Cytotoxicity, apoptosis, and viral replication in tumor cells treated withoncolytic ribonucleotide reductase-defective herpes simplex type 1 virus (hrR3) combined with ionizing radiation

The viral ribonucleotide reductase (rR)-defective herpes simplex type-1 (HS V-1) virus (hrR3) has been shown previously to preferentially replicate in and kill tumor cells. This selectivity is associated with tumor cell up-reg ulation of mammalian rR. Ionizing radiation (IR) is currently used in the t herapy of many malignancies, including glioblastoma, cervical carcinoma, an d pancreatic carcinoma. IR has been shown to up-regulate mammalian rR in tu mor cells and appears to increase the efficacy of at least one non-rR-delet ed HSV-1 strain in an in vivo tumor model. Here, we test the hypothesis tha t a single therapeutic radiation fraction will increase the replication and toxicity of hrR3 for malignant cell lines in vitro. PANC-1 pancreatic carc inoma, U-87 glioblastoma, and CaSki cervical carcinoma cell lines were trea ted with varying doses of IR and subsequently infected with hrR3 or KOS (wi ld-type HSV-1 strain). Cell survival was then measured using the 3-(4,5-dim ethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and trypan blue e xclusion cytometry. At 72 hours posttreatment, irradiation with 2 Gy reduce d survival from 100% to 76% in noninfected cells, from 61% to 48% in KOS-in fected cells, and from 39% to 27% in hrR3-infected PANC-1 cells. As such, a nalysis of variance indicated that the toxicity of the two modalities was a dditive. Similar additivity was seen in U-87 MG and CaSki cells. Absolute s urvival of hrR3-infected or KOS-infected PANC-1 cells decreased as a functi on of time after treatment (24-72 hours) and multiplicity of infection (MOI ) (0.05-5.0). However, the relative decrease in survival with the addition of IR to hrR3 or KOS in PANC-1 cells was not markedly affected by altering MOI (0.05-5.0), time (24-72 hours), radiation dose (2-20 Gy), or cell cultu re conditions (confluent/growth arrested). We used fluorescence-activated c ell sorter analysis with the cationic lipophilic dye DiOC(6) to quantify a reduction in mitochondrial membrane potential that is associated with apopt osis. Fluorescence-activated cell sorter analysis indicated increased apopt osis in both hrR3- and IR-treated cells at 48-72 hours, with hrR3 alone pro ducing the most induction. Viral yields from PANC-1 cells after irradiation and infection were examined. No significant differences were seen between irradiated and nonirradiated cells in viral replication, with hrR3 producin g single-step titers of 3.1 +/- 0.9 x 10(5) and 4.0 +/- 1.2 x 10(5) plaque- forming units/mL in nonirradiated and irradiated cells. Thus, complementary toxicity was seen between IR and hrR3 or KOS, regardless of cell type, tim e, MOI, IR dose, or culture conditions, without evidence of augmented apopt osis or viral replication.