Faculty Publications

An accurate biomarker for detection of ovarian cancer may reduce cancer-related mortality. Using a previously developed microarray-based technique, we evaluated differences in DNA methylation profiles in a panel of 56 genes using sections of serous papillary adenocarcinomas and uninvolved ovaries (n = 30) from women in a high-risk group. Methylation profiles were also generated for circulating DNA from blood of patients (n = 33) and healthy controls (n = 33). Using the most differentially methylated genes for naive Bayesian analysis, we identified ten of these profiles as potentially informative in tissues. Various combinations of these genes produced 69% sensitivity and 70% specificity for cancer detection as estimated under a stratified, fivefold cross-validation protocol. in plasma, five genes were identified as informative; their combination had 85% sensitivity and 61% specificity for cancer detection. These results suggest that differential methylation profiling in heterogeneous samples has the potential to identify components of a composite biomarker that may detect ovarian cancer in blood with significant accuracy. (J Mol Diagn 2009 11:60-65; DOI: 10.2353/jmoldx.2009.080072)

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 beta-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland. (C) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CRFB target genes (c-FOS, EGR-1, MKPI, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for M M therapy. (Am J Pathol 2009, 175:2197-2206; DOI: 10.2353/ajpath.2009.090400)

My experience in Tanzania has not only made me a better person, but also a better nurse. Creating this organization has been an awesome challenge. It is through Duluti Initiative, Inc. that I can give of myself and create opportunities for others who share in this mission. The mission of Duluti Initiative, Inc. is to decrease mortality and morbidity of the people of Tanzania, Africa by providing funds and access to healthcare. Here is how you can get involved. Donate. $15, for example, will supply a woman in our Expectant Mothers program with nutritious fruits and vegetables for one month. Spread the word! Contact us! http://duluti.org/. We would love to know your thoughts. Send us an e-mail about fundraising or other ways to contribute. Use the search engine www.goodsearch.com. Simply click the box that indicates which charity the search is for, enter duluti and click verify. For every search you conduct, Duluti Initiative, Inc. receives one penny. If 100 people use this search engine and conduct 10 searches per day for one year, at the end of the year Duluti Initiative, Inc. will receive a check for $3,650! Imagine what a contribution you can make! The best part is, all it costs is a little of your time. My experience in Tanzania has reinforced the importance of having strong nursing assessment skills, being able to think critically, and having a willingness to listen. Nursing is a "portable career" that we can take to any location on the planet and make a difference in someone's life. As nurses, we often take what we do for granted--but those that we help, do not.

Background-Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. Methods and Results-We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine beta-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE(-/-) Cbs(-/-) mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE(-/-) Cbs(-/-) mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE(-/-) Cbs(-/-) mice and another severe HHcy mouse model (Tg-S466L Cbs(-/-)) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6(Chi) and Ly-6C(mid) monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs(-/-) and Tg-hCBS apoE(-/-) Cbs(-/-) mice. These changes were exacerbated in Tg-S466L Cbs(-/-) mice with aging. Addition of L-homocysteine (100 to 500 mu mol/L), but not L-cysteine, maintained the Ly-6(Chi) subset and induced the Ly-6C(mid) subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6C(mid) subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. Conclusion-HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P) H oxidase-mediated oxidant stress. (Circulation. 2009;120:1893-1902.)

Estrogen plays vital roles in human health and diseases. Estrogen mediates its actions almost entirely by binding to estrogen receptors (ER), alpha and beta which further function as transcription factors. Selective estrogen receptor modulators (SERMs) are synthetic molecules which bind to ER and can modulate its transcriptional capabilities in different ways in diverse estrogen target tissues. Tamoxifen, the prototypical SERM, is extensively used for targeted therapy of ER positive breast cancers and is also approved as the first chemo-preventive agent for lowering breast cancer incidence in high risk women. The therapeutic and preventive efficacy of tamoxifen was initially proven by series of experiments in the laboratory which laid the foundation of its clinical use. Unfortunately, use of tamoxifen is associated with de-novo and acquired resistance and some undesirable side effects. The molecular study of the resistance provides an opportunity to precisely understand the mechanism of SERM action which may further help in designing new and improved SERMs. Recent clinical studies reveal that another SERM, raloxifene, which is primarily used to treat post-menopausal osteoporosis, is as efficient as tamoxifen in preventing breast cancers with fewer side effects. Overall, these findings open a new horizon for SERMs as a class of drug which not only can be used for therapeutic and preventive purposes of breast cancers but also for various other diseases and disorders. Major efforts are therefore directed to make new SERMs with a better therapeutic profile and fewer side effects.

BACKGROUND:: The standard of care for patients with advanced renal cell carcinoma (RCC) has changed to favor targeted therapy over immunotherapy. Differences in patterns of progression between patients treated with these 2 modalities, and the impact of disease stabilization on outcome, were investigated. METHODS:: Patients who progressed on first line antivascular therapy (AVT) or interferon were identified, and their medical records reviewed. RESULTS:: A total of 162 patients met inclusion criteria for this analysis. Patients in the AVT group had better baseline performance status, fewer liver metastases, and more responses (CR + PR) compared with the interferon group. Both groups were equally likely to develop distant metastases; however, for patients in the AVT group, these new metastases were more likely to arise in the setting of controlled disease at baseline sites (18% vs 4%, P = .012). There was no difference in anatomic sites of progression between the 2 groups. Patients responding (CR + PR) to AVT trended toward longer progression-free survival (PFS) compared with patients with stable disease (SD) (P = .06). No difference between responders and SD was seen in the interferon group. CONCLUSIONS:: Patients with RCC treated with antivascular therapy were more likely to progress at new sites in the setting of stable disease at baseline sites, suggesting that AVT may be more effective at controlling existing sites of disease than it is at preventing new metastases. Patients with SD on AVT had shorter PFS compared with responders (CR + PR). Whether this relationship extends to overall survival requires further study. Cancer 2009. (c) 2009 American Cancer Society.

L-Buthionine sulfoximine (BSO) is a potent inhibitor of glutathione biosynthesis and studies have shown that it is capable of enhancing the apoptotic effects of several chemotherapeutic agents. Previous studies have shown that long-term antihormonal therapy leads to acquired drug resistance and that estrogen, which is normally a survival signal, is a potent apoptotic agent in these resistant cells. Interestingly, we have developed an antihormone-resistant breast cancer cell line, MCF-7:2A. which is resistant to estrogen-induced apoptosis but has elevated levels of glutathione. In the present study, we examined whether BSO is capable of sensitizing anti hormone-resistant MCF-7:2A cells to estrogen-induced apoptosis. Our results showed that treatment of MCF-7:2A cells with 1 nM E2 plus 100 mu M BSO combination for 1 week reduced the growth of these cells by almost 80-90% whereas the individual treatments had no significant effect on growth. TUNEL and 4',6-diamidino-2-phenylindole (DAPI) staining showed that the inhibitory effect of the combination treatment was due to apoptosis. Our data indicates that glutathione participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to estrogen-induced apoptosis. (C) 2009 Published by Elsevier Ltd.

Supv3L1 is a conserved and ubiquitously expressed helicase found in numerous tissues and cell types of many species. In human cells, SUPV3L1 was shown to suppress apoptotic death and sister chromatid exchange, and impair mitochondrial RNA metabolism and protein synthesis. In vitro experiments revealed binding of SUPV3L1 to BLM and WRN proteins, suggesting a role in genome maintenance processes. Disruption of the Supv3L1 gene in the mouse has been reported to be embryonic lethal at early developmental stages. We generated a conditional mouse in which the phenotypes associated with the removal of exon 14 can be tested in a variety of tissues. Disruption mediated by a Mx1 promoter-driven Cre displayed a postnatal growth delay, reduced lifespan, loss of adipose tissue and muscle mass, and severe skin abnormalities manifesting as ichthyosis, thickening of the epidermis, and atrophy of the dermis and subcutaneous tissue. Using a tamoxifen-activatable Esr1/Cre driver, Supv3L1 disruption resulted in growth retardation and aging phenotypes, including loss of adipose tissue and muscle mass, kyphosis, cachexia, and premature death. Many of the abnormalities seen in the Mx1-Cre mice, such as hyperkeratosis characterized by profound scaling of feet and tail, could also be detected in tamoxifen-inducible Cre mice. Conditional ablation of Supv3L1 in keratinocytes confirmed atrophic changes in the skin and ichthyosis-like changes. Together, these data indicate that Supv3L1 is important for the maintenance of the skin barrier. In addition, loss of Supv3L1 function leads to accelerated aging-like phenotypes.

Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of beta-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation. (C) 2009 Elsevier B.V. All rights reserved.

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (deltaAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that deltaAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, deltaAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of deltaAg in the presence and absence of replicating and nonreplicating HDV RNAs. When deltaAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both deltaAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, deltaAg moved to the nucleoplasm, but nucleolin was unchanged. When deltaAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of deltaAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of deltaAg altered at specific locations considered to be essential for protein function. These studies provide evidence that deltaAg does not interact directly with either Pol II or nucleolin and that forms of deltaAg which support replication are also capable of prior nucleolar transit.

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy- thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine beta-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy- thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N- Hcy- protein levels. Liver N- Hcy- protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N- Hcy- protein is an important metabolite associated with Hcy pathophysiology in the mouse.-Jakubowski, H., Perla-Kajan, J., Finnell, R. H., Cabrera, R. M., Wang, H., Gupta, S., Kruger, W. D., Kraus, J. P., and Shih, D. M. Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice. FASEB J. 23, 1721-1727 (2009)

ABSTRACT: The link between estrogen and the development and proliferation of breast cancer is well documented. Estrogen stimulates growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Interestingly, there is strong evidence that estrogen induces apoptosis in breast cancer and other cell types. Forty years ago, before the development of tamoxifen, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in post-menopausal women. While the mechanisms by which estrogen induces apoptosis were not completely known, recent evidence from our laboratory and others demonstrates the involvement of the extrinsic (Fas/FasL) and the intrinsic (mitochondria) pathways in this process. We discuss the different apoptotic signaling pathways involved in E2 (17beta-estradiol)-induced apoptosis, including the intrinsic and extrinsic apoptosis pathways, the NF-kappaB (nuclear factor-kappa-B)-mediated survival pathway as well as the PI3K (phosphoinositide 3-kinase)/Akt signaling pathway. Breast cancer cells can also be sensitized to estrogen-induced apoptosis through suppression of glutathione by BSO (L-buthionine sulfoximine). This finding has implications for the control of breast cancer with low-dose estrogen and other targeted therapeutic drugs.

In the present study we measure the reliability and validity of the Reflective Function Rating Scale (RFRS) by comparing its results with reflective function (RF) scores obtained using the Adult Attachment Interview (AAI). As part of a randomized control trial comparing three different treatments for borderline personality disorder, eleven therapists were asked to rate the reflective functioning of 49 patients using the RFRS, 43 (87.8%) were female. Subject age ranged from 18 to 51 years (M = 30.88). The AAI a semistructured interview focused on early attachment relationships. We conducted a principal component factor analysis with varimax rotation on our sample. The factor scores were then correlated with a subset of 32 RF scores obtained from the AAI. Our preliminary findings indicate reliability and validity of the RFRS by demonstrating that its factor subscales represent cohesive constructs and by relating it in predicted ways to RF scores obtained from the AAI. (PsycINFO Database Record (c) 2009 APA, all rights reserved).