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

Abstract

Human 15-lipoxygenase (h15-LO) is present on chromosome 17p13.3 in close proximity to the tumor-suppressor gene, p53. 15-LO is implicated in antiinflammation, membrane remodeling, and cancer development/metastasis. The murine BALB/c embryo fibroblast cell line, (10)1val, expresses p53 in mutant (mt) conformation when grown at 39 degrees C and in wild-type conformation when grown at 32 degrees C. Transfection of h15-LO promoter constructs (driving luciferase reporter) into (10)1val cells and into p53-deficient (10)1 cells resulted in a marked increase in h15-LO promoter activity in (10)1val cells at 39 degrees C, but not at 32 degrees C, or as compared with (10)1 cells. Transfection of h15-LO promoter deletion constructs, however, resulted in total loss of activity in both cell types at 32 degrees C and 39 degrees C. Cotransfection of (10)1 cells with h15-LO promoter (driving luciferase reporter) along with increasing levels of a mt p53 expression vector demonstrated dose-dependent capacity of mt p53 to induce 15-LO promoter activity. No effect was observed with wild-type p53. In contrast to h15-LO promoter activity, (10)1val cells had significantly lower levels of endogenous (murine) 12/15-LO (mouse analog of h15-LO) mRNA and protein when grown at 39 degrees C compared with cells grown at 32 degrees C. Our data support the hypothesis that loss of a tumor-suppressor gene (p53), or "gain-of-function activities" resulting from the expression of its mutant forms, regulates 15-LO promoter activity in man and in mouse, albeit in directionally opposite manners. The studies define a direct link between 15-LO activity and an established tumor-suppressor gene located in close chromosomal proximity.

Figure 1

Schematic linear map of human 15-LO promoter–luciferase plasmids and their transcription activity in (10)1 and (10)1val cells. Plasmids containing various lengths of human 15-LO promoter 5′ of the luciferase gene were constructed as described in Materials and Methods. The sequence numbers in the map correspond with those published by Kelavkar et al. (49), with the major transcription start site set to +1. Plasmid DNA (1 μg) was transfected into (10)1 cells by FuGENE 6 Transfection reagent. The cells were grown in appropriate medium containing 10% FBS at 37, 32, and 39°C, respectively, for 24 h, depending on individual experiments, before being harvested for determination of luciferase activity. Each experiment was repeated at least three times with duplicate samples. The values shown on the right are the averages ±SD of the relative promoter activities.

Figure 2

Effects of wt or mt p53 on the 15-LO promoter in (10)1 cells. (10)1 cells were cotransfected with LOPB5 (1.0 μg) along with wt or mt p53 expression plasmid DNA (0.1 μg). The amount of DNA in each transfection was normalized with empty vector. The cells were grown in appropriate medium containing 10% FBS for 24 h before being harvested for determination of luciferase activity. Each experiment was repeated twice with triplicate samples. The values are expressed as a percentage of the luciferase activity in lysates from cells cotransfected with the control CMV-Neo-Bam vector only and are the means (±SD) of activities from triplicate samples in a representative experiment.

Figure 3

Effects of different amounts of p53 on human 15-LO promoter activity in (10)1 cells. (10)1 cells were cotransfected with LOPB5 (1.0 μg) and various amounts of wt or mt p53 expression plasmid. The DNA for each transfection was brought up to 2.0 μg with the CMV-Neo-Bam control vector. The cells were grown with 10% FBS in medium at appropriate temperatures for 24 h and harvested for luciferase assay. Each experiment was repeated three times with duplicate samples. Values are expressed as percentages of the control activity (0%) in lysates from cells cotransfected with LOPB5 and the CMV-Neo-Bam control vector and are means ± SD (bars) of luciferase activities from duplicate samples in a representative experiment.

Figure 4

Expression of wt and mt p53 in transfected (10)1 cells. (10)1 cells were transfected with wt or mt p53 expression plasmid DNA (1.0 μg) without any 15-LO promoter vector. The cells were grown and lysates were prepared as described in Fig. 3. Western blotting was conducted as described in Materials and Methods. Lanes: 1, (10)1; 2, wt p53; 3, p53–175 (mt p53); and 4, Escherichia coli-expressed p53 protein (standard).

Figure 5

Expression of mt p53 reduces 12/15-LO protein. (10)1 and (10)1val cells were plated and incubated at 37°C for 24 h before being shifted to either 39 or 32°C for another 24 h. Nuclear and cytoplasmic proteins were extracted for Western blotting as described in Materials and Methods. The 75-kDa protein band reactive with 12-LO antiserum is indicated. (10)1val cells have predominantly wt p53 at 32°C but have mutant p53 at 39°C. (10)1 cells contain no p53 protein.

Figure 6

Expression of mt p53 decreases the steady-state level of 12/15-LO mRNA. (10)1 and (10)1val cells were plated and incubated at 37°C for 24 h before being shifted to either 39 or 32°C for another 24 h. Total RNA was extracted for Northern blotting as described in Materials and Methods. The blot first was probed with a ³²P-radiolabeled insert of the mouse 12/15-LO gene. After being stripped, the membrane was reprobed with mouse α-actin.