In vitro model for liposome-mediated adenomatous polyposis coli gene transfer in a duodenal model.
OBJECTIVE
Duodenal adenomas arise in more than 90 percent of patients with familial adenomatous polyposis (FAP). Management of severe duodenal disease remains difficult and controversial. This study investigates transfer of functional wild-type adenomatous polyposis coli (APC) gene under conditions of varying pH and bile concentrations into a somatic duodenal cancer cell line (HUTU-80) as a prelude to in vivo gene therapy in the management of severe duodenal disease.


METHODS
In vitro transfection of human APC gene was performed on a human duodenal adenocarcinoma cell line (HUTU-80) by use of a liposomal vector. Different concentrations (5 percent and 10 percent) of human bile and varying pH (6 percent and 8 percent) were used during APC gene transfer to assess their effects on transfection efficiency. The effect of bile on the proliferation of HUTU-80 cells was evaluated by means of a colorimetric chemosensitivity assay with sulforhodamine B (SRB). The duration of APC transgene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
Exogenous APC transgene was expressed in HUTU-80 cell line for 6 days after transfection. Differing pH did not affect APC gene transfer into the duodenal cell line with similar transgene expression to controls, but APC transfection efficiency was reduced semiquantitatively in the presence of bile. Coculturing with human bile (5 percent and 10 percent) did not affect the proliferation of HUTU-80.


CONCLUSIONS
This study demonstrates transfer of APC gene into a duodenal epithelial cell line with prolonged transgene expression. Liposome-mediated APC gene transfer to the duodenum is feasible even in the presence of bile and varying pH, raising the potential of future gene therapy for this extremely difficult to treat condition.
Lee
Jack
J
Cancer Research United Kingdom, Colorectal Cancer Unit, St. Mark's Hospital, Middlesex, Harrow, United Kingdom.
Hargest
Rachel
R
Wasan
Harpreet
H
Phillips
Robin K S
RK
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D001646:Bile; D003470:Culture Media; D004379:Duodenal Neoplasms / Q000235:genetics* / Q000473:pathology*; D004386:Duodenum / Q000166:cytology / Q000473:pathology*; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D017491:Genes, APC; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D003470:Culture Media; D008081:Liposomes