Mutant p53 expression in fallopian tube epithelium drives cell migration

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

Tumorigenesis and peritoneal colonization from fallopian tube epithelium

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system.

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.

Induction and antagonism of antiviral responses in respiratory syncytial virus-infected pediatric airway epithelium.

Airway epithelium is the primary target of many respiratory viruses. However, virus induction and antagonism of host responses by human airway epithelium remains poorly understood. To address this, we developed a model of respiratory syncytial virus (RSV) infection based on well- differentiated pediatric primary bronchial epithelial cell cultures (WD-PBECs) that mimics hallmarks of RSV disease in infants. RSV is the most important respiratory viral pathogen in young infants worldwide. We found that RSV induces a potent antiviral state in WD-PBECs that was mediated in part by secreted factors, including interferon lambda-1 (IFNλ1)/IL-29. In contrast, type I interferons were not detected following RSV infection of WD-PBECs., Interferon (IFN) responses in RSV-infected WD-PBECs reflected those in lower airway samples from RSV-hospitalized infants. In view of the prominence of IL-29, we determined whether recombinant IL-29 treatment of WD-PBECs before or after infection abrogated RSV replication. Interestingly, IL-29 demonstrated prophylactic, but not therapeutic, potential against RSV. The absence of therapeutic potential reflected effective RSV antagonism of IFN-mediated antiviral responses in infected cells. Our data are consistent with RSV non-structural proteins 1 and/or 2 perturbing the Jak-STAT signaling pathway, with concomitant reduced expression of antiviral effector molecules, such as MxA/B. Antagonism of Jak-STAT signaling was restricted to RSV-infected cells in WD-PBEC cultures. Importantly, our study provides the rationale to further explore IL-29 as a novel RSV prophylactic.

Survival for oesophageal, stomach and small intestine cancers in Europe 1999-2007: Results from EUROCARE-5

Background: European regional variation in cancer survival was reported in the EUROCARE-4 study for patients diagnosed in 1995-1999. Relative survival (RS) estimates are here updated for patients diagnosed with cancer of the oesophagus, stomach and small intestine from 2000 to 2007. Trends in RS from 1999-2001 to 2005-2007 are presented to monitor and discuss improvements in patient survival in Europe. Materials and methods: EUROCARE-5 data from 29 countries (87 cancer registries) were used to investigate 1- and 5-year RS. Using registry-specific life-tables stratified by age, gender and calendar year, age-standardised 'complete analysis' RS estimates by country and region were calculated for Northern, Southern, Eastern and Central Europe, and for Ireland and United Kingdom (UK). Survival trends of patients in periods 1999-2001, 2002-2004 and 2005-2007 were investigated using the 'period' RS approach. We computed the 5-year RS conditional on surviving the first year (5-year conditional survival), as the ratio of age-standardised 5-year RS to 1-year RS. Results Oesophageal cancer 1- and 5-year RS (40% and 12%, respectively) remained poor in Europe. Patient survival was worst in Eastern (8%), Northern (11%) and Southern Europe (10%). Europe-wide, there was a 3% improvement in oesophageal cancer 5-year survival by 2005-2007, with Ireland and the UK (3%), and Central Europe (4%) showing large improvements. Europe-wide, stomach cancer 5-year RS was 25%. Ireland and UK (17%) and Eastern Europe (19%) had the poorest 5-year patient survival. Southern Europe had the best 5-year survival (30%), though only showing an improvement of 2% by 2005-2007. Small intestine cancer 5-year RS for Europe was 48%, with Central Europe having the best (54%), and Ireland and UK the poorest (37%). Five-year patient survival improvement for Europe was 8% by 2005-2007, with Central, Southern and Eastern Europe showing the greatest increases (≥9%). Conclusions Survival for these cancer sites, particularly oesophageal cancer, remains poor in Europe with wide variation. Further investigation into the wide variation, including analysis by histology and anatomical sub-site, will yield insights to better monitor and explain the improvements in survival observed over time.

Sediment distribution, hydrolytic enzyme profiles and bacterial activities in the guts of Oneirophanta mutabilis, Psychropotes longicauda and Pseudostichopus - what do they tell us about digestive strategies of abyssal holothurians?

This paper describes inter-specific differences in the distribution of sediment in the gut compartments and in the enzyme and bacterial profiles along the gut of abyssal holothurian species — Oneirophanta mutabilis, Psychropotes longicauda and Pseudostichopus villosus sampled from a eutrophic site in the NE Atlantic at different times of the year. Proportions of sediments, relative to total gut contents, in the pharynx, oesophagus, anterior and posterior intestine differed significantly in all the inter-species comparisons, but not between inter-seasonal comparisons. Significant differences were also found between the relative proportions of sediments in both the rectum and cloaca of Psychropotes longicauda and Oneirophanta mutabilis. Nineteen enzymes were identified in either gut-tissue or gut-content samples of the holothurians studied. Concentrations of the enzymes in gut tissues and their contents were highly correlated. Greater concentrations of the enzymes were found in the gut tissues suggesting that they are the main source of the enzymes. The suites of enzymes recorded were broadly similar in each of the species sampled collected regardless of the time of the year, and they were similar to those described previously for shallow-water holothurians. Significant inter-specific differences in the gut tissue concentrations of some of the glycosidases suggest dietary differences. For example, Psychropotes longicauda and Pseudostichopus villosus contain higher levels of chitobiase than Oneirophanta mutabilis. There were no seasonal changes in bacterial activity profiles along the guts of O. mutabilis and Pseudostichopus villosus. In both these species bacterial activity and abundance declined between the pharynx/oesophagus and anterior intestine, but then increased along the gut and became greatest in the rectum/cloaca. Although the data sets were more limited for Psychropotes longicauda, bacterial activity increased from the anterior to the posterior intestine but then declined slightly to the rectum/cloaca. These changes in bacterial activity and densities probably reflect changes in the microbial environment along the guts of abyssal holothurians. Such changes suggest that there is potential for microbial breakdown of a broader range of substrates than could be otherwise be achieved by the holothurian itself. However, the present study found no evidence for sedimentary (microbial) sources of hydrolytic enzymes.

BRCA1 Suppresses Osteopontin-mediated Breast Cancer

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor alpha, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorage independent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

An automated machine vision system for the histological grading of cervical intraepithelial neoplasia (CIN)

The histological grading of cervical intraepithelial neoplasia (CIN) remains subjective, resulting in inter- and intra-observer variation and poor reproducibility in the grading of cervical lesions. This study has attempted to develop an objective grading system using automated machine vision. The architectural features of cervical squamous epithelium are quantitatively analysed using a combination of computerized digital image processing and Delaunay triangulation analysis; 230 images digitally captured from cases previously classified by a gynaecological pathologist included normal cervical squamous epithelium (n = 30), koilocytosis (n = 46), CIN 1 (n = 52), CIN 2 (n = 56), and CIN 3 (n=46). Intra- and inter-observer variation had kappa values of 0.502 and 0.415, respectively. A machine vision system was developed in KS400 macro programming language to segment and mark the centres of all nuclei within the epithelium. By object-oriented analysis of image components, the positional information of nuclei was used to construct a Delaunay triangulation mesh. Each mesh was analysed to compute triangle dimensions including the mean triangle area, the mean triangle edge length, and the number of triangles per unit area, giving an individual quantitative profile of measurements for each case. Discriminant analysis of the geometric data revealed the significant discriminatory variables from which a classification score was derived. The scoring system distinguished between normal and CIN 3 in 98.7% of cases and between koilocytosis and CIN 1 in 76.5% of cases, but only 62.3% of the CIN cases were classified into the correct group, with the CIN 2 group showing the highest rate of misclassification. Graphical plots of triangulation data demonstrated the continuum of morphological change from normal squamous epithelium to the highest grade of CIN, with overlapping of the groups originally defined by the pathologists. This study shows that automated location of nuclei in cervical biopsies using computerized image analysis is possible. Analysis of positional information enables quantitative evaluation of architectural features in CIN using Delaunay triangulation meshes, which is effective in the objective classification of CIN. This demonstrates the future potential of automated machine vision systems in diagnostic histopathology. Copyright (C) 2000 John Wiley and Sons, Ltd.

Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

Advanced Glycation End Products (AGEs) accumulate in the Reproductive Tract of Men with Diabetes.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable affects of diabetes on sperm function. Specifically, a recent study has found significantly higher sperm nuclear DNA (nDNA) fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and 9 non-diabetic subjects. CML immunoreactivity was found through out the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells (cytoplasm and nuclei) of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggests that these compounds may play a hitherto unrecognized role in male infertility.

Cochlin in the eye: Functional implications

Aqueous humor is actively produced in the ciliary epithelium of the anterior chamber and has important functions for the eye. Under normal physiological conditions, the inflow and outflow of the aqueous humor are tightly regulated, but in the pathologic state this balance is lost. Aqueous outflow involves structures of the anterior chamber and experiences most resistance at the level of the trabecular meshwork (TM) that acts as a filter. The modulation of the TM structure regulates the filter and its mechanism remains poorly understood. Proteomic analyses have identified cochlin, a protein of poorly understood function, in the glaucomatous TM but not in healthy control TM from human cadaver eyes. The presence of cochlin has subsequently been confirmed by Western and immunohistochemical analyses. Functionally, cochlin undergoes multimerization induced by shear stress and other changes in the microenvironment. Cochlin along with mucopolysaccharide deposits have been found in the TM of glaucoma patients and in the inner ear of subjects affected by the hearing disorder DNFA9, a late onset, progressive disease that also involves alterations in fluid shear regimes. In vitro, cochlin induces aggregation of primary TM cells suggesting a role in cell adhesion, possibly in mechanosensation, and in modulation of the TM filter.