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
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Abstract |
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Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; mouse-ß2-microglobulin (Mß-2); RTPCR, reverse transcriptionpolymerase chain reaction; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction |
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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 (811) and expression can be detected prior to the appearance of visible papillomas (810). 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 (1215). 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 2030 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 RTPCR 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.
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Materials and methods |
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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 15 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 1924 animals per dose group, and experiments with Tg.AC mice used 912 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, RTPCR products were separated by electrophoresis on a 2% SeaKem GTG agarose gel (FMC Bioproducts, Rockland, ME) prepared with 1x Tris-borateEDTA, 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 (PBSBSA) 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 PBSBSA to remove excess antibody. Cells were resuspended in 5 ml PBSBSA and triturated eight times, and 45 ml PBSBSA were added to each tube. The cells were centrifuged and resuspended in PBSBSA at a concentration of 45x106 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 12x105 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 RTPCR 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.
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Results |
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The results for animals treated with 2.5 µg TPA are shown in Figure 1A and B. 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 1A
). 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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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 D). 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 1C
). 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 90100% among the three older groups of mice and 60% in mice that were 5 weeks old at treatment start (Figure 1D
).
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 I. In the 10 week old mice, a significantly higher papilloma multiplicity (16.9) per mouse was observed (P
0.05, MannWhitney U test) than in same aged animals treated with 5 µg TPA (Table I
); 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 I
). In both age groups treated with 10 µg TPA, all animals developed papillomas (Table I
, legend).
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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 1E). The average maximum multiplicity was 4.3 papillomas per mouse and the tumor incidence was 100% in the twice wounded mice (Figure 1E and F
).
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 I). Tumor incidence was only slightly higher in the older mice than in the younger mice treated with UV (Table I
).
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, 90100% 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 II, 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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At various times after the last TPA dose, mice of different ages at treatment start were euthanized, keratinocytes harvested and RTPCR 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 2A, 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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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 2C, lane 6), but when the cells were fractionated on the basis of buoyant density, transgene expression was detected in the denser keratinocyte fractions (Figure 2C
, lanes 25).
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 RTPCR. The left panel of Figure 3A 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 3A
, right panel) and RNA was extracted from the fractions for ras transgene expression analysis.
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Discussion |
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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 II).
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 2A).
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 I). 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 I
).
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 46-fold less than the 68 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 (4244). 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 2B). Transgene expression was detected 9 days after the start of TPA treatment specifically in the denser fractions of isolated keratinocytes (Figure 2C
). 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 3A
) and expression of the ras transgene (Figure 3B
). 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.
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Notes |
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Acknowledgments |
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References |
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