Quantitative real time PCR of deoxycytidine kinase mRNA by Light Cycler PCR in relation to enzyme activity and gemcitabine sensitivity.
Deoxycytidine kinase (dCK) is essential for the phosphorylation of gemcitabine and can predict response to gemcitabine in vivo. Conventional Competitive Template-Reverse Transcriptase-Polymerase Chain Reaction (CT-RT-PCR) was correlated with real time PCR using a Light Cycler (LC) with SYBR-Green detection to enable rapid and sensitive detection of dCK mRNA expression. We used cDNA from human xenografts to establish a relation between dCK activity and gemcitabine sensitivity. A significant correlation of LC-PCR was found with CT-RT-PCR (Pearson: r = 0.956; P < 0.0001), enzyme activity (Pearson: r = 0.972; P = 0.003) and gemcitabine sensitivity (Pearson: r = 0.695; P = 0.048). The LC-PCR was also applied to needle biopsy specimens. In bladder tumors a similar correlation was found, while esophageal tumors with a high dCK expression responded to gemcitabine treatment. The LC is a rapid and reliable method for quantitation of dCK mRNA levels in tumors to predict clinical gemcitabine sensitivity.
Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
Jennifer
J
Sigmond
Judith R
JR
Kroep
Willem
W
Loves
Giovanni
G
Codacci-Pisanelli
Godefridus J
GJ
Peters
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism* / Q000494:pharmacology*; D018076:DNA, Complementary / Q000032:analysis; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology*; D003842:Deoxycytidine Kinase / Q000096:biosynthesis* / Q000235:genetics; D004938:Esophageal Neoplasms / Q000473:pathology*; D020869:Gene Expression Profiling; D006801:Humans; D008027:Light; D010766:Phosphorylation; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014183:Transplantation, Heterologous; D016896:Treatment Outcome; D001749:Urinary Bladder Neoplasms / Q000473:pathology*; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D018076:DNA, Complementary; D012333:RNA, Messenger; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; D000093542:Gemcitabine