title: Retrovirus-mediated multidrug resistance gene (MDR1) overexpression inhibits chemotherapy-induced toxicity of granulosa cells.
abstract: OBJECTIVE
  To protect granulosa cells from chemotherapy-induced toxicity by retrovirus-mediated multidrug resistance (MDR1) gene transfection.
  
  
  METHODS
  Laboratory study.
  
  
  METHODS
  Academic research laboratory in a university hospital.
  
  
  METHODS
  None.
  
  
  METHODS
  KK15 immortalized murine granulosa cell line transiently transduced with sf91m3 retrovirus vector carrying MDR1 complementary DNA that encodes P-glycoprtoein (P-gp); transduced cells selected with colchicine and treated with doxorubicin or paclitaxel for 24-72 hours; expression and function of MDR1 and the messenger RNA (mRNA) expression of selected steroidogenesis enzymes evaluated by flow cytometry, cell viability assays, Western blot, and reverse-transcriptase polymerase chain reaction (RT-PCR).
  
  
  METHODS
  Viability of sf91m3-transduced KK15 cells after treatment with doxorubicin and paclitaxel.
  
  
  RESULTS
  The sf91m3-transduced KK15 demonstrated high expression of biologically active MDR1, as shown by flow cytometry analysis and immunoblotting using P-gp monoclonal antibody and Rhodamine 123 efflux assays. The sf91m3-transduced KK15 exhibited statistically significant resistance to toxicity of 10 μM paclitaxel. The MDR1-transduced KK15 cells were also protected from doxorubicin toxicity (10 nM to 2.5 μM), as shown by cell viability assay. Both flow cytometry and cell viability assays showed that the protection of KK15 from doxorubicin toxicity was lost at 5 μM of doxorubicin; equivalent to 500 times LD50. The sf91m3-transduced KK15 showed normal mRNA expression of a panel of selected steroidogenesis enzymes.
  
  
  CONCLUSIONS
  Retroviral gene delivery of human MDR1 inhibited chemotherapy-induced granulosa cell toxicity and offered chemoprotection in an in vitro model.
authors.affiliation: Department of Obstetrics and Gynecology, University of Wisconsin, Madison, Wisconsin 53791, USA. salih@wisc.edu
authors.forename: Sana M
authors.initials: SM
authors.lastname: Salih
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006107:Granulosa Cells / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D018411:Spodoptera / Q000235:genetics / Q000821:virology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents