Joe M. Angel

The nature and identification of quantitative trait loci: a community’s view

This white paper by eighty members of the Complex Trait Consortium presents a communitys 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?

Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications

For more than 60 years, the chemical induction of tumors in mouse skin has been used to study mechanisms of epithelial carcinogenesis and evaluate modifying factors. In the traditional two-stage skin carcinogenesis model, the initiation phase is accomplished by the application of a sub-carcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor-promoting agent. The initiation protocol can be completed within 1–3 h depending on the number of mice used; whereas the promotion phase requires twice weekly treatments (1–2 h) and once weekly tumor palpation (1–2 h) for the duration of the study. Using the protocol described here, a highly reproducible papilloma burden is expected within 10–20 weeks with progression of a portion of the tumors to squamous cell carcinomas within 20–50 weeks. In contrast to complete skin carcinogenesis, the two-stage model allows for greater yield of premalignant lesions, as well as separation of the initiation and promotion phases.

Constitutive activation and targeted disruption of signal transducer and activator of transcription 3 (Stat3) in mouse epidermis reveal its critical role in UVB-induced skin carcinogenesis

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, that is, 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 ultraviolet-photoproduct cells, including those located in the 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 were significantly greater than wild-type controls. In contrast, Stat3-deficient mice were resistant to UVB skin carcinogenesis. These results show 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.

The Polycomb-group gene eed regulates thymocyte differentiation and suppresses the development of carcinogen-induced T-cell lymphomas

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 17Rn51989SB 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 pleiotrophic effects dictated by developmental stage and cellular context.

Differential gene expression in epidermis of mice sensitive and resistant to phorbol ester skin tumor promotion

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.

Evidence That Gsta4 Modifies Susceptibility to Skin Tumor Development in Mice and Humans

BACKGROUND 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. METHODS 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. RESULTS 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). CONCLUSIONS Gsta4/GSTA4 is a novel susceptibility gene for NMSC that affects risk in both mice and humans.

A locus that influences susceptibility to 1,2‐dimethylhydrazine–induced colon tumors maps to the distal end of mouse chromosome 3

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.

Confirmation of the mapping of a 12-O-tetradecanoylphorbol-13-acetate promotion susceptibility locus, Psl1, to distal mouse chromosome 9

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.

Tlag2, an N-methyl-N-nitrosourea susceptibility locus, maps to mouse chromosome 4

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.

Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis.

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.