Masato Mitsuhashi^1,2, Katsuya Endo², Kazuhiko Obara², Hiroshi Izutsu², Taishi Ishida³, Norio Chikatsu³ and Atsushi Shinagawa3

¹Hitachi Chemical Research Center, Inc., Irvine, CA, 92617, U.S.A., ²Hitachi Chemical Co., Ltd., Hitachi, 317-0077, Japan, and ³Hitachi Ltd., Hitachi General Hospital, Hitachi, 317-8555, Japan.

Abstract:

Apoptosis was induced in heparinized human whole blood by 3 different ways (radiation, bleomycin, or etoposide), and various mRNA were quantified using the method we reported (Clin. Chem. 2006; 52:634–642). We found that cyclin-dependent kinase inhibitor 1A (p21) and p53 upregulated modulator of apoptosis (PUMA) were the most sensitive and universal mRNA markers of apoptosis in leukocytes. In order to define positive and negative responses, a synthetic RNA was spiked into the lysis buffer and the fold increase was calculated. As a result, 837/880 (95.1%) of data points stayed between 0.75 and 1.5 fold increase, and 874/880 (99.3%) were within 0.5–2.0 fold increase. When blood samples from 40 healthy adults were stimulated with 22 different drugs, more than 75% of the samples responded to bleomycin (1 μΜ), idarubicin (2 μΜ), vincristine (1 μΜ), daunorubicin (2 μΜ), cytarabine (10 μΜ), to induce p21 and/or PUMA mRNA, and approximately 25% showed no induction. Significant correlation was found between p21 and PUMA mRNA responses, and between daunorubicin and cytarabine, idarubicin, and vincristine for both p21 and PUMA. The quantification of drug-induced mRNA in whole blood will be considered as ex vivo, and is a suitable platform for biomarker screening as well as a model system for drug sensitivity tests in future.