Tyrosinase mutants are capable of prodrug activation in transfected nonmelanotic cells.
Tyrosinase has been suggested as a prodrug-converting enzyme for the treatment of melanoma. We hypothesized that tyrosinase expression in transfected nonmelanotic cells can be used in a gene therapy paradigm of prodrug activation. To verify our hypothesis, we used the following tyrosinase variants: (a) a full-length human tyrosinase clone (T); (b) a mutant lacking the COOH-terminal cytoplasmic domain (TdeltaC); (c) a mutant lacking the COOH-terminal transmembrane and cytoplasmic domains (TdeltaTC); and (d) a fusion with the eight COOH-terminal amino acids of lysosome-associated membrane protein-1 (TL). Expression of mutant and wild-type tyrosinases was induced by transfection in nontumorigenic human cells of epithelial origin (293HEK, MCF-10A adenoma, and NHDF-Ad human dermal fibroblasts) as well as in tumor cells (9L gliosarcoma, MCF7 adenocarcinoma, and HT-1080 fibrosarcoma). When compared with the wild-type tyrosinase transfectants, truncated mutant expression resulted in higher mRNA levels that paralleled higher enzyme activity of the truncated mutants. Two model tyrosinase prodrugs, hydroxyphenyl-propanol (HPP) and N-acetyl-4-S-cysteaminylphenol (NAcSCAP) inhibited proliferation and caused cell death of transfected cells in a dose-dependent manner. Effects of prodrug treatment were compared for tumorigenic cells and their nontumorigenic counterparts. Two truncated mutants (TdeltaC and TdeltaTC) showed low endogenous cytotoxicity and efficiently suppressed proliferation and induced cytotoxicity in transfected tumor cells in the presence of NAcSCAP. Overall, these results indicate that the developed tyrosinase mutants hold promise as prodrug activation systems for tumoral gene therapy.
Simonova
M
M
Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown 02129-2060, USA.
Wall
A
A
Weissleder
R
R
Bogdanov
A
A
D000230:Adenocarcinoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D001711:Biotransformation; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D005354:Fibrosarcoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D018316:Gliosarcoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008543:Melanins / Q000096:biosynthesis; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010636:Phenols / Q000493:pharmacokinetics / Q000494:pharmacology; D011355:Prodrugs / Q000493:pharmacokinetics*; D020005:Propanols / Q000493:pharmacokinetics / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D008543:Melanins; D010636:Phenols; D011355:Prodrugs; D020005:Propanols; D012333:RNA, Messenger; C417567:3-(4-hydroxyphenyl)propanol; D003543:Cysteamine; C066420:N-acetyl-4-S-cysteaminylphenol; D014442:Monophenol Monooxygenase