To assess whether HMGB1 release is specific to Ad-TK (+GCV)-induced GL26 cell-killing or a more general phenomenon, we treated GL26 (glioma), LLc1 (lung carcinoma), GL261 (glioma), or B16-F10 (melanoma) cells in vitro with three different cell-killing modalities currently in use in the clinic or clinical trials for brain tumors: either Ad-TK (+GCV), radiation, or chemotherapy (temozolomide). Here, TKT is linked to melanoma.