Human osteocalcin: a strong promoter for nitric oxide synthase gene therapy, with specificity for hormone refractory prostate cancer.
BACKGROUND
Gene therapy has been identified as a promising treatment strategy for hormone refractory prostate cancer (HRPC). We report, for the first time, the use of the human osteocalcin (hOC) promoter to control inducible nitric oxide synthase (iNOS) transgene expression in HRPC.


METHODS
Human prostate carcinoma cells (PC3, DU145, LNCaP), colon cancer cells (HT29) and human microvascular endothelial cells (HMEC-1) were transfected in vitro with constitutively driven CMV/iNOS or hOC/iNOS plasmid DNA by cationic lipid vector. End points of these experiments were Western blotting, NO(.) generation using the Greiss test to measure accumulated nitrite, and clonogenic assay.


RESULTS
Transfection of the hOC/iNOS plasmid increased iNOS protein and total nitrite levels in PC3 and DU145 cells, but not LNCaP or HT29. Transfection with CMV/iNOS or hOC/iNOS resulted in no additional cytotoxicity in androgen-dependent LNCaP cells or in the non-prostate cell lines. However, transfection with either construct resulted in a greatly reduced cell survival (to 10-20%) in the androgen-independent PC3 and DU145 cell lines.


CONCLUSIONS
Utilising the tumour-type specific properties of the hOC promoter in tandem with the iNOS gene, we have demonstrated target cell specificity, and transgene activation, in the androgen-independent prostate cancer cell lines (PC3 and DU145), an effect absent in normal and androgen-dependent cells. Furthermore, the levels of NO(.) generated are comparable with those seen generated with constitutively (CMV)-driven iNOS. The data obtained from this study provide a basis for future development of hOC/iNOS gene therapy.
McCarthy
Helen O
HO
School of Pharmacy, McClay Research Centre, Queen's University, Lisburn Road, Belfast, Northern Ireland, UK. h.mccarthy@qub.ac.uk
Coulter
Jonathan A
JA
Worthington
Jenny
J
Robson
Tracy
T
Hirst
David G
DG
D016923:Cell Death; D045744:Cell Line, Tumor; D003587:Cytomegalovirus; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008297:Male; D009376:Neoplasms, Hormone-Dependent / Q000201:enzymology / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D009573:Nitrites / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000235:genetics*; D014162:Transfection
D009573:Nitrites; C485184:enhanced green fluorescent protein; D015675:Osteocalcin; D049452:Green Fluorescent Proteins; D052247:Nitric Oxide Synthase Type II