Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus- thymidine kinase in combination with acyclovir Kristoffer Valerie, 1,2 David Brust, 1 Julie Farnsworth, 1,2 Cyrus Amir, 1–3 Mohiuddin M. Taher, 1 Chad Hershey, 1 and Jeffrey Feden 1 1 Department of Radiation Oncology, 2 Massey Cancer Center, and 3 Department of Biostatistics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298. Adenovirus expressing herpes simplex virus-thymidine kinase (HSV-TK) sensitizes internal rat glioma cells to radiation in combination with acyclovir (ACV). However, relatively high concentrations of ACV (10 M) are required to obtain significant radiosensitization. Serum levels rarely reach more than the lower micromolar range, preventing the full use of this genetic approach to radiosensitize cells in vivo. To better use the lower concentrations of ACV available in sera, we constructed an adenovirus expressing a mutant HSV-TK (HSV-TK(75)) isolated for its 20 times greater sensitivity to ACV than wild-type (wt) HSV-TK. We demonstrate that rat RT2 glioma cells infected with adenovirus AdCMV-TK(75) and exposed to either ACV or ganciclovir become more sensitive to lower concentrations (1–3 M) of the drugs compared with cells infected with AdCMV-TK(wt), which expresses wt HSV-TK. Most importantly, the RT2 cells become more sensitive to low doses (2– 4 Gy) of 60 Co radiation than cells infected with an adenovirus expressing wt HSV-TK. This sensitization is accompanied by an increased rate of apoptosis. In summary, we show that infection of rat glioma cells with an adenovirus expressing a mutant HSV-TK sensitizes the cells to low doses of radiation after exposure to ACV at lower concentrations than those required for wt HSV-TK. This finding suggests that this mutant adenovirus may improve the in vivo efficacy of HSV-TK-based cancer gene therapy approaches. Cancer Gene Therapy (2000) 7, 879 – 884 Key words: Apoptosis; cancer gene therapy; Fischer 344 rat; RT2 glioma cells; suicide gene. C ancer gene therapy approaches based on herpes simplex virus-thymidine kinase (HSV-TK) show promise for the treatment of glioma. 1–3 We demon- strated 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 clin- ically used anti-herpetic drug acyclovir (ACV) also radiosensitizes tumor cells in combination with HSV-TK (our unpublished observations). 5,6 However, one limit- ing 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. To determine whether a mutant form of HSV-TK performs better than wild-type (wt) HSV-TK to radio- sensitize cells, we made an adenovirus expressing a mutant form of HSV-TK that is 20 times more sensi- tive to ACV than is wt HSV-TK and tested whether infected rat RT2 glioma cells become more sensitive to radiation when exposed to low concentrations of ACV. 7 We found that cells infected with the mutant HSV- TK(75) adenovirus expressed HSV-TK protein at a level that was similar to that seen for cells infected with the wt HSV-TK adenovirus. However, cells infected with the mutant were significantly more sensitive to ACV in the 1- to 3-M range and, most importantly, these cells also were significantly more sensitive to radiation than were cells expressing wt HSV-TK. MATERIALS AND METHODS Cell culture RT2 cells (a rat glioma cell line derived from Fischer 344 rats 8 ) that form tumors with features typical of human glioblastoma 9 were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum supplemented with penicillin/strepto- mycin. 4 Infected cells were exposed to Zovirax/ACV (manu- factured 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. Cell viability was deter- mined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazo- lium bromide (MTT) assay. 10 Terminal deoxynucleotidyltrans- Received August 6, 1999; accepted November 30, 1999. Address correspondence and reprint requests to Dr. Kristoffer Valerie, Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0058. E-mail address: kvalerie@hsc.vcu.edu Cancer Gene Therapy, Vol 7, No 6, 2000: pp 879–884 879 © 2000 Nature America, Inc. 0929-1903/00/$15.00/+0 www.nature.com/cgt