Nancy Wu

Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

MIZ1, A NOVEL ZINC FINGER TRANSCRIPTION FACTOR THAT INTERACTS WITH MSX2 AND ENHANCES ITS AFFINITY FOR DNA

Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull. We demonstrated previously that individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h). The mutation behaves as a dominant positive in transgenic mice. The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted us to undertake a yeast two hybrid screen for Msx2-interacting proteins. Here we present a functional analysis of one such protein, designated Miz1 (Msx-interacting-zinc finger). Miz1 is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein, Nfi-1. Together these proteins define a new, highly conserved protein family. Analysis of Miz1 expression by Northern blot and in situ hybridization revealed a spatiotemporal pattern that overlaps that of Msx2. Further, Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor. Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter. The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype.

A phylogenetically conserved cis-regulatory module in the Msx2 promoter is sufficient for BMP-dependent transcription in murine and Drosophila embryos

To understand the actions of morphogens, it is crucial to determine how they elicit different transcriptional responses in different cell types. Here, we identify a BMP-responsive enhancer of Msx2, an immediate early target of bone morphogenetic protein (BMP) signaling. We show that the BMP-responsive region of Msx2 consists of a core element, required generally for BMP-dependent expression, and ancillary elements that mediate signaling in diverse developmental settings. Analysis of the core element identified two classes of functional sites: GCCG sequences related to the consensus binding site of Mad/Smad-related BMP signal transducers; and a single TTAATT sequence, matching the consensus site for Antennapedia superclass homeodomain proteins. Chromatin immunoprecipitation and mutagenesis experiments indicate that the GCCG sites are direct targets of BMP restricted Smads. Intriguingly, however, these sites are not sufficient for BMP responsiveness in mouse embryos; the TTAATT sequence is also required. DNA sequence comparisons reveal this element is highly conserved in Msx2 promoters from mammalian orders but is not detectable in other vertebrates or non-vertebrates. Despite this lack of conservation outside mammals, the Msx2 BMP-responsive element serves as an accurate readout of Dpp signaling in a distantly related bilaterian – Drosophila. Strikingly, in Drosophila embryos, as in mice, both TTAATT and GCCG sequences are required for Dpp responsiveness, showing that a common cis-regulatory apparatus can mediate the transcriptional activation of BMP-regulated genes in widely divergent bilaterians.

Benign retroperitoneal schwannoma: a case series and review of the literature.

OBJECTIVES To present our experience with four retroperitoneal schwannomas treated by surgical excision and review the current literature. Retroperitoneal schwannomas are rare, benign tumors and infrequently present to the urologist. METHODS From 1997 through 2002, the charts of 164 patients with a diagnosis of benign retroperitoneal soft tissue mass were reviewed. Of those, four had a pathologic diagnosis of retroperitoneal schwannoma. RESULTS Three of the 4 patients were women, with a median age of 54 years (range 46 to 80). The average tumor size was 13.7 cm (range 8.8 to 20). All patients underwent magnetic resonance imaging, computed tomography, or ultrasonography, and 3 of the 4 patients underwent a computed tomography-guided fine needle aspiration biopsy (all were either inaccurate or inconclusive). All patients underwent complete tumor excision with free margins of resection and tolerated surgery without any complications. None of the patients have had any evidence of recurrence at a mean follow-up of 26 months (range 10 to 48). CONCLUSIONS Retroperitoneal schwannomas are difficult to diagnose preoperatively. Computed tomography-guided fine needle aspiration biopsy does not appear to provide an accurate preoperative diagnosis. The surgical approach should focus on complete excision of the mass. Patients undergoing complete surgical resection tend to do well without evidence of early recurrence.

Fine needle aspiration diagnosis of extracranial glioblastoma multiforme: Case report and review of the literature

Background Hitherto uncommon, the incidence of extracranial metastases of primary brain malignancies may increase, with improved treatment methods and longer patient survival. Fine needle aspiration biopsy is a simple, safe and reliable method to diagnose metastatic malignancy. It has definite advantages over tissue biopsy, which is more invasive and is of higher risk to the patient. Ours is a case of glioblastoma multiforme, which metastasized to the scalp and was diagnosed on fine needle aspiration biopsy. Only a few articles document the cytological features of extracranial glioblastoma multiforme, diagnosed by fine needle aspiration biopsy. Case presentation We report the case of an elderly female who presented with focal neurological symptoms. She was diagnosed radiologically with an intracranial lesion in the left temporal region, which was subsequently resected. Histology revealed a glioblastoma multiforme confirmed by immunohistochemistry. The tumor recurred subsequently and the patient was treated with chemotherapy, intraoperatively. At a later stage, she presented with a scalp mass on which fine needle aspiration biopsy was performed. The cytomorphological features aided by immunohistochemistry supported a diagnosis of metastatic glioblastoma multiforme. The mass was later resected and histology confirmed the fine needle aspiration diagnosis of glioblastoma multiforme. Conclusion Reports of extracranial metastases of primary brain tumors are few. When they do occur, the primary cause is implantation during surgery or biopsy. However, spontaneous metastases to other organs do occur rarely. We believe fine needle aspiration biopsy to be very useful in the diagnosis of metastatic glioblastoma multiforme. The ability to use a cellblock for immunohistochemical studies is greatly advantageous and helpful in differentiating this tumor, from other malignancies that can occur in the scalp. A detailed discussion of the material obtained from fine needle aspiration biopsy of metastatic glioblastoma multiforme is presented, as well as a review of previous accounts in the literature.

Generation and characterization of tamoxifen-inducible Pax9-CreER knock-in mice using CrispR/Cas9

Pax9 encodes a paired‐box homeodomain (Pax) transcription factor and is critical for the development of multiple organs. Using CrispR/Cas9‐mediated homologous directed repair (HDR), we generated a new Pax9‐CreER knock‐in mouse line in which the CreERT2 fusion protein is produced after synthesis of endogenous Pax9 protein. We found that tdTomato reporter expression in Pax9‐CreER;tdTomato reporter mice is detectable in a similar pattern to the endogenous Pax9 expression, faithfully recapitulating the Pax9 expression domains throughout the embryo and in the adult mouse. At early embryonic stages, the tdTomato reporter is expressed first in the pharyngeal pouch region and later in the craniofacial mesenchyme, somites, limbs, and lingual papillae in the adult tongue. These results demonstrate that this new Pax9‐CreER knock‐in mouse line can be used for lineage tracing and genetic targeting of Pax9‐expressing cells and their progeny in a temporally and spatially controlled manner during development and organogenesis.

Fine needle aspiration (FNA) biopsy of palpable breast masses

One of the limitations preventing the widespread use of fine‐needle aspiration (FNA) is that it requires skill to obtain an adequate sample and well prepared smears. In this study, a new monolayer technique, the Cyto‐Tek MonoPrep (MP) system, which obviates the need for smear preparation, was evaluated against conventional smear (CS) preparation for palpable breast lesions.

T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

Kv1.3 is a voltage-gated potassium channel expressed on T cells that plays an important role in T cell activation. Previous studies have shown that blocking Kv1.3 channels in human T cells during activation results in reduced calcium entry, cytokine production, and proliferation. The aim of the present study was to further explore the effects of Kv1.3 blockers on the response of different human T cell subsets under various stimulation conditions. Our studies show that, unlike the immune suppressor cyclosporine A, the inhibitory effect of Kv1.3 blockers was partial and stimulation strength dependent, with reduced inhibitory efficacy on T cells under strengthened anti-CD3/CD28 stimulations. T cell responses to allergens including house dust mites and ragweed were partially reduced by Kv1.3 blockers. The effect of Kv1.3 inhibition was dependent on T cell subsets, with stronger effects on CCR7- effector memory compared to CCR7+ central memory CD4 T cells. Calcium entry studies also revealed a population of CD4 T cells resistant to Kv1.3 blockade. Activation of CD4 T cells was accompanied with an increase in Kv1.3 currents but Kv1.3 transcripts were found to be reduced, suggesting a posttranscriptional mechanism in the regulation of Kv1.3 activities. In summary, Kv1.3 blockers inhibit T cell activation in a manner that is highly dependent on the T cell identity and stimulation strength, These findings suggest that Kv1.3 blockers inhibit T cells in a unique, conditional manner, further refining our understanding of the therapeutic potential of Kv1.3 blockers.

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1 -/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.

Abstract 3279: Potential beneficial effects of the BRCA1 mutation carrier state on bone strength

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We previously suggested, based on observations in a mouse model in which we conditionally inactivated Brca1 in ovarian granulosa cells, that mutant mice are exposed to higher estradiol levels and also have prolonged estradiol exposure unopposed by progesterone due to an increase in the proestrus phase of their estrus cycle relative to metestrus. We sought to examine the biological significance of increased hormonal stimulation in homozygous Brca1 conditional knockout mice and to test the hypothesis that the effect is also present in mice with a heterozygous mutation such as present in human BRCA1 mutation carriers. We use endometrial cellular proliferation as an indicator for short-term effects of a Brca1mutation. Long-term effects were examined by measurements of long bone parameters using micro-computed tomography. The relative effects of heterozygous versus homozygous Brca1 mutations were evaluated by measuring the expression of enzymes involved in estradiol biosynthesis in granulosa cells. Mutant mice synchronized in the proestrus phase of their estrus cycle showed an increase in endometrial stromal cell proliferation in (P = .01). Mutant mice also showed an increase in trabecular bone density (P = .01) and femoral length (P = .02). Protein expression of aromatase and Hsd3B was higher in granulosa cells of mice carrying a heterozygous mutation in their ovarian granulosa cells compared to wild type littermates (P = .005 and .006 respectively). Our results provide a potential explanation for epidemiological observations to the effect that height is associated with increased risk for breast and ovarian cancer. The results also suggest that BRCA1 mutations, while associated with increased cancer risk, may also have some inherent advantages such as increased bone strength that may have contributed to the maintenance of mutated BRCA1 alleles in the human gene pool. Heterozygous Brca1 mutations such as present in human BRCA1 mutation carriers are not silent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3279. doi:1538-7445.AM2012-3279