Quantitative polymerase chain reaction of lysyl oxidase mRNA in malignantly transformed human cell lines demonstrates that their low lysyl oxidase activity is due to low quantities of its mRNA and low levels of transcription of the respective gene.
Lysyl oxidase (EC 1.4.3.13), an extracellular copper amino oxidase, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We developed here a polymerase chain reaction (PCR) method for the quantification of lysyl oxidase mRNA in which a synthetic RNA is used as an internal standard for coamplification with the targeted mRNA. The amount of lysyl oxidase mRNA when studied by Northern blot analysis and the number of lysyl oxidase mRNA molecules when determined by the quantitative PCR method were found to be markedly low in various malignantly transformed cell lines relative to control cell lines, quantitative PCR indicating values of about 2-10% of those in the controls. No difference was found in the number of beta-actin mRNA molecules between the transformed cells and the controls. Nuclear runoff experiments indicated that most if not all of the decrease in the number of lysyl oxidase mRNA molecules can be explained by diminished transcription of the respective gene.
Hämäläinen
E R
ER
Collagen Research Unit, University of Oulu, Finland.
Kemppainen
R
R
Kuivaniemi
H
H
Tromp
G
G
Vaheri
A
A
Pihlajaniemi
T
T
Kivirikko
K I
KI
D000328:Adult; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D017931:DNA Primers; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D008249:Protein-Lysine 6-Oxidase / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D012333:RNA, Messenger; D008249:Protein-Lysine 6-Oxidase