Downregulation of H19 improves the differentiation potential of mouse parthenogenetic embryonic stem cells.

Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer.

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target.

Met amplification and tumor progression in Cdkn2a-deficient melanocytes.

While many genetic alterations have been identified in melanoma, the relevant molecular events that contribute to disease progression are poorly understood. Most primary human melanomas exhibit loss of expression of the CDKN2A locus in addition to activation of the canonical mitogen-activated protein kinase signaling pathway. In this study, we used a Cdkn2a-deficient mouse melanocyte cell line to screen for secondary genetic events in melanoma tumor progression. Upon investigation, intrachromosomal gene amplification of Met, a receptor tyrosine kinase implicated in melanoma progression, was identified in Cdkn2a-deficient tumors. RNA interference targeting Met in these tumor cells resulted in a significant delay in tumor growth in vivo compared with the control cells. MET expression is rarely detected in primary human melanoma but is frequently observed in metastatic disease. This study validates a role for Met activation in melanoma tumor progression in the context of Cdkn2a deficiency.

Somatic pairing of chromosome 19 in renal oncocytoma is associated with deregulated EGLN2-mediated [corrected] oxygen-sensing response.

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase EGLN2 [corrected] a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of EGLN2 [corrected] in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.

Targeted disruption of the mouse Asna1 gene results in embryonic lethality.

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.

Generation and characterization of a transgenic mouse model with hepatic expression of human CYP2A6.

The aim of this study was to prepare and characterize a transgenic mouse model in which CYP2A6, a human P450 enzyme, is expressed specifically in the liver. CYP2A6, which is mainly expressed in human liver, is active toward many xenobiotics. Our transgene construct contained the mouse transthyretin promoter/enhancer, a full-length CYP2A6 cDNA, and a downstream neomycin-resistance gene for positive selection in embryonic stem cells. Hepatic expression of the CYP2A6 transgene was demonstrated by immunoblotting, whereas tissue specificity of CYP2A6 expression was confirmed by RNA-PCR. The transgenic mouse was further characterized after being backcrossed to the B6 strain for six generations. Hepatic microsomes from homozygous transgenic mice had activities significantly higher than those of B6 mice toward coumarin. The in vivo activity of transgenic CYP2A6 was also determined. Systemic clearance of coumarin was significantly higher in the transgenic mice than in B6 controls, consistent with the predicted role of CYP2A6 as the major coumarin hydroxylase in human liver. The CYP2A6-transgenic mouse model should be valuable for studying the in vivo function of this polymorphic human enzyme in drug metabolism and chemical toxicity.

Targeted disruption of Mig-6 in the mouse genome leads to early onset degenerative joint disease.

Degenerative joint disease, also known as osteoarthritis, is the most common joint disorder in human beings. The molecular mechanism underlying this disease is not fully understood. Here, we report that disruption of mitogen-inducible gene 6 (Mig-6) in mice by homologous recombination leads to early onset degenerative joint disease, which is revealed by simultaneous enlargement and deformity of multiple joints, degradation of articular cartilage, and the development of bony outgrowths or osteophyte formation within joint space. The osteophyte formation appears to be derived from proliferation of mesenchymal progenitor cells followed by differentiation into chondrocytes. Absence of the Rag2 gene does not rescue the joint phenotype, excluding a role for the acquired immune system in the development of this disease. Our results provide insight into the mechanism of osteoarthritis by showing that loss of Mig-6 leads to early onset of this disease, implying that this gene or its pathway is important in normal joint maintenance. Because of the striking similarity of osteoarthritis in humans and mice, the Mig-6 mutant mouse should provide a useful animal model for studying the mechanism of this disease and for testing drugs or therapies for treating osteoarthritis.

Spectral karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal instability in vitro.

The Ink4a/Arf locus is functionally linked to the Rb and p53 pathways through the action of its two gene products. Mouse models null for this locus show rapid onset of cancer with a preponderance of lymphomas and sarcomas. We report on a study of cell lines derived from sarcomas arising in Ink4a/Arf null mice. The cytogenetics of these lines was monitored over the course of serial passage. Results indicate that early passage cells are relatively normal. However, after multiple passages chromosomal instability becomes apparent as evidenced by increasing tetraploidy and aneuploidy, and the concomitant loss of clonality. To further evaluate the effect of Ink4a/Arf-deficiency on chromosomal stability in vitro, we isolated Ink4a/Arf deficient primary murine embryonic fibroblasts (MEFs), serially passaged them, and analyzed their chromosomal stability by spectral karyotyping (a 24-color chromosome paint-FISH technique). We found that chromosomal instability in Ink4a/Arf deficient MEFs developed with the same timing as seen in cell lines derived from Ink4a/Arf deficient sarcomas. Thus, chromosomal instability seen in Ink4a/Arf deficient tumors in vitro may be unrelated to the original phenotype of the tumor in vivo. Therefore, interpretation of cytogenetic data from cell lines derived from Ink4a/Arf deficient tumors should be done on early passage cells.

Transgenic mice with a hypomorphic NADPH-cytochrome P450 reductase gene: effects on development, reproduction, and microsomal cytochrome P450.

A mouse model with a hypomorphic NADPH-cytochrome P450 reductase (Cpr) gene (designated Cpr(low) allele) was generated and characterized in this study. The Cpr gene in these mice was disrupted by the insertion of a neo gene in intron 15, which led to 74 to 95% decreases in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart. In the liver, a pattern of pericentral distribution of CPR protein was preserved in the Cpr(low/low) mice, despite an overall reduction in CPR expression. Genotype distribution in F2 pups indicated limited embryonic lethality associated with the Cpr(low) allele, a finding that confirms the role of CPR-dependent enzymes in development. Adult male homozygotes had decreased body weight and decreased heart, lung, and kidney weights, whereas homozygous Cpr(low) females, which had increased serum testosterone and progesterone and decreased copulatory activities, were infertile. Furthermore, adult Cpr(low/low) mice had decreased plasma cholesterol, and some mice developed mild centrilobular hepatic lipidosis. In addition, despite apparently compensatory increases in total microsomal cytochrome P450 content in the liver and kidney, the decreases in CPR expression were accompanied by reductions in systemic clearance of pentobarbital, as well as in hepatic microsomal metabolism of acetaminophen and testosterone. These phenotypes illustrate the potential impact of a globally decreased CPR activity in human adults, and this novel knock-in mouse model provides a unique opportunity for further explorations of the in vivo roles of CPR and CPR-dependent enzymes.

Disruption of the Diaphanous-related formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42.

BACKGROUND: Mammalian Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during growth factor-induced cytoskeletal remodeling and cell division. While both p140 mDia1 (herein called Drf1) and p134 mDia2 (Drf3) have been shown to bind in vitro to activated RhoA-C, and Drf3 has also been shown to bind to Cdc42, little is known about the cellular function of these GTPase effector pairs. Thus, we have begun targeting the murine Drf genes to address their various contributions to small GTPase signaling in cytoskeletal remodeling and development. RESULTS: Drf1 +/+, +/-, and -/- cell lines were derived from embryonic stem cells. While some Drf1 +/- lines had fewer actin stress fibers, several Drf1 +/- and -/- cells were more motile and had more abundant lamella and filopodia. Because the apparent "gain-of-function" corresponded with elevated levels of Drf3 protein expression, we hypothesized that the effects on the actin cytoskeleton were due to Cdc42 utilization of Drf3 as an effector. In this study, we found that inactive Drf3 variants and microinjected Drf3 antibodies interfered with Cdc42-induced filopodia. In addition, we observed that Drf3 contains a previously unidentified CRIB-like motif within its GTPase binding domain (GBD). By fluorescent resonance energy transfer (FRET) analysis, we demonstrate that this motif is required for Cdc42 binding and Drf3 recruitment to the leading edge and, surprisingly, to the microtubule organizing center (MTOC) of migrating fibroblasts. CONCLUSIONS: Our observations extend the role of the mammalian Drfs in cell signaling and demonstrate that Cdc42 not only activates Drf3, but guides the effector to sites at the cell cortex where it remodels the actin cytoskeleton.