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JMD 2004, Vol. 6, No. 3
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

Establishment of Real-Time Polymerase Chain Reaction Method for Quantitative Analysis of Asparagine Synthetase Expression

Tamotsu Irino*, Toshiyuki Kitoh{dagger}, Kenichi Koami*, Terumi Kashima*, Kouichi Mukai{ddagger}, Eiji Takeuchi{ddagger}, Teruaki Hongo§, Tatsutoshi Nakahata, Sheldon M. Schuster|| and Mitsuhiko Osaka*

From the Division of Cancer Research, * Shiga Medical Center Research Institute, Moriyama, Shiga, Japan; the Department of Pediatrics, {dagger} Shiga Medical Center for Children, Moriyama, Shiga, Japan; the Department of Pathology, {ddagger} Shiga Medical Center, Moriyama, Shiga, Japan; the Department of Pediatrics, § Hamamatsu University of Medicine, Hamamatsu, Shizuoka, Japan; the Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the Department of Biochemistry and Molecular Biology, || University of Florida, College of Medicine, Gainesville, Florida

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.




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