(Received for publication, April 25, 1995; and in revised form, June 23, 1995)
From the
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 (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, 9, 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 X-linked,
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, 14, 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-c-Ha-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 purpose, we developed
a PCR ()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 also studied the amount of
lysyl oxidase mRNA by Northern blotting and the production of this mRNA
in in vitro nuclear runoff experiments.
The cDNA for the pro1 chain of type III
procollagen(23, 24) contained an internal TaqI restriction site in the amplified region and was thus
digested with TaqI, religated, and the deleted PCR fragment
amplified with primers 1 and 2. All the PCR products with deletions
were cloned into Bluescript SK, and plasmid isolation was performed by
the CsCl method(25) . The constructs were sequenced to verify
their identity and orientation.
Each PCR cycle in the
lysyl oxidase and type III procollagen pro1 chain amplification
reactions included 90 s of denaturation at 94 °C, 80 s of annealing
at 60 °C, and 90 s of extension at 72 °C. The conditions for
-actin were the same, except that the annealing temperature was
58 °C.
Figure 1:
Northern blot analysis of
lysyl oxidase mRNA in malignantly transformed and control cell lines.
Total cytoplasmic RNA, 20 µg, was hybridized to a P-labeled cDNA probe, HLO20(5) , for human lysyl
oxidase. The malignantly transformed cell lines are SV40 virus
transformed WI-38 cells (VA-13), melanoma cells (G-361), fibrosarcoma
cells (HT-1080), choriocarcinoma cells (JEG-3), and embryonal
rhabdomyosarcoma cells (RD). The control cell lines are embryonal lung
(WI-38, HEL) and skin (HES) fibroblasts and adult fibroblasts(9011).
The 4.3-kilobase band corresponding to the major species of the human
lysyl oxidase mRNA is shown.
Figure 2:
Autoradiogram of lysyl oxidase mRNA (568
bp) and the internal standard cRNA (508 bp)-specific PCR products from
one control(9011) and one transformed (HT-1080) cell line. 0.93 µg
of total cellular RNA from the 9011 cells and 10.96 µg of RNA from
the HT-1080 cells were reverse transcribed in the presence of 8.44
10
molecules of the internal standard cRNA. Serial
1:2 dilutions (from right to left) were performed,
the PCR amplifications were carried out, and the products were
separated on 6% PAGE, dried, and
autoradiographed.
The quantitative PCR method developed here for human lysyl oxidase mRNA follows the principles described by Wang et al.(27) . The specific mRNA and an internal standard cRNA containing a 60-bp deletion are coamplified in the same reaction and with the same primers, and the PCR products are separated by PAGE and quantified. The numbers of endogenous mRNA molecules can then be computed from the curve determined for the internal standard.
The
data obtained with the quantitative PCR indicated that the number of
lysyl oxidase mRNA molecules per picogram of total RNA was about
340-440 in the four control cell lines, the mean being about 8%
of the mean for the values of about 2000-6600 molecules/pg
determined for the mRNA molecules that encode -actin and about 60%
of the mean for the values of 380-980 molecules/pg for those
encoding the pro
1 chain of type III procollagen. The
last-mentioned value does not indicate a ratio of the number of mRNA
molecules for lysyl oxidase to the number for its polypeptide substrate
because type III procollagen represents only about 15-20% of the
total collagen synthesized by cultured skin fibroblasts (see (28) and references therein). Thus, the number of lysyl oxidase
mRNA molecules may be about one-tenth of the number for its polypeptide
substrates.
The number of lysyl oxidase mRNA molecules in malignantly transformed cell lines was very low, both when measured by Northern blotting and when assayed with the quantitative PCR. The latter indicated values of about 2-10% of those in the controls. Lysyl oxidase activity in malignantly transformed cell lines has previously been reported as about 10% that in controls(16) . The present assays indicated an even lower percentage, but as the activity levels in the transformed cells were below the limit of accurate measurement both in the previous study (16) and here, the actual levels probably do not differ greatly. The magnitude of the decrease in the number of lysyl oxidase mRNA molecules thus appears to be very similar to that in enzyme activity, indicating that the low enzyme activity is due to a pretranslational mechanism.
Nuclear runoff experiments indicated that most if not all of the decrease in the lysyl oxidase mRNA levels can be explained by diminished transcription of the respective gene. However, as the runoff values for the transformed cells were not quite as low as those for the mRNA molecules by comparison with the control cells, the data do not exclude the possibility that decreased stability of the lysyl oxidase mRNA may have contributed to the low mRNA and enzyme activity levels.
The present data obtained with several human tumor cell lines are in good agreement with those reported for the rrg mRNA, which appears to be identical to the lysyl oxidase mRNA in NIH 3T3 cells transformed by LTR-c-Ha-ras (see Introduction). Thus, the findings reported for the LTR-cHa-ras-transformed NIH 3T3 cells appear to be similar in a number of tumor cell types. It is of particular interest that the data obtained with the NIH 3T3 cells suggest that the lysyl oxidase gene may have a tumor suppressor activity(17, 18) . However, the mechanisms by which this gene may achieve this activity are currently unknown. It is likewise unknown what cis-acting elements in the lysyl oxidase gene and trans-acting factors in the cells are responsible for the low lysyl oxidase mRNA levels in the tumor cells. The promoter region and downstream sequences in the lysyl oxidase gene contain a number of potential binding sites for various transcription factors (12) , but the elements involved in the decrease in the transcription of the gene in transformed cells remain to be identified.