Fred Hutchinson Cancer Research Center C1-015, 1100 Fairview Avenue North, PO Box 19024, Seattle, WA 98109-1024, USA
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Abstract |
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Abbreviations: DMBA, dimethylbenz[a]anthracene; DNAPK, DNA-dependent protein kinase; dsbs, double-stranded breaks; SCID, severe combined immunodeficient; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction |
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SCID mice are prone to spontaneous T cell lymphomagenesis but have not shown predisposition to other spontaneous tumor types (3). Following a single dose of ionizing radiation or N-ethyl-N-nitrosourea, SCID mice developed T cell lymphomas with nearly 100% penetrance, but did develop other tumor types (10,11). One possible explanation for the lack of predisposition to other tumor types was that the tumor suppressor gene p53, which normally responds to DNA damage to initiate cell-cycle arrest or apoptosis, might be providing a protective effect. However mice which were mutant in both DNAPK and p53, e.g. scid/scid p53/ mice, did not show a generalized tumor predisposition, but exclusively developed lymphomas (11,12). A second possibility is that the DNA repair defect of SCID mice may not be important early in tumorigenesis, e.g. at tumor initiation, but could contribute to genetic instability associated with tumor progression. To test this idea we induced skin tumors in SCID mice with carcinogen treatment and measured tumor growth rate and malignant progression.
Two-stage chemical induction of skin cancer in mice is a well studied model, useful for discerning effects of host genetics, physiology and somatic tumor genetics on the stages of cancer development (13). A single application of carcinogen such as 7,12-dimethylbenz[a]anthracene (DMBA) results in tumor initiation. This is followed by twice weekly treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). After several weeks, benign papillomas develop and their number and growth rate can be measured. Following a latency of several months, a small percentage of these progress to malignant carcinomas which can also be quantified. Stage-specific somatic genetic alterations have been identified using this model. Tumor initiation is characterized by point mutations in the H-ras proto-oncogene, with >90% of papillomas and carcinomas having mutationally activated H-ras (14). Point mutation and deletion of the p53 tumor suppressor gene is a rate limiting step for conversion of papillomas to carcinomas (15,16). Amplification of the H-ras locus and deletion of loci on chromosome 4 are also observed in late stage spindle cell carcinomas (17,18). Thus, chromosomal rearrangements involving breakage and repair of dsbs are critical events during skin tumor progression. We hypothesized that the DNA dsb repair defect of SCID mice might increase the frequency of these events leading to an increase in tumor progression.
One important caveat to the use of mutants to study function is pleiotropism. Thus, in addition to the cellular based DNA repair defect, SCID mice are immunodeficient and the immune system could conceivably have both a positive (19) and negative (20) influence on tumor development. Differences in tumorigenesis observed in SCID mice could be attributed at the cellular level to the DNA repair defect or at the physiological level to immunodeficiency.
Therefore, to examine one of these distinct SCID phenotypes in isolation we used two additional mouse models. Rag1 and Rag2 knockout mice are deficient in the recombination-activating genes, Rag1 and Rag2 respectively (21,22). Both of the proteins encoded by these genes are required for initiating the process of V(D)J recombination during lymphocyte maturation. They do so by inducing dsbs at recombination signal sequences at the junction of the V, D and J segments. The coding sequences of these breaks are subsequently rejoined by DNAPK into functional V(D)J recombinants. Thus, Rag1 and Rag2 knockout mice lack mature T and B lymphocytes, and so share the immune defect of SCID mice, but have no known defects in DNA repair.
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Materials and methods |
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For the Rag1 skin tumor experiment, C57BL/6J Rag1/ mice were crossed to NIH mice to generate B6xNIH F1 Rag1+/ mice. These offspring were crossed together to generate B6xNIH F2 Rag1/ and Rag1+/+ mice. Mice were genotyped with respect to Rag1 gene status by PCR of toe-derived DNA (23). There were 23 Rag1/ and 25 wild-type littermates used.
For the Rag2 skin tumor experiment, 129 Rag2/ mice were crossed to NIH mice to generate 129xNIH F1 Rag2+/ mice. These offspring were crossed together to generate 129xNIH F2 Rag2/ and +/+ mice and again genotyped for Rag2 status by PCR (24). There were 29 Rag2/ and 28 control littermates used.
Tumor induction
The dorsal skins of 7-week-old mice were shaved and treated with a single application of DMBA (25 µg in 200 µl acetone) (Sigma) followed 1 week later by twice weekly applications of TPA (200 µl of 104 M acetone solution) (Sigma). TPA treatment was stopped after 15 weeks. The number and size of papillomas on each mouse was recorded at biweekly intervals. Mice were killed if moribund or following the detection of carcinomas, and the tumors fixed in formalin for histological analysis. Carcinomas were identified by visual inspection and confirmed by microscopic examination of hematoxylin and eosin (H&E) stained sections.
TPA-induced apoptosis
The dorsal skin of mice was shaved and treated with a single dose of TPA (200 µl of 104 M acetone solution), DMBA (25 µg in 200 µl acetone) or acetone alone and the skin sections subsequently fixed in formalin, processed and stained with H&E. Mice that were in the hair regeneration phase were not used. Apoptotic bodies in cells lining the hair follicles were identified by morphology and quantified per hair follicle. At least 30 hair follicles were counted in three skin sections from each animal. There were four to eight animals per group and all sections were read without knowledge of the genotype of the animal.
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Results |
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Both the rate of appearance and the total number of papillomas were significantly reduced in scid/scid compared with control mice of the same genetic background (Figure 1A). By 29 weeks, the scid/scid mice averaged two papillomas per mouse versus 11 papillomas per mouse for the control mice. These papillomas were monitored for conversion to carcinomas through 50 weeks of age. The incidence of squamous cell carcinomas in scid/scid mice was 0.23 carcinomas/mouse and for wild-types was 0.50 carcinomas/mouse. The conversion frequency of papillomas to carcinomas in scid/scid mice was 5.6% (three carcinomas/53 papillomas) and for control mice was 7.5% (20 carcinomas/267 papillomas). Thus, malignant progression was unaffected by DNAPK deficiency.
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We conclude that mice deficient in DNAPK activity are resistant to DMBA/TPA-induced skin tumor development. As this result was contrary to predictions, we wished to examine each of the two major SCID phenotypes in isolation, to identify the mechanism of this tumor resistance. Earlier work had shown that immunodeficient nude mice were resistant to chemically induced skin tumor development (25,26). As the immune system is very active in the epidermis, we first tested whether the immunodeficiency in SCID mice contributed to skin tumor resistance.
Skin tumor formation is unaltered in Rag1 and Rag2 deficient mice
We treated groups of Rag1/ and wild-type littermates as well as Rag2/ and wild-type littermates on the dorsal skin with DMBA/TPA (see Materials and methods). In contrast to the scid/scid mice, the Rag1 and Rag2 deficient mice did not develop lymphomas. The rate of appearance and total number of papillomas did not differ between Rag1/ mice and wild-type littermates nor between Rag2/ and their control littermates (Figure 1C and D). We monitored the papillomas of Rag1 deficient mice through 50 weeks for malignant conversion. The incidence of carcinomas in Rag1/ mice was 0.22 carcinoma/mouse and for controls was 0.24 carcinomas/mouse. The conversion frequency of papillomas to carcinomas for Rag1/ mice was 8% (five carcinomas/60 papillomas) and for wild-types was 10% (six carcinomas/60 papillomas). The Rag2/ mice were terminated at 25 weeks of age and progression was not measured.
This result indicates that the absence of T and B lymphocytes neither enhanced nor inhibited papillomagenesis or malignant progression. This also indicated that the resistance of SCID mice to papillomagenesis was unrelated to their immunodeficiency and suggested instead that the cellular defect in DNA dsb repair was responsible.
The tumor promoter used in these studies, TPA, has a multitude of effects, some of which are to induce DNA strand breaks, sister chromatid exchange and other structural and numerical chromosomal aberrations (2729). The mechanism by which TPA induces these events is not understood but it is believed that they may be important for tumor promotion (28). As SCID cells are sensitive to killing by other agents that induce DNA dsbs such as bleomycin and radiation (6,30), we tested whether epidermal cells of SCID mice were hypersensitive to killing by TPA. This would address whether the reduced skin tumor yield in SCID mice was due to cytotoxicity of TPA.
Increased TPA-induced apoptosis and cell killing in the epidermis of SCID mice
TPA induced a modest increase in apoptotic bodies in the hair follicles of control mice at 48 and 72 h post-treatment (Figure 2). This was accompanied by marked epithelial hyperproliferation and leukocyte infiltration, as has been well documented. TPA-treated SCID epidermis showed a 410-fold increase in the number of apoptotic cells relative to wild-type mice at all time points examined (Figure 2
). Apoptotic cells were primarily located in the bulb region at the base of the hair follicle and to a lesser extent in outer root sheath cells along the hair shaft. This is the same region where cells undergo p53-dependent apoptosis following ionizing radiation (31). In addition, TPA-treated SCID skin showed extensive cell killing of the basal and suprabasal keratinocytes with complete degeneration of the epidermis in some areas at 24 and 48 h. By 72 h, the SCID epidermis had partially recovered and displayed the normal degree of hyperplasia. There was no difference between SCID and wild-type mice in the number of apoptotic cells following DMBA treatment or acetone alone.
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Discussion |
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Immunodeficiency and skin cancer
There are two conflicting theories on the role of the immune system in skin cancer. Briefly, the immunosurveillance theory proposes that tumors arise as a result of a failure of the immune system to recognize and eliminate the nascent tumor cells (20). Tumors produce antigens that should be recognized by the immune system leading to their elimination. The immunostimulation theory suggests that the immune system can, in certain instances contribute to tumor growth (19). Weakly antigenic tumors may recruit immune cells that fail to eliminate the tumor and instead produce cytokines that stimulate tumor growth (19). The evidence to support both theories has recently been reviewed (32). The murine skin contains a large variety of immune cell types (32). Treatment with tumor promoters such as TPA induces a rapid infiltration of additional immune cells and an inflammatory response. This leads to release of cytokines as well as reactive oxygen that can indirectly induce chromosome damage in the target epidermal cells (32,33). These effects could enhance skin tumor formation.
Nude mice have a defect in the thymic stromal environment, an area that is necessary for T cell maturation (34). Consequently, these mice lack functionally mature T lymphocytes and are immunocompromised. Using a tumor induction protocol similar to that used here, it was shown that nude mice were resistant to chemically induced skin papillomas (25,26), similar to our results with SCID mice. Superficially, this suggested that a normal immune response might accelerate tumor growth and the deficient immune response in nude and SCID mice results in fewer tumors. However the nude mutation is also pleomorphic, leading as well to a lack of hair and other epidermal abnormalities. Thus, it is not possible to unambiguously attribute skin tumor resistance of either nude or SCID mice to their immune defect.
Here we used Rag knockout mice, whose only known defect is the lack of mature T and B lymphocytes, to specifically address the role of T and B cells in skin tumorigenesis. In two separate experiments, using both Rag1 and Rag2 deficient mice, we observed neither an increase nor a decrease in papilloma induction, nor altered progression to carcinoma relative to control littermates. This result demonstrates that T or B lymphocyte mediated immunity does not play a prominent role, either positive or negative, in DMBA/TPA-induced skin carcinogenesis.
Spontaneous tumorigenesis is not increased in nude, Rag1/, Rag2/ or scid/scid mice, with the notable exception of T cell lymphomas in scid/scid mice (3,11,21,22,35). The latter is likely attributable to the specific defect in V(D)J end joining in immature T lymphocytes, and not to their immune defect (36). The scid mutation also has no effect on intestinal adenoma formation. Min/+ mice are genetically predisposed to adenoma formation due to a mutation in the Apc gene (37). Min/+ scid/scid mice were shown to develop the same number of adenomas as Min/+ mice, indicating that the growth of these lesions is also T and B cell independent (38). Thus, despite clear evidence for immunorecognition of tumors in mice, a significant inhibitory role for T and B cells in autochthonous tumor development has not been established. The use of mutant mice with specific immune defects remains a powerful tool to address the role of immunity in cancer.
DNAPK and cancer
With the exception of T cell lymphomas, SCID mice are not predisposed to tumor development in other tissues. This is the case even after whole body irradiation or treatment with chemical carcinogens (10,11,39) and despite the fact that multiple cell types are hypersensitive to DNA damage. Thus, the defect in DNA dsb repair due to DNAPKcs deficiency is not a major predisposing factor for carcinogenesis. The lymphomagenesis observed in SCID mice is probably a particular consequence of the arrested maturation of T cells due to their inability to complete V(D)J T cell receptor recombination (36). In other tissues, the unrepaired DNA breaks that may result from DNAPK deficiency are apparently not efficiently converted to carcinogenic lesions. However, in one setting, DNAPK deficiency did increase tumor progression, apparently by increasing chromosomal alterations. In a previous study, we showed that spontaneous lymphomagenesis was accelerated in scid/scid p53+/ mice relative to scid/scid or p53+/ mice alone (11). We used the wild-type allele from p53+/ mice as a target for measuring the effect of the scid mutation on the frequency of loss of heterozygosity (LOH). Importantly, selection for loss of the allele was not artificially imposed but occurred naturally during tumor development. By Southern blot analysis, 100% of the tumors examined from scid/scid p53+/ mice had lost the wild-type p53 allele. By comparison, the frequency of LOH of p53 in tumors from p53+/ mice was 5070% (40,41). Thus, in at least one setting, mutant DNAPK appeared to enhance LOH of a tumor suppressor gene, leading to enhanced tumorigenesis.
DNAPK mediates rejoining of only a subset of DNA dsbs. For example, the generation and rejoining of dsbs are intermediates in meiotic recombination and we and others have shown that SCID mice have a normal frequency of meiotic recombination (42; K.V. and C.J.K., unpublished observations). SCID cells are also able to repair a substantial fraction of damage-induced dsbs, presumably via other end joining or homologous recombination pathways (5). As DNA dsbs are likely intermediates in the generation of certain chromosomal aberrations, it will be informative to determine if defects in these other pathways of dsb repair and recombination will result in tumor predisposition.
TPA, DNAPK and apoptosis
SCID mice and cells deficient in DNAPK are sensitive to killing by agents that induce DNA dsbs such as radiation and bleomycin (6). TPA was reported to induce DNA strand breaks in leukocytes, sister chromatid exchange and structural and numerical chromosome aberrations in mouse keratinocytes, and is recombinogenic in mammalian cells (2729,43). However, the mechanism for these effects, as well as the consequences in terms of tumor promotion are not known. There was a large increase in the number of apoptotic cells of the hair follicles as well as extensive killing of epidermal keratinocytes in SCID mice after a single application of TPA, compared with wild-type mice. This response has similarities to that observed in SCID epidermis following
irradiation (6) and suggests that TPA induces DNA strand breaks that are normally repaired by a DNAPK-dependent pathway. In the absence of DNAPK these lesions remain unrepaired leading to excessive cell death. This result adds support to the idea that chromosome aberrations induced by TPA are important for its tumor promoting activity (28). It is not known whether papillomas arise from cells of the interfollicular epidermis or from the hair follicle. The fact that both cell types from SCID mice were hypersensitive to killing by TPA suggests that the reduction in papillomas seen in these mice was due to excessive loss of initiated incipient tumor cells. It is noteworthy that p53-deficient mice also show reduced papilloma formation (16,44). However, unlike wild-type or SCID mice, hair follicle cells of p53-deficient mice fail to undergo apoptosis in response to DNA damage (31, and unpublished observations). Furthermore, hair follicle cells of SCID mice undergo normal p53-dependent apoptosis indicating that the p53 pathway remains functional in these mice (31). Thus, the mechanism for reduced papillomagenesis is different between p53-deficient and SCID mice.
These studies with SCID mice highlight the fact that not all types of genetic damage are uniformly carcinogenic. Tissue specific and environmental factors can also influence the effect of DNA repair defects on tumor predisposition. It will be informative to compare results from SCID mice to other mouse models of DNA repair defects or genomic instability to establish which specific types of genomic damage are relevant to tumor progression in which tissues.
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Acknowledgments |
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Notes |
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References |
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