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Identification of Epidermal Cell Subpopulations with Increased Ornithine Decarboxylase Activity following Treatment of Murine Epidermis with 12- O-Tetradecanoylphorbol-13-acetate Fredika M. Robertson, Susan K. Gilmour, Alan H. Conney, et al. Cancer Res 1990;50:4741-4746. Published online August 1, 1990.

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[CANCER RESEARCH 50,4741-4746,

August 1. 1990]

Identification of Epidermal Cell Subpopulations with Increased Ornithine Decarboxylase Activity following Treatment of Murine Epidermis with 12-0-Tetradecanoylphorbol-l 3-acetate1 Fredika M. Robertson,2 Susan K. Gilmour, Alan H. Conney, Mon-Tuan Huang, Andrew J. Beavis, Jeffrey D. Laskin, Oili A. Hietala, and Thomas G. O'Brien Departments of Surgery [F. M. R., A. J. B.Â¡and Environmental and Community Medicine fJ. D. L.J, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey, 08903; Department of Chemical Biology and Pharmacognosy, Rutgers University, Piscataway, New Jersey 08855 [A. H. C., M-T. H.]; Department of Biochemistry, University of Oulu, Oulu, Finland [O. A. H.J; and The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 fS. K. G., T. C. O.J

ABSTRACT Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12O-tetradecanoylphorbol-13-acetate(TPA). Using flow cytometry in com bination with polyclonal antibodies to ODC, we examined the levels of O1H '-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-I mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.

INTRODUCTION ODC3 (EC 4.1.1.17) is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway (1). It has been well documented that application of TPA to mouse skin results in rapid and transient induction of this enzyme and the subsequent accumulation of polyamines (2, 3). Modulation of ODC activity and subsequent polyamine metabolism by TPA is believed to be an important, if not essential, part of the tumor promotion process (4). The induction of ODC activity in mouse skin has been used to rank order phorbol esters as tumor promoters in the initiation-promotion model of carcinogenesis (5). Further more, compounds that block the rise of ODC in response to tumor promoters have been shown to inhibit tumor formation. These agents, which vary widely in mechanism of action and structure, include DL-a-difluoromethylornithine, retinoic acid, prostaglandin synthesis inhibitors, antioxidants, and protein kinase C inhibitors (6-16). Although ODC induction has been used as a biochemical marker for examining the activity of potential tumor-promoting agents, the precise role of this enzyme in the process of tumor Received 1/8/90; revised 4/4/90. 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. 1This project was supported in part by National Cancer Institute Grant ES01664 (T. G. O.) and New Jersey State Commission on Cancer Research Grant 88-593 CCR (F. M. R.). 3 To whom requests for reprints should be addressed, at Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903. 'The abbreviations used are: ODC, ornithine decarboxylase; TPA, 12-0tetradecanoylphorbol-13-acetate; PCS, fetal calf serum; PBS, phospate-buffered saline; FITC, fluorescein isothiocyanate.

promotion remains unclear. Animals treated with optimal ini tiation and promotion protocols have relatively few papillomas and carcinomas, despite the fact that very large numbers of cells are exposed to the initiator and promoter. This suggests that tumor formation is a rare event occurring only in a specific subpopulation of epidermal cells exposed to these chemicals. Observations from a number of laboratories have suggested that specific epidermal subpopulations respond differentially to tu mor promoters. For example, Raick (17), as well as KleinSzanto and Slaga (18), reported the development of a dark keratinocyte population following exposure to TPA. Morris and Argyris (19) identified a specific TPA-sensitive subpopu lation of cells that had an increased proliferation rate due to decreased cell cycle length. These observations suggest that populations of keratinocytes within the epidermis may respond acutely to exposure to tumor promoters by an initial increase in ODC activity followed by rapid proliferation. A small number of these tumor promoter-sensitive cells continue to divide and may constitute the cell pool from which papillomas and carci nomas arise. Because a number of laboratories, including our own, have found that there are specific subpopulations of keratinocytes that have greater sensitivity to the effect of TPA (17-21), we were interested in examining the ODC activity within individual cells isolated from mouse epidermis following acute and chronic treatment with TPA. Using flow cytometry and an ODCspecific polyclonal antibody, we have developed sensitive tech niques to examine the temporal sequence of ODC induction as well as to evaluate O DC-associated protein levels within epi dermal subpopulations. The method described in the present study provides a means to identify cells that have inducible ODC at early time periods following exposure to TPA. Al though the flow cytometric method has greater sensitivity than the immunohistochemical method in detection of early in creases in ODC levels, it does not have the magnitude of sensitivity of the biochemical method. However, relative in creases and decreases in ODC levels detected by flow cytometry are similar to those reported using the biochemical assay. The flow cytometric ODC detection system described in the present study has the advantage of providing insight into properties of subpopulations of cells that may be the precursors of neoplasia that cannot be accomplished using the biochemical method. MATERIALS

AND METHODS

Chemicals. Propidium iodide, Triton X-100, and trypsin (bovine pancreatic type XI) were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase (CLS type II) and Pronase were purchased from Worthington Biochemicals (Freehold, NJ). TPA was purchased from LC Services (Woburn, MA). Eagle's minimum essential medium,

FCS, and PBS were purchased from GIBCO (Grand Island, NY). Harris modified acid hematoxylin stain was obtained from Fisher Scientific 4741


StlBPOPULATION-SPECIFIC

(Springfield, NJ). Rabbit polyclonal antiserum directed against ODC was prepared as previously described (22). FITC-conjugated goat antirabbit IgG secondary antibody was purchased from Cooper Laborato ries (Malvern, PA). Isolation of Mouse Epidermal Cells. Mice used in these studies were 8-week-old female CD-I mice (25-30 g) obtained from Charles River Breeding Laboratories (Wilmington, MA). Mice were at the resting phase of hair growth at the beginning of the experiments. The dorsal hair of the mice was shaved with electric clippers 24-48 h prior to the first treatment. TPA (17 nmol), dissolved in 0.2 ml of acetone, was applied directly to the shaved area. Control mice received 0.2 ml of acetone alone. For short term experiments, animals received a single application of 17 nmol TPA and were then sacrificed at either 4 or 24 h, and cells were isolated. For long term experiments, mice were treated twice weekly for 3 weeks and sacrificed 4 h following the final treatment with 17 nmol TPA. Alternatively, animals were treated twice weekly for 22 weeks with 17 nmol TPA and maintained without TPA treatment for an additional 1-3 weeks. Experiments were repeated at least 6 times, with similar results. Skin samples from the treated areas were excised, cut into small pieces, and floated (epidermal side down) in a culture dish containing 0.25% trypsin in PBS (pH 7.3). After l h at 37Â°Cand subsequent 45min incubation at 25Â°C,the epidermal layer was scraped from the dermal layer using fine edged forceps and a scalpel blade. Ten ml of Eagle's minimum essential medium supplemented with 10% FCS were then added to the cultures to inhibit trypsin activity. A single-cell suspension of isolated epidermal cells was obtained by forcefully pipet ting the suspension several times. Viability of the cells was assessed by propidium iodide exclusion (23) and was found to be greater than 95%. Cells were isolated from papillomas by a modification of a procedure of Yuspa et al. (24). Briefly, papillomas were excised and placed in culture dishes containing 0.35% collagenase in PBS without calcium and magnesium, for 30 min at 37Â°C.The tissue was centrifuged and washed twice with PBS. The tissue was then placed in 1% trypsin in PBS (pH 7.3), minced with scissors, and incubated at 37Â°Cfor 1 h. Cells were disaggregated from the tissue using fine-edged forceps. Isolated cells were obtained as described above. Cells were centrifuged, washed twice with PBS, and then placed in a solution of 0.05% Pronase in Hanks' balanced salt solution for 30 min at 37Â°C.To stop the enzymatic activity, 2% FCS was added to the cell suspension, cells were filtered through 1SO-^m mesh to remove debris and washed twice, and viability was assessed as described above. Flow Cytometry. Epidermal cells were analyzed using a Coulter Profile analytical flow cytometer adjusted to an output of 15 mW at 488 nm excitation/525 nm emission. Forward angle and ninety-degree laser light scatter data were collected, as well as log intensity of integrated green fluorescence (510-550 nm). For each analysis, 20,000 events were accumulated. The flow cytometer was optically aligned daily and coefficients of variation for forward angle light scatter and integrated green fluorescence were less than 2, using fluorescently labeled alignment particles. The forward angle light scatter gain was set at 10 and the photomultiplier voltage for detection of green fluores cence was adjusted to 950 V for each analysis. The optical alignment procedures combined with the consistency of settings for collected signals assured that data from individual experiments could be com pared. Immunofluorescence. Polyclonal antiserum against androgen-stimulated mouse kidney ODC was prepared as described (22). Disaggregated epidermal cells (I x IO6) were fixed with 2% paraformaldehyde and permeabilized with 0.05% Triton X-100 for 15 min at 4Â°C.Cells were

ODC ACTIVITY

rescence as a function of relative cell number or dual-parameter histo grams, i.e., cell size versus fluorescence. Data depicted as green fluores cence represent the amount of ODC-specific antibody binding and are shown on a 3-decade logarithmic scale. Data in tabular form (Table 1) are presented as the mean channel number of cells that bound the ODC-associated immunoreactive protein. Biochemical Assay of ODC. At appropriate times following exposure of the dorsal skin to acetone or TPA, CD-I mice were sacrificed by cervical dislocation and epidermis from individual mice was separated by a brief heat treatment. ODC from soluble extracts (30,000 x g) was determined by measuring the release of I4CO2from DL-[l-'4C]-ornithine hydrochloride, as previously described (2).

RESULTS Effect of TPA on Mouse Epidermis. We used light microscopy to characterize the morphological alterations induced by TPA on cells within mouse epidermis. Female CD-I mice were treated with acetone or 17 nmol TPA for the described time periods. Hair was removed from the dorsal skin using a depi latory agent (Nair) and skin was removed and disaggregated as described above. Cytospin preparations of epidermal cells were prepared, stained with acid hematoxylin stain, and examined microscopically. We found that cell populations isolated from the epidermis of mice at 4 h after topical treatment with acetone consisted of approximately 95-98% keratinocytes and 2-5% leukocytes, when examined on the basis of morphology alone (Fig. I/I). There was no apparent increase in cell number at 4 h following 17 nmol TPA treatment; however, at 24 h following TPA treatment there was a 2-3-fold increase in absolute cell number. Polymorphonuclear cells that had infiltrated into the epidermis were present in low numbers at 4 h following TPA treatment (Fig. IB), and at 24 h after TPA treatment the polymorphonuclear cells comprised 15-20% of the total epi dermal cell population (Fig. 1C). Cells isolated at 24 h following 17 nmol TPA treatment were also more heterogeneous with respect to both size and granularity, as was reported by our laboratory previously (21). Ornithine Decarboxylase Antibody Binding by Mouse Epider mal Cells. We were interested in examining the ODC activity within epidermal cells present in control and TPA-treated mouse epidermis and establishing the temporal sequence of ODC induction following TPA exposure. Biochemical analysis of ODC activity has shown the peak enzyme induction to be about 4.5 h after TPA treatment (2); however, immunohistochemical detection methods were not sensitive enough to detect ODC activity after a single topical application of TPA (25). We therefore used flow cytometric techniques to measure ODC antibody binding. Using a specific antibody directed against Table 1 Numerical analysis of flow cytometric detection of ODC-immunoreactive protein Intensity of fluorescence (mean channel number)" TreatmentAcetone17

increase*3.01.16.5

then washed twice with PBS and incubated with a 1:500 dilution of ODC antibody in PBS containing 0.1% gelatin. After 30 min at 4Â°C, h17nmol TPA. 4 the cells were washed 3 times in PBS and incubated an additional 30 min with a 1:500 dilution of FITC-conjugated goat anti-rabbit whole molecule IgG secondary antibody. The cells were then washed 3 times in PBS and analyzed on the flow cytometer. Control cultures were incubated with FITC-conjugated goat anti-rabbit IgG secondary anti body and normal rabbit serum to determine the amount of nonspecific antibody binding. Data are shown as representative histograms, with the format of either single-parameter histograms showing green fluo

h17nmol TPA, 24 weeklyfor nmol TPA, 2x 3 weeksLog38.678.941.3108.3Linear2.9X.63.118.9Relative Â°Green fluorescence of ODC-immunoreactive protein bound by epidermal cells was collected on a logarithmic scale and mean channel numbers were determined from these histograms. Relative linear fluorescence values were cal culated from a nomogram (Coulter Cytometry'. Hialeah, FL). * Relative increase in intensity of green fluorescence as compared to the amount of ODC-immunoreactive protein bound by epidermal cells isolated from acetonetreated CD-I mice.

4742


StlBPOPULATlON-SPECIFIC

ODC ACTIVITY

detect basal levels of ODC present in cells isolated from ace tone-treated mice. There was a 3-fold increase in ODC-associated antibody binding following exposure of mouse skin to 17 nmol TPA for 4 h, when compared to ODC levels detected in cells isolated from acetone-treated animals (Fig. 2). ODC an tibody binding in cells isolated from mouse epidermis at 24 h was not significantly different from levels detected in cells isolated from acetone-treated mice (Fig. 3). This observation correlated with the biochemical detection of diminished ODC activity 24 h following exposure to TPA (2). Previous studies using biochemical and immunochemical de tection methods have shown that the level of ODC activity in mice treated with multiple applications of TPA is significantly higher than that observed following a single application of TPA (2, 25). We were therefore interested in determining the effects of long term tumor promoter treatment on ODC activity of epidermal cells and comparing the results with the ODC activity after a single application of TPA. Mice were treated with 17 nmol TPA twice a week for 3 weeks and then sacrificed at 4 h following the final topical application of TPA. Epidermal cells were isolated as described and analyzed for the amount of ODC antibody binding. The ODC antibody binding of cells isolated from mice treated for 3 weeks with 17 nmol TPA was 2.5-fold greater than that detected 4 h after a single application of TPA (Fig. 4, Table 1). When cells isolated from papillomas were analyzed for ODC-specific antibody binding, we found no sig nificant difference between the amount of ODC antibody bind-

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1.0

10

100

GREEN FLUORESCENCE Fig. 2. Flow cytomctric histogram of ODC-specific antibody binding by epi dermal eells isolated from CD-I mice after treatment with acetone (/?) or 4 h following exposure to 17 nmol TRA (C). A, amount of nonspecific antibody binding.

Fig. I. Light micrograph of acid hematoxylin-stained epidermal cells isolated from CD-I mice that were treated with acetone (A) orTPA (17 nmol) for4 h (B) or 24 h (C). Note the increasing numbers of polymorphonuclear cells at 4 and 24 h following TPA exposure. All photographs are at x 1000 magnification.

androgen-stimulated mouse kidney ODC (22), the amount of ODC in epidermal cells isolated at 4 and 24 h following topical application of TPA to mice was analyzed. These results were then compared with ODC-specific antibody binding by epider mal cells isolated from mice treated twice weekly for 3 weeks with 17 nmol TPA and sacrificed at 4 h following final TPA treatment, as well as those bearing papillomas that had initially been treated twice weekly with 17 nmol TPA for 22 weeks and subsequently not exposed to TPA for at least 1 week. Flow cytometric detection provided sufficient sensitivity to

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GREEN FLUORESCENCE Fig. 3. Flow cytometric histogram of ODC-specific antibody binding by cells isolated from CD-I mice after treatment with acetone (A), 24 h following treatment with 17 nmol TPA (B). or 4 h following exposure to 17 nmol TPA (C).

4743


SUBPOPULATION-SPECIFIC

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GREEN FLUORESCENCE Fig. 4. Flow cytometric histogram of ODC-specific antibody binding of epi dermal cells isolated from CD-I mice 4 h following a single application of 17 nmol TPA (A) or following 3 weeks of twice weekly application of 17 nmol TPA (B).

ODC ACTIVITY

from mice 4 h following exposure to 17 nmol TPA. Epidermal cells isolated from acetone-treated mice bound significantly less ODC-specific antibody (Fig. 5B). Flow Cytometric Characterization of Mouse Epidermal Cell Subpopulations Binding ODC-specific Antibodies. We were in terested in characterizing the specific subpopulations of epider mal cells that bound high amounts of immunoreactive ODC. Using flow cytometry, single-cell suspensions of epidermal cells were analyzed using laser light scatter properties. A comparison of cell size (forward angle light scatter) and log green fluores cence revealed one population of intermediate-size cells that bound ODC-specific antibodies isolated from CD-I mice at 4 h following a single topical application of 17 nmol TPA (Fig. 6A). In contrast, cells isolated from papillomas were smaller than those present following 4-h exposure to TPA (Fig. 6B). Table 2 shows the levels of ODC detected using the biochem ical assay method in epidermis isolated from littermates of the CD-I mice used for flow cytometric detection of ODC-immunoreactive protein. Although the flow cytometric method is not as sensitive as the biochemical assay at any of the time points analyzed, the relative increases and decreases in ODC-immunoreactive protein levels detected using the cytometric assay correlate with those using the biochemical detection method. Furthermore, the flow cytometric assay method provides infor mation about specific subpopulations with varying levels of ODC activity, as shown in Fig. 6. This type of analysis is not possible using the biochemical method. The immunochemical method does provide this type of heterogeneity information; however, ODC levels are not detectable by immunocytochemistry until 3 weeks of twice weekly application of TPA. DISCUSSION The epidermis is composed of a number of different cell types including keratinocytes and dendritic bone marrow-derived im mune cells including both Langerhans cells and Thy-1* cells.

B

Fig. 5. Fluorescence micrograph of ODC-specific antibody binding by epider mal cells isolated from CD-I mice 4 h following a single application of 17 nmol TPA (A) and ODC-specific antibody binding by epidermal cells isolated from acetone-treated mice (B).

ing within cells isolated from' papillomas

Fig. 6. Dual-parameter flow cytometric analysis of cell size (.v-axis) in relation to ODC-specific antibody binding (}'-axis) by epidermal cells isolated from CD1 mice 4 h following a single application of 17 nmol TPA (A) or by cells isolated from papillomas (III

and that in cells

isolated from mouse epidermis following 3 weeks of twice weekly applications of 17 nmol TPA. Since we were able to detect high levels of immunoreactive ODC within cells isolated from papillomas that had not been treated recently with TPA, this suggested that the cells within the papillomas constitutively expressed ODC. The flow cytometric analysis of ODC antibody binding by murine epidermal cells was confirmed using fluorescence mi croscopy. Fig. 5A is a fluorescent micrograph of cells isolated

Table 2 ODC activity in murine epidermis using the biochemical detection method

Treatment"Acetone

specific activity (nmol in 60 min/mg protein)0.12

17 nmol TPA. Ix 1.26 17 nmol TPA, 2x weekly 13.9Level for 3 weeksODC Â°All mice were sacrificed 4.5 h following TPA treatment.

4744


of induction10.5

116


Within each cell type, there is considerable heterogeneity. For example, the keratinocyte population within the epidermis con sists of both basal cells that are small and dense and larger, less dense, suprabasal cells located within the spinous and granular layers of the epidermis. Papillomas and carcinomas are believed to arise clonally from a specific subpopulation of basal keratinocytes that develop an altered phenotype after treatment with an initiator and pro moter (26-28). Epidermal tumors have been shown to have high levels of ODC, with carcinomas having a greater amount of enzyme activity than papillomas (2, 4). The constitutive expression of ODC by cells within papillomas suggests that specific keratinocytes expressing high levels of ODC after TPA treatment may be precursors of papillomas and carcinomas (25, 29). In studies of the enzymatic properties of ODC, O'Brien et al. (29) found that the enzyme activity in crude extracts of papil lomas differed in structural and functional properties when compared with ODC in extracts from normal epidermis. This result suggested that a tumor-associated isozyme of ODC may be regulated differently than ODC present in normal epidermis and could account for the apparently longer half-life of ODC and the constitutively high expression of this enzyme in papil lomas (30). The method used most often to measure ODC activity is the biochemical analysis of the release of I4CO2 from [l-'4C]ornithine hydrochloride (2, 4, 5). This assay system has been used extensively to characterize the induction of ODC following exposure of mouse epidermis to TPA (4). It has the advantage of sufficient sensitivity to detect ODC induction at early time points, 4-5 h following TPA treatment, but it provides infor mation solely about the total amount of active enzyme present in extracts of whole tissue. This method does not, therefore, allow assessment of the relative amounts of ODC within subpopulations of epithelial cells that may have varying amounts of ODC activity. Recently, Gilmour et al. (25) developed immunohistochemical techniques that allowed localization of ODC within cells in tissue sections of epidermis. Using specific polyclonal antibod ies to ODC and immunoperoxidase techniques, these investi gators demonstrated that mouse epidermal keratinocytes were heterogeneous in the expression of immunoreactive ODC fol lowing chronic treatment with TPA. The most intense antibodystaining was localized in the suprabasal keratinocytes surround ing the hair follicles. Although this study suggested a role for these cells in the tumor promotion process, ODC activity could not be detected at early time points, such as 4 h following a single TPA treatment that has been shown to cause a substantial ODC induction using the biochemical detection method (2, 4, 5). In the present studies, we developed sensitive methods to detect cellular heterogeneity in ODC activity following acute and chronic TPA treatment of mouse epidermis. The majority of the ODC antibody binding in cells isolated from mouse epidermis treated with a single application of TPA or with multiple applications was localized in larger cells that may correspond to more differentiated cells located in the suprabasal layers of the epidermis. However, in cells isolated from papil lomas, a subpopulation of small keratinocytes bound significant amounts of O DC-specific antibody. These findings lend further support to the hypothesis that there may be clonal expansion of specific subpopulations of keratinocytes within the epidermis that are very sensitive to the modulation of ODC activity and

ODC ACTIVITY

the subsequent alterations in polyamine metabolism induced by TPA treatment. Our observations agree with those of Gilmour et al. (25) and extend the ability to detect cellular heterogeneity patterns of ODC activity following exposure of mouse skin to TPA. The flow cytometric method described here, in conjunction with both the immunohistochemical and biochemical methods of ODC analysis, will allow identification and further character ization of specific subpopulations of epidermal keratinocytes that may be susceptible to neoplastic transformation in the murine multistage model of carcinogenesis. REFERENCES 1. Tabor. C. W., and Tabor, H. Polyamines. Annu. Rev. Biochem., 53: 749790, 1984. 2. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of the polyamine-biosynthetic enzymes in mouse epidermis by tumor promoting agents. Cancer Res.. 35: 1662-1670, 1975. 3. Pegg, A. E. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J., 234: 249-262. 1986. 4. O'Brien. T. G. The induction of ornithine decarboxylase as an early, possibly obligatory event in mouse skin carcinogenesis. Cancer Res., 36: 2644-2653, 1976. 5. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of polyaminebiosynthetic enzymes in mouse epidermis and their specificity for tumor promotion. Cancer Res., 35: 2426-2433, 1975. 6. Weeks, C. E., Herrmann, A. L., Nelson, F. R., and Slaga, T. J. Alphadifluoromethyl-ornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits tumor promoter-induced polyamine accumulation and carcinogenesis in mouse skin. Proc. Nati. Acad. Sci. USA, 79: 6028-6032, 1982. 7. Takigawa, M., Verma, A. K., Simsiman, R. C., and Boutwell, R. K. Polya mine biosynthesis and skin tumor promotion: inhibiton of 12-O-tetradecanoylphobol-13-acetate-promoted mouse skin formation by the irreversible inhibitor of ornithine decarboxylase, a-difluoromethylornithine. Biochem. Biophys. Res. Commun., 105: 969-976, 1982. 8. Takigawa, M., Verma, A. K., Simsiman, R. C., and Boutwell, R. K. Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by a-difluoromethylornithine. Cancer Res., 43:37323738, 1983. 9. Verma, A. K.. Shapas, B. G., Rice, H. M., and Boutwell, R. K. Correlation of the inhibition by retinoids of tumor promoter-induced mouse epidermal ornithine decarboxylase activity and of skin tumor promotion. Cancer Res., 59:419-425,1979. 10. Loprinzi, C. L., Verma, A. K., Boutwell, R. K., and Carbone, P. P. Inhibition of phorbol ester-induced human epidermal ornithine decarboxylase activity by oral compounds: a possible role in human chemoprevention studies. J. Clin. Oncol., 3: 751-757, 1985. 11. Verma, A. K., Ashendel, C. L.. and Boutwell, R. K. Inhibition by prostaglandin synthesis inhibitors of the induction of epidermal ornithine decarboxylase activity, the accumulation of prostaglandins, and tumor promotion caused by 12-O-tetradecanoyIphorbol-l 3-acetate. Cancer Res., 40: 308-315, 1980. 12. Verma, A. K., Rice. H. M., and Boutwell, R. K. Prostaglandins and skin tumor promotion: inhibition of tumor promoter-induced ornithine decarbox ylase activity in epidermis by inhibitors of prostaglandin synthesis. Biochem. Biophys. Res. Commun., 79: 1160-1166, 1977. 13. Kozumbo, W. J., Seed, J. L., and Kensler, T. W. Inhibition by 2(3)-rerr-butyl4-hydroxyanisole and other antioxidants of epidermal ornithine decarboxyl ase activity by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 43:25552559, 1983. 14. Perchellet, J-P., Owen, M. D., Posey, T. D., Orten, D. K., and Schneider, B. A. Inhibitory effects of glutathione level-raising agents and D-a/pAo-tocopherol on ornithine decarboxylase induction and mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis (Lond.), 6: 567573, 1985. 15. Gupta, A. K., Fischer, G. J., Elder, J. T., Nickoloff, B. J., and Vorhees, J. J. Sphingosine inhibits phorbol-induced inflammation, ornithine decarboxylase activity and activation of protein kinase C in mouse skin. J. Invest. Dermatol., 91: 486-491, 1988. 16. Enkvetchakul, B., Merrill, A. H., and Birt, D. F. Inhibition of the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate in mouse skin by sphingosine sulfate. Carcinogenesis (Lond.). 10: 379-381, 1989. 17. Raick. A. N. Ultrastructural, histological and biochemical alterations pro duced by 12-O-tetradecanoylphorbol-13-acetate on mouse epidermis and their relevance to skin tumor promotion. Cancer Res., 33: 269-286, 1973. 18. Klein-Szanto. A. J. P., and Slaga, T. J. Numerical variation of dark cells in normal and chemically induced hyperplastic epidermias with age and effi ciency of tumor promoter. Cancer Res., 41: 4437-4440, 1981. 19. Morris, R., and Argyris, T. S. Epidermal cell cycle time and transit times during hyperplastic growth induced by abrasion or treatment with 12-Otetradecanoylphorbol-13-acetate. Cancer Res., 43: 4935-4942, 1983.

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20. Reiners, J. J., and Slaga, T. J. Effect of tumor promoters on the rate and commitment to terminal differentiation of subpopulations of murine keratinocytes. Cell, 32: 247-255, 1983. 21. Robertson, F. M., Laskin, D. L., and Laskin. J. D. Production of reactive oxygen intermediates following application of the tumor promoter, 12-0tetradecanoylphorbol-13-acetate (PMA). Proc. Am. Assoc. Cancer Res., 29: 154, 1988. 22. Pulkka, A., Taskinen, T.. Aaltonen, H., Ramberg. J.. and Pajunen. A. Studies on the degredation of ornithine decarboxylase by the immunoblotting tech nique. Biochem. Int., //: 845-851. 1985. 23. Roth, G., and Valet, G. Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry. Cytometry. 9:316-324,1988. 24. Yuspa, S. H., Morgan, D.. Lichti, U., Spangler, E. F., Michael. D., Kilkenny, A., and Hennings, H. Cultivation and characterization of cells derived from mouse skin papillomas induced by an initiation-promotion protocol. Carcinogenesis (Lond.). 7: 949-958, 1986. 25. Gilmour, S. K., Aglow, E., and O'Brien, T. G. Heterogeneity of ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin and in epidermal tumors. Carcinogenesis (Lond.), 7: 943-947. 1986. 26. Yuspa, S. H., Hennings, H., Kulesz-Martin, M., and Lichti. U. The study of

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