Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

Abstract

Apoptosis was induced in heparinized human whole blood by 3 different ways (radiation, bleomycin, or etoposide), and various mRNA were quantifi ed 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 defi ne 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. Signifi cant correlation was found between p21 and PUMA mRNA responses, and between daunorubicin and cytarabine, idarubicin, and vincristine for both p21 and PUMA. The quantifi cation 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.

Ex vivo simulation of IgG Fc and T-cell receptor functions: An application to inflammatory bowel disease

“…[11][12][13] In brief, 50 L of blood sample was applied to 96-well filter plates in order to trap leukocytes. Sixty microliters of lysis buffer containing a cocktail of specific reverse primers was applied to the filter plates, and the resultant cell lysates were transferred to oligo(dT)-immobilized microplates (GenePlate, RNAture, Irvine, CA) 17 for poly(A) ϩ mRNA purification.…”

“…In the first half of this study, we developed a unique system to assess the overall function of the Fc␥R and the TCR under physiological ex vivo conditions using an assay system we previously described. [11][12][13] We then used this technology to assess the functions of the Fc␥R and the TCR in inflammatory bowel diseases (IBD) 14 because leukocytes play a role of pathogenesis of IBD by migrating to the disease site. Although the primary disease site is small intestine and colon in IBD, this study specifically focused on the functions of the Fc␥R and the TCR on peripheral-blood leukocytes as surrogate markers of IBD.…”

mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans

“…Differences in Cycle threshold (C t ) between the target and control mRNA (β actin) (ΔC t ) were used to quantify the relative amount of each target, calculated as 2 ÀΔC t . Primers used for gene expression assays have been previously described [25][26][27][28].…”

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

“…We recently found that mRNAs for p21 and PUMA 4 (p53 up-regulated modulator of apoptosis) were the most predictive mRNA markers of leukocyte apoptosis (5 ) and that the induction of these mRNAs was detectable in human whole blood within 1-4 h of drug incubation (5 ). p21 is an inhibitor of cyclindependent kinase (6 ), and increased p21 production consequently causes cell cycle arrest (7 ).…”

“…6) also showed weak p21 mRNA responses to ACR, but these ACR responses corresponded to CRs. Because high doses of AraC and DNR failed to induce p21 and PUMA mRNA responses in case 2, we also analyzed other BAX family mRNAs, as is described in our previous report (5 ). This study demonstrated that high doses of AraC, DNR, and MIT did not induce apoptosis-related mRNAs (GADD, SUMO, Apaf1, Bfl1, BclII, Bim, Bik, Bid, Bad, Bcl-xs, Bak, and Bax) (data not shown).…”