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Carcinogenesis, Vol. 22, No. 4, 651-659, April 2001
© 2001 Oxford University Press

CARCINOGENESIS

Age-dependent skin tumorigenesis and transgene expression in the Tg.AC (v-Ha-ras) transgenic mouse

Michael StJ. Battalora1,4, Judson W. Spalding1,6, Carl J. Szczesniak1, James E. Cape1, Rebecca J. Morris3, Carol S. Trempus1, Carl D. Bortner2, Byung M. Lee5 and Raymond W. Tennant1

1 Laboratory of Environmental Carcinogenesis and Mutagenesis and
2 Laboratory of Signal Transduction, National Institutes of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709,
3 Lankenau Medical Research Center, 100 Lancaster Avenue West of City Line, Wynnewood, PA, 19096, USA and
4 BASF Aktiengesellschaft, Department of Toxicology Z470, 67056, Ludwigshafen, Germany and
5 College of Pharmacy, Sung Kyun Kwan University, Suwon, Kyunggi-Do 440-746, South Korea


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transgenic Tg.AC (v-Ha-ras ) mice develop skin tumors in response to specific carcinogens and tumor promoters. The Tg.AC mouse carries the coding sequence of v-Ha ras, linked to a {zeta}-globin promoter and an SV40 polyadenylation signal sequence. The transgene confers on these mice the property of genetically initiated skin. This study examines the age-dependent sensitivity of the incidence of skin papillomas in Tg.AC mice exposed to topically applied 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, full thickness skin wounding or UV radiation. Skin tumor incidence and multiplicity were strongly age-dependent, increasing with increasing age of the animal when first treated at 5, 10, 21 or 32 weeks of age. Furthermore, the temporal induction of transgene expression in keratinocytes isolated from TPA-treated mouse skin was also influenced by the age of the mice. Transgene expression was seen as early as 14 days after the start of TPA treatment in mice that were 10–32 weeks of age, but was not detected in similarly treated 5-week old mice. When isolated keratinocytes were fractionated by density gradient centrifugation the highest transgene expression was found in the denser basal keratinocytes. Transgene expression could be detected in the denser keratinocyte fraction as early as 9 days from start of TPA treatment in 32-week old mice. Using flow cytometry, a positive correlation was observed between expression of the v-Ha-ras transgene and enriched expression of the cell surface protein ß1-integrin, a putative marker of epidermal stem cells. This result suggests that, in the Tg.AC mouse, an age-dependent sensitivity to tumor promotion and the correlated induction of transgene expression are related to changes in cellular development in the follicular compartment of the skin.

Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; mouse-ß2-microglobulin (Mß-2); RT–PCR, reverse transcription–polymerase chain reaction; TPA, 12-O-tetradecanoylphorbol-13-acetate.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Transgenic mouse models can be useful tools for assessing the carcinogenic potential of chemicals (1–3). The Tg.AC (v-Ha-ras) transgenic mouse, which carries the coding sequence of v-Ha-ras linked to a {zeta}-globin promoter and an SV40 polyadenylation signal sequence, develops skin papillomas in response to topically applied genotoxic and non-genotoxic carcinogens and tumor promoters (2,4–6). Thus, in the context of the two-stage mouse skin initiation/promotion model of carcinogenesis, these animals are considered to be `genetically initiated' (4,7).

In order to gain a clearer understanding of the mechanistic basis for the response of Tg.AC mice to carcinogens, the critical early molecular changes associated with neoplastic development, including induction of the ras transgene, must be defined. While ras transgene expression is not detected in untreated Tg.AC mouse skin, previous studies indicate that all skin tumors induced in Tg.AC mice express the ras transgene (8–11) and expression can be detected prior to the appearance of visible papillomas (8–10). In situ hybridization studies show that transgene expression occurs in a discrete focal hyperplasia associated with the hair follicle that appears to be a papilloma precursor (10). This follicular location suggests that these early ras transgene-expressing foci may develop in epidermal stem cells (12–15). These epidermal stem cells can also be collected in the denser basal keratinocyte fractions of isolated skin keratinocytes (16,17) and are rich in ß1-integrin (18,19). These characteristics allow this discrete population of keratinocytes to be studied selectively.

During the development of carcinogenicity studies with Tg.AC mice, it was noted that animals 20–30 weeks of age developed a high multiplicity of papillomas in response to a minimum of four topical treatments of TPA. This result could not be reproduced when the experiment was repeated with mice that were <10 weeks old. Based on these findings, more extensive studies were conducted to test whether the skin tumor response in Tg.AC mice increased with age. We report here on the effects of ageing on the induction of skin papillomas in Tg.AC mice by short-term dosing with TPA, full thickness wounding and UV radiation. Skin papilloma incidence and multiplicity were measured and compared for animals of different ages.

We also report here further studies that were designed to determine whether the temporal induction of transgene expression was also age dependent. Transgene expression was examined by RT–PCR in isolated keratinocytes or in basal keratinocytes fractionated by density gradient centrifugation from mice of different ages. In addition, populations of isolated keratinocytes were examined for ß1-integrin, a cell surface adhesion protein that is enriched in epidermal stem cells (18,19). The results of these studies will lead to a better understanding of the timing and mechanism of induced skin carcinogenesis in the Tg.AC transgenic mouse.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
Homozygous female transgenic Tg.AC mice were obtained from a US National Toxicology Program-maintained colony at Taconic Farms (Germantown, NY). FVB/N mice, the non-transgenic parent strain of the Tg.AC mouse line, were also from Taconic Farms. Mice were 4–5 weeks of age when received and were maintained for at least 1 week before use. The female mice were housed four or five per cage, in polycarbonate shoebox-style cages containing hardwood bedding (Beta Chips, Northeastern Products, Warrensbury, NY) and covered with spun-bonded polyester cage filters. Room temperature was 22 ± 1°C humidity ranged from 40 to 60 percent and fluorescent lighting was on a 12 h light–dark cycle. Purina Pico Chow No. 5058 (Ralston Purina, St Louis, MO) and tap water were provided ad libitum. Animals were identified with tattoo numbers on their tails (Animal Identification and Marking System, Piscataway, NJ). Husbandry practices complied with NIH Guidelines for Humane Care and Use of Laboratory Animals.

Chemicals
12-O-tetradecanoylphorbol-13-acetate (TPA) and 7,12-dimethylbenz[a]anthracene (DMBA) were from Sigma (St Louis, MO). Both chemicals were dissolved in spectral grade acetone (Fisher Scientific, Raleigh, NC) and were delivered topically to the dorsal skin surface in a total volume of 0.2 ml.

Tumor induction
The dorsal skin of each mouse was shaved with electric clippers 1–5 days prior to the start of treatment. FVB/N mice were initiated with a single dose of 10 µg DMBA, and 1 week later, promotion began with twice weekly treatments of 2.5 µg TPA for 20 weeks.

Three methods were used to induce tumors in Tg.AC mice: (i) chemical induction with topical application of TPA, two times a week for 2 weeks (e.g. Monday and Thursday). The fourth dose of TPA occurs on the tenth day after the first dose using this dose regimen; (ii) full thickness wounding, by surgical incision and (iii) UV radiation exposure. To accomplish full-thickness wounding, mice were anesthetized with methoxyflurane (Pitman-Moore, Mundelein, IL) and a 3 cm long, full-thickness incision was made in the skin parallel to the midline of the dorsal surface. The incisions were immediately closed with five to seven wound clips, which were removed 7 days later under anesthesia. UV radiation-induced tumors were generated as previously described (20). Mice were exposed to UV lamps three times in 1 week (Monday, Wednesday and Friday), for a cumulative exposure of 26 kJ/m2 UVA/UVB.

The dorsal skin of the mouse was observed weekly, and incidence and tumor multiplicity (papillomas per mouse) at the site of application (SOA) were recorded as previously described (5). Experiments with FVB/N mice used 19–24 animals per dose group, and experiments with Tg.AC mice used 9–12 animals per dose group. Papillomas were recorded if they persisted for three consecutive observations and were 1 mm or greater in size. When tumor multiplicity reached 30 papillomas on any one mouse, that mouse was assigned a maximum number of 30 even if more papillomas appeared, because at that point, papillomas began to coalesce and accurate counting became problematic. This only occurred in a few of the older 21 or 32 week old mice receiving the highest dose of TPA.

Transgene induction experiments
At various times after TPA or acetone treatment, one or two mice were euthanized by CO2 narcosis and the dorsal skin was removed. Epidermal cells were isolated as previously described with minor variations (21). The subcutaneous fat was removed and the skin was floated dermis side down on a solution containing 0.25% trypsin (Gibco BRL, Grand Island, NY) in PBS (Ca2+/Mg2+ free) for 2 h at 32°C. Then the epidermis (including hair follicles) was removed by firmly scraping with a scalpel into minimum essential medium (S-MEM) (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL). The suspension of epidermis and hair was stirred for 20 min in S-MEM with 10% FBS and filtered through a 100 µ nylon mesh. The filtered solution that passed through the mesh was centrifuged at 230 g for 7 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in PBS to wash away the medium. Following a second centrifugation, the supernatant was decanted and the pellet was flash frozen in alcoholic dry ice and stored at –70°C until used for RNA extraction.

Total RNA was isolated after briefly thawing the frozen pellet on ice. Then TriReagent (Molecular Research Center, Cincinnati, OH) was added according to the manufacturer's protocol. Tissues were homogenized with a polytron (Brinkman Instrument, Westbury, NY) for a total of 30 s divided into three 10 s intervals, with 10 s between each homogenization to prevent heating. The homogenate was extracted with chloroform, and the RNA was precipitated with isopropanol. This was followed by one wash with 75% percent ethanol. RNA pellets were resuspended in diethylpyrocarbonate (DEPC)-treated water and quantified spectrophotometrically.

Total RNA was assayed for ras transgene expression essentially as described elsewhere (8,22). Briefly, 1 µg total RNA was reverse transcribed using AMV-RT (Promega, Madison, WI) at 42°C. A 2 µl aliquot of the RT reaction mixture was added to the PCR mix containing primers for mouse-ß-2-microglobulin (Mß-2) and the remainder was added to a separate PCR mix containing ras transgene-specific primers. Following amplification, RT–PCR products were separated by electrophoresis on a 2% SeaKem GTG agarose gel (FMC Bioproducts, Rockland, ME) prepared with 1x Tris-borate–EDTA, stained with ethidium bromide and photographed under UV light.

Isolation of keratinocytes
Keratinocytes were generated as described above using skins from four to five mice. The number of viable cells was counted, cells divided into six 2 ml aliquots of 15 million cells each and gently layered on top of 27 ml preformed Percoll gradients in 30 ml Oak Ridge tubes as previously described (16). The Percoll gradient was made by diluting Percoll (Sigma, St Louis, MO) to 61.5% with S-MEM and 10% FBS and centrifuging at 20 500 g for 35 min. Density marker beads (Sigma) were included in the gradient mixture to indicate densities of 1.061, 1.074, 1.087 and 1.098 g/ml. The tubes were slowly accelerated to 237 g and centrifuged for 30 min without braking. Then five fractions were separated, based on the density marker beads, using a blunt-ended 18 gauge pipetting needle (Popper & Sons, New Hyde Park, NY). The cells from like-fractions from the six tubes were combined. The keratinocytes were then washed with S-MEM containing 10% FBS to remove the Percoll and counted. Following a final wash with PBS, the cells were pelleted and frozen as described above until RNA was extracted.

Flow cytometry
Keratinocytes were isolated from the skin of TPA-treated Tg.AC mice as described above in `transgene induction experiments', except that the cells were filtered twice through a 70 µ filter after the last centrifugation. The cells were then resuspended in PBS with 0.1% bovine serum albumin (PBS–BSA) and brought to a concentration of 1x106 cells/ml. A 10 ml aliquot of this solution was mixed with 10 ml RPMI-1640 (GibcoBRL) containing a rat anti-stromal monoclonal antibody that recognizes murine ß1-integrin (23). The hybridoma was from the American Type Culture Collection (CRL-2179) and was cultured according to their instructions. The medium was filter sterilized and used undiluted for staining the keratinocytes. Another aliquot of keratinocytes (10 ml) was mixed with 10 ml RPMI medium without the anti-ß1-integrin antibody as a control. The cells were mixed by hand and allowed to incubate at room temperature for 20 min, then centrifuged and the supernatant was removed. Both aliquots of cells were resuspended in 10 ml S-MEM and were triturated 10 times, and the tubes containing the cells were then covered with aluminium foil in a darkened room. A 50 µl aliquot of FITC-labeled secondary antibody (Goat anti-rat antibody; BD PharMingen, San Diego, CA) was added to both tubes and gently mixed. The cells were held at room temperature for 20 min, and washed with 40 ml PBS–BSA to remove excess antibody. Cells were resuspended in 5 ml PBS–BSA and triturated eight times, and 45 ml PBS–BSA were added to each tube. The cells were centrifuged and resuspended in PBS–BSA at a concentration of 4–5x106 cells/ml.

The keratinocytes were separated using a FACSVantage (Becton Dickinson, San Jose, CA). The cells were initially passed through a 35 µm strainer cap filter to remove aggregates. Propidium iodide (PI; Sigma) was added to the sample to a final concentration of 10 µg/ml to eliminate cells which had lost membrane integrity, thus serving as a vital dye. The control cells, which were stained only with the secondary antibody, were used as a comparison to determine if the primary antibody had attached to the other aliquot of cells. Cells were excited using a 488 nm argon laser, and the FITC and PI fluorescence were detected at 530 and 575 nm, respectively. Gates were set on a FITC versus PI dot plot to simultaneously collect the 20% ß1-integrin FITC-fluorescent cells. Approximately 1–2x105 cells of the ß1-integrin brightest and the ß1-integrin dimmest cells were collected. Cells were pelleted after collection, flash frozen as described above, and stored at –80°C until used for RNA extraction.

Since the number of available cells for RNA extraction was small in comparison with the previous experiments for detecting transgene induction, total RNA was extracted from the cell pellets using the Stratagene Micro RNA Isolation kit (Stratagene, La Jolla, CA) following the manufacturer's protocol. RNA pellets were resuspended in 20 µl DEPC-treated water. Aliquots of 3 µl were used in RT–PCR analysis for expression of the v-Ha-ras transgene and Mß-2 as a housekeeping gene, essentially as described above. After 25 PCR cycles, aliquots of the reactions amplifying the ras transgene were placed in tubes containing fresh PCR mix, and a nested (sense) primer, combined with the antisense primer used in the first reaction, was used to enhance signal detection through further amplification. The nested sense primer had the sequence 5'-ACTACCTACAGAGATTTA-3' (Genosys Biotechnologies, The Woodlands, TX) and was used at 1 pmol per reaction; all other conditions were as described for the first round of PCR. The nested amplification products for the ras transgene were 272 base pairs (bp) for contaminating DNA/unprocessed RNA and 207 bp for processed mRNA. After 15 PCR cycles, 10 µl aliquots of the nested reactions were electrophoresed along with 10 µl of Mß-2 reactions on a 2% agarose gel containing 0.5 µg/ml ethidium bromide and products were visualized under UV light.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Tumor induction
Tg.AC mice from the same shipment were randomly distributed among 12 experimental groups of 10–12 mice per group, and received one of the following treatment regimens: four treatments with 2.5 µg TPA; four treatments with 5.0 µg TPA; or a full-thickness surgical skin incision. The frequency of the topical TPA applications was two times a week for 2 weeks, and the treatments were started when the mice reached 5, 10, 21 or 32 weeks of age.

The results for animals treated with 2.5 µg TPA are shown in Figure 1A and BGo. Mice that were 5 or 10 weeks of age at start of treatment developed far fewer tumors over a 20 week period than mice that were 21 or 32 weeks old at start of treatment. The average maximum tumor multiplicity among mice that were 5, 10, 21 or 32 weeks old at start of treatment was 0.4, 0.6, 9.7 or 12.4 papillomas per mouse respectively (Figure 1AGo). The time to first tumor (latency period) was significantly reduced among mice that were 21 or 32 weeks old compared with that in the two younger age groups.



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  		Fig. 1. TPA- or wound-induced papilloma response in female homozygous Tg.AC mice. Four doses of TPA were topically applied in 0.2 ml acetone. Frequency of dosing was twice a week (e.g. Monday and Thursday) over a 10 day period. Groups of 10–12 mice were 5 ({blacksquare}), 10 ({blacktriangleup}), 21 (•) or 32 (+) weeks old at start of treatment. Papilloma multiplicity and incidence was monitored over a 20 week period from the time of first treatment. (A) The mean number of papillomas per mouse for mice treated with 2.5 µg TPA, and (B) the incidence. The mean number of papillomas per mouse for the group treated with (C) 5.0 µg TPA, and (D) the incidence. (E) The mean number of papillomas that occurred in response to full-thickness wounding, and (F) the incidence. The increase in papilloma response and incidence in mice wounded at 5 weeks of age and again, a second time, at 21 weeks of age ({blacklozenge}) is also depicted (E and F).

 
The incidence of mice with tumors was also age dependent at this dose of TPA (Figure 1BGo). Papillomas were induced in 100% of the mice that were 21 or 32 weeks old at start of treatment, while tumors occurred in only 30% of the mice that were 5 and 10 weeks old.

A much more robust but similar age dependent pattern of response was observed in the groups of mice dosed four times with 5 µg TPA (Figure 1C and DGo). Mice that were 5, 10, 21 or 32 weeks of age at start of treatment developed a maximum multiplicity of 2.4, 5.9, 18.3 or 27.1 papillomas per mouse respectively (Figure 1CGo). The latency period among the different age groups was also age dependent. Papillomas began to appear as early as 2 weeks after the first dose of TPA in the 32 week old group and maximum tumor multiplicity was reached much earlier among the mice that were 21 or 32 weeks old at treatment start. The incidence of mice with papillomas was 90–100% among the three older groups of mice and 60% in mice that were 5 weeks old at treatment start (Figure 1DGo).

Responses of Tg.AC mice, from a later experiment, that were 10 or 32 weeks of age and treated four times with 10 µg TPA are compared in Table IGo. In the 10 week old mice, a significantly higher papilloma multiplicity (16.9) per mouse was observed (P <= 0.05, Mann–Whitney U test) than in same aged animals treated with 5 µg TPA (Table IGo); however, in mice that were 32 weeks of age, the tumor multiplicity per mouse was similar for animals treated with either 5 or 10 µg TPA (Table IGo). In both age groups treated with 10 µg TPA, all animals developed papillomas (Table IGo, legend).


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  		Table I. Age-dependence of mean number of papillomas per mouse compared in Tg.AC mice treated with TPA, full-thickness skin wounding or UV radiation
 
Tg.AC mice also developed papillomas in an age-dependent manner in response to a 3 cm full-thickness surgical skin incision. The papillomas, which are induced by the wound repair process, occur along or immediately adjacent to the wound line. The average maximum tumor multiplicity achieved was less than 1.0 papilloma per mouse among the mice that were 5 or 10 weeks old at the time of wounding; but mice that were 21 or 32 weeks of age developed an average of 3.5 and 5.0 papillomas per mouse, respectively (Figure 1EGo). The maximum tumor multiplicity and time to first tumor was also achieved earlier among groups of mice of increasing age at the time of treatment. The incidence of mice with tumors increased with age and ranged from 40% for 5 week old mice to 100% for the 32 week old mouse group (Figure 1FGo).

Interestingly, when the mice that were wounded at 5 weeks of age were wounded again on the opposite side of the dorsal midline at 21 weeks of age, they developed papillomas along the new wound line with a similar incidence, time course and multiplicity as the mice that were wounded for the first time at 21 weeks of age (Figure 1EGo). The average maximum multiplicity was 4.3 papillomas per mouse and the tumor incidence was 100% in the twice wounded mice (Figure 1E and FGo).

The age-dependence of UV-induced papillomas was also examined in Tg.AC mice. Mice that were 10 or 32 weeks of age were exposed to UV three times in 1 week for 10 min per exposure (cumulative dose 26 kJ/m2 UVA/UVB). Mice that were 10 weeks old at the start of treatment developed an average maximum multiplicity of 2.4 papillomas per mouse, and mice that were 32 weeks at the start of treatment developed an average of 8.3 papillomas per mouse (Table IGo). Tumor incidence was only slightly higher in the older mice than in the younger mice treated with UV (Table IGo).

The kinetics of papilloma development in older mice (21 or 32 weeks old) in response to all three promotional stimuli were very similar, although the magnitude of the papilloma response varied. Papillomas were rarely observed 2 weeks after the start of TPA treatment, but by 4 weeks, 90–100% of the mice treated with TPA started to develop papillomas. After full-thickness skin wounding, the onset of papillomas was observed 3 weeks after the start of treatment. After UV radiation, the first papillomas were detected 4 weeks after the start of treatment. In all cases, the age of the mice affected the tumor latency, with the onset of tumors and maximum multiplicity developing earlier and more rapidly in older mice. For all experiments, the number of tumors reached a plateau from 7 to 12 weeks after the start of treatment.

Wild-type FVB/N mice, the parent strain of the Tg.AC mouse line, require a two-stage initiation/promotion dosing regimen to develop skin papillomas. The age-dependence of tumor development was examined in 9 or 31 week old FVB/N mice using a DMBA/TPA protocol. As summarized in Table IIGo, there was no significant difference in the response of wild-type FVB/N mice to DMBA/TPA at these two ages.


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  		Table II. The papilloma response of FVB/N mice as a function of the age at the start of two-stage carcinogenesis treatment
 
Transgene induction
Transgene expression is not readily detected in whole skin of Tg.AC mice by RT–PCR prior to the appearance of papillomas in treatment-induced skin (8,11). Therefore, to enhance detection of transgene expression, keratinocytes were isolated from the skin of Tg.AC mice of different ages that had been treated with TPA. Our intent was to define the parameters of age and TPA dose regimen that would result in the earliest detection of transgene expression after TPA treatment. This effort led toward increasing the sensitivity for transgene expression detection in subpopulations of isolated keratinocytes that also exhibited properties of epidermal stem cells.

At various times after the last TPA dose, mice of different ages at treatment start were euthanized, keratinocytes harvested and RT–PCR performed on the total RNA extracted from the isolated keratinocytes. Since all papillomas in Tg.AC mice characterized to date have been shown to express the transgene (11), mice with visible papillomas were not used as a source of keratinocytes for these experiments.

As shown in Figure 2AGo, transgene expression could not be detected in keratinocytes prepared 16 days after start of treatment from 5 week old mice that were treated with 5 µg TPA (lane 1) or 10 µg TPA (lane 2). However in mice that were treated with 10 µg TPA when they were 10 weeks old (lane 3) or 32 weeks old (lane 4) transgene expression could be detected as early as 14 days after start of treatment.



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  		Fig. 2. (A) RT–PCR for transgene mRNA from keratinocytes from Tg.AC mice. Mice were treated four times over a period of 10 days with 5 (lane 1) or 10 µg (lanes 2–4) TPA and then killed 16 days (lanes 1 and 2) or 14 days (lane 3 and 4) after the start of treatment. Lanes 1 and 2 used RNA samples from mice that were 5 weeks old at the start of treatment. Lane 3 sample was from mice that were 10 weeks old at the start of treatment, and lane 4 sample was from mice that were 32 weeks old at the start of treatment. Total RNA was isolated from keratinocytes (lanes 1–4) and assayed by RT–PCR with v-Ha-ras transgene specific primers or ß-2 microglobulin (Mß-2) specific primers. Lane 5 is a positive control RNA sample from a Tg.AC mouse skin tumor. Lane 6 is a 100 bp DNA molecular weight marker. RT–PCR products for the ras transgene gave a DNA-derived band at 279 bp and a RNA-derived band at 214 bp (upper panel). The RT–PCR product for Mß-2 was 212 bp (lower panel). (B) RT–PCR for transgene mRNA from keratinocytes from 25 week old female Tg.AC mice treated four times with 10 µg TPA over a period of 10 days and killed 14 days after the start of treatment. Lanes 1 through 5 used RNA isolated from the least dense to the most dense cells. Lane 6 is a positive control RNA sample from a Tg.AC mouse skin tumor. Lane 7 is a 100 bp DNA molecular weight marker. RT–PCR products for the ras transgene gave a DNA-derived band at 279 bp and a RNA-derived band at 214 bp (upper panel). The RT–PCR product for Mß-2 was 212 bp (lower panel) and indicates the integrity of the mRNA samples. Other details are given in (A) and Materials and methods. (C) RT–PCR for transgene mRNA from keratinocytes from 32 week old Tg.AC mice treated two times over a period of five days with 10 µg TPA and killed 9 days after the start of treatment. Lane 6 used RNA from keratinocytes before separation by buoyant density fractionation. Lanes 1–5 used RNA samples from the least dense to the most dense cells. Lane 7 is a positive control RNA sample from a Tg.AC mouse skin tumor. Lane 8 is a water blank, and lane 9 is 100 bp MW ladder (GibcoBRL). The RT–PCR product for Mß-2 was 212 bp (lower panel). Other details are given in (A) and Materials and methods.

 
Previous work indicated that early expression of the v-Ha-ras transgene or oncogenic c-Ha-ras could be located in cells of the hair follicle (10,13,14) and that these cells resembled epidermal stem cells (15). Furthermore, Morris et al. (16) had demonstrated that an enrichment of a subpopulation of incipient epidermal stem cells could be obtained by density gradient fractionation of isolated keratinocytes. Therefore it seemed likely that transgene expression could be detected with greater sensitivity if this subpopulation of keratinocytes could be further isolated and examined. To test this possibility, keratinocytes from TPA-treated mice were fractionated by buoyant density centrifugation through a Percoll gradient and RNA from the fractions tested by RT–PCR for transgene expression. Figure 2BGo shows the results for relative transgene expression in fractionated keratinocytes obtained 14 days after the start of four applications of 10 µg TPA to 25 week old mice. The highest level of transgene expression is in one of the denser cell fractions (Figure 2BGo, lane 4).

Based on the results of the previous experiments, we next tried to maximize age and TPA dose conditions in order to facilitate very early detection of transgene expression. Thirty-two week old mice were treated twice with 10 µg TPA (Monday and Friday) and were killed 9 days after the start of treatment. Transgene expression could not be detected in isolated keratinocytes (Figure 2CGo, lane 6), but when the cells were fractionated on the basis of buoyant density, transgene expression was detected in the denser keratinocyte fractions (Figure 2CGo, lanes 2–5).

Previous studies by Jones and Watt (18) and Jones et al. (19) indicated that keratinocytes with stem cell-like characteristics are rich in the cell surface marker ß1-integrin. This suggests that ß1-integrin expression might correlate with transgene expression, and that ß1-integrin could be used as a co-segregating marker for transgene expression in keratinocytes. Flow cytometry was used to sort keratinocytes from TPA-treated skin of Tg.AC mice, and cells with the highest and lowest level of ß1-integrin were collected as two distinct populations, and analyzed for the expression of the ras transgene by RT–PCR. The left panel of Figure 3AGo demonstrates that incubation of keratinocytes with an antibody to ß1-integrin in combination with a FITC-labeled secondary antibody caused a shift to the right, as compared with secondary antibody alone. The 20% ß1-dimmest and 20% brightest cells were sorted (Figure 3AGo, right panel) and RNA was extracted from the fractions for ras transgene expression analysis.



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  		Fig. 3. (A) Flow cytometric analysis of ß1-integrin expression in keratinocytes isolated from TPA-treated Tg.AC mice: 10 week old female Tg.AC mice were treated four times over a period of 2 weeks with 10 µg TPA and killed 14 days after the start of treatment. Keratinocytes were isolated as described in Materials and methods, and analyzed for expression of ß1-integrin by flow cytometry in the absence (pink) or presence (green) of primary ß1-integrin antibody to ensure specificity (left panel). The keratinocytes expressing ß1-integrin were then examined on an anti-ß1-integrin (FITC) versus propidium iodide dot plot to simultaneously isolate the 20% brightest and 20% dimmest ß1-integrin expressing cells by flow cytometry (right panel). (B) RT–PCR for ras transgene expression in ß1-bright and ß1-dim keratinocytes. Total RNA was extracted from ß1-integrin bright and dim keratinocyte populations and assayed for ras gene expression as described in Materials and methods. Product sizes for the ras transgene (upper panel) following PCR amplification using nested primers were 272 bp for contaminating DNA and 207 bp for processed mRNA. The RT–PCR product for the housekeeping gene Mß-2 was 212 bp (lower panel). Lanes 2, 4 and 6 show ras transgene-expressing ß1-integrin bright cells, and lanes 3, 5 and 7 show very faint or no transgene expressing ß1-integrin dim cells. Lane 8 is a positive control for transgene expression (SCC), and lanes 1 and 9 are water blanks. Molecular weight markers are 100 bp DNA ladder (Gibco BRL).

 
Figure 3BGo shows that the population of keratinocytes with the highest level of ß1-integrin (lanes 2, 4 and 6) expressed the transgene, whereas keratinocytes with the lowest level of ß1-integrin (lanes 3, 5 and 7) only faintly expressed the transgene or not at all. This result confirms that higher ß1-integrin expression correlates with transgene expression in keratinocytes from Tg.AC mice induced with the tumor promoter TPA.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Skin carcinogenesis has been studied extensively in non-transgenic mice using a two-stage (initiation/promotion) dosing regimen. Consistent with the two-stage model of carcinogenesis, tumors develop in these animals after sequential topical treatment with a single dose of the initiator (commonly, DMBA) followed by repetitive dosing with TPA or croton oil. In those studies, an age-dependent increase in the tumor response has only been rarely observed. In contrast, this study describes a strongly age-dependent sensitivity to tumor promotion in genetically initiated Tg.AC transgenic mice treated with TPA, a full-thickness wound incision or UV light (Figure 1Go, Table IGo).

In most experiments with non-transgenic mice, the papilloma response either decreases with age or is virtually the same as the response in younger mice. For example, Berenblum and Shubik (24) noted that a delay between initiation and promotion of either 3 or 43 weeks resulted in the same tumor yield in Swiss mice. Similar results were observed in STS mice by Boutwell (25), in NMRI mice by Loehrke et al. (26) and in SENCAR mice by Morris et al. (27). In contrast, a decrease in tumor induction was observed by Roe et al. (28) in Swiss mice and by Van Duuren et al. (29) and Stenbaeck et al. (30) in ICR/Ha mice. Morris et al. (27) also observed decreased papilloma multiplicity in CD-1 mice; however, the tumor incidence was virtually identical among mice initiated at 8 or 60 weeks of age and promoted 1 week later.

Several reports have shown an increased tumor response after a delay between initiation and promotion. Hennings and Boutwell (31) noted that delaying wounding 16 weeks after initiation led to a more pronounced tumor response compared with a delay of only 6 weeks. Delaying promotion has also been reported to lead to an increased tumor response with the promoter chrysarobin (32). Similar findings were noted with the promoter mezerein (33). However, a more extensive study with mezerein showed an increased papilloma response in some experimental groups when promotion with mezerein was delayed, but this observation was not reproducible with all doses of mezerein (34). In summary, these studies with non-transgenic mice strains do not present consistent and reproducible findings with respect to the relationship between tumor development and age of carcinogen exposure.

The age-dependent increase in tumor response reported in the present study with Tg.AC mice has been consistent and reproducible for over 20 successive generations. This is in marked contrast to the results from the conventional studies cited above. On the other hand, our study with the wild-type FVB/N parent mouse strain agrees with the results observed in the usual two stage (initiation/promotion) model (i.e. there was no difference in tumor yield among the FVB/N mice treated at 9 or 31 weeks of age, Table IIGo).

These data suggest that the presence and expression of the inducible v-Ha-ras transgene in Tg.AC mice alter the animal's response to tumor promoters; in particular, the transgene appears to confer an age-dependent increased sensitivity on this response. This notion is supported by the fact that transgene expression is strongly associated with tumor formation, and that induced temporal transgene expression in keratinocytes from mice exposed to TPA is also age-dependent, when responses in different aged mice exposed to similar dose regimens are compared (Figure 2AGo).

In this study, the response of Tg.AC mice to TPA was also dose-dependent. All animals showed an increase in tumor formation when the dose was increased 2-fold from 2.5 to 5 µg TPA (Table IGo). An additional dose increase to 10 µg TPA led to increased tumor development in 10 week old but not in 32 week old animals (Table IGo).

The same pattern of age-dependent response was also observed in the wound induced mice. Although the actual tumor multiplicity was much lower than that observed in the TPA or UV treated mice, it should be noted that the total wound area was about 1.5 cm2, which is 4–6-fold less than the 6–8 cm2 areas that were exposed to TPA or UV. In this context, as Argyris (35) pointed out, the wound repair process induces a papilloma response that is equal to or greater than that induced by classical promoters such as TPA.

The mechanism of the age-dependence of tumor development and ras transgene induction is not clear at present. One possibility is that normal developmental changes in keratinocytes are co-opted by the molecular mechanisms that regulate the induction of transgene expression, thus stimulating tumor formation in older Tg.AC mice. Several observations support this possibility. For example, newborn skin of the NMRI mouse strain is particularly resistant to induction of DNA synthesis by TPA, but this response increases steadily from 1 week after birth to 7 weeks after birth (36). Similar findings have been noted in BALB/c mice (37). Furthermore, the skin of newborn mice is resistant to the operationally defined Stage 1 skin tumor promoting activity of TPA (38). Ashcroft et al. (39) noted that older mice have an increased angiogenic response to skin wounding, and that ageing also influences the inflammatory response and extracellular matrix components of mouse skin. These findings suggest that a growth factor, cytokine or transcription factor(s) that is developmentally regulated could influence the target cells of skin tumorigenesis in Tg.AC mice. There is strong evidence that an early inflammatory response gene, GM-CSF, is essential for papilloma induction. A recent report (40) showed that suppression of GM-CSF dramatically reduced TPA induced papilloma multiplicity in Tg.AC mice.

Another possible factor influencing the age-dependence of tumor development in Tg.AC mice could be that changes have occurred in the methylation status of the transgene. Recently it was shown that hypomethylation of the transgene correlates with development of skin tumors in Tg.AC mice (41). Subtle changes in the methylation status of the transgene could occur during ageing and influence tumorigenesis in these mice.

It should be noted that previous studies using oncogenic mice with a ras transgene did not report an age-dependence of papilloma induction similar to that reported here (42–44). For example, Greenhalgh et al. (44) reported a progressive decrease in tumor development with age in mice with a ras transgene under the control of a human keratin 1 promoter.

The generality of the findings reported here for other tumor promoting agents remains to be explored. Presently, it is not clear if this mouse responds in an age-dependent manner to all carcinogenic stimuli, or only to stimuli that have been defined as skin tumor promoters such as TPA, wounding and UV radiation. This question should be explored in future studies.

Another significant finding of these studies is that the ras transgene in Tg.AC mice is more highly expressed in dense basal keratinocytes than in unfractionated keratinocytes (Figure 2BGo). Transgene expression was detected 9 days after the start of TPA treatment specifically in the denser fractions of isolated keratinocytes (Figure 2CGo). This is the earliest that we have been able to detect transgene expression after treatment onset. This is a significant finding, and suggests that the target cells of epidermal skin carcinogenesis are of a denser basal cell nature (16,17). Furthermore, a strong correlation was observed between a high level of expression of ß1-integrin, a cell surface marker abundant in putative epidermal stem cells (18,19) (Figure 3AGo) and expression of the ras transgene (Figure 3BGo). These results suggest that early transgene expressing keratinocytes represent epidermal progenitor cells. The induction of transgene expression after TPA exposure combined with co-localization with expression of ß1-integrin may provide a marker for latent neoplastic cells and aid in their further characterization in both Tg.AC and wild-type FVB/N mice.


   Notes
 
6 To whom correspondence should be addressed Email: spalding{at}niehs.nih.gov Back


   Acknowledgments
 
This work was supported in part by NIH Grant CA54293 awarded to R.J.M. The authors thank Nancy Mitchell, Stan Stasiewicz and Candace Roberts for their help in preparing the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 17, 2000; revised December 4, 2000; accepted December 21, 2000.


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