and 24hrs after the inhibitor was added, cells were infected with ICP34.5- virus for 24 hrs. (b) Virus recovery (mean + SD) in GBM-XD456 cells 24 and 48 hours post-infection with ICP34.5- or chimeric C154 virus in normoxia with (Inhibitor) and without (Control) a single 10μM dose of p38 MAPK inhibitor 24 hours prior to infection. (c) Activated Hsp27 (p-Hsp27) in GBM-XD456 cells 48 hour post-transfection with Hsp27 SiRNA or scrambled siRNA (control).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: (a) Activated p38 MAPK (p-p38 MAPK) and Hsp27 (p-Hsp27) in GBM-XD456 cells (control) infected for 24 hours with ICP34.5- virus at 10 MOI. At time point 0 hours (virus infection only), a single dose of p38 MAPK inhibitor was added at 10μM. At 2, 4, 6 and 24hrs after the inhibitor was added, cells were infected with ICP34.5- virus for 24 hrs. (b) Virus recovery (mean + SD) in GBM-XD456 cells 24 and 48 hours post-infection with ICP34.5- or chimeric C154 virus in normoxia with (Inhibitor) and without (Control) a single 10μM dose of p38 MAPK inhibitor 24 hours prior to infection. (c) Activated Hsp27 (p-Hsp27) in GBM-XD456 cells 48 hour post-transfection with Hsp27 SiRNA or scrambled siRNA (control).
Mentions: Since p38 MAPK signaling was greater in normoxia, we next sought to determine if inhibiting p38 MAPK activation in normoxia could decrease virus yields. GBM-XD456 cells were treated with a single 10μM dose of p38 MAPK inhibitor (SB 203580). At 2, 4, 6 or 24 hours after the inhibitor was added, cells were infected with ICP34.5- virus for 24 hours and p-p38 MAPK and p-Hsp27 were assessed by western blotting. Adding inhibitor 2, 4 or 6 hours prior to infection resulted in decreased p-p38 MAPK signal with the greatest decrease at 6 hours (61% of the control (total p38 MAPK); Figure 7a). By 24 hours the signal intensity of p-p38 MAPK was back to 81% of the control. Hsp27 was inhibited at all time points with greatest inhibition at 24 hours (52% of the control). The results demonstrate that activation of p38 MAPK and Hsp27 was inhibited by a single dose of SB 203580.

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