Effects of mutant p53 expression on human 15-lipoxygenase-promoter activity and murine 12/15-lipoxygenase gene expression: Evidence that 15-lipoxygenase is a mutator gene

Plasmids and Cell Culture.

The human p53 expression plasmids and the empty vector used in this study have been described previously (9). All of the p53 constitutive expression constructs were produced with a cytomegalovirus (CMV) promoter–enhancer expression vector. Briefly, a wt p53 cDNA was inserted into pCMV-Neo-Bam vector to express p53 (originally named pC53-SN3). Other vector-expressing mt human p53 was constructed similarly. p53–175 is a mt human p53 cDNA expression plasmid containing a substitution of histidine for arginine at amino acid 175.

p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts (40), contains large deletions in both p53 alleles, and, consequently, is deficient in the p53 protein. The (10)1val cell line was developed from (10)1 cells by transfection with an expression vector, producing a temperature-sensitive p53 protein (24, 40). At 32°C, the p53 protein is predominantly in the wt conformation, whereas at 39°C, most of the p53 protein takes on a mt form. Cells were cultured in DMEM containing 10% FBS/2 mM glutamine/100 units/ml of penicillin/100 μg/ml of streptomycin/25 μg/ml of fungizone in 5% CO₂ at 37°C.

Preparation of Protein Extracts.

The (10)1 and (10)1val cells were plated 24 h before the duplicate plates of cells were shifted to either 39 or 32°C. After 24 h at the appropriate temperature, cells were trypsinized from the plate, washed once with ice-cold PBS containing 1 mM PMSF, and centrifuged at 1,000 × g for 4 min. The cell pellet then was processed for lysate protein extraction. In all buffers, protease inhibitors were added just before use: PMSF and benzamidine at 1 mM each, aprotinin and leupeptin at 10 μg/ml, and pepstatin A at 1 μg/ml. Protein concentrations were determined by the Bio-Rad protein assay. The proteins were stored in aliquots at −70°C.

SDS/PAGE and Western Blotting.

For detection of 12/15-lipoxygenase (12/15-LO) protein, a 25-μg sample of cell lysate protein (2 × 10⁶ cells) extract was mixed with loading buffer, boiled for 5 min, and then loaded onto a 10% polyacrylamide gel containing 0.1% SDS. The gel was run at 200 V for 40 min. The separated proteins were transferred onto a nitrocellulose membrane by electroblotting. Equivalent loading was checked by Ponceau S staining of the blot. After being blocked with 5% nonfat dry milk in PBS, the nitrocellulose membrane was incubated with polyclonal 12-lipoxygenase (LX12A) antiserum (Oxford Biomedical Research, Oxford, MI) for 1 h at room temperature. After incubation with donkey anti-sheep peroxidase second antibody (1:10,000 dilution), the 12/15-LO protein was visualized by using the Luminol/Enhancer (enhanced chemiluminescence, ECL) solutions as described by the manufacturer.

For detection of p53 protein, 25 μg of cell lysate protein was mixed with loading buffer, boiled for 5 min, loaded onto a 10% polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane as described above. The membrane was blocked overnight with 5% nonfat dry milk in PBS and incubated for 1 h at room temperature with the p53 antibody (1:1,500 dilution). After incubation with rabbit anti-sheep peroxidase second antibody (1:5,000 dilution), the p53 protein was visualized by using the Luminol/Enhancer as described above.

Isolation and Analysis of mRNAs.

The (10)1 and (10)1val cells were plated in 10-cm-diameter plates for 24 h at 37°C before being shifted to either 39 or 32°C for an additional 24 h. The cells then were harvested, and RNA was extracted for Northern blot analysis as described previously (49). Briefly, 25 μg of total RNA was electrophoresed on a 1% agarose/6% formaldehyde gel and transferred to a Duralon-UV membrane, which was then UV cross-linked and subjected to prehybridization and then hybridization with the 12/15-LO cDNA probe. After hybridization, the membrane was washed and exposed to x-ray film for autoradiography. The same membrane then was stripped and rehybridized with α-actin probe as an internal standard. The DNA probe used to analyze 12/15-LO mRNA was a 106-bp DNA fragment from the coding region of a rat 12/15-LO gene (4). A 1.5-kb full-length cDNA clone of rat cytoplasmic α-actin (Stratagene) was used as the α-actin probe.

Construction of Plasmids for Transient Expression.

The 5′ flank sequence of the human 15-LO gene was PCR-amplified by using different primers. The products were subcloned into a luciferase reporter vector, pGL2-basic (Promega). The integrity of all constructs was determined by restriction enzyme site mapping, and all constructs produced by PCR were sequenced by using fluorescent methodology.

To identify DNA sequence regulating 15-LO gene expression, the region from −628 to −23 bp (+1 is adenine in the ATG start codon) was amplified by PCR from the 3-kb fragment cloned in LOPB-5, using the adapter as sense and an antisense primer. The sequence of the primers were as follows: adapter as sense primer, 5′-TTAAAGGTACCGCAGACAACAGGGAGGC-3′ (KF-1); antisense primer, 5′-TTTAACCCAAGCTTCTGGTGGAGAAGAAGGGTGG-3′ (HR-1). The 605-bp PCR product was purified and restricted with KpnI and HindIII and cloned into a plasmid pGL2 containing the luciferase reporter gene (Promega). The resulting plasmid LOPB5 and the other plasmids as described below then were transiently transfected into the (10)1 and (10)1val cells. The luciferase activity assay demonstrated that the LOPB5 clearly had promoter activity in the cells tested. To determine further the site of the minimal 15-LO promoter region, a series of deletion mutants was constructed by using the previously described procedure. Therefore, individual fragments of various sizes encompassing the promoter region were subcloned in the plasmid pGL2. Three antisense primers, 5′-TTTAACCCAAGCTTTACAGCAGGCAGGCGAGG-3′ (HR-2) and 5′-TTAACCCAAGCTTCAAGGGGCAGATTCA-3′ (HR-3), and a sense primer, 5′-TTAAAGGTACCGTGGTACACACGT GC-3′ (KF-2), were used to generate PCR-amplified fragments of various sizes.

A 318-bp fragment, namely LOKB1 (−628 to −310 nt) with KF-1 and HR-2, a 338-bp fragment, LOKB2 (−361 to −23 nt) with KF-2 and HR-1, and a 196-bp fragment, LOKB3 (−361 to −165 nt) with KF-2 and HR-3, were generated and cloned in the reporter plasmid as described (49).

Transient Transfections and Dual-Luciferase and Protein Assays.

Cells were plated at 40% confluency in 12-well plates. After 24 h, the plasmid DNA (1 μg) was transfected into the cells by FuGENE 6 Transfection reagent (Boehringer Mannheim). Luciferase plasmids were cotransfected with coreporter pRL-SV40 vector promoter upstream of Rluc (Promega) into (10)1 and (10)1val cells to monitor transfection efficiency. The cells were grown for 24 h before being harvested for assays. The cell extracts were prepared with the Passive lysis buffer (Promega). The cell lysate was analyzed for protein concentration (Bio-Rad) as well as for dual-luciferase reporter assay by using a luminometer with autoinjector (Turner design), as described by the manufacturer (Promega). The experiments were performed in duplicate or triplicate. Simultaneously individual expression of pRL-SV40 was checked for inhibition or increased expression by wt as well as mt p53.

Transcriptional Activity of the Human 15-LO Promoter in (10)1 Cells.

Human 15-LO is not normally expressed in murine fibroblasts. Hence, we studied the effects of p53 by using cloned human promoter as described in Materials and Methods. Toward this end, human 15-LO promoter–luciferase reporter plasmids were constructed with varying lengths of 5′ promoter sequences (Fig. 1). (10)1 cells were used in the transient transfection assays because of their negative p53 background, which is ideal for studying the role of p53 in the transcriptional regulation of exogenously transfected gene promoters (19, 30). As shown in Fig. 1, the reporter construct (LOPB5) of 600 bp containing human 15-LO promoter sequences gave maximal transcriptional activity in (10)1val cells grown at 39°C and none at 32°C as compared with (10)1 cells transfected and grown under similar conditions. Stepwise deletion of 5′ sequences from the promoter resulted in a concomitant decrease in the luciferase activity in both (10)1 and (10)1val cells grown at 32^o and 39°C, respectively, except by the LOKB2 construct.

Human 15-LO Promoter Activity in (10)1 and (10)1val Cells.

Expression of wt p53 had no effect on the expression of murine 12/15-LO mRNA and protein in (10)1val cells grown at 32°C whereas, surprisingly, at 39°C there was decreased expression (Figs. 5 and 6). To test whether similar effects are seen in p53 interaction with the human 15-LO promoter activity, the promoter–luciferase reporters, namely, LOPB5, LOKB1, LOKB2, and LOKB3, were transiently transfected, in duplicate, into (10)1 and (10)1val cells at 37°C and then shifted to 32 or 39°C for 20 h. Transfection of 15-LO basal promoter and deletion constructs lacking regions essential for promoter activity into p53-deficient (10)1 and (10)1val cells expressing mt or wt p53, depending on temperatures grown, indicates an increase in activity in (10)1val cells at 39°C with no effect at 32°C in the basal promoter, compared with (10)1 cells transfected similarly. However, in the deletion constructs there was a loss in activity in cells at both 32°C and 39°C, respectively, except by the LOKB2 construct (Fig. 1). These data were substantiated further by using (10)1 cells cotransfected with wt p53 and 15-LO promoter constructs and grown at 32°C and, simultaneously, mt 53 and 15-LO promoter constructs grown at 39°C (Fig. 2). These results together demonstrate that expression of p53 in the mt conformation decreased murine 12/15-LO transcriptional activity but increased the human 15-LO promoter luciferase activity. These differences reflect reciprocal regulation of these genes by p53 in mice vs. humans, highlighting the extreme care that must be exercised in interpreting mice data with respect to the biology of tumor-suppressor functions.

mt p53 Enhances Promoter Activity of Human 15-LO.

To study the effects and possible interactions, if any, of p53 expression with the human 15-LO gene promoter, (10)1 cells were transfected with LOPB5 along with various amounts of expression plasmids encoding wt or mt p53 (Fig. 3). Transfection with the wt p53 expression plasmid did not show any effect on the 15-LO promoter activity whereas that with the mt p53 expression plasmid at levels as low as 10 ng caused a significant increase of activity (4- to 5-fold compared with wt p53). Increasing amounts of mt p53 plasmid used in the transfection resulted in increased 15-LO promoter activity (Fig. 3). However, greater amounts of p53 plasmid did not increase the induction proportionally, indicating a saturable mechanism for this p53 function. Also, transfection with low or as high as 1.0-μg levels of wt p53 expression plasmid had no significant effect on the 15-LO promoter activity.

Western blot analyses of protein from transfected cell lysates were performed to confirm that wt and mt p53 proteins were produced in the cells transfected with the p53 expression vectors. With transfection of 1.0 μg of the p53 construct DNA, similar levels of wt and mt p53 were detected (Fig. 4). These results confirm that the significant effect on 15-LO promoter activity by mt p53 is because of mt p53 expression in the transfected cells and is unaffected by wt p53.

Expression of mt p53 Reduces Murine 12/15-LO protein.

To study the effect of p53 expression on endogenous 12/15-LO protein, we used the murine fibroblast (10)1 cell line, which is negative for p53 protein, and the derivative (10)1val cell line, which expresses a temperature-sensitive p53 protein. When the (10)1val cells are grown at 32°C, the protein is predominantly in the wt conformation, whereas at 39°C, most of the p53 protein is in the mutant conformation. Western blot analysis showed that (10)1val cells incubated at 39°C for 24 h had a dramatically decreased level of 12/15-LO protein compared with the cells incubated at 32°C (Fig. 5). The decrease in 12/15-LO protein is a consequence of mt p53 and not temperature effects, because shifting (10)1 cells to 32°C for the same time period had a minimal effect on the expression of 12/15-LO protein.

Expression of mt p53 Decreases the Steady-State Level of Murine 12/15-LO.

The decreased amount of 12/15-LO protein in cells expressing mt p53 could result from either pre- or posttranslational events. With the same cell lines and temperatures as described for Fig. 5, the effect of p53 expression on endogenous 12/15-LO mRNAs was determined by Northern blot analysis. As shown in Fig. 6, the steady-state level of 12/15-LO was decreased markedly by mt p53 expression, as reflected by a sharp reduction of the 12/15-LO mRNA in (10)1val cells when they are incubated at 39°C for 24 h rather than at 32°C. Under the same conditions, the temperature had no effect on the level of 12/15-LO mRNA in (10)1 cells. When reprobed with α-actin cDNA as an internal loading control, the same membrane showed similar levels of α-actin mRNA at either temperature in both cell lines, indicating that mt p53 selectively inhibits 12/15-LO gene expression. Thus, mt p53 expression in (10)1val cells at permissive temperatures effectively decreases 12/15-LO mRNA levels, thus reducing 12/15-LO protein at a translational level.

Mutant p53 has been shown previously to bind abnormally to DNA in vitro (50) and only recently to have specific binding (51) by different classes of human p53 mutants (52–54). Mutant p53 proteins also have been shown to stimulate the expression of some genes, such as the multidrug resistance (MDR) gene (28), and bind to MAR/SAR DNA elements (51). Investigations therefore are underway to understand the direct interaction between p53 and probable suppressors or binding proteins that stimulate the expression of h15-LO and repress the activity of murine 12/15-LO.

The present studies suggest that mt p53 stimulation may be a form of “squelching,” whereby one or more factors inhibitory or regulatory for the transcription of LOs are regulated secondarily. Consistent with this idea, similar results have been obtained by others (55–58). Our data with 15-LO and p53 studies propose an interesting prediction: tumors with mutant missense p53 protein may well be more aggressive or have a poorer prognosis than tumors with no p53 proteins (deletions impart only a “loss of function” mutation). Thus, it may be that a missense p53 mutation contributing a “gain of new function” is worse than no p53 gene product at all. Previous studies demonstrating that lipoxins inhibit human natural killer cell cytotoxicity (1), thereby potentially hampering tumor surveillance, suggest a synergizing (or even mediating?) effect for these 15-LO products in enhancing the tumor-promoting action of mt p53. 15-LO expression then would be the first demonstration of a “gain-of-function” mutation imparted by p53 mutagenesis, which would promote carcinogenesis. Additionally, activation of 15-LO may lead to the generation of non-arachidonate-derived oxidation products such as 13-HODE (linoleic acid as substrate), which have been implicated in tumor metastasis (59). Further, non-eicosanoid-related activities of 15-LO, such as peroxidation of membrane lipids, also may contribute to alterations in cellular functions (60) arising from its de novo induction in cells containing mutant p53.

Identification of targets such as 15-LO that seem to be regulated by mutant p53, secondarily acting as mutator genes, is critical to our understanding of human tumor biology and to the development of cancer therapies.