Antisense oligonucleotides selectively regulate aspartyl beta-hydroxylase and its truncated protein isoform in vitro but distribute poorly into A549 tumors in vivo.
Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.
Experimental Station E336, Bristol-Myers Squibb Company, Route 141 and Henry Clay Road, Wilmington, DE 19880-0400, USA. siew.ho@bms.com
Siew Peng
SP
Ho
Michael S
MS
Scully
Candice M
CM
Krauthauser
Eric J
EJ
Wexler
Mark D
MD
Stow
Joseph E
JE
Dinchuk
Janet S
JS
Kerr
Paul A
PA
Friedman
D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008175:Lung Neoplasms; D006899:Mixed Function Oxygenases / Q000235:genetics*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D020319:Oligodeoxyribonucleotides, Antisense; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase