Potent anticancer effects of lentivirus encoding a Drosophila melanogaster deoxyribonucleoside kinase mutant combined with brivudine.
OBJECTIVE
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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Department of Surgical Oncology, First Affiliated Hospital, Center of Experiment Technology and Medical Research, Lab 1, Cancer Institute, China Medical University, Shenyang, China.
Nian-Qu
NQ
Zhang
Lei
L
Zhao
Shuai
S
Ma
Ming
M
Gu
Xin-Yu
XY
Zheng
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000628:therapy*; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics / Q000628:therapy*; D003131:Combined Modality Therapy; D004331:Drosophila melanogaster / Q000201:enzymology*; D005260:Female; D005434:Flow Cytometry; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000627:therapeutic use; D009154:Mutation / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D000091402:Prohibitins; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013274:Stomach Neoplasms / Q000235:genetics / Q000628:therapy*; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D050505:Mutant Proteins; C000717331:PHB2 protein, human; C000717333:Phb2 protein, mouse; D000091402:Prohibitins; D012333:RNA, Messenger; C020235:brivudine; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D001973:Bromodeoxyuridine