brivudine and Colorectal-Neoplasms

Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have been reported to exert suicide gene effects in combined gene/chemotherapy of cancer. Here, we aimed to further evaluate the capacity of the mutanted enzyme and its potential for inhibiting cancer cell growth.. We altered the sequence of the last 10 amino acids of Dm-dNK to perform site-directed mutagenesis and constructed active site mutanted Dm-dNK (Dm-dNKmut), RT-PCR and western bloting studies were used to reveal the expression of lentivirus mediated Dm-dNKmut in a breast cancer cell line (Bcap37), a gastric cancer cell line (SGC7901) and a colorectal cancer cell line (CCL187). [3H]-labeled substrates were used for enzyme activity assays, cell cytotoxicity was assessed by MTT assays, cell proliferation using a hemocytometer and apoptosis induction by thenannexin-V-FITC labeled FACS method. In vivo, an animal study was set out in which BALB/C nude mice bearing tumors were treated with lentivirus mediated expression of Dm-dNKmut with the pyrimidine nucleoside analog brivudine (BVDU, (E)-5-(2-bromovinyl)-(2-deoxyuridine).. The Dm-dNKmut could be stably expressed in the cancer cell lines and retained its enzymatic activity. Moreover, the cells expressing Dm-dNKmut exhibited increased sensitivity in combination with BVDU, with induction of apoptosis in vitro and in vivo.. These findings underlined the importance of BVDU phosphorylated by Dm-dNKmut in transduced cancer cells and the potential role of Dm-dNKmut as a suicide gene, thus providing the basis for future intensive research for cancer therapy.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Bromodeoxyuridine; Cell Proliferation; Colorectal Neoplasms; Combined Modality Therapy; Drosophila melanogaster; Female; Flow Cytometry; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutant Proteins; Mutation; Phosphotransferases (Alcohol Group Acceptor); Prohibitins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Stomach Neoplasms; Tumor Cells, Cultured

Topics: Adverse Drug Reaction Reporting Systems; Aged, 80 and over; Antimetabolites, Antineoplastic; Antiviral Agents; Bromodeoxyuridine; Capecitabine; Colorectal Neoplasms; Deoxycytidine; Drug Interactions; Drug-Related Side Effects and Adverse Reactions; Fatal Outcome; Female; Fluorouracil; Herpes Zoster; Humans; Switzerland

Herpesvirus saimiri (HVS) has the capacity to incorporate large amounts of heterologous DNA and can infect many different human cell types. To develop its potential as a gene therapy vector, we cloned herpes simplex virus thymidine kinase (TK) gene into the HVS genome in the form of an enhanced green fluorescent protein (EGFP) fusion protein, using a cosmid-based approach. At multiplicity of infection = 100 over 90% of human leukemic K562 and Jurkat cells were transduced with HVS/EGFP-TK. Conditions of no selective pressure expression were maintained at > 92% per cell division. Expression of the EGFP-TK fusion protein rendered transfected leukaemic cells sensitive to cytotoxic treatment with the prodrugs ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2'deoxyuridine (BVDU) at concentrations as low as 10 ng/ml. The viral vector was also screened against a panel of colorectal and pancreatic carcinoma cell lines. All cell lines were transduced but showed a range of sensitivity to infection. Three of the most easily transduced cell lines: Mia PaCa, HCT116 and SW948 transduced with HVS/EGFP-TK were effectively ablated by subsequent treatment with GCV or BVDU. Our results show that in its current form HVS/EGFP-TK could be utilized as an antitumour agent, or it could be developed further by inclusion of a therapeutic gene, with TK presence ensuring a mechanism of controlled removal of modified cells when no longer necessary. These results suggest that HVS/EGFP-TK has a great potential for a number of gene therapy applications.

Topics: Animals; Antiviral Agents; Aotidae; Base Sequence; Bromodeoxyuridine; Cells, Cultured; Colorectal Neoplasms; Ganciclovir; Genetic Vectors; Green Fluorescent Proteins; Humans; Jurkat Cells; K562 Cells; Kidney; Leukemia; Molecular Sequence Data; Pancreatic Neoplasms; Recombinant Fusion Proteins; Simplexvirus; Thymidine Kinase; Transfection