Chien Chen

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of theHPRT promoter in single cell-derived lines. Reactivation ofHPRT correlated with complete promoter demethylation. In contrast, the 61 5aCdr-treated clones that failed to reactivateHPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated (and unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRTreactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivateHPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.

Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

ABSTRACT Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

Delayed Treatment of Papillary Thyroid Carcinoma Arising from Struma Ovarii in a Patient with History of Bilateral Salpingo-Oophorectomy: A Case Report

OBJECTIVE We present a case of papillary thyroid carcinoma arising from struma ovarii treated erroneously as ovarian adenocarcinoma for more than 3 years. METHODS We report clinical, surgical, laboratory, and imaging findings of the study patient and review the relevant literature. RESULTS A 64-year-old woman was treated for ovarian adenocarcinoma for more than 3 years before it was determined that she likely had papillary thyroid carcinoma arising from struma ovarii. This is the first reported case of thyroid carcinoma arising from struma ovarii in a patient with a history of bilateral salpingo-oophorectomy. Possible etiologies include residual ovarian tissue after oophorectomy, ectopic thyroid, or metastatic thyroid cancer. CONCLUSIONS It is important to include struma ovarii and thyroid carcinoma arising from struma ovarii in the differential diagnosis, even with a history of bilateral salpingo-oophorectomy. This case emphasizes the importance of effective communication among the pathologist, oncologist, and surgeon to ensure timely initiation of appropriate therapy and reduced patient morbidity.

Rapid onsite evaluation: A comparison of cytopathologist and pulmonologist performance.

Rapid onsite evaluation (ROSE) has several potential benefits but also can prolong procedures if one must wait for a cytopathologist, and it can involve a considerable time commitment on the part of the cytopathologist. At the University of Arkansas for Medical Sciences, interventional pulmonologists have routinely reviewed cytology specimens. This study was performed to determine prospectively how accurately pulmonologists could perform ROSE and whether they could contribute to the efficiency of the process.

Fine-needle aspiration diagnosis of thyroid blastomycosis.

OBJECTIVE To describe an elusive case of blastomycosis involving the thyroid gland, which was ultimately diagnosed by use of ultrasound-guided fine-needle aspiration (FNA). METHODS We present a case report, including clinical features, results of laboratory studies, and radiographic, computed tomographic, and ultrasonographic findings. In addition, the treatment and the utility of FNA of the thyroid relative to the diagnosis of blastomycosis are discussed. RESULTS An 18-year-old woman with no significant past medical history and with a competent immune system presented initially to her family physician because of headaches, lymphadenopathy, blurry vision, and fatigue. Radiography of the chest showed findings considered consistent with pneumonia, for which amoxicillin was prescribed. Subsequently, an ophthalmologist diagnosed anterior uveitis and initiated topical corticosteroid therapy. Worsening symptoms prompted performance of computed tomography of the chest, which suggested thyroid involvement. Ultimately, FNA of a thyroid nodule led to the cytologic diagnosis of blastomycosis. The patient was treated successfully with amphotericin for blastomycosis of the eye, lung, and thyroid. CONCLUSION Physicians should consider the potential presence of blastomycosis when a lung lesion does not improve with typical treatment interventions. Disseminated blastomycosis can be diagnosed with use of FNA of the thyroid.

Extranodal Rosai-Dorfman disease in the scrotum of a 13-month male: a unique anatomic presentation.

To the Editor: Rosai–Dorfman disease (RDD), formerly known as sinus histiocytosis with massive lymphadenopathy, is usually characterized by substantial bilateral, painless lymphadenopathy, fever, polyclonal gammopathy, and leukocytosis. Extranodal involvement occurs in up to 41% of systemic cases, with skin being the most common site. RDD confined only to the skin (primary cutaneous RDD) is rare, with fewer than 40 cases reported in the literature, but has been described in a wide array of anatomic sites. Although testicular involvement by RDD has been documented occasionally, involvement of scrotal skin by cutaneous RDD in an infant has not yet been described in the literature. A 13-month-old white male presented with a several month history of scrotal swelling and bilateral inguinal lymphadenopathy without any associated systemic symptoms. On physical examination, a 2-cm, ulcerated lesion on the right scrotum was appreciated (Fig. 1). The preoperative clinical diagnosis was that of a scrotal ulcer/mass with associated lymphadenopathy, and the mass was subsequently excised without complication. Histologic examination revealed a dense dermal infiltrate of histiocytic cells, with ulceration of the overlying epidermis. Most of these cells had abundant lightly eosinophilic cytoplasm, round nuclear contours, and single, distinct nucleoli. Admixed lymphoid aggregates with conspicuous plasma cells and scattered granulocytes were present surrounding the sheets of histiocytes (Figs. 2–4). The histiocytic cells were uniformly strongly positive for S100 protein and CD45 (leukocyte common antigen) and variably positive for CD68 by immunohistochemistry; FIGURE 1. Well-circumscribed, ulcerative lesion on right scrotum.

Orthotopic VX rabbit tongue cancer model with FDG-PET and histologic characterization

We present our experience with the use of an immunocompetent medium‐sized animal model of tongue cancer that may be suitable for imaging and surgical studies.

Sestamibi Localization of a Parathyroid Adenoma in the Presence of a Thymoma: a Case Report and Review of the Literature

Sestamibi Localization of a Parathyroid Adenoma in the Presence of a Thymoma: a Case Report and Review of the Literature Preoperative localization in the surgical management of primary hyperparathyroidism is complicated by widely variable parathyroid gland anatomy, coexisting thyroid pathology, incidence of supernumerary and ectopic parathyroid glands, and limitations of targeted imaging studies. Technetium 99mTcsestamibi scans have decreased accuracy, sensitivity and specificity in the presence of multi-gland parathyroid disease. A number of reports suggest that thymomas, which also show increased sestamibi uptake, are potential sources of false positives on 99mTc-sestamibi scans. Herein, we describe a thymoma in association with a parathyroid adenoma complicating localization of hyperfunctional parathyroid tissue. We review the literature of concurrent pathology between parathyroid adenomas and thymomas paying particular attention to how these cases present on preoperative 99mTcsestamibi scans.