Identification of five novel modifier loci of Apc(Min) harbored in the BXH14 recombinant inbred strain.

Every year thousands of people in the USA are diagnosed with small intestine and colorectal cancers (CRC). Although environmental factors affect disease etiology, uncovering underlying genetic factors is imperative for risk assessment and developing preventative therapies. Familial adenomatous polyposis is a heritable genetic disorder in which individuals carry germ-line mutations in the adenomatous polyposis coli (APC) gene that predisposes them to CRC. The Apc ( Min ) mouse model carries a point mutation in the Apc gene and develops polyps along the intestinal tract. Inbred strain background influences polyp phenotypes in Apc ( Min ) mice. Several Modifier of Min (Mom) loci that alter tumor phenotypes associated with the Apc ( Min ) mutation have been identified to date. We screened BXH recombinant inbred (RI) strains by crossing BXH RI females with C57BL/6J (B6) Apc ( Min ) males and quantitating tumor phenotypes in backcross progeny. We found that the BXH14 RI strain harbors five modifier loci that decrease polyp multiplicity. Furthermore, we show that resistance is determined by varying combinations of these modifier loci. Gene interaction network analysis shows that there are multiple networks with proven gene-gene interactions, which contain genes from all five modifier loci. We discuss the implications of this result for studies that define susceptibility loci, namely that multiple networks may be acting concurrently to alter tumor phenotypes. Thus, the significance of this work resides not only with the modifier loci we identified but also with the combinations of loci needed to get maximal protection against polyposis and the impact of this finding on human disease studies.

The armadillo repeat domain of Apc suppresses intestinal tumorigenesis.

The adenomatous polyposis coli (APC) gene is known to act as a tumor suppressor gene in both sporadic and hereditary colorectal cancer by negatively regulating WNT signaling. Familial adenomatous polyposis (FAP) patients develop intestinal polyps due to the presence of a single germline mutation in APC. The severity of the FAP phenotype is a function of the position of the APC mutation, indicating a complex role for APC that extends beyond the canonical WNT pathway. APC encodes a large protein with multiple functional domains, including an armadillo repeat domain that has been linked to protein-protein interactions. To determine the effect of the armadillo repeat domain on intestinal tumorigenesis, we generated a congenic mouse line (Apc ( Δ242 )) carrying a gene trap cassette between exons 7 and 8 of the murine Apc gene. Apc ( Δ242/+) mice express a truncated Apc product lacking the armadillo repeat domain as part of a fusion protein with ß-geo. Expression of the fusion product was confirmed by X-gal staining, ensuring that Apc ( Δ242 ) is not a null allele. In contrast, Apc ( Min/+) mice produce a truncated Apc product that contains an intact armadillo repeat domain. On the C57BL/6J background, Apc ( Δ242/+) mice develop more polyps than do Apc ( Min/+) mice along the entire length of the small intestine; however, polyps were significantly smaller in Apc ( Δ242/+) mice. In addition, polyp multiplicity in Apc ( Δ242/+) mice is affected by polymorphisms between inbred strains. These data suggest that the armadillo repeat domain of the Apc protein suppresses tumor initiation in the murine intestine while also promoting tumor growth.

A Tcf4-GFP reporter mouse model for monitoring effects of Apc mutations during intestinal tumorigenesis.

Apc mutations cause intestinal tumorigenesis through Tcf4 activation. However, direct techniques for studying Tcf4 activation in vivo are limited. Here, we describe the development of a Tcf4-GFP reporter mouse model for directly studying Tcf4 activation. We first developed a GFP reporter construct (Tcf4-GFP) and transfected it into SW480 cells that have constitutively activated Tcf4. Reporter activity increased 47-fold. Next, we created transgenic (Tg) mice by transducing the construct into C57BL/6J mice. Fluorescence microscopy did not detect GFP in intestinal sections, but flow cytometry showed 5% of crypt cells to be GFP(+). We then established cross-bred mice (Tg x Apc(Min/+)), which have a germline Apc mutation and sustained Tcf4 activation. Here, fluorescence microscopy showed GFP(+) cells at or near the base of normal-appearing crypts. In adenomas, in which Apc is inactivated, GFP(+) signal was even greater. Immunostaining for the Tcf4 target genes survivin (BIRC5) and cyclin D1 (CCND1) showed that their expression also paralleled GFP positivity. We conclude that GFP directly reports Tcf4 activation in vivo and tracks the predicted increases in Tcf4 activation that result from Apc inactivation, and that Apc mutation contributes to survivin and cyclin D1 overexpression through Tcf4 activation. Our Tcf4 mouse should be useful in studying the effects of chemopreventive agents on Wnt signaling and changes in proliferative crypt cell populations-including stem cells-during intestinal tumorigenesis.

Diversity in secreted PLA2-IIA activity among inbred mouse strains that are resistant or susceptible to Apc Min/+ tumorigenesis.

The secreted phospholipase A2 type IIA (Pla2g2a) gene was previously identified as a modifier of intestinal adenoma multiplicity in Apc Min/+ mice. To determine if intestinal secreted phospholipase A2 (sPLA2) activity was also attenuated in susceptible strains, we developed a sensitive assay to directly quantitate sPLA2 activity in the murine intestinal tract utilizing a fluorescent BODIPY-labeled phospholipid substrate. Here, we report assay conditions that distinguish between secreted and cytosolic PLA2 enzyme activities in extracts of intestinal tissue. The small intestine exhibited higher activity levels than the large intestine. Consistent with predictions from the sPLA2-IIA gene sequence in inbred strains, we detected low levels of enzyme activity in inbred strains containing sPLA2-IIA mutations; these strains were also associated with greater numbers of intestinal polyps. Additionally, the assay was able to distinguish differences in levels of sPLA2 activity between neoplasia-resistant strains, which were then shown by sequencing to carry variant wild-type sPLA2-IIA alleles. Immunohistochemical analyses of intestinal tissues were consistent with sPLA2-IIA activity levels. This approach enables further studies of the mechanisms of sPLA2 action influencing the development and tumorigenesis of the small intestine and colon in both mice and humans.

[Letter to the Editor] The need for improved education and training in research antibody usage and validation practices.

Address correspondence to Leonard P. Freedman, Global Biological Standards Institute, 1020 19th Street, NW, Suite 550, Washington, DC, 20036. E-mail: lfreedman@gbsi.org.

A conserved tissue-specific homeodomain-less isoform of MEIS1 is downregulated in colorectal cancer.

Colorectal cancer is one of the most common cancers in developed nations and is the result of both environmental and genetic factors. Many of the genetic lesions observed in colorectal cancer alter expression of homeobox genes, which encode homeodomain transcription factors. The MEIS1 homeobox gene is known to be involved in several hematological malignancies and solid tumors and recent evidence suggests that expression of the MEIS1 transcript is altered in colorectal cancer. Despite this potential connection, little is known about the role of the gene in the intestines. We probed murine gastrointestinal tissue samples with an N-terminal Meis1 antibody, revealing expression of two previously described isoforms, as well as two novel Meis1 products. A 32 kD Meis1 product was expressed in the nuclei of non-epithelial cells in the stomach and colon, while a 27 kD product was expressed in the cytoplasm of epithelial cells in the proximal colon. Our data suggest that the 27 kD and 32 kD Meis1 proteins are both forms of the Meis1d protein, a homeodomain-less isoform whose transcript was previously identified in cDNA screens. Both the MEIS1D transcript and protein were expressed in human colon mucosa. Expression of the MEIS1D protein was downregulated in 83% (10/12) of primary colorectal cancer samples compared to matched normal mucosa, indicating that MEIS1D is a biomarker of colorectal tumorigenesis. The decreased expression of MEIS1D in colon tumors also suggests that this conserved homeodomain-less isoform may act as a tumor suppressor in human colorectal cancer.

Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis.

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.

The modifier of Min 2 (Mom2) locus: embryonic lethality of a mutation in the Atp5a1 gene suggests a novel mechanism of polyp suppression.

Inactivation of the APC gene is considered the initiating event in human colorectal cancer. Modifier genes that influence the penetrance of mutations in tumor-suppressor genes hold great potential for preventing the development of cancer. The mechanism by which modifier genes alter adenoma incidence can be readily studied in mice that inherit mutations in the Apc gene. We identified a new modifier locus of ApcMin-induced intestinal tumorigenesis called Modifier of Min 2 (Mom2). The polyp-resistant Mom2R phenotype resulted from a spontaneous mutation and linkage analysis localized Mom2 to distal chromosome 18. To obtain recombinant chromosomes for use in refining the Mom2 interval, we generated congenic DBA.B6 ApcMin/+, Mom2R/+ mice. An intercross revealed that Mom2R encodes a recessive embryonic lethal mutation. We devised an exclusion strategy for mapping the Mom2 locus using embryonic lethality as a method of selection. Expression and sequence analyses of candidate genes identified a duplication of four nucleotides within exon 3 of the alpha subunit of the ATP synthase (Atp5a1) gene. Tumor analyses revealed a novel mechanism of polyp suppression by Mom2R in Min mice. Furthermore, we show that more adenomas progress to carcinomas in Min mice that carry the Mom2R mutation. The absence of loss of heterozygosity (LOH) at the Apc locus, combined with the tendency of adenomas to progress to carcinomas, indicates that the sequence of events leading to tumors in ApcMin/+ Mom2R/+ mice is consistent with the features of human tumor initiation and progression.

Analysis of expansion of myeloid progenitors in mice to identify leukemic susceptibility genes.

The myeloid progenitor cell compartment (MPC) exhibits pronounced expansion in human myeloid leukemias. It is becoming more apparent that progression of myelodysplastic syndromes and myeloproliferative diseases to acute myelogenous leukemia is the result of defects in progenitor cell maturation. The MPC of bone marrow was analyzed in mice using a cell culture assay for measuring the relative frequency of proliferative myeloid progenitors. Response to the cytokines SCF, IL-3, and GM-CSF was determined by this assay for the leukemic mouse strain BXH-2 and ten other inbred mouse strains. Significant differences were found to exist among ten inbred mouse strains in the nature of their MPC in bone marrow, indicating the presence of genetic polymorphisms responsible for the divergence. The SWR/J and FVB/J strains show consistently low frequencies of myeloid progenitors, while the DBA/2J and SJL/J inbred strains show consistently high frequencies of myeloid progenitors within the bone marrow compartment. In addition, in silico linkage disequilibrium analysis was conducted to identify possible chromosomal regions responsible for the phenotypic variation. Given the importance of this cell compartment in leukemia progression and the soon to be released genomic sequence of 15 mouse strains, these differences may provide a valuable tool for research into leukemia.

Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HoxA9 in murine and human cell lines.

Coexpression of the homeodomain protein Meis1 and either HoxA7 or HoxA9 is characteristic of many acute myelogenous leukemias. Although Meis1 can be overexpressed in bone marrow long-term repopulating cells, it is incapable of mediating their transformation. Although overexpressing HoxA9 alone transforms murine bone marrow cells, concurrent Meis1 overexpression greatly accelerates oncogenesis. Meis1-HoxA9 cooperation suppresses several myeloid differentiation pathways. We now report that Meis1 overexpression strongly induces apoptosis in a variety of cell types in vitro through a caspase-dependent process. Meis1 requires a functional homeodomain and Pbx-interaction motif to induce apoptosis. Coexpressing HoxA9 with Meis1 suppresses this apoptosis and provides protection from several apoptosis inducers. Pbx1, another Meis1 cofactor, also induces apoptosis; however, coexpressing HoxA9 is incapable of rescuing Pbx-mediated apoptosis. This resistance to apoptotic stimuli, coupled with the previously reported ability to suppress multiple myeloid differentiation pathways, would provide a strong selective advantage to Meis1-HoxA9 coexpressing cells in vivo, leading to leukemogenesis.

Exclusion of Madh2, Madh4, and Madh7 as candidates for the modifier of Min 2 (Mom2) locus.

We recently identified the Modifier of Min 2 (Mom2) locus. Mom2 is a new modifier of intestinal tumorigenesis that resulted from a spontaneous mutation in a B6 Apc(Min)/+ mouse. The presence of one resistant Mom2(R) allele results in a significant reduction in small intestinal polyp number and colon polyp incidence in Apc(Min)/+ mice. Through linkage analysis, we previously localized Mom2 to a 14-cM region on mouse Chromosome (Chr) 18, distal to the Apc gene. This region is syntenic with human Chr 18q, which frequently undergoes loss of heterozygosity (LOH) in several human cancers, including colorectal cancer. Residing in this region are the Madh2 and Madh4 genes, which have both been implicated in human colorectal cancer. Based on meiotic recombinations within the Mom2 region in the derivation of our congenic animals, we have narrowed the location of the Mom2 locus and excluded Madh2, Madh4, and Madh7, as well as Mbd1, Mbd2, Dcc, and Tcf4, as candidates for the Mom2 gene.

Identification of the modifier of Min 2 (Mom2) locus, a new mutation that influences Apc-induced intestinal neoplasia.

Min (Multiple intestinal neoplasia) mice carry a dominant mutation in the adenomatous polyposis coli (Apc) gene and develop multiple adenomas throughout their intestinal tract (Moser et al. 1990; Su et al 1992). Polyp multiplicity in Min mice is greatly influenced by genetic background. A modifier locus, Mom1 (Modifier of Min 1), was identified and localized to distal mouse chromosome 4 (Moser et al. 1992; Dietrich et al. 1993), and accounts for some of the genetic variance in polyp multiplicity. Mom1 is a semidominant modifier of polyp size and multiplicity in Min mice (Gould and Dove 1997), and encodes the secretory type II nonpancreatic phospholipase A2 (Pla2g2a) gene (MacPhee et al. 1995; Cornier et al. 1997, 2000). We now report the identification of a second Modifier of Min 2 (Mom2) locus that is the result of a spontaneous mutation. One resistant Mom2 allele can suppress 88%-95% of polyps detected in Apc(Min)/+ mice, indicating that Mom2 acts in a dominant fashion. Linkage analysis has localized Mom2 to distal mouse chromosome 18. The effects of the Mom2 locus on reducing polyp multiplicity are stronger than the effects of the Mom1 locus, in both the small and large intestines. Some Apc(Min)/+ mice that carried one resistant Mom2 allele were tumor-free at 21 weeks of age, even in the absence of a resistant Mom1 allele. Thus, the resistant Mom2 allele can, in some cases, completely suppress the penetrance of the Apc(Min) mutation.

Analysis of reciprocal congenic lines reveals the C3H/HeJ genome to be highly resistant to ApcMin intestinal tumorigenesis.

Genetic background affects polyp development in the Multiple intestinal neoplasia (Apc(Min)) mouse model. The Modifier of Min 1 (Mom1) locus accounts for approximately 50% of the variation in polyp multiplicity. We generated reciprocal congenic lines, such that the recipient C57BL/6J (B6) strain carries a donor C3H/HeJ (C3H) Mom1 allele, and the recipient C3H strain carries a donor B6 Mom1 allele. Hybrid progeny from congenic females mated to B6 Apc(Min/+) males were analyzed. A single C3H Mom1 locus on the B6 background reduced small intestinal polyp numbers by 50% and colon polyp incidence by 66% compared to their susceptible B6 Mom1(S/S)Apc(Min/+) siblings. These findings show that the C3H genome contains a resistant Mom1(R) locus. The reciprocal congenic line, which carries the susceptible B6 Mom1(S) locus on the C3H background, reduced small intestinal polyp numbers by 80% and colon polyp incidence by 95% compared to B6 Mom1(S/S)Apc(Min/+) mice. These data demonstrate that unidentified modifiers in the C3H strain can suppress intestinal polyp multiplicity in Apc(Min/+) mice, and act in the absence of a resistant Mom1(R) locus.

A novel transgenic line of mice exhibiting autosomal recessive male-specific lethality and non-alcoholic fatty liver disease.

We have isolated a Meis1a transgenic mouse line exhibiting recessive male-specific lethality and non-alcoholic fatty liver disease (NAFLD), which coincides with pubescence and is androgen-dependent. The phenotype is due to disruption of an endogenous locus, since other Meis1a transgenic lines do not exhibit these phenotypes. Necropsy analysis revealed hepatic microvesicular steatosis in pubescent male homozygous mice, which is absent in transgenic females. The transgene insertion site was localized to chromosome 1 and further refined by cloning the flanking regions. Sequence analysis shows that the integration site disrupts a putative metallo-beta-lactamase gene with a 21.3 kb deletion encompassing exons 5-7.

Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos.

Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.