Treatment of colon cancer cells using the cytosine deaminase/5-fluorocytosine suicide system induces apoptosis, modulation of the proteome, and Hsp90β phosphorylation

Abstract

The bacterial cytosine deaminase (CD) gene, associated with the 5-fluorocytosine (5FC) prodrug, is one of the most widely used suicide systems in gene therapy. Introduction of the CD gene within a tumor induces, after 5FC treatment of the animal, a local production of 5-fluorouracil resulting in intratumor chemotherapy. Destruction of the gene-modified tumor is then followed by the triggering of an antitumor immune reaction resulting in the regression of distant wild-type metastasis. The global effects of 5FC on colorectal adenocarcinoma cells expressing the CD gene were analyzed using the proteomic method. Application of 5FC induced apoptosis and 19 proteins showed a significant change in 5FC-treated cells compared with control cells. The up-regulated and downregulated proteins include cytoskeletal proteins, chaperones, and proteins involved in protein synthesis, the antioxidative network, and detoxification. Most of these proteins are involved in resistance to anticancer drugs and resistance to apoptosis. In addition, we show that the heat shock protein Hsp90B is phosphorylated on serine 254 upon 5FC treatment. Our results suggest that activation of Hsp90B by phosphorylation might contribute to tumor regression and tumor immunogenicity. Our findings bring new insights into the mechanism of the anticancer effects induced by CD/5FC treatment. [Mol Cancer Ther 2007; 6(10):2747 -56]

Radiosensitization and Anti-tumour Effects of Cytosine Deaminase and Thymidine Kinase Fusion Suicide Gene in Human Adenoid Cystic Carcinoma Cells

“…The 5-FUTP is incorporated into RNA and interferes with RNA processing, whereas 5-FdUMP irreversibly inhibits thymidylate synthase and hence DNA synthesis. 7,8 Tumour cells expressing viral HSV-TK are able initially to phosphorylate ganciclovir, leading to accumulation of its cytotoxic metabolite, ganciclovir triphosphate, and subsequent incorporation into DNA and apoptosis. 9 -11 In addition to direct cytotoxicity to the transduced cells, it has been demonstrated that significant toxicity from these two converted pro-drugs can be transmitted to adjacent cells, a process known as the 'bystander' effect -the ability of suicide enzyme-expressing cells to sensitize neighbouring non-expressing bystander cells to the pro-drug.…”

“…As stated above, the cytotoxic effect of the CD/5-FC combination is mediated predominantly through inhibition of thymidylate synthase by 5-FdUMP. 7,8 This results in a redistribution of cells in early S phase, 24 as well as DNA strand breakage due to the disruption of deoxynucleotide pools. 25 The cytotoxicity of the HSV-TK/ganciclovir system results from inhibition of DNA polymerases by ganciclovir monophosphate, which leads to disruption of DNA synthesis.…”

Proteomics, a new tool to monitor cancer therapy?

“…A proteomic approach with 2-DE also allowed new insights into the mechanism of the most widely used suicide system in gene therapy [22]. Colon carcinoma cells were treated with the bacterial cytosine deaminase gene (CD) and 5-fl uorocytosine prodrug (5FC).…”

Enhancement of Anti-tumor Activity of Newcastle Disease Virus by the Synergistic Effect of Cytosine Deaminase

“…The cytosine deaminase (CD) gene product convert the prodrug 5-flucytosine (5-FC) into cytotoxic substance 5-fluorouracil (5-FU), which has anti-tumor activity (Topf et al, 1998;Pierrefite-Carle et al, 1999;Negroni et al, 2007). The CD/5-FC system demonstrates stronger bystander effect and the gap-junctional intercellular communications than other suicide gene system, which results in strong inhibition of tumor growth (Khatri et al, 2006;Stolworty et al, 2008;Dachs et al, 2009).…”