Murine Chromosome Research Articles

MiR-143 and miR-145

Vascular smooth muscle cells (VSMCs) are able to perform both contractile and synthetic functions, which are associated with changes in morphology, proliferation, and migration rates and are characterized by the specific expression of different marker proteins. Under normal physiological conditions, VSMC rarely proliferate in adult tissues, but undergo major phenotypic changes from the contractile to the synthetic in response to environmental cues, a phenomenon known as switching, or phenotypic modulation.1,2 Phenotypic switching is accompanied by production of abundant cytokines, extracellular matrix, and an increased rate of proliferation and migration. Therefore, the transition of VSMCs from a differentiated phenotype to a dedifferentiated state plays a critical role in the pathogenesis of cardiovascular diseases such as hypertension, vascular injury, and arteriosclerosis.2,3 However, the molecular mechanisms involved in phenotypic switching remain elusive. The last decade has witnessed an exciting discovery that led to a revolution in our understanding of the extensive regulatory gene expression networks modulated by small, untranslated RNAs, microRNAs (miRNAs).4 miRNAs comprise a novel class of endogenous, small RNAs of ≈20 to 25 nucleotides. Although the mature miRNA is very small, it is derived from a transcriptional product of a few hundred to a few thousand nucleotides. This process of maturation is known as miRNA biogenesis, extensively reviewed by Kim.5 Biogenesis of miR-143 and miR-145 is pictorially presented in Figure 1. Functionally, miRNAs are noncoding RNAs that negatively regulate gene expression. In the current, generally accepted model, they act mostly by inducing an inhibition of the translation of their target mRNAs, and, in a minority of cases, via their degradation.6,7 Very recently, however, Bartel's team challenged this view by showing that, in a vast majority of cases, mammalian microRNAs act by destabilizing their target mRNAs and decreasing their levels. …

The intestinal factor Tff3 and a miRNA network regulate murine caloric metabolism

Genetic impairment of the genes coding for three mammalian trefoil peptides resulted in severe gastrointestinal malfunctions. The trefoil peptides also appear involved in caloric metabolism. Monitoring global miRNA expression of Tff3 deficient mice points to an interplay of Tff3 with a miRNA regulatory network. We identified 21 miRNAs that were deregulated when compared to the wild type strain. In silico evaluation indicated that the majority of the 21 miRNA were connected with the metabolic pathway “glycolysis/gluconeogenesis’’ (p=0.032), a signaling pathway including nine target genes Aldh9a1, Aldh2, Pck1, Aldoc, Pgam2, Pck2, Adh4, Adh5, and Fbp1. Association of Tff3 with this metabolic pathway is further supported by the observation that the body mass of adult Tff3 KO mice (five months) showed a clearly reduced weight. Furthermore, the majority of the identified 21 miRNA genes are localized on murine chromosomes 2 and 5 in three clusters (2A1, 2B, 5B3) suggesting a coordinated expression control and function.

Primordial germ cell proliferation is impaired in Fused Toes mutant embryos

Over the first 4days of their life, primordial germ cells invade the endoderm, migrate into and through the developing hindgut, and traverse to the genital ridge where they cluster and ultimately inhabit the nascent gonad. Specific signal–receptor combinations between primordial germ cells and their immediate environment establish successful migration and colonization. Here we demonstrate that disruption of a cluster of six genes on murine chromosome 8, as exemplified by the Fused Toes (Ft) mutant mouse model, results in severely decreased numbers of primordial germ cells within the early gonad. Primordial germ cell migration appeared normal within Ft mutant embryos; however, germ cell counts progressively decreased during this time. Although no difference in apoptosis was detected, we report a critical decrease in primordial germ cell proliferation by E12.5. The six genes within the Ft locus include the IrxB cluster (Irx3, -5, -6), Fts, Ftm, and Fto, of which only Ftm, Fto, and Fts are expressed in primordial germ cells of the early gonad. From these studies, we have discovered that the Ft locus on mouse chromosome 8 is associated with cell cycle deficits within the primordial germ cell population that initiates just before translocation into the genital ridge.

Distinct genetic regions modify specific muscle groups in muscular dystrophy

Phenotypic expression in the muscular dystrophies is variable, even with the identical mutation, providing strong evidence that genetic modifiers influence outcome. To identify genetic modifier loci, we used quantitative trait locus mapping in two differentially affected mouse strains with muscular dystrophy. Using the Sgcg model of limb girdle muscular dystrophy that lacks the dystrophin-associated protein γ-sarcoglycan, we evaluated chromosomal regions that segregated with two distinct quantifiable characteristics of muscular dystrophy, membrane permeability and fibrosis. We previously identified a single major locus on murine chromosome 7 that influences both traits of membrane permeability and fibrosis in the quadriceps muscle. Using a larger cohort, we now found that this same interval strongly associated with both traits in all limb skeletal muscle groups studied, including the gastrocnemius/soleus, gluteus/hamstring, and triceps muscles. In contrast, the muscles of the trunk were modified by distinct genetic loci, possibly reflecting the embryological origins and physiological stressors unique to these muscle groups. A locus on chromosome 18 was identified that modified membrane permeability of the abdominal muscles, and a locus on chromosome 3 was found that regulated diaphragm and abdominal muscle fibrosis. Fibrosis in the heart associated with a region on chromosome 9 and likely reflects differential function between cardiac and skeletal muscle. These data underscore the complexity of inheritance and penetrance of single-gene disorders.

TH2 heterogeneity: Does function follow form?

T(H)2 immune responses are required for the 2 fundamental pathological processes characteristic of allergic disease: IgE-mediated hypersensitivity and eosinophilic inflammation. The 3 established T(H)2 cytokines, IL-4, IL-5, and IL-13, each play a nonredundant role in allergic disease pathology. The recent explosion of T(H) subpopulations combined with the wide availability of polychromatic cytokine staining has facilitated the discovery of T(H)2 lineage heterogeneity. In this article we review T(H)2 heterogeneity and ask the following question: At what point do these subpopulations graduate from in vitro curiosities to immunologically robust therapeutic targets? We propose criteria to establish a T-cell subset as a biologically relevant entity and address the evidence to support these T(H)2 subpopulations having a unique function or specific contribution to allergic pathology or host defense.

An Allelic Variant of Crry in the Murine Sle1c Lupus Susceptibility Interval Is Not Impaired in Its Ability To Regulate Complement Activation

The Sle1c subinterval on distal murine chromosome 1 confers loss of tolerance to chromatin. Cr2, which encodes complement receptors 1 and 2 (CR1/CR2; CD35/CD21), is a strong candidate gene for lupus susceptibility within this interval based on structural and functional alterations in its protein products. CR1-related protein/gene Y (Crry) lies 10 kb from Cr2 and encodes a ubiquitously expressed complement regulatory protein that could also play a role in the pathogenesis of systemic lupus erythematosus. Crry derived from B6.Sle1c congenic mice migrated at a higher m.w. by SDS-PAGE compared with B6 Crry, as a result of differential glycosylation. A single-nucleotide polymorphism in the first short consensus repeat of Sle1c Crry introduced a novel N-linked glycosylation site likely responsible for this structural alteration. Five additional single-nucleotide polymorphisms in the signal peptide and short consensus repeat 1 of Sle1c Crry were identified. However, the cellular expression of B6 and B6.Sle1c Crry and their ability to regulate the classical pathway of complement were not significantly different. Although soluble Sle1c Crry regulated the alternative pathway of complement more efficiently than B6 Crry, as a membrane protein, it regulated the alternative pathway equivalently to B6 Crry. These data fail to provide evidence for a functional effect of the structural alterations in Sle1c Crry and suggest that the role of Cr2 in the Sle1c autoimmune phenotypes can be isolated in recombinant congenic mice containing both genes.

Calpain-10 is a component of the obesity-related quantitative trait locus Adip1

Calpain-10 is a component of the obesity-related quantitative trait locus Adip1

Calpain-10 is a component of the obesity-related quantitative trait locus Adip1

We previously mapped Adip1, an obesity quantitative trait locus (QTL), to the central portion of murine chromosome 1 containing the calpain-10 (Capn10) gene. Human studies have associated calpain-10 (CAPN10) variants with type 2 diabetes and various metabolic traits. We performed a quantitative hybrid complementation test (QHCT) to determine whether differences attributed to Adip1 are the result of variant Capn10 alleles in LG/J and SM/J mice. We crossed LG/J and SM/J to wild-type (C57BL/6J) and Capn10 knockout (Capn10(-/-)) mice to form four F(1) hybrid groups: LG/J by wild-type, LG/J by Capn10(-/-), SM/J by wild-type, and SM/J by Capn10(-/-). We performed a two-way ANOVA with the experimental strain, tester strain, and their interaction as the factors. Significant interaction indicates a quantitative failure to complement. We found failure to complement for fat, organ, and body weights, and leptin, female free fatty acid, and triglyceride levels. Capn10(-/-) resulted in heavier weights and higher serum levels in LG/J crosses but not in SM/J crosses. For glucose tolerance and insulin response tests, the Capn10(-/-) allele resulted in lower glucose levels in crosses with SM/J but had no effect in the LG/J crosses. Differences between the LG/J and SM/J Capn10 alleles are the likely source of some of the QTL effects mapped to Adip1 in the LG/J-by-SM/J cross. Capn10 plays an important role in regulating obesity and diabetes in mice.

Effects of long-term memantine on memory and neuropathology in Ts65Dn mice, a model for Down syndrome

Memantine is a partial NMDA receptor antagonist that has been shown to improve learning and memory in several animal models, and is approved for the treatment of Alzheimer's disease (AD). Chronic treatments using memantine in animal models of Alzheimer's disease show disease-modifying effects and suggest a potential neuroprotective function. The present study assessed the effects of both short- and long-term memantine treatment in a mouse model of Down syndrome (DS), the Ts65Dn mouse. The Ts65Dn mouse contains a partial trisomy of murine chromosome 16, and exhibits hippocampal-dependent memory deficits, as well as progressive degeneration of basal forebrain cholinergic neurons (BCFNs). Ts65Dn mice were treated with memantine for a period of 6 months, beginning at 4 months of age. At the end of treatment the mice underwent memory testing using novel object recognition and water radial arm maze tasks, and then histologically analyzed for markers of neurodegeneration. Memantine treatment improved spatial and recognition memory performance in the Ts65Dn mice, though not to the level of normosomic littermate controls. Despite these memory improvements, histological analysis found no morphological signs of neuroprotection of basal forebrain cholinergic or locus coeruleus neurons in memantine-treated Ts65Dn mice. However, memantine treatment of Ts65Dn mice gave rise to elevated brain-derived neurotrophic factor expression in the hippocampus and frontal cortex, suggesting a mechanism of behavioral modification. Thus, our findings provide further evidence for memory facilitation of memantine, but suggest pharmacological rather than neuroprotective effects of memantine both after acute and chronic treatment in this mouse model.

Cartilage remodeling genes on murine chromosome 15 involved in sex-affected pathways controlling the effector phase of arthritis (88.19)

Abstract Gender differences in susceptibility to many autoimmune diseases including rheumatoid arthritis (RA) are well documented. To dissect sex-affected inflammatory mechanisms in murine models for RA, we generated BALB/c.DBA/2-Pgia8 congenic strain. The strain carries chromosome 15 sex-specific locus Pgia8 that controls arthritides induced with aggrecan, collagen type II, B. burgdorferi, and pristane. We report here that the locus also controls collagen antibody-induced arthritis (CAIA), which stresses the locus role in the effector inflammatory phase of the disease. CAIA severity in congenic males was 30% lower (p < 0.005) than in wild-type BALB/c males (locus effect). Severity in congenic males was only 50% of that in congenic females (sex-related effect). Differential expression analysis of total RNA isolated from paws showed that only dozen of genes within the locus were differentially expressed in wild-type versus congenic mice, and their expression was significantly affected by sex. In inflammatory conditions, expression pattern for the gene encoding for Cthrc1 (collagen triple helix repeat containing) was highly correlated with CAIA severity, r=0.84. In basal conditions, Cthrc1 expression correlated with the concentration of IL-6 message. Taken together, these data could indicate that Cthrc1 controls cartilage remodeling differently in males and females and might provide a converging point for regulation of different types of arthritides including RA.

Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFβ-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.

Angelman Syndrome: Finding the Lost Arc

Angelman syndrome is a neurodevelopmental disorder caused by mutations in the maternally inherited UBE3A gene, which encodes a ubiquitin ligase. Greer et al. (2010) now identify a UBE3A substrate called Arc that promotes endocytosis of neuronal AMPA receptors, providing insight into synaptic defects that may underlie the cognitive deficits in Angelman syndrome.

Quantitative trait locus analysis of ovarian cysts derived from rete ovarii in MRL/MpJ mice

MRL/MpJ (MRL) is a model mouse for autoimmune diseases such as dermatitis, vasculitis, arthritis, and glomerulonephritis. In addition to these immune-associated disorders, we found that older MRL mice develop ovarian cysts originating from the rete ovarii, which is lined by ciliated or nonciliated epithelium and considered remnants of mesonephric tubules. Ovarian cysts, which are reported to have several sources, are associated with female infertility, but information regarding the genetic etiology of ovarian cysts originating from the rete ovarii is rare. In this study, to elucidate the genetic background of development of ovarian cysts, we performed quantitative trait locus (QTL) analysis using 120 microsatellite markers, which cover the whole genome of murine chromosomes, and 213 backcross progenies between female MRL and male C57BL/6N mice. The quantitative trait measured was the circumferences of rete ovarii or ovarian cysts. As a result, suggestive linkages were detected on Chrs 3, 4, 6, and 11, but significant linkages were located on Chr 14 by interval mapping. We thereby designated the 27.5-cM region of Chr 14 "MRL Rete Ovarian Cysts (mroc)." The peak regions of Chrs 4 and 14 in particular showed a close additive interaction (p < 0.00001). From these results we concluded that multiple loci on Chrs 3, 4, 6, 11, and 14 interact to result in development of ovarian cysts in MRL mice.

Preferential Loss of Porcine Chromosomes in Reprogrammed Interspecies Cell Hybrids

Fusion of terminally differentiated somatic cells with pluripotent embryonic stem cells has been proposed as model for reprogramming the somatic cell genome, and may contribute to our understanding of the underlying mechanisms of this epigenetic process. We established an interspecies cell fusion model using murine embryonic stem cells (ESCs) and porcine fibroblasts. These inter-species fusion experiments yielded much lower conversion efficiency rates than murine intraspecies fusion. Nevertheless, two double-resistant mouse-pig hybrid clones could be generated. Reactivation of the porcine OCT4 gene, an essential pluripotent stem cell marker, and demethylation of the porcine OCT4 promoter in hybrid clone 1, suggested successful reprogramming of porcine chromosomes. A rapid loss of porcine chromosomes was observed during the selection phase. Spectral karyotyping (SKY) analysis showed that fusion-hybrid clone 1 carried a tetraploid mouse chromosome complement with only few pig chromosomes and/or chromosomal fragments. Hybrid clone 2 had a diploid set of murine chromosomes complements and also contained an interspecies chromosome fusion product. Interspecies cell fusion results in hybrid cells that retained the complement of mouse chromosomes and preferentially lose porcine chromosomes during colony expansion. Neither species-specific chromosomal segregation nor reprogrammed diploid porcine cells were observed. These findings indicate that generation of reprogrammed pluripotent diploid cells by cell fusion may require additional supporting provisions.

An NZW-derived interval on chromosome 7 moderates sialadenitis, but not insulitis in congenic nonobese diabetic mice.

Autoimmune lymphocytic infiltration of the salivary glands, termed sialadenitis, is a pathologic feature of Sjögren's syndrome (SjS) that is also prominent in nonobese diabetic (NOD) mice. Genetic factors regulate sialadenitis, and a previous (NOD x NZW)F2 study detected linkage to murine chromosome (Chr) 7. The locus, subsequently annotated as Ssial3, maps to the distal end of Chr7 and overlaps a region associated with type 1 diabetes susceptibility in NOD mice. To examine whether Ssial3 could contribute to both diseases, or was specific for SjS, we generated a congenic mouse strain that harbored an NZW-derived Chr7 interval on the NOD genetic background. This congenic strain exhibited reduced sialadenitis compared with NOD mice and confirmed Ssial3. This reduction, however, did not ameliorate saliva abnormalities associated with SjS-like disease in NOD mice, nor were congenic mice protected against insulitis (lymphocytic infiltration of the pancreatic islets) or diabetes onset. Thus, the Ssial3 locus appears to have a tissue-specific effect for which the NZW allele is unable to prevent other autoimmune traits in the NOD mouse. Anomalous increases for antinuclear Ab production and frequency of marginal-zone B cells were also identified in congenic mice, indicating that the NZW-derived Chr7 interval has a complex effect on the NOD immune system.

Identification of novel susceptibility genes in ozone-induced inflammation in mice

Ozone (O(3)) remains a prevalent air pollutant and public health concern. Inf2 is a significant quantitative trait locus on murine chromosome 17 that contributes to susceptibility to O(3)-induced infiltration of polymorphonuclear leukocytes (PMNs) into the lung, but the mechanisms of susceptibility remain unclear. The study objectives were to confirm and restrict Inf2, and to identify and test novel candidate susceptibility gene(s). Congenic strains of mice that contained overlapping regions of Inf2 and their controls, and mice deficient in either major histocompatibility complex (MHC) class II genes or the Tnf cluster, were exposed to air or O(3). Lung inflammation and gene expression were assessed. Inf2 was restricted from 16.42 Mbp to 0.96 Mbp, and bioinformatic analysis identified MHC class II, the Tnf cluster and other genes in this region that contain potentially informative single nucleotide polymorphisms between the susceptible and resistant mice. Furthermore, O(3)-induced inflammation was significantly reduced in mice deficient in MHC class II genes or the Tnf cluster genes, compared with wild-type controls. Gene expression differences were also observed in MHC class II and Tnf cluster genes. This integrative genetic analysis of Inf2 led to identification of novel O(3) susceptibility genes that may provide important, new therapeutic targets in susceptible individuals.

Identification of sister chromatids by DNA template strand sequences

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells. Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo.

Impact of Growth Factor Independence 1 in Human T-Cell Lymphomas; Pathogenic Potential Identified by Insertional Mutagenesis in a Murine T-Cell Lymphoma Model.

Abstract 5047 IntroductionThe transcriptional repressor and oncogene Growth factor independence 1 (Gfi1) has a major oncogenic potential and is aberrantly expressed in murine lymphomas and several human cancers. Gfi1 is a key regulator of stem cell quiescence and plays a significant role in T-cell development, lineage commitment, and influences development of maturate granulocytes and monocytes. The genomic locus on murine chromosome 5 encoding Gfi1 is a frequent integration locus activated in T-cell lymphomas induced by the SL3-3 Murine Leukemia Virus (MLV) as well as other MLVs, indicating that Gfi1 is essential in development of these tumors. In the SL3-3 induced T-cell lymphoma model, retroviral insertions in the Gfi1 3'UTR have been demonstrated to decouple microRNA-mediated posttranscriptional regulation of protein expression (Dabrowska et al, 2009) further supporting its role in lymphomagenesis. In human cancers, Gfi1 protein expression has been observed in HTLV-1 induced ATLL and SCLC but no knowledge exists on how Gfi1 contributes to initiating and maintaining human T-cell lymphomas. MethodsGfi1 gene and protein expression patterns were determined in precursor and mature human T-cell lymphomas by real time PCR and Western blot analysis. Furthermore, localization and expression patterns of the Gfi1 protein was determined in the human T-cell lymphomas by immunohistochemical staining with Gfi1 antibodies and compared to similar staining of MLV induced tumors. ResultsOur results demonstrated that Gfi1 mRNA and protein levels vary significantly among the human T-cell lymphomas, and do not always show a direct proportional pattern. Thus, Gfi1 mRNA expression can be relatively high without resulting in a corresponding high protein expression, suggesting that a microRNA mediated posttranscriptional regulation exists in some tumors but may be disrupted in others. Furthermore, an additional Gfi1 protein variant was identified in one of the T-cell lymphoma entities. Immunohistochemical staining demonstrated varying Gfi1 protein expression in both nucleus and cytoplasm in the T-cell lymphomas and different distributions of the protein within the tumor and tumor cells were observed among samples. Staining of normal human tonsil demonstrated Gfi1 protein to be localized in the cytoplasm. We hypothesise that regulation of Gfi1 may include shuttling between cytoplasm and nucleus and that lymphomagenesis enables unlimited nuclear access. ConclusionOur data shows that deregulated Gfi1 expression plays a major role in the development of MLV induced lymphomas and strongly indicates that retroviral insertional mutagenesis in murine models of human NHLs can be used to identify new genes involved in lymphomagenesis and, by use of functional assays, their impact on human lymphomas can be evaluated. DisclosuresNo relevant conflicts of interest to declare.

Caspase-9: A Candidate Susceptibility Factor for Murine Alkylator-Induced Leukemia.

Abstract 1273Poster Board I-295Therapy-related acute myeloid leukemia (tAML) is caused by exposure to chemotherapies and radiotherapies and has a poor prognosis. To better understand the genetic factors involved in secondary leukemogenesis, we studied alkylator-induced leukemias in murine models. We performed genome-wide mRNA profiling and found that differential expression of apoptosis-related genes was correlated with strain-dependent differences in tAML susceptibility. To identify genetic variants associated with these gene expression changes, we examined copy number variants (CNVs), differences in the DNA copy number of genomic regions greater than one kilobase in length, across 20 different inbred murine genomes. Our studies indicate that CNVs are a major contributor to natural variation among inbred laboratory mouse strains, and our array-based expression data show that the presence of certain CNVs correlates with differences in apoptotic gene expression. One CNV identified on murine chromosome 4 is correlated with altered Caspase-9 (Casp9) expression levels in murine hematopoietic stem/progenitor cells. Casp9 is a gene downstream from extrinsic and intrinsic death-inducing signals crucial for the initiation of cellular apoptosis, and we propose that it may be an important factor that influences tAML susceptibility. Sequencing the CNV region confirmed in two tAML susceptible strains (DBA/2J and PL/J) the presence of a 1,705 bp CNV loss in Casp9 intron 6. In these strains, Casp9 expression is undetectable in flow sorted kit+/lineage- (KL) hematopoietic stem/progenitor cells as measured by microarray profiling, and confirmed in independent samples by qRT-PCR using assays targeting Casp9 exons 2-3 or exons 8-9. Full-length Casp9 cDNA clones could be isolated from mRNA libraries prepared from KL cells, but 35% of the transcripts isolated from DBA/2J and PL/J mice were a novel isoform lacking exon 2 that results in a frameshift and an early stop codon in exon 4. This premature translation termination codon is predicted to trigger nonsense-mediated mRNA decay, leading to the degradation of the novel isoform and thus preventing its translation. This mechanism may account for the low Casp9 expression levels in DBA/2J and PL/J mice. In addition, Casp9 cDNA sequencing also identified an unusually high SNP density in exon 2 for the full-length isoforms in these strains. At least two of the six SNPs result in the formation of putative exonic splice enhancers (ESE), sequences that enhance the splicing of the exon in which they reside. The presence of these novel ESEs could explain the excision of exon 2 and the creation of the novel Casp9 isoform in DBA/2J and PL/J mice. We hypothesize that cells with relatively low Casp9 expression would be more resistant to alkylator-induced apoptosis. Cells that fail to undergo apoptosis after genotoxic stress may be more likely to accumulate mutations and initiate leukemias. Preliminary data from flow cytometric apoptosis assays (by Annexin V staining) in flow sorted KL bone marrow cells after treatment with ENU, an alkylating agent, suggest that PL/J cells may be more resistant to ENU-induced apoptosis, compared to C57BL/6J cells with normal Casp9 expression (5.3±1.4% vs. 9.7±1.8% post-ENU AnnexinV+ cells, respectively). These results suggest that differences in Casp9 expression levels may play a role in influencing individual susceptibility to tAML, and that inherited genetic factors may explain the observed expression differences. Ultimately, our understanding of the role that genetics plays in determining susceptibility to secondary leukemias may allow us to define a process by which individuals who are more susceptible can be successfully identified and screened from potential treatments that are known to induce these cancers. DisclosuresNo relevant conflicts of interest to declare.

Epistatic interactions govern chemically‐induced lung tumor susceptibility and Kras mutation site in murine C57BL/6J‐ChrA/J chromosome substitution strains

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.