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[Cancer Research 63, 3037-3042, June 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Inhibition of the Development of Metastatic Squamous Cell Carcinoma in Protein Kinase C {epsilon} Transgenic Mice by {alpha}-Difluoromethylornithine Accompanied by Marked Hair Follicle Degeneration and Hair Loss

Deric L. Wheeler, Kristin J. Ness, Terry D. Oberley and Ajit K. Verma1

Departments of Human Oncology [D. L. W., K. J. N., A. K. V.] and Pathology and Laboratory Medicine [T. D. O.], Medical School, University of Wisconsin, Madison, Wisconsin 53762, and Veterans Administration Hospital, Madison, Wisconsin 53705 [T. D. O.]


   ABSTRACT
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 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C {epsilon} (PKC{epsilon}) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3–4-fold more in PKC{epsilon} transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC{epsilon} transgenic mice was completely prevented by administration of the suicide inhibitor of ODC {alpha}-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC{epsilon} transgenic mice. DFMO treatment-associated hair loss in PKC{epsilon} transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC{epsilon} transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC{epsilon} transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
PKC,2 which is ubiquitous in eukaryotes, is a major intercellular receptor for the mouse skin tumor promoter TPA. PKC forms part of the signal transduction system involving the turnover of inositol phospholipids and is activated by DAG, which is produced as a consequence of this turnover. On the basis of the structural similarities and cofactor requirements, the PKC isoforms have been grouped into three subfamilies of enzymes: the conventional PKCs ({alpha}, ßI, ßII, and {gamma}), which are dependent on DAG/TPA, Ca2+, and PS; the novel PKCs ({delta}, {epsilon}, {theta}, and {eta}), which require only PS and DAG/TPA; and atypical PKCs ({zeta}, {lambda}, and {iota}), which retain only the PS dependence but have no requirement for DAG/TPA or Ca2+ for activation (PKCµ, which is usually classified as a nPKC, is not easily grouped with any of the other isoforms; Refs. 1, 2, 3 ). At least six PKC isoforms ({alpha}, {delta}, {epsilon}, {eta}, {zeta}, and µ) are expressed in mouse skin (4 , 5) . To determine the in vivo functional specificity of PKC{alpha}, -{delta}, and -{epsilon} in TPA signaling to mouse skin tumor formation, we generated transgenic mice that overexpressed T7-epitope-tagged PKC{alpha}, -{delta}, or -{epsilon} in their epidermis (4, 5, 6) . The expression of individual PKC isoforms was directed to the basal cells of the epidermis and hair follicles using the human cytokeratin 14 (K14) promoter (4, 5, 6) . Overexpression of PKC{alpha} did not affect the induction of skin tumors elicited by the initiation (DMBA)-promotion (TPA) protocol (6 , 7) . However, the overexpression of PKC{delta} suppressed the formation of both skin papillomas and carcinomas (5) , whereas PKC{epsilon} transgenic mice developed papilloma-independent mSCCs (4 , 8) .

Despite different skin tumor promotion susceptibilities, both PKC{delta} and PKC{epsilon} transgenic mice superinduced epidermal ODC after TPA treatment (6) . We reported recently that PKC{delta}-mediated signals to ODC induction and skin tumor suppression are unrelated (9) . We now present data in this communication indicating that TPA-superinduced ODC activity and subsequent accumulation of putrescine in PKC{epsilon} transgenic mice are not only essential for the development of mSCC but also for the growth and maintenance of hair follicles where the precursor cells for mSCC may reside.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Materials.
TPA was purchased from Alexis Corporation (San Diego, CA). DMBA was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI).

Mice.
Transgenic mice were maintained by mating hemizygous transgenic mice with wild-type FVB/N mice (4, 5, 6) . The mice were housed in groups of 3–4 in plastic bottom cages in light-, humidity-, and temperature-controlled rooms; food and water were available ad libitum. The animals were kept in a normal rhythm of 12-h light and 12-h dark periods. The transgene was detected by PCR analysis using genomic DNA isolated from 1-cm tail clips (4, 5, 6) .

Tumor Induction Experiments.
Mouse skin tumors were induced by the initiation-promotion regimen (4) . For mouse skin tumor initiation, a single 100-nmol dose of DMBA in 0.2 ml of acetone was applied topically to the shaved backs of mice. Two weeks after initiation, TPA in 0.2 ml acetone was applied twice weekly to the skin for the duration of the experiment. Tumor multiplicity was observed every week. Carcinomas were recorded grossly as downward-invading lesions, which were confirmed histologically (8) .

Assay of ODC Activity.
For the assay of ODC activity from mouse epidermis, mice were sacrificed by cervical dislocation at the appropriate time after treatment, and the epidermis from individual mice was separated from the dermis by a brief heat treatment (57°C for 30 s). Epidermal preparations were homogenized in 50 mM Tris-HCl buffer (pH 7.2) containing 0.1 mM pyridoxal phosphate, 1 mM DTT, and 0.1 mM EDTA. The epidermal extracts were centrifuged at 30,000 x g for 15 min to give a soluble supernatant. Soluble epidermal ODC activity was determined by measuring the release of 14CO2 from DL-[1-14C]ornithine (9) .

Polyamine Assay.
The polyamines putrescine, spermidine, and spermine were analyzed from 2% perchloric acid epidermal extracts (10) . The derivatized polyamines were separated and quantitated by high-performance liquid chromatography on a Waters 8 x 10 Novapak C18 cartridge using a gradient for a period of 30 min with fluorescent detection (excitation, 340 nm; emission, 515 nm).

Real-Time Quantitative PCR.
Total RNA was isolated using the RNeasy RNA isolation kit (Qiagen), DNase treated, and 1 µg was used to prepare cDNA using Ready-to-Go reverse transcription-PCR beads (Amersham). Quantitative reverse transcription-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye as described using the iCycler detection system (Bio-Rad). We also quantified transcripts of the 18 s RNase as an endogenous RNA control, and each sample was normalized on the basis of its 18 s content.

Histological Analysis.
The tissue to be examined was excised promptly after euthanasia and immediately placed in 10% neutral-buffered formalin (8) . The tissue was fixed for at least 1 h in formalin and then embedded in paraffin. Four-µm sections were cut for H&E staining. Skin sections were analyzed by a board-certified anatomical pathologist (T. D. O.).


   Results
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Sensitivity of PKC{epsilon} Transgenic Mice to TPA-induced Polyamine Biosynthesis.
PKC{epsilon} transgenic mice were evaluated for their sensitivity to TPA-induced ODC gene expression, ODC activity, and polyamine levels (Fig. 1)Citation . In this experiment, PKC{epsilon} transgenic mice and their wild-type littermates were initiated by applying 100 nmol DMBA in 0.2 ml of acetone to their shaved backs. One week later, TPA either at a 5-nmol dose (Fig. 1, A, B, and D)Citation or at the indicated doses (Fig. 1C)Citation was applied topically to the skin. The levels of ODC mRNA, ODC activity, and polyamine levels were determined at 3, 5, and 7 h, respectively, after TPA treatment. As compared with wild-type littermates, PKC{epsilon} transgenic mice elicited increases in the steady-state levels of ODC mRNA (2.5 fold; Fig. 1ACitation ), ODC activity (3–4 fold; Fig. 1, B and CCitation ), and putrescine levels (3–4 fold; Fig. 1DCitation ). TPA treatment did not alter the accumulation of epidermal spermidine, whereas spermine levels were decreased in both wild-type and PKC{epsilon} transgenic mice (Fig. 1D)Citation .



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  		Fig. 1. Effects of TPA on epidermal ODC gene expression, ODC activity, and polyamine levels in PKC{epsilon} transgenic mice. PKC{epsilon} transgenic mice and their wild-type littermates were shaved and initiated with 100 nmol of DMBA. Either vehicle acetone (0.2 ml) or TPA at 5 nmol dose (A, B, and D) or at various doses (C) in 0.2 ml acetone were applied once to the skin 1 week after DMBA initiation. Total skin RNA was isolated 3 h after TPA (5nmol) treatment and analyzed for ODC mRNA levels by real-time quantitative PCR as described in "Materials and Methods." The fold-expression was determined by normalizing to the acetone control group. Epidermal ODC activity (B and C) and polyamine levels (D) were determined at 5 and 7 h after treatment, respectively. ODC activity: each value is the mean of duplicate determinations of ODC activity of soluble epidermal extract from three mice; bars, ±SE. Polyamine levels: each value is either an average from two mice (variation is <10%) or mean; bars, ±SE. of determinations for the three mice. RNA level: each value is an average from two mice.

 
DFMO Inhibits the Formation of mSCC in PKC{epsilon} Transgenic Mice.
DFMO is an irreversible inhibitor of ODC (11) . To determine whether TPA-induced ODC activity plays a role in the development of mSCC in PKC{epsilon} transgenic mice, we compared the effects of inhibition of TPA-induced ODC activity by DFMO on skin tumor formation between PKC{epsilon} transgenic mice and their wild-type littermates. In this experiment (Fig. 2A)Citation , the mice were initiated with a single 100-nmol dose of DMBA in 0.2 ml of acetone. Beginning 1 week later the mice were treated with 5-nmol of TPA twice weekly for the indicated weeks. The control mice were given tap water, whereas the experimental groups were given 0.5% DFMO in their drinking water. The results of the effects of DFMO in the drinking water on carcinoma formation in PKC{epsilon} transgenic mice are summarized in Fig. 2ACitation . The carcinoma incidence in the wild-type mice was 0% in both the tap water control and DFMO experimental groups (Fig. 2, A–C)Citation . The PKC{epsilon} transgenic mice on tap water formed no papillomas but had a carcinoma incidence of ~50% (Fig. 2, A and B)Citation . The PKC{epsilon} transgenic mice receiving DFMO elicited complete suppression of carcinoma formation (Fig. 2A)Citation . As reported before (9) , DFMO treatment dramatically suppressed papilloma formation in wild-type mice (Fig. 2, B and C)Citation .



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  		Fig. 2. DFMO inhibits the development of mSCC in PKC{epsilon} transgenic mice. PKC{epsilon} transgenic mice and their wild-type littermates were shaved and initiated with 100 nmol of DMBA in 0.2 ml acetone. Starting 1 week later, mice were treated with 5 nmol TPA in 0.2 ml acetone twice weekly. The mice were given either tap water or 0.5% DFMO in the tap water. The papilloma, and the carcinoma incidence and multiplicity were recorded weekly. Carcinomas were recorded as downward invading lesions, which were confirmed histologically. The carcinoma data (A) is expressed as percentage of the effectual total. The effectual total is defined as the number of mice in each group at the time of appearance of the first carcinoma in any group. The photographs of representative mice at 20 weeks of skin tumor promotion; B, mice on tap water; C, wild-type mice on DFMO-containing water; and D, PKC{epsilon} transgenic mice on DFMO-containing water. Each photograph depicts littermates. Tg, transgenic mice; wt, wild-type mice.

 
DFMO Treatment Leads to Hair Loss Marked by Regression of the Hair Follicle in PKC{epsilon} Transgenic Mice.
The possibility was explored that DFMO administration during skin tumor promotion by TPA in PKC{epsilon} transgenic mice may result in hair loss (Fig. 2D)Citation by disrupting hair follicle growth. In this experiment (Fig. 3)Citation , dorsal skin samples were obtained for histological examination of both the follicular and interfollicular epidermis at 72 h after the 2nd, 5th, 12th, and 20th weeks of TPA promotion. After 2 weeks of skin tumor promotion, TPA-induced epidermal hyperplasia in the interfollicular epidermis was comparable in wild-type mice on either DFMO or tap water controls (Fig. 3, A and B)Citation . However, the interfollicular epidermis of the PKC{epsilon} transgenic mice on DFMO was severely hyperplastic. Furthermore, the hair follicle had increased hyperproliferation as noted by the narrowing of the hair follicle lumen (Fig. 3, C and D)Citation . After 5 weeks of tumor promotion, the PKC{epsilon} transgenic mice elicited severe hyperplasia in both the follicular and interfollicular epidermis. In addition, the transgenic mice on DFMO exhibited a disorganization of the hair follicle and hyperplasia of the bulb region with an increase in undifferentiated cells predominately beneath the bulge region (data not shown). At 12 weeks of chronic TPA treatment, PKC{epsilon} transgenic mice had complete hair loss in the TPA-treated area. Histological analysis of the skin sections from the transgenic mice on DFMO revealed loss of the hair follicle with remnants remaining as indicated by the presence of sebaceous glands, rudimentary hair follicles, and follicular cysts (Fig. 3H)Citation . In addition, PKC{epsilon} transgenic mice on DFMO exhibited severe focal hyperplasia of the interfollicular epidermis relative to mice not on DFMO. Similar degeneration of the hair follicle and intense hyperplasia of interfollicular epidermis was also observed at the 20th week of TPA promotion in the PKC{epsilon} transgenic animals (data not shown).



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  		Fig. 3. Changes in follicular and interfollicular epidermis during skin carcinogenesis. PKC{epsilon} transgenic mice and their wild-type littermates were shaved and initiated with 100 nmol DMBA in 0.2 ml of acetone. Starting 1 week later, mice were treated with 5 nmol TPA in 0.2 ml acetone twice weekly. The mice were given either tap water or 0.5% DFMO in the tap water. Mice were sacrificed at 2 (A–D) and 12 (E–H) weeks of tumor promotion, and skin specimens were fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin. Four µM-thick sections were cut for H&E staining. Note the hyperplastic hair follicles of PKC{epsilon} transgenic mice after 2 weeks of tumor promotion with 0.5% DFMO in their drinking water (C and D). Micrographs G and H show that the dorsal skin of PKC{epsilon} transgenic mice on DFMO do not have visible hair follicles. E, epidermis; HF, hair follicle; HFL, hair follicle lumen; D, dermis; S, sebaceous gland; M, muscle. A–D, cross-sections; E–H, longitudinal sections. Magnification: x200.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
ODC, which decarboxylates ornithine to form putrescine, is the key enzyme in mammalian polyamine biosynthesis. TPA-induced ODC activity and the resultant accumulation of putrescine have been shown to be essential in mouse skin tumor promotion by TPA (12) . In this context, the findings using DFMO, an enzyme-activated irreversible inhibitor of ODC, are noteworthy (11, 12, 13) . DFMO, when given in the drinking water, prevented carcinogenesis in almost every experimental animal model of cancer (11, 12, 13, 14, 15, 16) . DFMO-associated toxic side effects at the evaluated doses (0.5–1% w/v) included gastrointestinal upset and reversible hearing loss. We now present that DFMO, when given in the drinking water (0.5%) during skin tumor promotion by TPA, completely prevented the development of mSCC in PKC{epsilon} transgenic mice and led to marked alopecia accompanied by hair follicle loss and skin ulceration (Fig. 2D)Citation . These results imply that the PKC{epsilon}-mediated signal to ODC induction is essential for the maintenance of hair follicles and the development of mSCC in PKC{epsilon} transgenic mice.

The functional specificity of PKC isoforms ({alpha}, {delta}, and {epsilon}) toward ODC induction by TPA is yet unclear. In a transient transfection assay, both PKC{alpha} and PKC{delta} induced the transcription from the ODC promoter (-72/+130) reporter construct in vitro (17) . Similarly, PKC{delta} activation was found to induce ODC message (3-fold) and ODC activity. TPA treatment failed to induce ODC in keratinocytes isolated from PKC{delta} knockout mice (9) . Furthermore, mice that overexpress PKC{delta} in their epidermis superinduced ODC activity, whereas overexpression of PKC{alpha} in vivo did not (6 , 9) . TPA induced both the message and activity of ODC to a greater extent in PKC{epsilon} transgenic mice than their wild-type littermates (Fig. 1)Citation . Our results illustrated in Fig. 1Citation and reported before (9) indicate that signaling to ODC via PKC may exhibit functional redundancies.

The evidence indicating the importance of ODC and resultant accumulation of putrescine in hair growth have been conflicting (18, 19, 20, 21) . Fischer et al. (15) reported that DFMO treatment resulted in a mild decrease in hair growth. Other studies have shown that systemic DFMO treatment resulted in hair loss in dogs (19) . In contrast, transgenic mice, which have targeted overexpression of ODC in the hair follicle and outer root sheath, elicit hair loss and excessive skin wrinkling possibly because of increased formation of follicular cysts (20 , 21) . When ODC activity in these ODC-overexpressing transgenic mice was inhibited using DFMO, these mice maintained normal-appearing hair coats (21) . In the current paper, FVB/N mice treated with TPA for 20 weeks exhibited little hair loss, whereas PKC{epsilon} transgenic mice exhibited mild hair loss (Fig. 2B)Citation . When the FVB/N mice were give 0.5% DFMO in their drinking water during tumor promotion, little effect in hair loss was noted. However, PKC{epsilon} transgenic mice exhibited severe hair loss (Fig. 2D)Citation , which on histological examination revealed almost complete loss of hair follicles (Fig. 4, G and H)Citation . These results indicate that PKC{epsilon} and ODC may play, in concert, pivotal roles in the development, growth, and maintenance of the hair follicle.



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  		Fig. 4. Proposed mechanism of cellular origins of carcinomas in PKC{epsilon} transgenic mice. The mouse skin tumors (A and B) were elicited by initiation with a single dose of 100 nmol DMBA in 0.2 ml acetone followed by twice-weekly treatments of 5 nmol TPA in 0.2 ml acetone for 20 weeks. A, photograph of a wild-type mouse with a carcinoma derived from an existing papilloma (papilloma-dependent carcinoma). B, photograph of a PKC{epsilon} transgenic mouse with a carcinoma, which derived without obvious appearance of a papilloma. (papilloma-independent carcinoma). C, paraffin section of skin from an 8-week-old untreated female wild-type mouse (FVB/N). This picture represents the anatomical structure of a normal hair follicle. D, a schematic representation of the hair follicle showing the epidermis, sebaceous gland, hair shaft, outer root sheath, inner root sheath, and the bulge region containing the hair follicle stem cells. E, histopathology of a wild-type papilloma after 12 weeks of skin tumor promotion. Note the hyperplastic epidermis and the presence of hair follicles in the lower compartment of the papilloma (*). F, shown is the diagrammatic presentation of a papilloma and a possible mechanism for the development of carcinomas from existing papillomas. I, in this scenario, an initiated cell (i.e., stem cell) from the hair follicle may migrate upwards and give rise to a papilloma with potential to convert to a carcinoma. Alternatively (II), papilloma-dependent carcinomas may derive from a cell in the hyperplastic epidermis (*). Both of these proposed mechanisms would give the appearance on gross examination of a carcinoma derived from the papilloma, although the cellular origins differ. G, histopathology of a premalignant lesion in PKC{epsilon} transgenic mice after 8 weeks of skin tumor promotion has been reported before (8) . Shown is the hyperplastic hair follicle with an area of premalignancy (denoted by {blacktriangleup}) stemming from the hair follicle. H, schematic representation of a possible mechanism for the development of papilloma-independent carcinoma. Carcinomas in PKC{epsilon} transgenic mice appear to solely derive from the hair follicle because the loss of the hair follicle coincides with the subsequent inhibition of the development of carcinomas (Fig. 3)Citation . E, epidermis; SG, sebaceous gland; HS, hair shaft; ORS, outer root sheath; IRS, inner root sheath; SC, stem cell; Ca, carcinoma; C and G, x200; E, x40.

 
ODC expression has been linked to hair follicle cycling (22) . For example, ODC is abundantly expressed in the lower part of the follicular bulb in the anagen (growth phase) of the hair cycle, whereas no expression is detected in either the catagen (regression phase) or telogen (resting phase) phases of the hair cycle. As compared with the wild-type littermates, PKC{epsilon} transgenic mice exhibited hyperplasia of the hair follicle (Fig. 4, B, D, and F)Citation during skin tumor promotion by TPA. DFMO treatment, in conjunction with TPA promotion, led to almost complete loss of the hair follicle with an extreme thickening of the interfollicular epidermis in PKC{epsilon} transgenic mice (Fig. 4, G and H)Citation . The target cells for skin papillomas and carcinomas have been postulated previously either to be the basal keratinocytes of the interfollicular epidermis or the cells that form the outer root sheath of the hair follicle (23, 24, 25) . The results presented here (Figs. 2Citation and 3)Citation additionally strengthen our findings that the carcinomas in PKC{epsilon} transgenic mice originate from cells in the hair follicle (8) . A proposed mechanism of the cellular origins of carcinomas in wild-type and PKC{epsilon} transgenic mice during two-stage chemical carcinogenesis is illustrated in Fig. 4Citation .

This histological analysis of the epidermis during skin tumor promotion in PKC{epsilon} transgenic mice revealed a dramatic and persistent hyperplasia in both the interfollicular epidermis and the hair follicle (Fig. 3)Citation . These results led to two key questions: (a) why does hyperplasia of the hair follicle in PKC{epsilon} transgenic mice lead to hair loss; and (b) why is hyperplasia of the hair follicle followed by hair follicle regression? For the first question it seems plausible that heavy cell growth in the lower portion of the hair follicle may lead to actual physical blocking of the hair follicle lumen, and this may block the growth and protrusion of the hair shaft. Alternatively, the severe hyperproliferation seen in the hair follicle in conjunction with the undifferentiated cells beneath the bulge region may suggest that these cells are unable to properly differentiate and, thus, lack the ability to form cells giving rise to the hair shaft resulting in loss of hair. For the second question, one possible explanation is that the progenitor cells leading to the hyperplasia of the hair follicle may have a finite proliferative potential and, thus, exceed this potential leading to cell death.

In conclusion, we report that mice overexpressing PKC{epsilon} in basal keratinocytes and the hair follicle are more sensitive than their wild-type littermates to TPA-induced ODC activity and subsequent accumulation of putrescine. TPA-induced ODC activity and putrescine are: (a) essential for the maintenance and growth of hair follicles; and (b) linked to the development of mSCC in PKC{epsilon} transgenic mice. PKC{epsilon} transgenic mice provide a useful model to investigate human SCCs. The SCCs developed in PKC{epsilon} transgenic mice, like human SCC, are papilloma-independent, poorly differentiated, and have metastatic potential (8) . DFMO treatment completely prevented the development of SCCs in PKC{epsilon} transgenic mice, implying that DFMO may be a useful agent in the chemoprevention of human SCCs. The toxic side effects (severe hair loss) observed in PKC{epsilon} transgenic mice after DFMO treatment were not noticed in either preclinical (11 , 13, 14, 15, 16) or clinical chemoprevention studies (26, 27, 28, 29) .


   ACKNOWLEDGMENTS
 
We thank Nancy Dreckschmidt for the breeding of mice; Toshi Kinoshita for processing, sectioning, and staining of the skin sections; and Todd Brown for assistance in illustration.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Human Oncology K4/532, CSC, 600 Highland Avenue, University of Wisconsin Comprehensive Cancer Center, Madison, WI 53792. Back

2 The abbreviations used are: PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMBA, 7,12-dimethylbenz(a)anthracene; DFMO, {alpha}-difluoromethylornithine; ODC, ornithine decarboxylase; DAG, diacylglycerol; PS, phosphatidylserine; mSCC, metastatic squamous cell carcinoma; SCC, squamous cell carcinoma. Back

Received 12/20/02. Accepted 4/25/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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