Abstract
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.
Highlights
Lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ⑀-amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin
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
No signal for lysyl oxidase mRNA was seen in RNA from the transformed cell lines, including SV40-transformed WI-38 cells (VA-13), melanoma cells (G-361), fibrosarcoma cells (HT-1080), choriocarcinoma cells (JEG-3), and embryonal rhabdomyosarcoma cells (RD)
Summary
Lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ⑀-amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin (for reviews, see Refs. 1 and 2). Lysyl oxidase activity is markedly low in the culture medium of many malignantly transformed human cell lines (16). The cDNA-derived amino acid sequence of the mouse ras recision gene, rrg (17), has been found to match that of rat lysyl oxidase (18), suggesting that rrg and lysyl oxidase are identical. The purpose of this work was to explore further the reasons for the low lysyl oxidase activity observed in the culture medium of malignantly transformed cells. For this purpose, we developed a PCR1 method for the quantification of lysyl oxidase mRNA in which a synthetic RNA (cRNA) is used as an internal standard for coamplification with the target mRNA. We studied the amount of lysyl oxidase mRNA by Northern blotting and the production of this mRNA in in vitro nuclear runoff experiments
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