Overexpression of a dominant-negative ornithine decarboxylase in mouse skin: effect on enzyme activity and papilloma formation

Lisa M. Shantz1,4, Yongjun Guo3, Janet A. Sawicki3, Anthony E. Pegg1,2 and Thomas G. O'Brien3

1 Departments of Cellular and Molecular Physiology H166 and
2 Department of Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA and
3 Lankenau Institute for Medical Research, Wynnewood, PA 19096, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A transgenic mouse line expressing a truncated form of the ornithine decarboxylase (ODC) dominant-negative mutant K69A/C360A under the control of the keratin 6 promoter has been established (K6/ODCdn mice). These mice were backcrossed onto both the DBA/2J and C57BL/6J backgrounds for subsequent tumorigenesis experiments utilizing an initiation/promotion protocol. In short-term experiments, expression of the ODCdn protein product was induced in the epidermis within 24 h after application of the tumor promoter tetradecanoyl phorbol acetate (TPA) to the skin, and ODC activity in the epidermis of K6/ODCdn mice was reduced by at least 75% compared with littermate controls. However, in tumorigenesis experiments utilizing a variety of initiator (7,12-dimethylbenz[a]anthracene; DMBA) and promoter (TPA) concentrations, K6/ODCdn mice formed at least as many tumors as their littermate controls regardless of background strain. In experiments utilizing chrysarobin, a tumor promoter with a different mechanism of action than TPA, again there was no significant difference in tumor formation between K6/ODCdn mice and littermate controls. Similarly, when K6/ODCdn mice were crossed with K5/ODC mice, a transgenic line described previously which forms tumors without application of a promoting agent, double transgenic mice formed as many tumors as mice expressing the K5/ODC transgene alone. Analysis of epidermis following multiple TPA applications revealed a dramatic spike in ODC activity in both K6/ODCdn mice and non-transgenic mice after six applications, and western blot analysis suggested a stabilization of endogenous wild-type ODC in K6/ODCdn transgenic mice. ODC activity, endogenous protein and polyamines were also elevated in tumors from K6/ODCdn mice. The accumulation of endogenous ODC protein is most probably the result of competition from the transgene-derived ODCdn protein for binding of antizyme, which is known to regulate ODC activity by stimulating degradation of the ODC protein.

Abbreviations: DFMO, {alpha}-difluoromethylornithine; DMBA, 7,12-dimethylbenz[a]anthracene; K6, keratin 6; ODC, ornithine decarboxylase; ORS, outer root sheath; TPA, tetradecanoyl phorbol acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous work from this laboratory has demonstrated that overexpression of an ornithine decarboxylase (ODC) transgene in murine skin greatly increases the susceptibility of this tissue to tumor development (1–3). In addition to enhanced susceptibility to carcinogenesis, ODC overexpression also caused other phenotypic changes including alopecia, excessive nail growth and age-related progressive skin wrinkling. In skin, perhaps because of its high ornithine content (4), upregulation of ODC leads to substantial elevations in putrescine and spermidine levels that presumably mediate the profound phenotypic alterations observed.

The experiments described here were designed to inhibit endogenous ODC activity by expression of the ODC mutant, K69A/C360A. This ODC mutant has been shown previously to behave in a dominant-negative manner, and intracellular expression of this mutant can both reduce ODC activity and reverse transformation of cultured cells (5–8). In order to test whether a similar approach would be effective in vivo, we produced a transgenic mouse expressing the K69A/C360A ODC mutant under the control of a bovine cytokeratin 6 promoter/regulatory region. This promoter construct has been used successfully to target ODC expression to the outer root sheath (ORS) keratinocytes of the hair follicle, the presumptive target cells for carcinogens in mouse skin (1–3). During normal epidermal differentiation, cytokeratin K6 and its partner K16 are expressed specifically in the ORS of the hair follicles. During hyperproliferative states such as that caused by treatment with tetradecanoyl phorbol acetate (TPA), cytokeratin K6 is expressed throughout the interfollicular epidermis as well as in the ORS (9–12). This transgenic approach to lower intracellular ODC activity has advantages over the use of ODC inhibitors such as {alpha}-difluoromethylornithine (DFMO), as inhibition of enzyme activity does not depend on the continued presence of the inhibitor, and there is no question as to whether the ODC protein is the only target.

The ODCdn protein used in these experiments has been truncated to 425 amino acids by the insertion of a stop codon. It has been shown previously that truncation of ODC at this point results in a protein with a dramatically extended half-life (13), and this is thought to be due to the resistance of the truncated form of ODC to degradation. The wild-type form of ODC has an extremely short half-life (20–30 min), and its degradation is mediated by the protein antizyme, which binds to the monomeric form of ODC, preventing formation of the enzymatically active homodimer, and then targets ODC to the 26S proteasome where it undergoes ubiquitin-independent degradation (14–19). We used the K6/ODCdn transgenic model to determine whether overexpression of the dominant-negative mutant ODC would inhibit endogenous ODC activity following application of a tumor promoter, and also to study whether the decrease in ODC activity brought about by expression of this mutant could influence tumor development in skin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Taq polymerase used in PCR reactions was from Promega (Madison, WI). dNTPs were from Amersham Pharmacia Biotech (Piscataway, NJ). L-[1-14C]Ornithine (52 mCi/mmol) was from DuPont-New England Nuclear (Boston, MA). TPA was purchased from CalBiochem (San Diego, CA). 7,12-Dimethylbenz[a]anthracene (DMBA) was from Kodak Laboratory Chemicals (Rochester, NY). Chrysarobin was a generous gift from Dr John DiGiovanni (University of Texas M.D. Anderson Cancer Center, Smithville, TX). Other biochemical reagents used were from Sigma Chemical Co. (St Louis, MO) and Bio-Rad (Richmond, CA).

Production and characterization of transgenic mice
Transgenic mice were generated by DNA microinjection of fertilized B6C3F2 oocytes using standard techniques (20). The transgene was a 20 kb fragment derived from a vector containing a bovine K IV* (K6) minimal promoter/regulatory region (1) upstream of a murine ODC cDNA which had been engineered previously to contain point mutations to alanine at lysine 69 and cysteine 360 residues, as well as an introduced stop codon at position 425 (5). The fragment to be used in microinjection was first purified and then precipitated and resuspended in microinjection buffer (10 mM Tris–HCl pH 7.4, 0.25 mM EDTA). Genomic DNA was isolated from the tails of potential transgenic mice and subjected to PCR analysis to identify mice bearing the transgene using primers described previously (1). PCR was confirmed by Southern blot analysis (data not shown). Transgenic mice were designated K6/ODCdn.

Breeding of K6/ODCdn mice onto the DBA/2J or C57BL/6J background
For carcinogenesis experiments on mice expressing the K6/ODCdn transgene, founder males and transgenic male progeny were bred with either DBA/2J or C57BL/6J females (Jackson Laboratories, Bar Harbor, ME) for five generations. The transgene is therefore maintained in the hemizygous state. Neither the founders nor subsequent backcross progeny had any obvious phenotypic abnormalities. DBA/2J and C57BL/6J mice were chosen for these studies because they exhibit different degrees of sensitivity to the skin tumor initiation/promotion protocol using DMBA and TPA, with DBA/2J mice being very sensitive and C57BL/6J resistant (21). All mice used in the experiments described were at least fifth generation transgenics, ensuring that ~97% of genes are derived from the appropriate background (20).

Biochemical analyses
ODC was assayed at 37°C by measuring the release of 14CO2 from L-[1-14C]ornithine as described previously (22). To assess dominant-negative ODC activity, mixing experiments were performed as follows: different volumes of extracts from the K6/ODCdn epidermis were added to tubes containing a given amount of wild-type ODC extract. Units of dominant-negative activity were calculated from the difference of the expected ODC activity (sum of wild-type ODC units and units measured separately in the dominant-negative extract) and the observed ODC activity of the mixture. For polyamine analysis, samples were acid extracted using 10% TCA and analyzed for polyamines using reverse phase HPLC analysis as described previously (23).

Induction of endogenous ODC and the dominant-negative transgene in response to TPA
The dorsal area of each mouse was shaved with surgical clippers and the mice were allowed to rest for 24 h. At t = 0, either acetone or 6.8 nmol TPA in acetone (total vol = 200 µl) was applied. At time points between 0 and 88 h, mice were killed and a depilatory agent was applied to the shaved area of the back. After 5 min, the depilatory agent was removed by rinsing with cold tap water. The skin was excised from the back with scissors and placed in ice water for 5 min, transferred to a 55°C water bath for 20 s, then immediately returned to ice water for another 5 min. The skin was placed epidermis side up on a glass plate kept on ice. The skin was immobilized and the epidermis was scraped off using a razor blade and flash frozen in liquid nitrogen. The dermis was also flash frozen (1).

The samples were resuspended in buffer A (25 mM Tris–HCl pH 7.5, 2.5 mM DTT, 0.1 mM EDTA) for subsequent analysis. The epidermal samples were sonicated on ice for 30 s. The dermal samples were homogenized for 30 s on ice using a Polytron homogenizer. All samples were centrifuged at 30 000 g for 30 min at 4°C. Each supernatant sample was assayed in duplicate for ODC activity and total protein. One sample from each time point (100 µg total protein) was also subjected to SDS–PAGE and western blot analysis. ODC proteins were detected using a purified rabbit polyclonal antibody against mouse ODC (6), and quantified using a fluorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Immunocytochemical analysis
Tissues were fixed overnight in Fekete's solution (60% ethanol, 3.2% formaldehyde and 0.75 M acetic acid), embedded in paraffin and 5 µm sections were cut for immunocytochemistry. Sections were incubated with a 1:500 dilution of rabbit ODC antiserum and specific staining was detected with an ABC Vectastain kit (Vector Laboratories, Burlingame, CA).

Tumorigenesis experiments
At the fifth backcross generation to C57BL/6J or DBA/2J backgrounds, mice were used for tumorigenesis experiments. For these experiments, 6–8-week-old mice were initiated by treatment with a single dose of DMBA (either 400 or 800 nmol for C57BL/6J mice, 200 nmol for DBA/2J mice) followed by twice weekly applications of either 17 nmol TPA for C57BL/6J mice, or 2.0 and 6.8 nmol for DBA/2J mice. The number of tumors that developed (beginning at 7 weeks of TPA treatment) was counted at weekly intervals for 16–26 weeks after initiation. In another experiment, C57BL/6J hemizygous K6/ODCdn females were mated with hemizygous K5/ODC males (2) and newborn pups treated with 200 nmol DMBA as described. At weaning, mice bearing the K5/ODC transgene, ~50% of which should also carry the K6/ODCdn transgene, were identified by their loss-of-hair phenotype and by PCR as described previously (2). These mice were observed for tumor development over a period of 24 weeks. Genotyping was unknown to the individual who counted the tumors. All animal protocols were approved by the Animal Care and Use Committees of the Pennsylvania State University College of Medicine and The Lankenau Institute for Medical Research. Statistical analysis carried out on the data from the tumorigenesis experiment used the non-parametric Wilcoxon Rank Sum test. Analysis was performed in the Statview package (SAS).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Time course of ODC and ODCdn transgene induction in response to TPA
The objective of these experiments was to determine the induction of both endogenous ODC and the dominant-negative K69A/C360A transgene over an 88 h time period in response to multiple applications of 6.8 nmol of TPA administered topically to DBA/2J K6/ODCdn mice and their littermate controls (males and females ~7 weeks old). The concentration of TPA chosen was that found to produce papillomas in 100% of DBA/2J mice (21,24), and the 88 h time course approximates the timing of a twice weekly TPA promotion protocol.

The ODC activities in the epidermis of both K6/ODCdn transgenics and normal littermates are shown in Figure 1Go. As mentioned above, it has been shown previously that endogenous ODC is induced sharply several hours after TPA application (25). The K6 promoter, which drives expression of the transgene, is also induced by TPA, but more slowly over a 24–36 h period (2,26). Therefore, in order to examine whether expression of the transgene can block ODC induction after repeated TPA applications, some mice received a second dose of TPA at 72 h. As expected, ODC activity in the epidermis of control mice spiked 6 h after the first TPA application, but was reduced almost to untreated levels by ~12 h. A similar induction was seen in response to the second TPA application (Figure 1Go). In contrast, ODC activity in the epidermis of transgenic mice was only 27% compared with that of littermate controls after 6 h (activity of 74 versus 268 pmol/30 min/mg in transgenics and controls, respectively). After the second TPA application, ODC activity in transgenics was also significantly lowered (63 versus 173 pmol/30min/mg protein in controls). Similar results were seen on the C57BL/6J background treated with 17 nmol of TPA (data not shown).



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Fig. 1. Time course of ODC activity in epidermis of K6/ODCdn mice (Tg) and normal DBA/2J littermates after application of TPA to the skin. K6/ODCdn mice (filled square) and littermate controls (square) on the DBA/2J background (6–8-week-old) were treated with 6.8 nmol TPA and killed at the indicated times. The epidermis was prepared and ODC activity assays were performed as described in the Materials and methods. n = 3 mice per time point for each group, and the results are expressed as the mean ODC activity in pmol/30 min/mg protein ± SD.

 
Western blots were used to measure expression of both endogenous and transgenic ODC proteins in the epidermis of DBA/2J transgenic mice (Figure 2Go). Like the endogenous ODC activity, wild-type ODC protein (molecular weight of 51 kDa) is visible only transiently after TPA application, particularly after the second application (see 76 and 80 h). On the other hand, the transgene-derived ODCdn protein, which is truncated from 461 to 425 amino acids and has a molecular weight of ~47 kDa, is clearly induced over time in response to TPA treatment, with maximal induction 36 h after TPA (Figure 2Go). In western blots of non-transgenic epidermis, no ODC is visible at any time point tested, despite the obvious induction in ODC activity (data not shown).



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Fig. 2. Time course of wild-type endogenous ODC protein and transgene-derived ODCdn protein expression in epidermis of K6/ODCdn mice and normal DBA/2J littermates after application of TPA to the skin. Cytosolic extract (100 µg per sample) was prepared from epidermis of the same mice described in Figure 1Go, separated on a 10% SDS PAGE gel, and western blot analysis for ODC protein was performed as described in the Materials and methods. The band for wild-type endogenous ODC corresponds to a molecular weight of 51 kDa, while the truncated transgene-derived dominant-negative mutant band corresponds to a molecular weight of 47 kDa.

 
The same response of transgene expression was also observed in western blots of dermal samples, although endogenous ODC was not visible at any time in these samples (data not shown). This may seem a contradiction, as the expression of the K6 promoter is limited to hair follicles and interfollicular epidermis. However, although the cells of the ORS are epidermal, the hair follicle extends into the dermis and the entire follicle cannot be isolated with the epidermal fraction upon scraping the epidermis from the dermis. Thus, transgene expression is seen in both fractions.

Immunocytochemical analysis of mice treated once with TPA and killed 36 h later, confirmed that ODC protein in the hair follicles and interfollicular epidermis of the K6/ODCdn transgenic mice (Figure 3AGo) is much more highly expressed than that in non-transgenic littermates (Figure 3BGo). Based on the levels of wild-type ODC and ODCdn proteins detected in western blots, this difference is presumably due to induction of the transgene by TPA. However, although it is clear that expression of the transgene is induced by TPA treatment, ODCdn protein expression is visible in the hair follicles from 2 days of age in transgenic pups, while there is virtually no staining in control littermates (day 23, Figure 3C and DGo). Therefore, the reduction in ODC activity in transgenic mice after one TPA application is primarily due to this constitutive expression of the transgene-derived ODCdn protein, as initial transgene induction occurs more slowly than endogenous ODC induction (see Figures 1 and 2GoGo).



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Fig. 3. Expression of ODC protein in the skin of K6/ODCdn transgenic mice and littermate controls. Immunostaining of ODC in hair follicles and interfollicular epidermis of 6-week-old transgenic (A) and control (B) mice on the DBA/2J background 36 h after treatment with 6.8 nmol TPA. Staining is much more intense in the transgenic mice due to induction of transgene expression by TPA. In the absence of TPA induction, transgene-derived ODCdn protein is still easily visualized in the hair follicles of 23-day-old transgenic mice (C), although no staining is visible in the interfollicular epidermis. Untreated 23-day-old control mice (D), show no staining for ODC protein. [The dark spots in (D) represent hair shafts present in the follicles in this cross section of the skin.]

 
Activity of the ODCdn subunit
The preceding results are consistent with a mechanism in which monomers of the ODCdn protein form heterodimers with endogenous wild-type ODC monomers or homodimers with each other. We have shown previously that the combination of wild-type ODC and ODCdn proteins in vitro results in a redistribution of monomers such that wild-type and mutant monomers combine to form heterodimers with reduced enzyme activity (5,8). To test the functionality of the in vivo-expressed ODCdn protein, mixing experiments were carried out with wild-type ODC and epidermal extracts from C57BL/6J K6/ODCdn mice treated with TPA once, twice or four times. There was very little inhibitory activity in the extract from mice treated once with TPA but in extracts from mice treated two and four times there was a clear dose-dependent increase in inhibitory activity (Table IGo). Extracts from non-transgenic mice treated with acetone or once or four times with TPA always gave the expected (additive) enzymatic activity in mixing experiments (data not shown). The results demonstrate that during repetitive TPA treatments (analogous to a tumor promotion protocol), sufficient ODCdn subunits are expressed to maintain endogenous wild-type ODC activity at low levels.


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Table I. Mixing experiments with wild-type (wt) ODC and extracts (ext) from C57BL/6J K6/ODCdn epidermis
 
Tumorigenesis experiments using an initiation/promotion protocol
Having established that the K6/ODCdn transgene is expressed at high levels in an apparently functionally active form during repetitive tumor promoter treatment, the susceptibility of K6/ODCdn mice and non-transgenic littermates to an initiation/promotion protocol was determined. In this experiment, 6-week-old DBA/2J mice were initiated with 200 nmol DMBA and subsequently promoted with either acetone or 6.8 nmol TPA twice weekly. Tumors were counted every week from 6 to 16 weeks. The results of the final tumor counts are shown in Table IIGo. Papillomas appeared after 7 weeks in both transgenic mice and their littermate controls, and there was no significant difference in the number of papillomas over a 16-week period in the DBA/2J background. Acetone-treated mice formed no papillomas during the course of the experiment (data not shown). A second carcinogenesis experiment was performed using an initiating dose of 200 nmol DMBA and a promoting dose of 2 nmol TPA twice per week. After 26 weeks of promotion, again there was no difference in the number of papillomas formed in transgenic mice and their littermate controls (Table IIGo). Initiating doses of 400 and 800 nmol DMBA were chosen for C57BL/6J K6/ODCdn mice as the genetic background is relatively resistant to an initiation/promotion protocol compared with other strains (24,27). In contrast to the DBA/2J background, after 20 weeks of twice weekly TPA applications, the tumor yield (tumors/mouse) was significantly greater in K6/ODCdn mice versus non-transgenic littermates (Table IIGo).


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Table II. Tumorigenesis experiments with K6/ODCdn mice and littermate controls
 
Previous studies have suggested that activation of protein kinase C mediates the induction of ODC by phorbol esters (28). However, other classes of tumor promoters, which do not activate protein kinase C, such as the anthrone chrysarobin, induce ODC effectively in mouse epidermis (29), suggesting alternative pathways for ODC induction by these agents. In addition, the induction of endogenous ODC by chrysarobin occurs with peak induction at about 36 h after application of the promoter (30). This is similar to the timing of the K6 cytokeratin response to tumor promoter application. In contrast, the induction of endogenous ODC and the K6-driven ODCdn transgene by TPA do not follow similar kinetics, with the endogenous induction being much more rapid (4–6 h) compared with induction of the transgene (24–36 h) (Figure 2Go). To study whether tumor promotion by chrysarobin would be inhibited more effectively by the ODCdn transgene, transgenic DBA/2J mice and their littermate controls were treated with 200 nmol DMBA followed by weekly treatments with 220 nmol chrysarobin. After 26 weeks of promotion, tumor yields were 0.23 ± 0.42 [standard deviation (SD); n = 13] for transgenics and 0.76 ± 1.25 (SD; n = 13) for controls. These values are not statistically different (P = 0.13), suggesting that the inability of the dominant-negative transgene to inhibit tumorigenesis is not related to the mechanism or timing of ODC induction.

The ability of the K6/ODCdn subunit to modify tumorigenesis was also evaluated in a transgenic model. We have described the tumorigenic effect of a single dose of 200 nmol DMBA in newborn K5/ODC mice on a C57BL/6J background. These mice overexpress a truncated ODC with wild-type activity (2,3). The K5 promoter is a non-inducible promoter, which targets both the basal interfollicular keratinocytes and the ORS keratinocytes (2,31). Appropriate matings (see Materials and methods) between K5/ODC and K6/ODCdn mice produce four possible genotypes, two of which were used in a single-dose DMBA protocol: K5/ODC (+/–)/K6/ODCdn (+/–) and K5/ODC (+/–)/K6/ODCdn (–/–). As TPA treatment is not required to produce tumors in DMBA-treated K5/ODC mice, this experimental protocol avoids the strong inducing effect of TPA on the K6/ODCdn transgene. Twenty weeks after DMBA treatment, the tumor yields in mice of the two genotypes were identical: 10.5 ± 4.2 (SD; n = 23) tumors/mouse in K5/ODC (+/–) mice and 10.9 ± 5.9 (SD; n = 17) tumors/mouse in double transgenic K5/ODC (+/–)/K6/ODCdn (+/–) mice.

Effect of multiple TPA applications on epidermal ODC activity and protein expression
The experiments described in Figure 1Go show that during short-term TPA treatment, ODC activity is effectively reduced in ODCdn mice. However, in carcinogenesis experiments, ODCdn mice form at least as many tumors as their littermate controls. To understand these results more fully, a long-term TPA experiment was performed in which both ODCdn mice and littermate controls on the DBA/2J background were first initiated with 200 nmol DMBA and then promoted two times per week with TPA. After 1, 2, 6, 12 and 18 TPA applications, two mice were killed from each group. The epidermis was isolated and ODC activity and protein were measured. The results are presented in Figure 4Go. The results show that after six TPA applications there is a dramatic spike of both ODC activity (4000 versus 2500 pmol/30 min/mg in controls and transgenics, respectively) and wild-type ODC protein in the transgenics as well as the controls. This superinduction of ODC after multiple TPA applications has been documented by other investigators (28), and suggests that ODC activity in the skin of K6/ODCdn mice after multiple TPA applications is sufficiently elevated to support tumor formation, in spite of the presence of the dominant-negative ODC protein. After six TPA applications, the endogenous wild-type ODC protein, as measured by band density on western blot, is ~10-fold higher in transgenics compared with controls (Figure 4BGo). Similar results were seen in Figure 2Go, which shows that endogenous wild-type ODC protein is visible following TPA application to transgenic skin with a high ODCdn protein content, suggesting a stabilization of endogenous wild-type ODC protein in the presence of high levels of the ODCdn protein.



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Fig. 4. Effect of multiple TPA applications on ODC activity and protein expression in the epidermis of K6/ODCdn mice and normal DBA/2J littermate controls. K6/ODCdn mice and littermate controls on the DBA/2J background (6–8-week-old) were initiated with 200 nmol DMBA then treated with 6.8 nmol TPA for the indicated number of times. Mice were killed 24–48 h after TPA application, and ODC activity and the expression of both endogenous wild-type ODC protein and transgene-derived ODCdn protein were measured as described in the Materials and methods. (A) ODC activity in transgenic mice (filled circle) and littermate controls (filled square) treated with one application of 200 nmol DMBA and 1–18 applications of 6.8 nmol TPA. (B) Density of wild-type endogenous ODC (filled circle, transgenic mice; filled square, control mice) and transgene-derived ODCdn protein (circle) bands from the same mice quantitated from western blots of 100 µg cytosolic protein.

 
K6/ODCdn expression in tumors
The enhancement of tumorigenesis in C57BL/6J K6/ODCdn mice versus littermate controls and lack of effect in the DBA/2J strain were unexpected based on previous work from several laboratories that suggested upregulation of ODC was a necessary condition for tumor promotion (32–35). To attempt to explain this paradox, ODC protein and polyamine levels were measured in tumors from K6/ODCdn mice killed 2 weeks after the final TPA application. Figure 5Go shows a massive accumulation of the truncated ODC dominant-negative protein in the tumors of DBA/2J ODCdn mice, and also a clear accumulation of endogenous wild-type ODC. In contrast, the endogenous ODC is not visible in tumors from non-transgenic littermates. Tumor wild-type ODC levels were also elevated in C57BL/6J mice (data not shown). Perhaps more significantly, putrescine levels, as well as spermidine and spermine, were actually substantially increased in tumors derived from K6/ODCdn mice (Table IIIGo), confirming that tumor formation in the transgenic mice is accompanied by high levels of polyamines in spite of the presence of the dominant-negative ODC protein.



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Fig. 5. Expression of wild-type endogenous ODC protein and transgene-derived ODCdn protein in tumors of K6/ODCdn mice and normal DBA/2J littermates after 16 weeks, exposure to the DMBA/TPA initiation/promotion protocol. Mice were initiated with 200 nmol DMBA and promoted twice weekly with 6.8 nmol TPA. Two weeks after the final TPA application, mice were killed and tumors were isolated. Tumors were processed for western blot analysis as described in the Materials and methods. The results show the expression of ODC and ODCdn proteins in five transgenic mice and four littermate controls (100 µg cytosolic extract per sample).

 

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Table III. Polyamine levels in tumors from wild-type versus K6/ODCdn transgenic mice
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In considering an in vivo model in which to test the biologic effects of suppressing ODC function via a dominant-negative approach, a mouse model in which a transgene is driven by a strong keratin promoter offers several distinct advantages. First, some degree of tissue targeting can be achieved based on the known expression pattern of keratin genes (11,36). We have used the bovine K6 promoter/regulatory elements to target a truncated ODCdn transgene to hair follicles (10,11). Secondly, deleterious developmental effects can be avoided by choosing a keratin promoter that is expressed at high levels post-natally. Finally, keratin promoters in general are very strong, so high-level expression can be achieved. This is an important consideration for a dominant-negative approach in which a mutant competes with wild-type subunits in forming multi-subunit complexes. An additional advantage of the K6 promoter/regulatory sequence is that it is inducible by TPA (9,11,12), and the involvement of ODC induction in the two-stage model of initiation and promotion in mouse skin is well-characterized (4,25,32). The work described herein demonstrates that a functional dominant-negative ODC protein under the control of a K6 promoter/regulatory region can be expressed in skin. Presumably because of the restricted expression pattern of K6-driven transgenes in this tissue (ORS keratinocytes of hair follicles), there were no obvious phenotypic abnormalities observed in the K6/ODCdn mice, and expression was difficult to detect in unperturbed animals. However, treatment with the phorbol ester TPA was clearly able to induce expression of the ODCdn protein.

An important question to be answered was whether expression of the ODCdn protein would inhibit endogenous wild-type ODC. As ODC activity in unperturbed epidermis is very low, TPA was used to induce endogenous wild-type ODC activity (25). Because the K6-driven ODCdn subunit is also induced by TPA the lack of induction of measurable ODC activity in transgenic mice (Figure 1Go) indicates that sufficient ODCdn is expressed under these conditions to neutralize wild-type ODC activity. Western analysis confirmed that much more of the ODCdn protein is expressed after TPA treatment than the wild-type ODC protein.

Previous work has established that upregulation of an active, albeit truncated, transgene-derived ODC protein dramatically increases the susceptibility of mouse skin to tumor development following carcinogen exposure (1–3). If ODC upregulation is a necessary component of the tumor promotion stage of skin carcinogenesis, then inhibition of wild-type, endogenous ODC by expression of the ODCdn protein should inhibit tumorigenesis. However, when a classical DMBA/TPA initiation/ promotion protocol was used to test this hypothesis in two separate backgrounds, one resistant (C57BL/6J) and one sensitive (DBA/2J) to tumor promotion by TPA (21,27), an actual enhancement of tumor yield occurred in C57BL/6J mice, while no difference was seen in DBA/2J mice. Use of chrysarobin, a tumor promoter with a different mechanism of action and different time course of ODC induction than TPA (29), also showed no difference between transgenic and control DBA/2J mice. Finally, in a quite different model involving DMBA initiation of a double transgenic K5/ODC (+/–)/K6/ODCdn (+/–) C57BL/6J mouse, ODCdn expression again had no effect on tumor yield. Thus, in none of the tumorigenesis models did expression of the ODCdn protein inhibit neoplastic development.

Several possible explanations for this apparently paradoxical result can be proposed: (i) some property of ODC other than production of putrescine is important to tumorigenesis; (ii) high ODC activity is irrelevant to tumorigenesis; (iii) the residual enzymatic activity of the ODCdn protein is sufficient for papilloma formation; or (iv) the rapid exchange of subunits that is known to occur between wild-type and dominant-negative monomers (7,8) results in ODC activity that is sufficient to enhance tumorigenesis. The information available suggests that this final possibility is correct. First, the results in Figure 4Go show an ~4000-fold superinduction of ODC activity in the epidermis of non-transgenic animals after six TPA applications, and this induction is still 2500-fold in transgenic mice. Secondly, levels of wild-type ODC protein and of putrescine are elevated in transgenic tumors as well as in control tumors. These two observations support the concept that high ODC activity and putrescine production are important for tumorigenesis to occur. The increased tumor yield in K6ODCdn mice on the C57BL/6J background also supports this idea, as putrescine levels were actually higher in these mice compared with their littermate controls.

Given that the presence of the ODCdn protein reduces epidermal ODC activity in short-term experiments, what is the source of the unexpected spike in ODC activity in the transgenic animals after long-term TPA treatment? The amount of ODCdn protein in transgenics continues to increase through 12 TPA applications. However, by this time both the ODC activity and the endogenous wild-type ODC protein have substantially decreased in the epidermis. In addition, previous in vitro experiments have shown that the activity of the dominant-negative ODC protein is only 0.03% that of wild-type ODC (8). Taken together, these observations make it unlikely that the ODCdn protein would contribute significantly to total enzyme activity. The spike of ODC activity seen in the transgenic mice after six TPA applications is probably therefore the result of a rapid exchange between wild-type and dominant-negative monomers, resulting in a residual activity from wild-type homodimers (7,8).

As wild-type ODC in transgenic epidermis is at levels about 10 times those measured in the epidermis of non-transgenic littermates, the results suggest that the endogenous wild-type ODC protein is stabilized in the transgenic animals, leading to high ODC activity in spite of the presence of the ODCdn transgene. The reason for this apparent stabilization is currently under investigation. The most likely candidate is antizyme, which binds to ODC and targets it for degradation. As mentioned above, the mutant ODC is truncated to increase stability and facilitate its accumulation to high levels. However, this protein maintains its antizyme binding site (14), and has equal affinity for antizyme compared with wild-type ODC (16). Thus, antizyme may bind to the truncated ODC mutant, but this does not result in degradation because the signal to direct the ODC to the proteasome is absent (14). As a result, the endogenous wild-type ODC may be protected from antizyme-mediated proteasomal degradation because much of the available antizyme remains bound to the truncated ODC. Similar results have been shown in cell culture, where overexpression of a stable ODC with a mutation in the C-terminal region results in a dramatic increase in antizyme protein levels (37,38). Given the dramatic accumulation of truncated ODC in the skin and tumors of transgenic mice and the fact that the truncated and full-length ODCs bind antizyme with equal affinity, it is reasonable that most of the antizyme present would be bound to the truncated protein, resulting in increased stability of the endogenous ODC. Induction of antizyme protein would also be consistent with the increased wild-type ODC protein and putrescine levels seen in the tumors of K6/ODCdn mice.

In summary, we have developed an in vivo model in which a functionally active dominant-negative mutant of ODC is expressed in skin. In short-term experiments it can ablate the induced level of endogenous ODC caused by the tumor promoter TPA. In long-term carcinogenesis experiments, ODC activity and putrescine levels in the skin are not sufficiently reduced to inhibit tumorigenesis in a two-stage initiation/promotion model. The high-level expression of the dominant-negative ODC appears to result in a stabilization of the endogenous ODC protein, perhaps as a result of competition for binding of antizyme. These experiments emphasize the importance of the rapid turnover of the ODC protein in its regulation, and support the idea that overexpression of antizyme may be an extremely effective means of reducing ODC activity and subsequent tumorigenesis in response to tumor promotion. Our other recent results (39) also suggest that this is the case.


    Notes
 
4 To whom correspondence should be addressed Email: lms17{at}psu.edu Back


    Acknowledgments
 
We thank Paula Shoop and Suzanne Sass-Kuhn for excellent technical assistance. This work was supported by grants CA-82768 (to L.M.S.) and CA-18138 (to A.E.P.) from the NCI, ES-01664 (to T.G.O.) from NIEHS, and a Pennsylvania State University Cancer Center Research Grant (to L.M.S).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 26, 2001; revised January 8, 2002; accepted January 14, 2002.