Establishment of real-time polymerase chain reaction method for quantitative analysis of asparagine synthetase expression.
We established a real-time quantitative PCR (RQ-PCR) with which to measure abundance of the asparagine synthetase (AS) mRNA. The level of AS mRNA paralleled AS enzyme activity, as well as the AS protein level detected by Western blotting and by in situ immunostaining. Cytotoxicity tests in vitro showed that the AS mRNA level also synchronized with cellular resistance to L-asparaginase in cell lines. Cellular levels of AS enzyme activity correlated with resistance to L-asparaginase. These results indicate that the AS mRNA level is an index of resistance to L-asparaginase. RQ-PCR is superior to enzyme assays, Western blotting, and immunostaining in the following ways: less labor and time, accurate and reproducible quantitativity, and broad dynamic range. In addition, RQ-PCR could evaluate differences in L-asparaginase sensitivity although immunostaining could not. And in clinical samples, we analyzed eight pediatric leukemia cases by this RQ-PCR to evaluate whether this method was applicable to clinical laboratories and the expression level of AS mRNA in each case were predictable for the effectiveness of L-asparaginase treatment. Consequently, this method was useful enough in defining candidates for selective therapy that targets an AS deficiency.
Department of Pediatrics, Shiga Medical Center for Children, Moriyama 524-0022, Japan.
Tamotsu
T
Irino
Toshiyuki
T
Kitoh
Kenichi
K
Koami
Terumi
T
Kashima
Kouichi
K
Mukai
Eiji
E
Takeuchi
Teruaki
T
Hongo
Tatsutoshi
T
Nakahata
Sheldon M
SM
Schuster
Mitsuhiko
M
Osaka
D001215:Asparaginase / Q000494:pharmacology; D001217:Aspartate-Ammonia Ligase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000276:immunology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007938:Leukemia / Q000175:diagnosis* / Q000628:therapy; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis
D012333:RNA, Messenger; D001215:Asparaginase; D001217:Aspartate-Ammonia Ligase