Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes.
OBJECTIVE
To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.


METHODS
Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the "ping-pong" technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.


RESULTS
The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6 x 10(5) cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7 microgram/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4 ng.ml-1 x 10(6) cells-1 x 2 days-1.


CONCLUSIONS
The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.
Guan
J
J
Department of Gynecology and Obstetrics, People's Hospital, Peking University, Beijing 100044, China.
Ma
L
L
Wei
L
L
D016475:3T3 Cells; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D024201:Bystander Effect; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013937:Thymidine Kinase; D015774:Ganciclovir