Radiosensitization of rat glioma with bromodeoxycytidine and adenovirus expressing herpes simplex virus-thymidine kinase delivered by slow, rate-controlled positive pressure infusion

Abstract

Infection of rat RT2 glioma cells in vitro with an adenovirus (ADV-TK) expressing herpes simplex virus (HSV) thymidine kinase (TK) and subsequent exposure to 5-bromo-2Ј-deoxycytidine (BrdC), which is specifically incorporated into ADV-TK-infected cell DNA as 5-bromo-2Ј-deoxyuridine (BrdU), results in significant radiosensitization (sensitizer enhancement ratio: 1.4 -2.3) compared with Ad␤gal-infected cells. Cell killing correlated well with increased BrdU DNA incorporation and with apoptosis. Whereas radiation (4 Gy) alone was relatively ineffective in inducing apoptosis, treatment with HSV-TK/BrdC resulted in BrdC dose-(10 -100 M) and time-dependent (24 -48 hours) increases, and the combination of the two treatments produced a synergistic response (1.5-to 2-fold). To investigate the effects of the ADV-TK/BrdC treatment in vivo, RT2 cells were grown as soft tissue tumors in Fischer 344 rats and conditions for virus infusion were optimized by altering the volume and rate of infusion using a rate-controlled positive pressure device. We found that relatively large volumes (100 -150 L) of virus delivered at rates of Յ1 L/minute were optimal and gave uniform and reproducible results. Using these optimal infusion conditions, we were able to achieve 40% adenovirus infection in the tumor. Infection of RT2 tumors with ADV-TK and continuous administration of BrdC from an osmotic pump resulted in significant (.001 Ͻ P Ͻ .009) tumor regression 6 days after radiation (30 Gy delivered as 2 ϫ 5 Gy over 3 days) compared with controls. In situ staining of sectioned tumors with anti-BrdU antibody or by high-performance liquid chromatography analysis of extracted and hydrolyzed tumor DNA confirmed that we obtained efficient and specific incorporation of BrdU into tumor cells. These results suggest that adenovirus-mediated delivery of HSV-TK in combination with BrdC and radiation can potentially be an efficient combination modality for the treatment of gliomas. Cancer Gene Therapy (2000) 7, 778 -788 Key words: Cancer gene therapy; drug delivery; halogenated pyrimidines; osmotic pump; radiation.T he halogenated pyrimidines (HPs) 5-bromo-2Ј-deoxyuridine (BrdU) and 5-iodo-2Ј-deoxyuridine (IdU) have been studied extensively as radiosensitizers of various mammalian cells in culture. [1][2][3] Preclinical and clinical studies have revealed that both BrdU and IdU are relatively efficient radiosensitizers if incorporated at sufficient levels into DNA. 4,5 However, although some clinical success has been achieved with head and neck tumors, attempts to radiosensitize gliomas with HPs have been disappointing, perhaps due to poor incorporation of the halogenated nucleosides into the DNA of these types of tumor cells. 6 Therefore, improved or alternative approaches are needed to treat this disease. The thymidine kinase (tk) gene of herpes simplex virus (HSV) has been used extensively to sensitize cancer cells to the HSV-TK-specific drug ganciclovir (GCV), and quite promising results with glioma have been obtained using various animal tumor mode...

Transient exposure of carcinoma cells to RAS/MEK inhibitors and UCN-01 causes cell deathin vitroandin vivo

“…A piece of the tumor (f50% by volume) was removed and placed in a 10-cm dish containing 5 mL RPMI on ice. In parallel, the remainder of the tumor was placed in 5 mL Streck Tissue Fixative (Fisher Scientific) in a 50 mL conical tube for fixation; H&E staining of fixed tumor sections was done as described (31). The tumor sample that had been placed in RPMI was minced with a sterile disposable scalpel into the smallest possible pieces and then placed in a sterile disposable flask.…”

Inhibition of the Type III Epidermal Growth Factor Receptor Variant Mutant Receptor by Dominant-Negative EGFR-CD533 Enhances Malignant Glioma Cell Radiosensitivity

“…Replication-incompetent Ad was produced as described previously and successfully used to generate the Ad-EGFR-CD533 vector (21,22). Ad-EGFRvIII was constructed by transferring a SapI-XbaI fragment, spanning the coding sequence of EGFRvIII, into pZeroTGCMV, followed by recombination in bacteria (21,23). The pSL 1180-EGFRM was kindly provided by S. Batra (Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE).…”

“…The pSL 1180-EGFRM was kindly provided by S. Batra (Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE). An Ad expressing the bacterial lacZ reporter gene (AdLacZ) was used as control virus (21,23). Briefly, Ad-LacZ, Ad-EGFRvIII, and Ad-EGFR-CD533 were produced in 293 cells as described previously (21) and purified by double CsCl gradient centrifugation followed by dialysis against 13% glycerol in PBS (21,23).…”

Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus-thymidine kinase in combination with acyclovir

“…4 Infected cells were exposed to Zovirax/ACV (manufactured by Glaxo Wellcome, Chapel Hill, NC) or ganciclovir (GCV) (kindly provided by Syntex, Palo Alto, Calif) added to the medium. Cells were irradiated in medium with a 60 Co ␥ irradiator (Picker Zonegard V4 M60; Picker Interational, Cleveland, Ohio) set at 1 Gy/minute.…”

“…[1][2][3] We demonstrated recently that rat glioma cells are sensitized to radiation both in vitro and in vivo after infection with an adenovirus expressing HSV-TK and exposure to the halogenated pyrimidine bromodeoxycytidine. 4 The clinically used anti-herpetic drug acyclovir (ACV) also radiosensitizes tumor cells in combination with HSV-TK (our unpublished observations). 5,6 However, one limiting factor for efficient clinical use of HSV-TK and ACV is the extent to which HSV-TK can use the lower concentrations of ACV present in sera, which are only in the lower micromolar range.…”