Augmentation of melanoma-specific gene expression using a tandem melanocyte-specific enhancer results in increased cytotoxicity of the purine nucleoside phosphorylase gene in melanoma.
The lineage-specific human tyrosinase promoter has been used to successfully target gene expression at the transcriptional level to melanoma cells. The tyrosinase promoter, alone and in combination with a single, or a dual, tandem melanocyte-specific enhancer, was used to regulate expression of the firefly luciferase reporter gene. Transient transfections of these tissue-specific luciferase constructs in human and murine melanoma (Pmel, B16mel) and colon carcinoma (WiDr, MC38) cell lines resulted in melanoma-specific luciferase expression that was amplified 5- and 500-fold with the addition of a single or double enhancer, respectively, to the tyrosinase promoter. When the double enhancer-promoter construct expressed the highly toxic Escherichia coli purine nucleoside phosphorylase (PNP) gene, transfection of the same cell lines followed by administration of the prodrug 6-methyl purine deoxyriboside (6-MPDR) at a concentration of 50 microM caused melanoma-specific in vitro cell killing. Within 5 days after prodrug administration methylthiazol-tetrazolium (MTT) cytotoxicity assays showed that only 15 and 9% of Pmel and B16mel cells, respectively, remained viable compared with controls. This effect was highly specific, as 90 and 96% of WiDr and MC38 colon carcinoma cells remained viable 5 days after identical treatment. This effect was a direct result of increased tissue-specific conversion of 6-MPDR to the toxic metabolite 6-methylpurine (6-MP), as documented by HPLC analysis of culture supernatants. These results show that the dual tandem melanocyte-specific enhancer provides powerful amplification of the transcriptional targeting of gene expression afforded by use of the tyrosinase promoter. This amplification translates into increased, highly specific cytotoxicity to melanoma by the PNP/6-MPDR enzyme/prodrug system and, therefore, has potential efficacy in the use of gene therapy for the treatment of metastatic melanoma.
Park
B J
BJ
Metabolism Section, Surgery Branch, Clinical Sciences Division, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Brown
C K
CK
Hu
Y
Y
Alexander
H R
HR
Horti
J
J
Raje
S
S
Figg
W D
WD
Bartlett
D L
DL
D000818:Animals; D001483:Base Sequence; D002470:Cell Survival / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D008546:Melanoma, Experimental / Q000235:genetics* / Q000473:pathology; D051379:Mice; D014442:Monophenol Monooxygenase / Q000235:genetics; D011401:Promoter Regions, Genetic; D011684:Purine Nucleosides / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics*; D015854:Up-Regulation
D017931:DNA Primers; D011684:Purine Nucleosides; C032885:6-methylpurine 2'-deoxyriboside; D014442:Monophenol Monooxygenase; D011683:Purine-Nucleoside Phosphorylase