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Carcinogenesis |
Deutsches Krebsforschungszentrum, Research Program on Tumor Cell Regulation, 69120 Heidelberg, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LOX are a family of non-heme iron dioxygenases that regio- and stereospecifically insert molecular oxygen into polyunsaturated fatty acid subtrates generating 5S-, 8S-, 12S-, 12R-, or 15S-hydroperoxyeicosatetraenoic acids and, on reduction, the corresponding hydroxy derivatives (HETE) with arachidonic acid and 9S- or 13S-hydroperoxyoctadecadienoic acids and the corresponding hydroxy derivatives (HODE) with linoleic acid as a substrate (5) . In addition to well known LOX such as 5S-LOX, leukocyte-type (l) and platelet-type (p) 12S-LOX, as well as reticulocyte-type 15S-LOX-1, the mammalian LOX family has recently expanded to comprise several isozymes that have been found to be preferentially expressed in the epidermis of man and mice. These include the e12S-LOX and 12R-LOX, the mouse 8S-LOX and its human orthologue 15S-LOX-2, and eLOX-3, a LOX with yet unknown enzymatic activity. These novel isozymes may be regarded as a distinct epidermis-type subgroup of the LOX multigene family (6 , 7) .
On tumor induction in mouse skin the LOX isoforms 8S- and p12S-LOX have been found to be aberrantly overexpressed in papillomas and squamous cell carcinomas, leading to an accumulation of the corresponding metabolites 8S- and 12S-HETE (8 , 9) . Both LOX products have been shown to induce chromosomal damage in primary basal mouse keratinocytes (10) . Moreover, in tumors the amounts of these metabolites correlated with the formation of etheno adducts of DNA (11) , indicating that 8S- and 12S-HETE may generate an endogenous mutagenic potential that may possibly contribute to genetic instability of neoplastic cells.
The most abundant LOX isozyme in normal mouse skin is e12S-LOX (12) . Its mRNA is detected in all epidermal cell layers whereas the expression of the other LOX isoforms is restricted to the suprabasal layers of mouse epidermis (6) . Recombinant e12S-LOX generates 12S-HETE from arachidonic acid and 13S-HODE from linoleic acid and is unique in accepting, in addition, methyl linoleate as a substrate, indicating that more complex lipids rather than free polyunsaturated fatty acids are endogenous substrates of this isozyme. However, as compared with p12S- and l12S-LOX the catalytic activity of e12S-LOX is very low (12) .
Here, we show that the expression of e12S-LOX becomes completely down-regulated in the course of mouse skin carcinogenesis according to the initiation-promotion protocol. This down-regulation may indicate an anti- rather than a procarcinogenic effect of this LOX isozyme. To test this hypothesis, two transgenic mouse lines were generated that differentially expressed e12S-LOX under the control of the bovine pK6 known to be constitutively up-regulated in mouse skin tumors (13) . As compared with the wild type, low transgene expression correlated with a decreased skin tumor response paralleled by an accumulation of the linoleic acid derivative 13S-HODE. In contrast, high transgene expression coincided with an increased tumor response paralleled by an accumulation of the arachidonic acid derivative 12S-HETE as the predominant LOX product. These results indicate a complex interaction between different LOX isoforms and an opposite role of arachidonic acid and linoleic acid products in the modulation of skin carcinogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
B6D2F2, DBA/2, and NMRI mice were obtained from RCC/Life Technologies, Inc. (Füllinsdorf, Switzerland). Heterozygous and homozygous K6.e12S-LOX mice and the corresponding wild-type littermates were bred by RCC/Life Technologies, Inc. and at the central animal facility of the Deutsches Krebsforschungszentrum. All animals were kept under an artificial day/night rhythm and were fed Altromin standard food pellets (Altromin, Lage, Germany), with sterile water available ad libitum. Shaving of the back skin with electrical clippers was performed 3 days before treatment. For topical applications, compounds were dissolved in 0.1 ml of acetone and applied onto the shaved back skin.
Generation and Characterization of K6.e12S-LOX Transgenic Mice.
The coding region of e12S-LOX (14)
was subcloned by PCR into the ClaI site of the cytokeratin IV minilocus expression vector (15)
to generate pK6.e12S-LOX. DNA sequences in the construct were verified by automated DNA sequencing using the ABI Prism 310 genetic analyzer (Perkin-Elmer/Applied Biosystems, Weiterstadt, Germany). The vector backbone was removed by NotI digestion, followed by gel electrophoresis of the 12.8-kb insert fragment. Transgenic mice were generated by pronuclear microinjection of fertilized B6D2F2 oocytes with the purified insert fragment. Two-cell embryos were transferred to the oviducts of pseudopregnant foster DBA/2 mice. Transgenic mice were detected by PCR screening of genomic tail DNA using transgene-specific primers (upper primer, 5'-TGCCGGGAAGCTCCTCTCATAG-3'; lower primer, 5'-TTTCTGTTTGCGCAGCTTCACC-3'; annealing 56°C) to generate a 252-bp fragment and verified by Southern blot analysis. DNA was digested with BamHI to release the 5.7-kb e12S-LOX transgene and subjected to Southern blot using e12S-LOX cDNA as a probe. The founder mice were positive in both PCR and Southern blot analysis and were matched back to DBA/2. Homozygous K6.e12S-LOX mouse lines were obtained by breeding heterozygous offsprings of an individual founder line. Homozygosity was verified by Southern blot analyses and backcrossing with wild-type littermates.
Quantitative RT-PCR Analysis.
Control and TPA-treated mouse skin and papillomas from wild-type and transgenic mice were dissected and snap-frozen in liquid nitrogen or by using a cold table. Epidermis was removed by scraping with a scalpel. Total RNA was extracted from the pulverized frozen tissues by using RNA Clean (Hybaid, Ulm, Germany), and reverse transcription was performed with the Gene Amp RNA PCR kit (Perkin-Elmer). First-strand cDNA synthesis was carried out with 1 µg of total RNA in a 20-µl reaction mixture with oligo(dT) primer according to the manufacturer’s specifications. The reverse transcription mixture contained 1 mM of each dNTP, 2 µl 10x PCR buffer [500 mM KCl and 100 mM Tris-HCl (pH 8.3)], 4 µl of MgCl2 (25 mM), 1 µl of oligo(dT) primer (50 µM), 1 µl of RNase inhibitor (20 units/µl), and 1 µl of MuLV reverse transcriptase (50 units/µl). This mixture was incubated for 10 min at room temperature, 15 min at 42°C, and heated to 99°C for 5 min. Two microliters of a 1:10 dilution of these reactions served as templates for a real-time PCR using the FastStart DNA Master SYBR Green I Kit (Roche, Mannheim, Germany) with the LightCycler Instrument (Roche). The 20-µl reactions were set up according to the manufacturer’s specifications with 0.5 µM for each primer and 4 mM MgCl2. The temperature program consisted of an initial denaturation at 95°C for 10 min, followed by repeated denaturation at 94°C for 15 s, annealing (see Table 1
for annealing temperature, primers, and details) for 10 s, and elongation at 72°C for 20 s for 44 cycles. The identity of the PCR products was checked by melting curve analysis (95°C for 1 s, 65°C for 15 s, and subsequent heating at 95°C with a slope of 0.1°C/s) in the LightCycler and by gel electrophoresis. Determination of the copy number was done by simultaneously amplifying a standard curve using known plasmid concentrations ranging from 101 to 105 copies. For each amplicon the target sequence was cloned to give a specific plasmid for calibration. The fluorescence kinetics were transformed to the copy number using the second derivative maximum method provided by the instrument software. To ensure the fidelity of mRNA extraction and reverse transcription, all samples were subjected to PCR amplification with primers specific for the constitutively expressed gene ß-actin and normalized. At least one of the primers for each target was designed to span an intron/exon splice site and subsequently tested for the absence of any genomic amplification product. Genomic DNA and H2O served as negative controls in all experiments. To ensure reproducibility, LightCycler PCRs were repeated independently.
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Immunohistochemistry.
For Ki-67 staining skin was immersed in Tissue-Tek (Sakura Finetek, Europe, Zouterwoude, The Netherlands), snap-frozen in liquid nitrogen, and stored at -70°C. Five-micrometer cryosections were prepared from this frozen tissue. Slices were collected on SuperFrost Plus microscope slides (Neolab, Heidelberg, Germany), air dried, and stored at -70°C. Thawed sections were fixed in freshly prepared 1% paraformaldehyde in PBS and in absolute ethanol for 5 min each. Specimens were washed (10 min in PBS), and endogenous peroxidase was blocked (3% H2O2 in methanol for 10 min). After washing for 5 min each in aqua bidest and PBS, sections were incubated in block solution (1% ELISA-BSA in PBS; Sigma) for 1 h. Thereafter, rabbit antimouse Ki-67 antiserum (Dianova, Hamburg, Germany) diluted 1:50 in block solution was added for 16 h at 4°C. Specimens were rinsed in PBS (three times for 10 min) before the addition of the peroxidase-conjugated second antibody (goat antirabbit IgG-horseradish peroxidase; Dianova) diluted 1:100 in block solution for 1 h at room temperature. After rinsing with three changes of PBS, 10 min each, tissues were incubated in 0.07% 3,3'-diaminobenzidine and 0.16% hydrogen peroxide (Fast DAB tablets; Sigma) for 1 h at room temperature. Tissues were washed in aqua bidest, counterstained in Mayer’s hematoxylin, dehydrated through ethanol series, and mounted in eukitt (Kindler, Freiburg, Germany).
Induction of Mouse Skin Tumors by the Initiation-Promotion Protocol.
For tumor experiments mice were housed in Macrolon cages (four animals/type II cage) under an artificial day-night rhythm and controlled temperature (21 ± 1°C) and humidity (50–60% relative humidity). Experimental groups of 20 animals (transgenic mouse lines or the corresponding wild-type littermates) were initiated with a single epicutaneous application of 0.1 µmol of DMBA in 0.1 ml of acetone or 0.1 ml of acetone alone (initiation control). Starting 1 week later, the mice were treated twice weekly with 2.5 or 10 nmol of TPA, dissolved in 0.1 ml of acetone, or 0.1 ml of acetone alone (promotion control). This treatment was continued for 25 weeks. The tumor incidence (number of papilloma bearers/number of survivors, in percentage) and yield (papillomas/survivor) were recorded weekly. Papillomas were harvested 2 weeks after the last TPA treatment and snap-frozen in liquid nitrogen. For preparation of the tumors, great care was taken to avoid contamination with nonepithelial material. A section of each tumor sample was analyzed histologically and showed that more than 95% of the removed biopsy material was of epithelial origin.
Extraction and Fractionation of Lipids from Epidermis and Epidermal Tumors.
Aliquots of 100–300 mg of frozen epidermis or epidermal tumors were disintegrated for 30 s in a ball mill at liquid nitrogen temperature. For the extraction of lipids according to Bligh and Dyer (16)
, the pulverized tissue was suspended in 1 ml of methanol and homogenized by using a Douncer for 1 min. Homogenization was continued for another 2 min after the addition of 2 ml of trichloromethane. On centrifugation for 10 min at 15000 x g and 4°C, the supernatant was removed. The sediment was reextracted successively with 3 ml of methanol/trichloromethane equal to 2:1 (v/v) for 3 min, 2 ml of trichloromethane for 2 min, and 1 ml of methanol for another 2 min. The combined supernatants were washed with a 0.88% aqueous KCl solution (1/4 of the volume) and evaporated using a Speedvac (Saur, Reutlingen, Germany) centrifuge. Redissolved in 0.2 ml of n-hexane/methyl-tert-butylether/acetic acid equal to 100:3:0.3 (v/v/v), the lipid extracts were fractionated into free fatty acids, phospholipids, and other polar lipids by chromatography over aminopropyl silica cartridges (ICT; Ref. 17
) preequilibrated with 0.6 ml of acetone/water equal to 7:1 (v/v) and twice with 1 ml of n-hexane. After rinsing the loaded cartridges with 5 ml of n-hexane, 6 ml of n-hexane/trichloromethane/ethyl acetate equal to 100:5:5 (v/v/v) and 5 ml of trichloromethane/isopropanol equal to 2:1 (v/v), successively free fatty acids were eluted with 6 ml of trichloromethane/methanol/acetic acid equal to 100:2:2 (v/v/v) and the phospholipid fraction was eluted with 6 ml of methanol/trichloromethane/H2O equal to 10:5:4 (v/v/v). The fatty acid fraction was evaporated and subjected to RP-HPLC. The phospholipid fraction was cleaved by alkaline hydrolysis with 5% potassium hydroxide in methanol at 60°C for 30 min under argon protection. The hydrolysis was stopped by neutralization with glacial acetic acid, and the free fatty acids were extracted with ethyl acetate. After evaporation of the organic solvent the extracted fatty acids were dissolved in ethanol, and an aqueous 0.1 M ammonium formate buffer (pH 3.1) was added to obtain a 15% by volume ethanolic solution. A fatty acid-enriched fraction was obtained by solid-phase extraction of the samples on C18 silica cartridges with ethyl acetate according to Powell (18)
. The organic phase was evaporated, and the fatty acid fraction was subjected to RP-HPLC.
RP-HPLC Analysis of HETE and HODE.
RP-HPLC analysis of HETE and HODE was performed with a YMC-Pack ODS-AM column (25 x 0.46 cm, 5-µm particle size; YMC Europe, Schermbeck, Germany) with a 1-cm guard column. Products were eluted at 0.5 ml/min with a solvent system consisting of methanol/water/acetic acid equal to 86:14:0.01 (v/v/v). Elution was monitored at 236 nm and 205 nm. The products were identified and quantified by comparing retention times and peak areas with those of authentic standards. Retention times were 17.4 min for 13-HODE, 17.6 min for 9-HODE, 18.9 min for 15-HETE, 20.7 min for 12-HETE, and 21.3 min for 8-HETE. 13-HODE, 9-HODE, and 15-HETE were collected in a single fraction and further analyzed via SP-HPLC. 12-HETE was collected in a second fraction and directly subjected to CP-HPLC.
SP-HPLC Analysis of HETE and HODE.
The fraction containing 13-HODE and 9-HODE obtained from RP-HPLC was further analyzed by SP-HPLC using a Zorbax Sil column (25 x 0.46 cm; 5-µm particle size; Bischoff, Stuttgart, Germany). Products were eluted at 1.0 ml/min with a solvent system consisting of n-hexane/propan-2-ol/acetic acid/water equal to 98:2:0.1:0.025 (v/v/v/v). Elution was monitored at 236 nm and 205 nm. The products were identified and quantified by comparing retention times and peak areas with those of authentic standards. Retention times were 13.0 min for 15-HETE, 17.3 min for 13-HODE, and 26.8 min for 9-HODE.
CP-HPLC Analysis of HETE and HODE.
Fractions of HETE and HODE obtained from RP- and SP-HPLC were rechromatographed by CP-HPLC using a Chiracel OB column (25 x 0.46 cm; 5-µm particle size; Baker Instruments, Deerfield, IL) eluted at 0.5 ml/min with the solvent system n-hexane/propan-2-ol/acetic acid in various proportions: 97.5:2.5:0.05 (v/v/v) for 12-HETE, 9-HODE and 8-HETE; 99:1:0.05 (v/v/v) for 15-HETE and 13-HODE. Elution was monitored at 236 nm. The products were identified and quantified by comparing retention times and peak areas with those of authentic external standards. Retention times were 13 min for 12R-HETE, 16 min for 12S-HETE, 29 min for 9S-HODE, 48 min for 9R-HODE, 22 min for 8S-HETE, 28 min for 8R-HETE, 28 min for 15S-HETE, 35 min for 15R-HETE, 40 min for 13S-HODE, and 52 min for 13R-HODE.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Northern blot analyses of RNA from papillomas and carcinomas demonstrated a complete down-regulation of e12S-LOX mRNA in the course of tumor development (Fig. 1B)
. By using real-time RT-PCR a similar down-regulation of e12S-LOX was also observed in papillomas from wild-type littermates of the transgenic mouse lines described below (Fig. 1C)
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, inset. Microinjection of the linearized construct into pronuclei of B6D2F2 oocytes resulted in an offspring of 126 mice. Among them, four mice harbored the transgene according to Southern blot analysis of genomic tail DNA. Two founders, K6.e12S-LOX/9 and K6.e12S-LOX/50, showing different levels of transgene mRNA concentrations, were used to generate homozygous mouse lines. Homozygosity was confirmed by Southern blot and backcross analysis (data not shown). Gender distribution was similar in wild-type and transgenic mice. Fertility was found to be reduced in the high expressor line K6.e12S-LOX/50 as compared with the low expressor K6.e12S-LOX/9 and wild-type mice, respectively. Data from the homozygous transgenic lines are presented.
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. Low copy numbers of transgenic mRNA were detected in normal epidermis of the high expressor line K6.e12S-LOX/50, most probably due to pK6-directed expression of the transgene in suprabasal cells of the outer root sheath, but not in the low expressor line K6.e12S-LOX/9. After a single application of TPA, the pK6-driven expression of the transgene was induced. As shown in Fig. 2
, a pronounced difference between the low and the high expressor line was observed.
Skin Phenotype of Transgenic Mouse Lines.
No difference of skin morphology could be detected in H&E-stained sections of both untreated and TPA-treated epidermis from wild type as compared with transgenic mice. The same holds true for keratin 5 and involucrin expression according to immunohistochemical analysis. Depending on the expression level of the transgene Ki67 staining revealed a slightly increased proliferative index in normal and TPA-treated epidermis of transgenic mice (data not shown).
Susceptibility of K6.e12S-LOX Transgenic Mice for Tumor Promotion.
The K6.e12S-LOX transgenic mice were evaluated for their sensitivity toward skin tumor development using the two-stage approach of mouse skin carcinogenesis with DMBA as an initiator and the phorbol ester TPA as a tumor promoter. No tumors developed on DMBA treatment or TPA treatment alone in wild-type and transgenic mice. On combined treatment the tumor response depended both on the TPA dose and the expression level of the transgene. Transgenic mice of the K6.e12S-LOX/9 line developed less papillomas than wild-type mice as evidenced by a strongly reduced incidence with the low TPA dose and a clearly diminished tumor yield in the presence of both 2.5 nmol and 10 nmol of TPA (Fig. 3A, C, and D)
. On the other hand, the susceptibility of the line K6.e12S-LOX/50 was found to be increased over that of wild-type mice as shown by the tumor incidence in the presence of 2.5 nmol of TPA and the tumor yield induced by 10 nmol of TPA (Fig. 3, A and D)
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. As compared with the surrounding skin, transgenic e12S-LOX mRNA was found to be barely up-regulated in papillomas of K6.e12S-LOX/9 mice but strongly overexpressed in those of K6.e12S-LOX/50 mice. The levels of p12S-LOX were only moderately altered in tumors of wild-type, K6.e12S-LOX/9, and K6.e12S-LOX/50 mice, respectively. In K6.e12S-LOX/9 mice a striking observation was the strong increase of the steady-state concentration of l12S-LOX mRNA in tumors as compared with the surrounding skin. These animals showed a reduced susceptibility for papilloma formation as compared with wild-type and K6.e12S-LOX/50 mice.
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. Papillomas of K6.e12S-LOX/9 mice contained larger amounts of 13-HODE as compared with wild-type and K6.e12S-LOX/50 mice correlating with a massive up-regulation of l12S-LOX (Fig. 4, A and B)
. 8-HETE and 9-HODE levels were low and did not significantly differ between wild-type and transgenic mice (data not shown). The ratio of the S and R enantiomers of 12-HETE was about 80:20 or 87:13 in lipid extracts from papillomas of wild-type or K6.e12S-LOX/50 and K6.e12S-LOX/9 mice, respectively, pointing to a predominantly LOX-catalyzed formation of this metabolite. The same holds true for 13-HODE showing S:R ratios between 80:20 and 70:30. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we have analyzed the functional role of e12S-LOX in epidermal carcinogenesis. Whereas treatment of the animals with the phorbol ester TPA resulted in an up-regulation of 8S- and p12S-LOX mRNA levels (6) , expression of e12S-LOX mRNA was only transiently induced, followed by a down-regulation starting after about 8 h. Most strikingly, we observed a complete down-regulation of e12S-LOX mRNA in the course of epidermal tumor development, indicating a possible antitumorigenic effect of the enzyme. Down-regulation of distinct LOX in the course of tumor development is not without precedent. Thus, 15-LOX-2 expression has been found to be reduced in prostate cancer and high-grade prostatic intraepithelial neoplasia (22 , 23) . 15S-LOX-1 expression was also reduced in human colorectal cancer (24) , although this observation was confounded by another report (25) . These findings indicate that distinct LOX may cause antitumorigenic rather than protumorigenic activities (4) .
To test this hypothesis for e12S-LOX, we generated transgenic mice with targeted expression of this enzyme in papillomas induced according to the initiation-promotion protocol. Two transgenic lines were chosen for this study (i.e., a low expressor and a high expressor line). With the exception of a slight increase of the proliferative index in normal and TPA-treated epidermis, both lines did not show a distinct phenotype with respect to skin morphology and differentiation pattern. However, pronounced differences with respect to tumor development became apparent in that low transgene expression correlated with a decreased and high transgene expression with an increased susceptibility for papilloma development as compared with wild-type mice. Thus, depending on the expression level of the transgene, opposite effects on tumor development were observed.
Moreover, the papillomas of the low expressor line differed in their qualitative and quantitative LOX mRNA and LOX product profiles from those of the high expressor line and of wild-type mice. In tumors of wild-type mice, p12S-LOX mRNA was found to be moderately overexpressed. In contrast, large copy numbers of l12S-LOX mRNA prevailed in papillomas of low expressors whereas the transgenic e12S-LOX mRNA was most abundant in papillomas of high expressors. The mRNA profiles of other epidermal LOX isozymes including 8S-LOX, 12R-LOX, and e-LOX-3 did not show significant differences between wild-type and transgenic mice (data not shown). Thus, distinct 12S-LOX mRNA profiles correlated with different tumor susceptibilities. The mechanisms involved in the strong up-regulation of l12S-LOX mRNA in papillomas of low expressors remain to be established.
This correlation could be extended to the product profiles. 12S-HETE was the predominant arachidonic acid product in wild-type and high expressor mice whereas 13S-HODE was most abundant in papillomas from low expressor mice. 12S-HETE is the main arachidonic acid metabolite of p12S-LOX, and 13S-HODE is the predominant linoleic acid metabolite of e12S-LOX and l12S-LOX (12 , 26) . This substrate preference does not fully explain the product profiles observed in the papillomas of wild-type and transgenic animals. In fact, overall substrate availability primarily determined by the activity of distinct phospholipase A2 subtypes is known to be a limiting factor. Moreover, these enzymes have been shown to exhibit a distinct selectivity for the fatty acid released (27) . Interestingly, a coupling between phospholipases A2 subtypes and COX isozymes has been observed on stimulation of prostaglandin biosynthesis (28) . Thus, the strong accumulation of 13S-HODE in papillomas of low expressor mice being in line with the high expression of l12S-LOX might be supported by a preferential release of linoleic acid from cellular phospholipids. The strong accumulation of 12S-HETE in papillomas of wild-type mice may be attributed to the expression and activity of p12S-LOX and the preferential release of arachidonic acid through simultaneous activation of the arachidonic acid-selective cytosolic PLA2 by 12S-HETE in a mitogen-activated protein kinase-dependent pathway (29 , 30) . A similar coupling occurring between the highly expressed transgenic e12S-LOX in papillomas of high expressor mice may give rise to the accumulation of 12S-HETE.
On the basis of data obtained from different systems, 12S-HETE and 13S-HODE have been proposed to have opposite effects on tumorigenesis. Thus, 12S-HETE is thought to promote carcinogenesis due to an up-regulation of tumor cell adhesion molecules (31 , 32) , a stimulation of angiogenesis (33) and of tumor cell spreading (34) , and an inhibition of apoptosis (35) . In contrast, 13S-HODE is likely to have antitumorigenic effects due to an induction of apoptosis and cell cycle arrest (24 , 36) and an induction of differentiation (37 , 38) . Both metabolites may also play a role in experimental tumor induction in mouse skin. In fact, arachidonic acid has been shown to have a protumorigenic activity that is inhibited by linoleic acid, and accordingly 12S-HETE and 13S-HODE seem to be involved in these opposing effects (39 , 40) . Thus, 12S-HETE has been reported to stimulate keratinocyte proliferation and adhesion to fibronectin and to inhibit terminal differentiation of keratinocytes (40 , 41) , whereas 13S-HODE was found to reverse epidermal hyperproliferation in the skin of guinea pigs (42) , to counteract the inhibition of terminal differentiation by 12S-HETE, and to prevent keratinocyte adhesion to fibronectin (40) .
In summary, our studies support the notion (4) that LOX might be categorized as both pro- and antitumorigenic enzymes depending on the individual expression levels and the resulting product profiles. The differential functions of individual LOX for the carcinogenic process have to be considered in the development of novel cancer chemoprevention strategies targeting this family of proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant 10-1565-Ma3 from the Deutsche Krebshilfe (Bonn, Germany). 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Deutsches Krebsforschungszentrum, B0500, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: p.krieg{at}dkfz.de
4 The abbreviations used are: COX, cyclooxygenase; LOX, lipoxygenase(s); HPLC, high-performance liquid chromatrography; CP-HPLC, chiral-phase HPLC; DMBA, dimethylbenz[a]anthracene; e12S-LOX, epidermis-type 12S-LOX; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; K6, keratin 6; l12S-LOX, leukocyte-type 12S-LOX; pK6, K6 promoter; p12S-LOX platelet-type 12S-LOX; RP-HPLC, reverse-phase HPLC; SP-HPLC, straight-phase HPLC; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received 3/25/02. Accepted 6/11/02.
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) and papillomas (
) was reverse-transcribed to cDNA. PCR was run with specific primer sets for endogenous e12S-LOX and ß-actin as an internal control (see "Materials and Methods" and Table 1
) and K6.e12S-LOX/50 (
) mice were initiated by a single topical application of 100 nmol of DMBA, dissolved in 0.1 ml of acetone, and promoted by twice-weekly applications of 2.5 nmol (A and C) or 10 nmol (B and D) of TPA, dissolved in 0.1 ml of acetone. Papillomas were counted weekly and recorded as tumor incidence (number of papilloma bearers in percentage) and tumor yield (number of papillomas/surviving mice).
) and K6.e12S-LOX/50 mice (