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Abstract
Both p53 and pRb are inactivated in many tumor types. Inactivation can occur due to deletion, mutation or by binding to DNA viral oncoproteins. In the present study, we addressed the question of how inactivation of p53 or pRb affects the sensitivity of human cancer cells to the antifolate thymidylate synthase (TS) inhibitors, such as raltitrexed and nolatrexed by using human colorectal cancer RKO cells as an in vitro model. Functions of wild type p53 and pRb in these cells were disrupted by expression of human papilloma virus (HPV) type 16 oncoproteins E6 and E7, respectively. Cytotoxicity assays showed that inactivation of p53 or pRb conferred resistance to both TS inhibitors, and this enhanced resistance was able to be reversed by removing thymidine from the culture media, suggesting that the thymidine salvage pathway contributes to this augmented resistance. Furthermore, determination of the salvage enzyme thymidine kinase (TK) level and activity revealed that TK levels were higher in cells lacking p53 or pRb. These findings indicate that drugs activated through the thymidine salvage pathway, such as iododeoxyuridine (IdUrd) may selectively kill cells lacking p53 or pRb, due to increased thymidine salvage in these cells.
Although fluoropyrimidines are widely used in human cancer therapy, low response rates and dose-limiting toxicities such as myelosuppression are obstacles to successful chemotherapy with these agents. In order to protect bone marrow cells from fluoropyrimidine toxicity, we expressed a drug-resistant human TS variant, G52S, via retrovirus mediated gene transfer. Cytotoxicity assays and CFU-GM assays showed that expression of hTSG52S in MH3T3 cells and mouse bone marrow cells conferred significant resistance to fluoropyrimidines. In vivo gene transfer studies using a mouse model were also performed. Mice transplanted with marrow infected with hTSG52S showed protection from lethal 5-FU toxicity as compared with mock-infected animals. These data indicate that gene transfer of the human TS mutant G52S into marrow progenitor cells can efficiently protect these cells from fluoropyrimidine cytotoxicity.
