Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5'-nucleotidase mRNA measurement in cell lines and in patients with leukemia.
The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.
Månsson
E
E
Department of Medicine, Division of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
Liliemark
E
E
Söderhäll
S
S
Gustafsson
G
G
Eriksson
S
S
Albertioni
F
F
D015720:5'-Nucleotidase / Q000235:genetics*; D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D018076:DNA, Complementary / Q000032:analysis; D003842:Deoxycytidine Kinase / Q000235:genetics*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007223:Infant; D007938:Leukemia / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D008297:Male; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase; D003842:Deoxycytidine Kinase; D015720:5'-Nucleotidase