Abstract
Alternative splicing of the human β-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression ofhumbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbughas been associated with a variety of human cancers, and althoughhumbug 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 andhumbug 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 humbugtranscripts. 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 β-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.
Footnotes
- Abbreviations:
- BAH
- β-aspartyl (asparaginyl) hydroxylase
- kb
- kilobase
- ON
- oligonucleotide
- G3PDH
- glyceraldehydes-3-phosphate dehydrogenase
- H&E
- hematoxylin and eosin
- Received October 8, 2001.
- Accepted December 18, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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