Clinical Chemistry
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Clinical Chemistry 54: 673-681, 2008. First published February 21, 2008; 10.1373/clinchem.2007.091975
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(Clinical Chemistry. 2008;54:673-681.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Ex Vivo Simulation of the Action of Antileukemia Drugs by Measuring Apoptosis-Related mRNA in Blood

Masato Mitsuhashi1,2,a, Katsuya Endo2, Kazuhiko Obara2, Hiroshi Izutsu2, Taishi Ishida3, Norio Chikatsu3 and Atsushi Shinagawa3

1 Hitachi Chemical Research Center, Inc., Irvine, CA; 2 Hitachi Chemical Co., Ltd., Hitachi, Japan; 3 Hitachi Ltd., Hitachi General Hospital, Hitachi, Japan.

aAddress correspondence to this author at: Hitachi Chemical Research Center, Inc., 1003 Health Sciences Rd., Irvine, CA. Fax 949-725-2727; e-mail mmitsuhashi{at}HCRcenter.com.

Background: In conventional bioassays, isolated cells are suspended in culture media, incubated in vitro for several days, and then characterized with respect to any cellular changes. In developing new molecular tests under physiological ex vivo conditions, we quantified the production of mRNAs for p21 and PUMA (p53 up-regulated modulator of apoptosis), which are involved in cell cycle arrest and apoptosis, respectively.

Methods: We stimulated human whole blood with a chemotherapeutic drug (cytarabine, daunorubicin, mitoxantrone, aclarubicin, etoposide, or idarubicin) for 4 h and then quantified mRNA by assessing mRNA recovery and cDNA-synthesis efficiency in each sample. We also used immunoassay and flow cytometry to investigate nucleosome and annexin V, respectively, as apoptosis markers.

Results: Ex vivo mRNA analysis yielded more positive results than nucleosome and annexin V analyses. The concentrations of cytarabine- and daunorubicin-induced p21 and PUMA mRNAs were significantly lower in acute myelogenous leukemia (AML) patients than in healthy controls (P <0.0001), whereas idarubicin induced significantly greater responses in AML patients than in controls (P = 0.01). The patients had different mRNA-response patterns, which were largely classifiable into 4 groups. Prednisone enhanced cytarabine or mitoxantrone induction of p21 and PUMA mRNAs in 3 (2.6%) of 114 reactions. All 15 patients who achieved complete remission had received at least one drug that produced positive mRNA responses, whereas we observed a lack of mRNA response to the clinically used drugs in all 3 cases in which the therapy failed to induce any hematologic improvement.

Conclusion: This study introduced ex vivo mRNA analysis as a candidate platform for drug-sensitivity tests in leukemia.






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