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PDF file - 1781K, Effect of metformin on epidermal phospho-ULK1 in the absence or presence of TPA treatment.

Although it is well known that the majority of human cancers occur as the result of exposure to environmental carcinogens, it is clear that not all individuals exposed to a specific environmental carcinogen have the same risk of developing cancer. Considerable evidence indicates that common allelic variants of low-penetrance, tumor susceptibility genes are responsible for this interindividual variation in risk. We previously reported a skin tumor promotion susceptibility locus, Psl1, which maps to the distal portion of chromosome 9, that modified skin tumor promotion susceptibility in the mouse. Furthermore, Psl1 was shown to consist of at least two subloci (i.e., Psl1.1 and Psl1.2) and that glutathione S-transferase alpha 4 (Gsta4), which maps to Psl1.2, is a skin tumor promotion susceptibility gene. Finally, variants of human GSTA4 were found to be associated with risk of nonmelanoma skin cancer. In the current study, a combination of nested and contiguous C57BL/6 congenic mouse strains, each inheriting a different portion of the Psl1 locus from DBA/2, were tested for susceptibility to skin tumor promotion with 12-O-tetradecanoylphorbol-13-acetate. These analyses indicate that Psl1 is a compound locus with at least six genes, including Gsta4, that modify skin tumor promotion susceptibility. More than 550 protein-coding genes map within the Psl1 locus. Fine mapping of the Psl1 locus, along with two-strain haplotype analysis, gene expression analysis, and the identification of genes with amino acid variants, has produced a list of fewer than 25 candidate skin tumor promotion susceptibility genes.

In the present study, the ability of metformin to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was analyzed in mice maintained on either an overweight control diet or an obesity-inducing diet. Rapamycin was included for comparison, and a combination of metformin and rapamycin was also evaluated. Metformin (given in the drinking water) and rapamycin (given topically) inhibited development of both papillomas and squamous cell carcinomas in overweight and obese mice in a dose-dependent manner. A low-dose combination of these two compounds displayed an additive inhibitory effect on tumor development. Metformin treatment also reduced the size of papillomas. Interestingly, all treatments seemed to be at least as effective for inhibiting tumor formation in obese mice, and both metformin and rapamycin were more effective at reducing tumor size in obese mice compared with overweight control mice. The effect of metformin on skin tumor development was associated with a significant reduction in TPA-induced epidermal hyperproliferation. Furthermore, treatment with metformin led to activation of epidermal AMP-activated protein kinase (AMPK) and attenuated signaling through mTOR complex (mTORC)-1 and p70S6K. Combinations of metformin and rapamycin were more effective at blocking epidermal mTORC1 signaling induced by TPA consistent with the greater inhibitory effect on skin tumor promotion. Collectively, the current data demonstrate that metformin given in the drinking water effectively inhibited skin tumor promotion in both overweight and obese mice and that the mechanism involves activation of epidermal AMPK and attenuated signaling downstream of mTORC1. Cancer Prev Res; 7(1); 54–64. ©2013 AACR.

In the past 20 yr, several inbred strains have been derived from SENCAR outbred mice. These strains display different susceptibility to the induction of papillomas and progression to squamous cell carcinomas (SCC) in the skin after chemical carcinogenesis. In the present study, we showed that one of these strains SENCARB/Pt was highly susceptible to the development of N-methyl-N-nitrosourea (MNU)- and 7,12-dimethylbenz[a]anthracene (DMBA)-induced lymphomas. In contrast, the SSIN/Sprd inbred strain is completely resistant to T-cell lymphomagenesis by both carcinogens. Within 175 d after a single injection of 75 mg/kilogram body weight (kbw) of MNU, SENCARB/Pt mice exhibited a 91.6% incidence of lymphoma. In addition, during an independent tumorigenesis study with repeated doses of intragastric DMBA, SENCARB/Pt mice showed an incidence of 75% lymphoma development 300 d after the last treatment. Histopathological and flow cytometric parameters indicated that the lymphomas were of the T-cell lineage. In order to study the genetics of MNU-induced tumorigenesis, we generated F1 hybrid mice between SSIN/Sprd and SENCARB/Pt mice. Tumor incidence in MNU-injected F1 mice suggested that the high tumor incidence is a dominant trait. Loss of heterozygosity (LOH) analysis in these tumor samples revealed allelic imbalances on chromosomes 15 and 19. Given that these inbred strains are closely related, it is likely that a relatively small number of loci are responsible for the observed differences in susceptibility. Therefore, these SENCAR inbred strains constitute important new tools to study the genetic basis of resistance and susceptibility to chemically induced thymic lymphoma formation.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact potygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

Susceptibility to N-methyl-N-nitrosourea (MNU)-induced lymphomas is a multigenic trait. We previously mapped a resistance locus (Tlag1) to mouse chromosome 7 in genetic crosses of sensitive AKR with resistant C57L mice. Analysis of the MNU sensitivity of AKXL recombinant inbred strains that are homozygous for the AKR allele of Tlag1 suggested that at least two additional tumor susceptibility loci segregate in these crosses. A second susceptibility locus (Tlag2) now has been mapped to chromosome 4. Only those mice that inherited the susceptibility alleles at both Tlag1 and Tlag2 were sensitive to MNU induction of thymic lymphomas, suggesting that these two loci interact. Chromosome 4 has been associated with susceptibility to hematopoietic tumor development in several mouse models, suggesting that one or more genes mapping to this chromosome are important in lymphomagenesis in general.

While inheritance of mutated alleles of highly penetrant tumor suppressor genes such as retinoblastoma or p53 predisposes individuals to a greatly increased risk of developing cancer, epidemiological data indicate that the majority of sporadic tumors in humans result from interactions of environmental and host genetic factors. The host genetic factors are poorly penetrant tumor susceptibility genes that determine the likelihood that a cancer will arise from carcinogen exposure. The majority of colon tumors in humans are sporadic in nature. 1,2-dimethylhydrazine (DMH)–induced colon tumors in mice provide a useful animal model to identify genes that influence susceptibility to carcinogen-induced colon tumors in humans. A genome-wide scan of genetic crosses of relatively sensitive C57BL/6J with relatively resistant CBA mice treated with DMH revealed a linkage of DMH susceptibility with the distal end of mouse chromosome 3, suggesting that one or more tumor susceptibility genes may map to this region. Mol. Carcinog. 27:47–54, 2000. © 2000 Wiley-Liss, Inc.

It has been known for many years that there are dramatic differences in the susceptibility of mouse stocks and strains to two-stage skin carcinogenesis and that these differences are due to the animals' responsiveness to tumor-promoting agents. In earlier studies using several inbred mouse strains, we found that susceptibility to skin tumor promotion by phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) is a multigenic trait. To extend this work, we conducted a genome scan of (C57BL/6 × DBA/2)F1 × C57BL/6 mice previously scored for sensitivity to skin tumor promotion by TPA. As a result of this scan, we now report an association of increased TPA promotion susceptibility with inheritance of the DBA/2 alleles of markers on the distal portion of mouse chromosome 9. Additional linkage analyses using (C57BL/6 × DBA/2)F2 and B×D recombinant inbred mice confirmed this association and suggested that a TPA promotion susceptibility locus maps near D9Mit51 (LODw = 4.1). We designated this locus promotion susceptibility locus 1 (Psl1). Mol. Carcinog. 20:162–167, 1997. © 1997 Wiley-Liss, Inc.

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

The incidence of nonmelanoma skin cancer (NMSC) is equivalent to that of all other cancers combined. Previously, we mapped the 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion susceptibility locus, Psl1, to distal chromosome 9 in crosses of sensitive DBA/2 mice with relatively resistant C57BL/6 mice. Here, we used the mouse two-stage skin carcinogenesis model to identify the gene(s) responsible for the effects of Psl1.Interval-specific congenic mouse strains (n ≥ 59 mice per strain) were used to more precisely map the Psl1 locus. Having identified glutathione S-transferase α4 (Gsta4) as a candidate tumor promotion susceptibility gene that mapped within the delimited region, we analyzed Gsta4-deficient mice (n = 62) for susceptibility to skin tumor promotion by TPA. We used quantitative polymerase chain reaction, western blotting, and immunohistochemistry to verify induction of Gsta4 in mouse epidermis following TPA treatment and biochemical assays to associate Gsta4 activity with tumor promotion susceptibility. In addition, single-nucleotide polymorphisms (SNPs) in GSTA4 were analyzed in a case-control study of 414 NMSC patients and 450 control subjects to examine their association with human NMSC. Statistical analyses of tumor studies in mice were one-sided, whereas all other statistical analyses were two-sided.Analyses of congenic mice indicated that at least two loci, Psl1.1 and Psl1.2, map to distal chromosome 9 and confer susceptibility to skin tumor promotion by TPA. Gsta4 maps to Psl1.2 and was highly induced (mRNA and protein) in the epidermis of resistant C57BL/6 mice compared with that of sensitive DBA/2 mice following treatment with TPA. Gsta4 activity levels were also higher in the epidermis of C57BL/6 mice following treatment with TPA. Gsta4-deficient mice (C57BL/6.Gsta4(-/-) mice) were more sensitive to TPA skin tumor promotion (0.8 tumors per mouse vs 0.4 tumors per mouse in wild-type controls; difference = 0.4 tumors per mouse; 95% confidence interval = 0.1 to 0.7, P = .007). Furthermore, inheritance of polymorphisms in GSTA4 was associated with risk of human NMSC. Three SNPs were found to be independent predictors of NMSC risk. Two of these were associated with increased risk of NMSC (odds ratios [ORs] = 1.60 to 3.42), while the third was associated with decreased risk of NMSC (OR = 0.63). In addition, a fourth SNP was associated with decreased risk of basal cell carcinoma only (OR = 0.44).Gsta4/GSTA4 is a novel susceptibility gene for NMSC that affects risk in both mice and humans.

Human cancers result from a combination of exposure to carcinogens combined with individual susceptibility. Epidemiologic studies, as well as studies in animal models of cancer, have demonstrated a wide range of responses to carcinogen exposure and it is now clear that common allelic variants within the genome are responsible for this interindividual variation. Identification of specific genes that modify the response to carcinogen exposure has been difficult. However, new approaches in both human and animal studies have improved the likelihood that cancer modifier genes will be identified. Importantly, many of the modifier genes identified in animal models have been shown to modify cancer risk in humans, demonstrating the utility of animal studies. The identification of cancer modifier genes will ultimately lead to the development of new cancer risk models, the detection of individuals at high risk for cancer development, and improved prevention strategies.

In this study, the potential role of Stat3 in UVB-induced skin carcinogenesis was examined using skin-specific gain and loss of function transgenic mice, i.e., K5.Stat3C and K5Cre.Stat3fl/fl mice, respectively. The epidermis of Stat3-deficient mice was highly sensitive to UVB-induced apoptosis, whereas the epidermis of K5.Stat3C mice was more resistant to UVB-induced apoptosis. In particular, the status of Stat3 influenced the survival of UV-photoproduct cells, including those located in the bulge region of hair follicles. K5.Stat3C mice exhibited significantly increased epidermal proliferation and hyperplasia in response to UVB irradiation, whereas Stat3-deficient mice showed reduced epidermal proliferation and hyperplasia. Expression of target genes regulated by Stat3, such as cyclin D1 and Bcl-xL, was increased in epidermis of both control and UVB-irradiated K5.Stat3C mice, and downregulated in epidermis of both control and UVB-irradiated K5Cre.Stat3fl/fl mice. Following UVB irradiation, the formation of skin tumors in K5.Stat3C mice was accelerated and both the incidence and multiplicity of skin tumors was significantly greater than wild-type controls. In contrast, Stat3-deficient mice were resistant to UVB skin carcinogenesis. These results demonstrate that Stat3 plays an important role in the development of UVB-induced skin tumors through its effects on both survival and proliferation of keratinocytes during carcinogenesis.

Previous data from two-stage carcinogenesis studies in mouse skin demonstrated that genetic control of susceptibility to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), in crosses between susceptible DBA/2J and resistant C57BL/6J mice is a multigenic trait. Utilizing a cDNA microarray approach, we compared global gene expression profiles in the epidermis of these two mouse strains treated with TPA or vehicle (acetone). Gene expression in the epidermis was analyzed after the treatment to identify global effects of TPA, as well as potential candidate genes that modify susceptibility to skin tumor promotion. DBA/2J and C57BL/6J mice were treated topically four times with 3.4 nmol TPA or acetone over a 2-wk period, and RNA was extracted from epidermis 6 h after the final treatment. Labeled cDNA generated from each group was hybridized to commercial cDNA microarrays (Agilent) containing more than 8000 targets. More than 450 genes were significantly influenced, directly or indirectly, by TPA treatment in the epidermis of either strain. Notably, 44 genes exhibited differential expression between the tumor promotion sensitive and resistant mouse strains. Several genes that were differentially expressed in DBA/2J versus C57BL/6J epidermis after TPA treatment were located in chromosomal regions linked to TPA promotion susceptibility. Three genes, Gsta4, Nmes1 (MGC58382), and Serpinb2, located within promotion susceptibility loci Psl1 (chr 9), Psl2 (chr 2), and Psl3 (chr 1), respectively, were identified in this analysis as potential candidates for modifiers of susceptibility to skin tumor promotion by TPA.

International audience; This white paper by eighty members of the Complex Trait Consortium presents a community's view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

Genetic differences in susceptibility to two-stage skin carcinogenesis have been known for many years. Studies of genetic crosses of sensitive DBA/2 with resistant C57BL/6 mice suggested that multiple autosomal genes determine the sensitivity of these mice to 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion. Previous studies mapped one promotion susceptibility locus, Psl1, to distal chromosome 9. Analysis of TPA promotion susceptibility in (C57BL/6 x DBA/2)F(1) x C57BL/6 mice and B x D recombinant inbred mouse strains suggested tentative associations of promotion susceptibility with several other chromosomal regions. To confirm these associations (C57BL/6 x BxD27)F(2) mice analyzed for TPA promotion susceptibility were genotyped for polymorphic genetic markers mapping to chromosomal regions for which tentative associations had been previously detected. BxD27 mice are sensitive to TPA skin tumor promotion but carry the C57BL/6 allele of Psl1. Because Psl1 does not segregate in this cross, its effect on TPA promotion susceptibility is the same for all mice in the cross. The results of this analysis support the mapping of three novel promotion susceptibility loci to chromosomes 1, 2, and 19. Psl2 maps near D2Mit229 on distal chromosome 2, and inheritance of the dominant DBA/2 allele results in increased sensitivity to TPA. Psl3 maps near D1Mit511 on distal chromosome 1. Interestingly, inheritance of an allele from the resistant C57BL/6 parent results in increased sensitivity to TPA. Psl3 appears to have an additive affect, with heterozygous mice having a stronger response to TPA than mice homozygous for the DBA/2 allele and a weaker response to TPA than mice homozygous for the C57BL/6 allele. Psl4 maps near D19Mit38 on distal chromosome 19 and inheritance of the dominant C57BL/6 allele results in decreased TPA sensitivity. Analysis of the combined effects of these loci on TPA promotion susceptibility indicates that they contribute independently to the overall sensitivity to TPA.

Susceptibility to N-methyl-N-nitrosourea (MNU)-induced lymphomas is a multigenic trait. We previously mapped a resistance locus (Tlag1) to mouse chromosome 7 in genetic crosses of sensitive AKR with resistant C57L mice. Analysis of the MNU sensitivity of AKXL recombinant inbred strains that are homozygous for the AKR allele of Tlag1 suggested that at least two additional tumor susceptibility loci segregate in these crosses. A second susceptibility locus (Tlag2) now has been mapped to chromosome 4. Only those mice that inherited the susceptibility alleles at both Tlag1 and Tlag2 were sensitive to MNU induction of thymic lymphomas, suggesting that these two loci interact. Chromosome 4 has been associated with susceptibility to hematopoietic tumor development in several mouse models, suggesting that one or more genes mapping to this chromosome are important in lymphomagenesis in general.

Susceptibility to two-stage skin carcinogenesis in the mouse is affected by several genes. In addition, studies suggest that genes that modify the response of mice to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) also may influence histologic changes in the skin as the result of TPA treatment. One TPA susceptibility locus, Psl1, previously was mapped to distal chromosome 9. The mapping of this locus was confirmed by marker-based genotypic selection. Furthermore, Psl1 or a gene closely linked to Psl1 influenced epidermal hyperplasia and epidermal labeling index of mice treated with TPA.

The mouse Polycomb-group gene, embryonic ectoderm development (eed), appears to regulate cellular growth and differentiation in a developmental and tissue specific manner. During embryogenesis, eed regulates axial patterning, whereas in the adult eed represses proliferation of myeloid and B cell precursors. The present report demonstrates two novel functional activities of eed: alteration of thymocyte maturation and suppression of thymic lymphoma development. Mice that inherit the viable hypomorphic 17Rn5(1989SB) eed allele sustain a partial developmental block at or before the CD4(-)CD8(-)CD44(-)CD25(+) stage of thymocyte differentiation. Furthermore, mice that are homozygous or heterozygous for the hypomorphic eed allele have an increased incidence and decreased latency of N-methyl-N-nitrosourea-induced thymic lymphoma compared to wild-type littermates. These findings support the notion that Polycomb-group genes exert pleiotropic effects dictated by developmental stage and cellular context.

Cancer development is a multistep process that involves both the activation of protooncogenes and the inactivation of tumor suppressor genes. Furthermore, both epidemiological and experimental data indicate that a third class of genes, tumor susceptibility genes, control the propensity to develop carcinogen-induced tumors. Recent studies suggest that tumor susceptibility is determined by the combined effect of both sensitivity and resistance genes. The mouse skin model of multistage carcinogenesis is an excellent paradigm in which to study the genetics of cancer susceptibility. This is particularly true with regard to tumor promotion, a process that occurs in other organs and species including humans. We have studied the genetics of tumor promotion susceptibility in the mouse two-stage skin tumor model using crosses between sensitive DBA/2 or C3H and resistant C57BL/6 mice. Our results suggest that TPA promotion susceptibility is a multigenic trait. We have tentatively mapped one tumor susceptibility locus, Psl1, to mouse chromosome 9 and are currently identifying and characterizing candidate tumor susceptibility genes that map to this chromosomal region. The multistage model of carcinogenesis in mouse skin has, for more than 50 years, provided a conceptual framework from which to study the carcinogenesis process. Many concepts now currently applied to other tissues and model systems were originally derived from the mouse skin model. Because tumor promotion is an important component of carcinogenesis in humans, the identification of genes that modify response to tumor-promoting stimuli would be a significant advancement in our understanding of the genetic basis of susceptibility to cancer.

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with tumor promotion susceptibility by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a proteomic approach of two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. In these experiments, we examined epidermal protein lysates and identified proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following TPA treatment. Among 19 differentially expressed proteins identified using this methodology were two calcium-binding proteins, S100A8 and S100A9. Their differential expression was further examined and validated by one or more of the following methods: i) one-dimensional (1-D) Western blot analysis; ii) 2-D Western blot analysis; iii) immunohistochemical analysis; and iv) quantitative real-time PCR. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially up-regulated in epidermis of DBA/2 vs C57BL/6 mice following topical treatment with two other tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that S100A8/A9 could be linked to several inflammatory networks. Further analyses revealed significantly increased expression of several inflammation-related genes including TNF-α, NFκB and IL-22 in epidermis of TPA-treated DBA/2 mice. Follow-up studies confirmed that these three inflammation related genes were upregulated in epidermis of TPA-treated DBA/2 mice compared to similarly treated C57BL/6 mice. These data suggest that differential expression of inflammation related genes in epidermis contributes to TPA-induced inflammation and skin tumor promotion susceptibility in DBA/2 mice. Taken together, our present data provide further insight into potential molecular mechanisms for the differential susceptibility of DBA/2 and C57BL/6 mice in terms of both inflammation and skin tumor promotion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2011-2729

N-Methyl-N-nitrosourea (MNU) is a potent carcinogen that causes the development of murine thymic lymphomas. MNU-induced tumor incidence varies considerably among different inbred mouse strains. In particular, the AKR strain is highly susceptible, whereas the C57L strain is highly resistant to MNU-induced lymphoma formation. Crosses between AKR and C57L mice were established to investigate the genetic basis for the differential susceptibility of these inbred strains. A strong association between MNU-induced lymphoma development and coat color was observed in (AKR x C57)F2 and AKR x (AKR x C57)F1 progeny such that albino mice developed a higher tumor incidence than nonalbino animals. These data suggest that a locus on chromosome 7 influences tumor development. Analysis of four additional polymorphic loci (D7Rp2, Fes, Hbb, and Int-2) on chromosome 7 in AKR x (AKR x C57)F1 backcross mice revealed a significant linkage between high tumor incidence and homozygous inheritance of AKR alleles at the albino (tyrosinase) and Hbb loci. Thus, inheritance of at least one C57L allele at the albino or Hbb loci was associated with protection against MNU-induced lymphoma development. There was no association between tumor incidence and genotype at the D7Rp2, Fes, or Int-2 loci. Taken together, the data suggest that whereas C57L mice contain a dominant tumor suppressor gene on chromosome 7, in the AKR strain both alleles at this locus are defective resulting in enhanced susceptibility to MNU-induced lymphomagenesis.

The incidence of non-melanoma skin cancer (NMSC) is equivalent to that of all other human cancers combined, and genetic factors contribute to risk of the disease. Using genetic crosses of skin tumor promotion sensitive DBA/2 mice with relatively resistant C57BL/6 mice, loci that modify susceptibility to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) have been mapped to chromosomes (chr) 1, 2, 9, and 19. Here, we show that Glutathione S-transferase alpha 4 (Gsta4), which maps to skin tumor promotion susceptibility locus Psl1.2 on chr 9, was expressed at significantly different levels in the epidermis of C57BL/6 versus DBA/2 mice following treatment with diverse promoting agents. Gsta4 expression was dramatically upregulated in the epidermis of C57BL/6 mice, but not DBA/2 mice, following treatment with TPA, okadaic acid, chrysarobin, or ultraviolet light. Gsta4 deficient mice were more susceptible to skin tumor development in the two-stage skin carcinogenesis protocol providing compelling evidence that Gsta4 underlies the effect of Psl1.2 on susceptibility to skin tumor promotion by TPA. A number of polymorphisms were detected in the putative promoter region of Gsta4 in C57BL/6 versus DBA/2 mice, and ongoing studies are addressing the genetic basis of strain-specific expression of Gsta4 during tumor promotion. Finally, inheritance of polymorphisms in GSTA4 was associated with risk of NMSC in human populations, further supporting Gsta4/GSTA4 as a gene that modifies susceptibility to skin tumor development. Supported by NIH grant ES016623. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4210.

The inbred mouse strain CWD/LeAgl, which has a high incidence of spontaneous B-cell lymphomas, expresses both ecotropic MuLV and MCF viruses. Studies indicated that the MCF viruses expressed in CWD tumors were characteristic of nononcogenic MCF viruses and that ecotropic MuLV may be the etiological agent in spontaneous B-cell lymphomagenesis. Somatically acquired proviruses of approximately the same size were detected in several tumor DNAs suggesting that integration of proviral sequences into specific regions of the mouse genome may be an important step in lymphomagenesis of this strain.

Treatment of young AKR/J mice with N-methyl-N-nitrosourea (MNU) results in the induction of a high incidence of thymic lymphomas occurring between 4 and 6 months of age. The tumor incidence is higher and the latency period is shorter than that observed in other MNU-treated mouse strains. Analysis of tumor incidence in crosses of AKR/J with C57L/J mice indicates that several genes influence the incidence and latency of MNU-induced thymic lymphomas. One of these genes appears to be tightly linked to the albino locus of chromosome 7.

An immunobinding assay is described that tests antigens attached to nitrocellulose paper against antisera contained in microtiter plates. In comparison to a conventional microtiter plate radioimmunoassay, the nitrocellulose paper radioimmunoassay is clearly superior in both antigen attachment and antibody binding. Studies using bovine serum albumin and human IgG demonstrated superior antigen attachment extending from 5-fold in a physiological solution, to 50-fold in 50% fetal calf serum, to over 1000-fold in detergent solutions. With titrations using a rabbit anti-human IgG serum, the antibody binding in the nitrocellulose paper radioimmunoassay averaged over 5 times the binding in the microtiter plate radioimmunoassay. The nitrocellulose paper radioimmunoassay was also modified to quantitate human IgG. With this assay, 15 pg of human IgG inhibited the antibody binding by 50%. The nitrocellulose paper radioimmunoassay is easy to perform, and, since it combines the antigen-binding properties of the nitrocellulose paper with the convenience of assaying samples in microtiter plates, this assay should prove useful for investigating the many antigens that attach to nitrocellulose paper.

We have studied somatically acquired murine leukemia proviral integrations in primary N -methyl- N -nitrosourea (MNU)-induced thymic lymphomas of AKR/1 mice. The majority of MNU-induced lymphomas contain newly acquired murine leukemia proviral sequences. In contrast to spontaneous AKRA lymphomas which contain multiple integrations of mink cell focus-forming recombinant proviruses, MNU-induced lymphomas contain ecotropic-related proviruses. This conclusion was based on the demonstration that Eco RI- and Pvu II-digested DNA from MNU-induced lymphomas contains new 3′ proviral-cellular junction fragments that hybridize with the ecotropic-specific pAKV-4 and pAKV-5 hybridization probes. Also, Eco RI/ Pst I double digests of DNA from MNU-induced lymphomas revealed that the acquired proviruses do not contain an internal 3′ Eco RI site characteristic of mink cell focus-forming recombinant viruses. The proviral integration patterns suggest that MNU-induced lymphomas are clonal or oligoclonal in nature. This conclusion is supported by comparison of proviral integration patterns in lymphomas obtained from thymus and spleen of individual mice, and by analyses of T-cell receptor β-chain gene rearrangements. The frequent occurrence of ecotropic-related proviral sequences in MNU-induced lymphomas suggests that these newly acquired proviruses may play a role in tumor development.

The AKR mouse strain is characterized by a high incidence of spontaneous thymic lymphoma that appears in older animals (greater than 6 months of age) and is associated with novel provirus integrations of ecotropic and recombinant murine leukemia viruses (MuLVs). Treatment of 4- to 6-week-old AKR/J mice with the carcinogen N-methyl-N-nitrosourea (MNU) results in thymic lymphomas that arise as early as 3 to 4 months of age and contain novel somatically acquired MuLV provirus integrations. The AKR/J strain develops MNU-induced lymphoma with a higher incidence and shorter latency than has been observed for other inbred mouse strains. To determine whether provirus integrations of endogenous MuLV account for the enhanced susceptibility of the AKR strain, the incidence and latency of MNU-induced lymphoma development was compared in AKR/J and AKR.Fv-1b mice. The restrictive b allele of the Fv-1 locus restricts integration and replication of endogenous N-tropic MuLV; therefore, AKR-Fv-1b mice have a very low incidence of spontaneous lymphoma. In contrast, AKR.Fv-1b mice develop MNU-induced lymphomas with an incidence and latency similar to those of the AKR/J strain. Furthermore, thymic lymphomas from both strains express an immature CD4-8+ phenotype, indicating neoplastic transformation of the same thymocyte subset. Southern blot analysis confirmed that lymphoma DNA from AKR.Fv-1b mice did not contain somatically acquired provirus integrations. These results demonstrate that provirus integration does not contribute to the predisposition of AKR mice to develop a high incidence of early MNU-induced lymphomas. Nevertheless, MNU treatment stimulated high-level expression of infectious ecotropic MuLV in AKR.Fv-1b as well as in AKR/J mice, suggesting that viral gene products might enhance lymphoma progression.