Real-time PCR determination of IMPDH1 and IMPDH2 expression in blood cells.
BACKGROUND
Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide synthesis and is implicated in cell cycle control. Inhibition of this enzyme is associated with immunosuppressive, antiviral, and antitumor activity. IMPDH basal activity increases after initiation of immunosuppressive therapy.


METHODS
A real-time reverse-transcription PCR assay was developed and validated for mRNA quantification of the 2 human IMPDH isoforms. Target gene expressions were normalized to the geometric mean of 3 housekeeping genes. Assay utility was tested by analyzing patient samples and cultured cells exposed to immunosuppressive drugs such as the IMPDH inhibitor mycophenolic acid.


RESULTS
The assay was linear over 6 logs of cDNA input and demonstrated specific quantification of IMPDH1 and IMPDH2 expression in cultured cells and patient samples. Limits of detection and quantification were 10 and 10(3) copies of cDNA per reaction, respectively. Within-run and total between-day CVs were <15% for normalized expression. Changes in IMPDH1 and 2 expression were observed in patient samples after initiation of an immunosuppressive regimen that included calcineurin inhibitors, mycophenolate mofetil, and steroids.


CONCLUSIONS
This assay can be used to study the regulation of IMPDH expression and the involvement of the enzymes in immunological and malignant proliferative conditions. This may contribute to the processes of drug development and to the establishment of monitoring strategies for treatment effect and disease activity.
Bremer
Sara
S
Department of Medical Biochemistry, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway.
Rootwelt
Helge
H
Bergan
Stein
S
D001773:Blood Cells / Q000187:drug effects / Q000201:enzymology*; D065095:Calcineurin Inhibitors; D002478:Cells, Cultured; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000097:blood*; D007166:Immunosuppressive Agents / Q000494:pharmacology* / Q000627:therapeutic use; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000097:blood; D016030:Kidney Transplantation; D009173:Mycophenolic Acid / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D012156:Reticulocytes / Q000187:drug effects / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013256:Steroids / Q000494:pharmacology / Q000627:therapeutic use
D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D007527:Isoenzymes; D013256:Steroids; D007168:IMP Dehydrogenase; C488211:IMPDH1 protein, human; C501687:IMPDH2 protein, human; D009173:Mycophenolic Acid