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

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 ⑀-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 ␤-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.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). Molecular cloning and complete cDNA-derived amino acid sequences have been reported for the rat (3, 4), human (5, 6), and chick (7) enzymes, which were found to be synthesized in precursor forms of 411, 417, and 420 amino acids, respectively. The human lysyl oxidase gene is located on chromosome 5 (5, 6) and the mouse gene on chromosome 18 (8 -10), both genes consisting of seven exons (11,12). Increased lysyl oxidase activity has been reported in fibrotic disorders (1), while a deficiency is found in two Xlinked, recessively inherited human disorders, the occipital horn syndrome and Menkes syndrome, and in the X-linked recessively inherited mottled series of allelic mutant mice (13-15). In these X-linked disorders, the low enzyme activity appears to be secondary to abnormalities in copper metabolism.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 levels of rrg mRNA (17) and lysyl oxidase activity (18) are markedly decreased in NIH 3T3 cells transformed by LTR-cHa-ras compared with those in nontransformed NIH 3T3 or in cells after reversion following prolonged treatment with interferon-␤ (17). Transfection of the revertants with antisense rrg constructs leads to a transformed morphology again, and the cells become tumorigenic in nude mice (17).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 pu...

Autocrine Growth Factor Regulation of Lysyl Oxidase Expression in Transformed Fibroblasts

“…Although the relationship between low lysyl oxidase expression and a transformed phenotype has been confirmed in independent studies (13,16,17), there is surprisingly little information regarding the mechanism of down-regulation of lysyl oxidase in tumor cells. Lysyl oxidase promoter/reporter gene assays have so far not reproducibly identified transcriptional elements that confer clear tumor cell-specific effects on lysyl oxidase transcription (21)(22)(23).…”

“…Diminished levels of lysyl oxidase are consistently found in cancer cell lines as well as in transformed cell lines (12)(13)(14)(15)(16)(17), and restoration of a normal phenotype is consistently associated with the return of lysyl oxidase expression to normal levels (15,16,18). In particular, lysyl oxidase expression is diminished in cell lines transformed with ras or ras-dependent oncogenes.…”

“…As noted above lysyl oxidase is consistently down-regulated in cancer cells and in transformed cell lines, and studies support that lysyl oxidase transcription is diminished in transformed cells (17,20). It is important to realize, however, that the mechanisms responsible for diminished lysyl oxidase transcription in transformed cells are not well understood.…”

Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 in colorectal tumors

“…5 Low levels of LOX mRNA were detected in several human tumor cell lines, in SV 40 transformed WI-38 cells, melanoma cells, fibrosarcoma, choriocarcinoma and rhabdomyosarcoma cells as a result of transcriptional downregulation and decrease in mRNA stability. 6,7 Similarly, reduced lysyl oxidase mRNA levels were observed in ras-transformed NIH 3T3 cells, rat fibroblasts and osteoblast cells. 8 -10 The lower LOX activity of the transformed cells correlated with increased tumorigenicity in nude mice.…”

Lysyl Oxidase Is Required for Vascular and Diaphragmatic Development in Mice

“…Messenger RNA expression of all human lysyl oxidase family members appears to be most abundant in adult tissues by Northern blotting (Table I). Under normal conditions, human LOX mRNA expression is low in most tissues with 442 molecules/picogram embryonic skin fibroblast total RNA or about 8% of the mRNA molecules that encode for ␤-actin (29). LOXL1 mRNA expression appears greater than LOX mRNA expression in many adult mouse tissues, while expression of LOXL2, LOXL3, and LOXL4 mRNA appears to be low overall.…”