Biomedical subjects

Katherine F Roby

Publications and source records attributed to Katherine F Roby.

14 recordsLinked to original sources

PubMed2006

Design, synthesis, and antiproliferative and CDK2-cyclin a inhibitory activity of novel flavopiridol analogues.

The design and synthesis of a small library of 8-amidoflavone, 8-sulfonamidoflavone, 8-amido-7-hydroxyflavone, and heterocyclic analogues of flavopiridol is reported. The potential activity of these compounds as kinase inhibitors was evaluated by cytotoxicity studies in MCF-7 and ID-8 cancer cell lines and inhibition of CDK2-Cyclin A enzyme activity in vitro. The antiproliferative and CDK2-Cyclin A inhibitory activity of these analogues was significantly lower than the activity of flavopiridol. Molecular docking simulations were carried out and these studies suggested a different binding orientation inside the CDK2 binding pocket for these analogues compared to flavopiridol.

Adenosine Triphosphate↗

PubMed2006

Tumor necrosis factor (TNF) receptor type 2 is an important mediator of TNF alpha function in the mouse ovary.

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.

Animals↗

PubMed2006

Interleukin-1alpha-induced chemokines in mouse granulosa cells: impact on keratinocyte chemoattractant chemokine, a CXC subfamily.

IL-1 is well known to be involved in the immune system and have a role in ovarian inflammation as well as exhibiting inhibitory effects on steroidogenesis and folliculogenesis. Because multiple aspects of ovarian function have also been shown to involve cytokine/chemokine networks, IL-1alpha-induced chemokine gene expression in mouse granulosa cells was investigated. Granulosa cells from immature mice at 28 d of age were cultured with IL-1alpha (10 ng/ml). IL-1alpha induced abundantly and specifically keratinocyte chemoattractant (KC) chemokine, a CXC subfamily. KC chemokine mRNA and protein were increased 1-2 h after IL-1alpha and then gradually decreased. The KC promoter (-701/+30) containing three nuclear factor (NF)-kappaB sites was fully responsive to IL-1alpha, whereas deletions and mutants of the NF-kappaB sites lowered the responsiveness to IL-1alpha. The proximal NF-kappaB site (-69/-59) played a critical role in regulating IL-1alpha-induced KC chemokine promoter activity. Overexpression of the inhibitor of NF-kappaB (IkappaB) blocked KC promoter activity induced by IL-1alpha, whereas overexpression of p65, a component of NF-kappaB, increased promoter activity and mRNA of KC chemokine. In addition, FSH did not affect NF-kappaB signaling or IL-1alpha-induced KC chemokine promoter activity. Within 1-3 h after ip injection of lipopolysaccharide (100 mug/mouse), a product known to stimulate release of IL-1, KC chemokine was localized in the ovary to granulosa cells as well as the thecal-interstitial layer. The results of this study indicate that KC gene is a chemokine induced acutely by IL-1alpha via NF-kappaB signaling in mouse granulosa cells.

Animals↗

PubMed2006

CD80 in immune suppression by mouse ovarian carcinoma-associated Gr-1+CD11b+ myeloid cells.

An elevated number of Gr-1+CD11b+ myeloid cells has been described in mice bearing transplantable tumors, and has been associated with immune suppression. We examined the role of such myeloid suppressor cells in mice bearing the spontaneously transformed syngeneic mouse ovarian surface epithelial cell line, 1D8. We observed high levels of CD80 expression by Gr-1+CD11b+ cells from spleen, ascites, and tumor tissue of mice bearing 1D8 ovarian carcinoma, whereas CD40 and CD86 were absent. CD80 expression was not detected on Gr-1+CD11b+ cells from naïve mice. However, the expression of CD80 by Gr-1+CD11b+ cells from naïve mice was promoted by coculture with 1D8 cells. Because irradiated 1D8 cells, but not 1D8-conditioned medium, up-regulate CD80 expression by Gr-1+CD11b+ cells, this phenomenon likely requires direct interaction. Gr-1+CD11b+ cells derived from 1D8 tumor-bearing mice provided significant suppression of antigen-specific immune responses, but Gr-1+CD11b+ cells from naïve mice did not. Both short interfering RNA-mediated knockdown and genetic knockout of CD80 expression by Gr-1+CD11b+ cells of 1D8 tumor-bearing mice alleviated the suppression of antigen-specific immune responses. Suppression via CD80 on Gr-1+CD11b+ myeloid cells was mediated by CD4+CD25+ T regulatory cells and required CD152. CD80 knockout or antibody blockade of either CD80 or CD152 retarded the growth of 1D8 tumor in mice, suggesting that expression of CD80 on Gr-1+CD11b+ myeloid cells triggered by 1D8 ovarian carcinoma suppresses antigen-specific immunity via CD152 signaling and CD4+CD25+ T regulatory cells. Thus, CD80-dependent responses to myeloid suppressor cells may contribute to tumor tolerance and the progression of ovarian carcinoma.

Animals↗

PubMed2006

Integrative genomic analysis of protein kinase C (PKC) family identifies PKCiota as a biomarker and potential oncogene in ovarian carcinoma.

The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCiota (43.8%), PKCbeta1 (37.1%), PKCgamma (27.6%), PKCzeta (22.5%), and PKCtheta (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCiota and PKCtheta, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCiota expression correlated with tumor stage or grade, and PKCiota overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCiota protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCiota did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCiota expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCiota contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCiota may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer.

Biomarkers, Tumor↗

PubMed2006

Transcriptomic analysis of an in vitro murine model of ovarian carcinoma: functional similarity to the human disease and identification of prospective tumoral markers and targets.

Ovarian cancer is an aggressive disease of poor prognostic when detected at advanced stage. It is widely accepted that the ovarian surface epithelium plays a central role in disease etiology, but little is known about disease progression at the molecular level. To identify genes involved in ovarian tumorigenesis, we carried out a genome-wide transcriptomic analysis of six spontaneously transformed mouse ovarian surface epithelial (MOSE) cell lines, an in vitro model for human ovarian carcinoma. Loess normalization followed by statistical analysis with control of multiple testing resulted in 509 differentially expressed genes using an adjusted P-value < or = 0.05 as cut-off. The top 20 differentially expressed genes included 10 genes (Spp1, Cyp1b1, Btg1, Cfh, Mt1, Mt2, Igfbp5, Gstm1, Gstm2, and Esr1) implicated in various aspects of ovarian carcinomas, and other 3 genes (Gsto1, Lcn7, and Alcam) associated to breast cancer. Upon functional analysis, the majority of alterations affected genes involved in glutathione metabolism and MAPK signaling pathways. Interestingly, over 20% of the aberrantly expressed genes were related to extracellular components, suggestive of potential markers of disease progression. In addition, we identified the genes Pura, Cnn3, Arpc1b, Map4k4, Tgfb1i4, and Crsp2 correlated to in vivo tumorigenic parameters previously reported for these cells. Taken together, our findings support the utility of MOSE cells in studying ovarian cancer biology and as a source of novel diagnostic and therapeutic targets.

Animals↗

PubMed2005

Alterations in reproductive function in SRC tyrosine kinase knockout mice.

The role of Src tyrosine kinase in regulating reproductive processes in female mice was investigated using Src wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice. Ovarian Src kinase activity transiently increased in Src +/+ mice following eCG administration. Src knockout mice were infertile. Estrous cycles and vaginal opening in Src knockouts were variable and altered compared with Src +/+ and +/- mice. Follicle development was compromised in Src knockout mice as evidenced by reduced numbers of large pre-antral and antral follicles compared to Src +/+ mice. Corpora lutea were not observed in the ovaries of Src knockout mice; however, administration of eCG and hCG did result in ovulation. Serum LH and FSH on d 40 and 52 of age did not differ between Src wild-type and knockout females. Results from these studies reveal that female Src knockout mice are infertile due to reduced follicle development and anovulation.

Animals↗

PubMed2005

Tumor necrosis factor alpha (TNF) suppresses cAMP response element (CRE) activity and nuclear CRE binding protein in MA-10 mouse Leydig tumor cells.

TNF is known to suppress gonadotropin-induced steroid secretion by Leydig cells. However, the mechanisms by which this occurs are largely unknown. Because expression of many steroidogenic proteins is regulated by the PKA pathway, effects of TNF on CRE activity were examined using MA-10 mouse Leydig tumor cells. The cells were transfected with a CRE-luciferase construct, and stimulated with either LH or 8Br-cAMP in the presence or absence of TNF. TNF suppressed, LH-stimulated and 8Br-cAMP stimulated CRE activity. TNF also suppressed CRE activity stimulated with a PKA expression vector. Further experiments suggested that the effect of TNF on CRE activity was not mediated by the NF-kappaB pathway. TNF did not affect levels of either CREB or phospho-CREB in whole cell lysates; however, TNF decreased both CREB and phospho-CREB in nuclear extracts in a time-dependent manner. The decrease in nuclear CREB is likely to be a major mechanism of the suppressive effects of TNF on steroidogenesis in MA-10 Leydig cells.

Animals↗

PubMed2004

Tumor necrosis factor-alpha induces serum amyloid A3 in mouse granulosa cells.

TNF-alpha has significant inhibitory effects on steroidogenesis and folliculogenesis and is associated with several inflammatory responses. Because ovulation is an inflammatory reaction, the effects of TNF on the family of acute-phase proteins in granulosa cells were investigated. Granulosa cells from immature mice at 28 d of age were cultured in the presence of 10 ng TNF/ml for 24 h. Serum amyloid A3 (SAA3), a main acute-phase protein, was induced by TNF in granulosa cells. The other isoforms of serum amyloid proteins SAA1, SAA2, and SAA4 were neither expressed in granulosa cells nor induced by TNF. TNF did not induce SAA3 mRNA in granulosa cells from TNF receptor type 1 (TNFR1) knockout mice, although SAA3 mRNA was induced within 3 h after TNF treatment in wild-type cells. Two SAA3 promoters, -617/+73 and -198/+73, were responsive to TNF and to p65, a component of the TNF signaling molecule nuclear factor (NF)-kappaB. The -106/+73 promoter of SAA3 lacking a NF-kappaB-like site was not responsive to TNF or p65. In granulosa cells from TNFR1 knockout mice, the SAA3 promoter (-198/+73) was responsive to transfection with the p65 component of NF-kappaB, but neither TNF treatment nor overexpression of the p50 component of NF-kappaB increased promoter activity. Similar results were observed in the murine ovarian granulosa tumor cell line (OV3121-1). Overexpression of the inhibitor of NF-kappaB (called IkappaB) blocked SAA3 promoter activity induced by TNF and by p65 in OV3121-1 cells. Closer analysis of deletion mutants of the SAA3 promoter revealed the necessity of a NF-kappaB like site for responsiveness to TNF in the OV3121-1 cells. TNF rapidly increased p65 in OV3121-1 nuclei when compared with controls not treated with TNF. TNF also increased phospho-IkB and SAA3 in whole-cell homogenates as determined by Western blots. Thus, TNF likely increased SAA3 promoter activity and protein by activating NF-kappaB signaling via TNFR1 in mouse granulosa cells. SAA3 is a novel gene in granulosa cells with yet unknown functions in the ovary.

Animals↗

PubMed2004

Src tyrosine kinase regulates CYP17 expression and androstenedione secretion in theca-enriched mouse ovarian cells.

Src tyrosine kinase belongs to a non-receptor tyrosine kinase family and has been shown to be involved in G protein-coupled receptor desensitization and internalization. Stimulation of ovarian thecal cells with lutein-izing hormone (LH) activates adenylyl cyclase via a G protein-coupled LH receptor leading to an increase in cAMP. Subsequently, cAMP activates protein kinase A (PKA) that increases steroidogenesis. In order to evaluate the role of Src in thecal cell steroidogenesis, a pharmacological approach was utilized by treating a population of mouse ovarian theca-enriched cells (TEC) in vitro with two Src inhibitors, geldanamycin (GA) and herbimycin A (HA). Treatment of TEC with either GA or HA increased basal androstenedione secretion without alteration of cAMP. In the presence of forskolin, GA and HA treatment further increased androstenedione secretion. RT-PCR analysis of RNA from cells treated with GA for 8, 24, and 48 h revealed that GA increased cytochrome P450 17alpha-hydroxylase/lyase (CYP17) mRNA at 48 h. CYP17 promoter activity also increased after treatment of cells with GA and after co-transfection with a Src dominant negative plasmid. Inhibition of PKA using H89 blocked the effect GA and HA on androstenedione secretion. These results indicate that the pharmacological inhibitors of Src, GA and HA, tested in vitro increased thecal CYP17 promoter activity, CYP17 mRNA, and androstenedione secretion. In addition, GA and HA induced thecal androstenedione secretion may be cAMP independent but possibly requires PKA.

3-Hydroxysteroid Dehydrogenases↗

PubMed2003

Tumor necrosis factor alpha (TNF) increases granulosa cell proliferation: dependence on c-Jun and TNF receptor type 1.

TNF alpha has significant in vitro effects on steroidogenesis and folliculogenesis and reproductive alterations occur in TNF receptor type 1 (TNFR1) knockout mice. The present study investigated the effect of in vitro TNF on granulosa cell proliferation from immature mice at 28 d of age, with emphasis on intracellular signaling that regulates granulosa cell proliferation. TNF dose dependently increased granulosa cell proliferation and the proto-oncogene c-Jun protein. However, other Jun family members such as JunD was expressed constitutively and JunB was not expressed. In vitro TNF did not increase c-Jun and proliferation in granulosa cells from TNFR1 knockout mice. The time course of TNF-induced c-Jun revealed biphasic patterns of short-term (3 h) and long-term (24 h) induction. The time courses of Ser63- and Ser73-phospho c-Jun coincided with changes in total c-Jun. Among MAPK cascades, stress-activated protein kinase/c-Jun-NH(2)-teminal kinase signaling was increased transiently in TNF-treated cells, whereas p38MAPK and ERK1 and 2 were not changed. In addition, overexpression of nuclear factor-kappa B and addition of ceramide and 8-bromo-cAMP did not increase c-Jun or proliferation. Antisense oligonucleotides for c-Jun blocked cell proliferation induced by TNF. In conclusion, the above results demonstrate that TNF increased c-Jun by activating stress-activated protein kinase/c-Jun-NH(2)-teminal kinase signaling via TNFR1 in mouse granulosa cells, and the induced c-Jun resulted in increased cell proliferation.

Age Factors↗

PubMed2003

Src tyrosine kinase promotes survival and resistance to chemotherapeutics in a mouse ovarian cancer cell line.

The vast majority of ovarian cancers originate in the ovarian surface epithelium. Unfortunately, there is a lack of appropriate animal models for ovarian cancer research. Spontaneously transformed mouse ovarian surface epithelial cells may provide a faithful animal model for human ovarian cancer. One such cell line (ID8) has been partially characterized. ID8 cells demonstrate constitutive Src tyrosine kinase activation with resulting phosphatidylinositol-3-kinase activation and Akt and forkhead phosphorylation. In addition, focal adhesion kinase is constitutively phosphorylated at tyrosine 925, a Src phosphorylation site, resulting in increased Ras activation. These features are common to human ovarian cancer cell lines. Inhibition of Src enhances the cell killing effects of both paclitaxel and cisplatinum. Finally, Src inhibition restores sensitivity of a drug resistant ID8 cell line. The ID8 mouse ovarian cancer cell line presents new opportunities to study ovarian cancer progression and pre-therapeutic trials in an immune competent background.

Animals↗

PubMed2002

Estradiol enhances and estriol inhibits the expression of CYP1A1 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in a mouse ovarian cancer cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous pollutant and promoter of carcinogenesis. This study investigated the interaction between TCDD and different estrogens in a cancer cell line (ID8) derived from mouse ovarian epithelium. TCDD-induced ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P4501A1 (CYP1A1) expression in a dose- and time-dependent manner. Estrogen receptor (ER) alpha mRNAs were constitutively expressed, but ER beta and progesterone receptor (PR) mRNAs were not expressed. Induction of EROD by TCDD was completely inhibited by a alpha-naphthoflavone and phenanthroline, two aryl hydrocarbon receptor (AhR) antagonists. Progesterone and gonadotropins (FSH and LH) had no effect on the induction of EROD by TCDD. Congeners of 17beta-estradiol (E2) increased the induction of EROD activity by TCDD dose-dependently in the relative potency order: estrone (El)>E2> or = 4-hydroxyestradiol (4OHE2)> or = 2-hydroxyestradiol (2OHE2). In contrast, estriol (E3) decreased EROD activity induced by TCDD. E2 increased TCDD-induced CYP1A1 protein and mRNA whereas E3 decreased both the protein and mRNA. E2 did not alter luciferase activity induced by TCDD in cells transfected with a luciferase reporter containing dioxin response elements (DRE) or a CYP1A1 promoter. In contrast, E3 dose-dependently decreased the luciferase activity. A pure anti-estrogen (ICI 182780) inhibited the interaction between E2 and TCDD but did not block E3's effect on EROD activity. These results indicate that E2 may affect TCDD-induced CYP1A1 expression by a mechanism different from E3 in ID8 cells. It appears that the potentiation of E2 in the induction of CYP1A1 by TCDD occurs by a mechanism involving ER alpha since a specific ER antagonist blocked the potentiation. The inhibitory effect of E3 may be due to a rapid direct effect on EROD and a later suppression of CYP1A1 expression.

Animals↗

PubMedDate not supplied

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum inhibin concentrations and inhibin immunostaining during follicular development in female Sprague-Dawley rats.

Intact and hypophysectomized immature rats were pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0 or 32 microg/kg p.o.) and sacrificed throughout synchronized follicular development (0, 12, 24, 48, and 72 h after equine chorionic gonadotropin, eCG). TCDD administration to intact rats resulted in a premature elevation of serum FSH and LH by 12 h post-eCG. In intact rats pretreated with TCDD, the intensity of ovarian immunoreactivity for inhibin and the number of ovarian follicles staining for inhibin in midsaggital ovarian sections were decreased at the time of eCG administration (24 h post-TCDD) in comparison to controls. However, this decreased ovarian staining for inhibin was not associated with alterations in serum inhibin concentrations. Serum inhibin was suppressed in TCDD-treated rats when compared to intact controls only at 24 h post-eCG. Hypophysectomized animals exhibited no effect of TCDD on serum inhibin at any timepoint but did have decreased estradiol concentrations during follicular development. In summary, TCDD reduced serum concentrations of inhibin after the premature increases in FSH and LH suggesting that inhibin is not important in the initial elevation of FSH following exposure to TCDD.

Animals↗