Greg Rice

Integrin-linked kinase expression increases with ovarian tumour grade and is sustained by peritoneal tumour fluid.

Integrin‐linked kinase (ILK) is a serine threonine kinase, overexpression of which promotes tumour growth and invasion through deregulation of the cell cycle. This study demonstrates the relative expression of ILK in normal, benign, low‐grade, and high‐grade (borderline, grade I/II, and grade III) ovarian tumours of serous, mucinous, endometrioid, and clear cell types in order to assess its potential as a marker for epithelial ovarian cancer progression. Seventy‐three specimens including ten normal, ten benign, 14 borderline, 17 grade I/II, and 22 grade III were evaluated by immunohistochemistry. Immunoreactive ILK was not detectable in normal ovarian surface epithelium. All 53 carcinomas studied were positive and the staining intensity correlated significantly with the grade of the tumour. Ovarian cancer cell lines had high expression of ILK, while immortalized normal ovarian surface epithelial cell lines (HOSE) showed low basal expression of ILK by western blotting. Peritoneal tumour fluid (PTF) upregulated ILK expression in ovarian cancer cell lines but had no effect on HOSE cells. PTF‐induced up‐regulation of ILK expression in ovarian cancer cell lines correlated with the activation of the downstream protein kinase B (PKB/Akt) pathway. Collectively, these data demonstrate that ILK expression increases with ovarian cancer progression and that soluble factors in PTF mediate sustained overexpression of ILK in ovarian cancer cells. Suppression of ILK expression may therefore represent a novel and an efficient mechanism for controlling ovarian tumour growth. Copyright

Role of Integrin Receptors for Fibronectin, Collagen and Laminin in the Regulation of Ovarian Carcinoma Functions in Response to a Matrix Microenvironment

Integrins play an important role in cellular matrix interactions requisite for cancer cell adhesion, growth, migration and invasion. In this study, we have investigated the expression of integrin subunits α3, α6, αv and β1 in normal ovaries, benign ovarian tumors and ovarian carcinomas of different pathological grades. The expression of these integrins in ovarian cancer cell lines was also investigated, and their role in sustaining proliferation, adhesion, migration and invasion in cohort with the activation of signaling pathways in response to extracellular matrices (ECM) was evaluated. We demonstrate a differential expression pattern of α3, α6, αv and β1 integrin subunits in ovarian carcinomas compared to normal ovaries and benign ovarian tumors. Ovarian cancer cell lines (Hey, Ovcar3 and Peo.36) demonstrated significantly high expression of α3, α6, αv and β1 integrin subunits. A significant increase in proliferation and adhesion (P<0.05) in response to collagen 1 (Coll) and laminin (LM), ligands for integrin receptor α3β1 and α6β1 was observed in ovarian cancer cell lines. On the other hand, fibronectin (FN), a receptor for αvβ1 integrin, increased proliferation in all ovarian cancer cell lines studied but only enhanced adhesion in Hey cell line (P<0.05). Neutralizing antibodies against α3, α6, αv and β1 integrin subunits inhibited ECM-induced proliferation, but increased adhesion to ECM was inhibited by β1 integrin subunit antibody. No suppression of Coll, LM and FN-induced (Hey cells only) adhesion was observed in the presence of α3 or αv subunit antibodies but LM-induced adhesion was inhibited by blocking α6 subunit functions. LM, FN and Coll enhanced chemotactic migration in Hey cells, but direct invasion across ECM was observed only in the presence of LM and Coll. Blocking antibodies against α3, α6 and β1 integrin subunits inhibited both chemotactic migration and invasion of Hey cells in response to respective ECM. Adhesion of ovarian cancer cells to FN, Coll and LM activated Ras, Erk and Akt pathways. Neutralizing αv and β1 functions did not inhibit FN-induced activation of Ras and Erk pathways but inhibited the Akt pathway. On the other hand, antibodies against α6 and β1 subunits, but not α3 subunit, inhibited LM-induced activation of Ras but did not inhibit the downstream Akt pathway. Neutralizing β1 subunit function however, inhibited LM-induced Erk activation. Coll-induced activation of Ras, Erk and Akt pathways was inhibited by α3 and β1 integrin subunit antibodies. These results indicate that α3β1, αvβ1 and α6β1 integrin mediate proliferation, adhesion, migration and invasion of ovarian cancer cells in response to ECM and targeting these integrins to modulate integrin–ECM interactions in tumor cells may be a promising tool to reduce the dissemination of ovarian carcinoma in vivo.

A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

BackgroundC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.MethodsProtein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting.ResultsmCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP.ConclusionThe data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

Pre-labour Fetal Membranes Overlying the Cervix Display Alterations in Inflammation and NF-κB Signalling Pathways

To generate new insights into the process of fetal membrane rupture, we have developed a technique whereby fetal membranes overlying the cervix (i.e. supracervical site, SCS) are tagged in women undergoing term elective Caesarean section. The specific aim is to determine the effect of supracervical apposition on the release of inflammatory mediators and NF-kappaB signalling in pre-labour fetal membranes. Fetal membranes were collected from both the SCS and from a distal site (DS). The level of NF-kappaB proteins and its transcriptional co-activator protein CBP and p300 was determined by Western blotting and/or immunohistochemistry (IHC), and cytokine and prostaglandin release was quantified by enzyme immunoassay. Tissues obtained before labour at term possess an area of the fetal membranes (i.e. supracervical site) that exhibit decreased release of IL-1beta, IL-6, IL-8, TNF-alpha and PGE(2). IHC revealed that NF-kappaB signalling proteins, CBP and p300 were significantly increased in SC fetal membranes compared to distal membranes. In summary, data from this study confirm that supracervical fetal membranes display altered structural and biochemical characteristics.

Association between αvβ6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen‐dependent matrix degradation in ovarian cancer

Altered expression of αv integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS‐1, expressed αv integrin and associated β1, β3, and β5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed αvβ6 integrin. The expression of αvβ6 in OVCA 429 and OVCA 433 was far higher than αvβ3 and αvβ5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW‐uPA), pro‐metalloproteinase 2 and 9 (pro‐MMP‐9 and pro‐MMP‐2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2‐fold more plasminogen‐dependent [3H]‐collagen type IV degradation. Plasminogen‐dependent [3H]‐collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP‐9 or αvβ6 integrin, indicating the involvement of αvβ6 integrin, uPA and MMP‐9 in the process. The αvβ6 correlated increase in HMW‐uPA and pro‐MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP‐9, and MMP‐2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen‐dependent [3H]‐collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co‐expression of αvβ6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms. J. Cell. Biochem. 84: 675–686, 2002.

Increased expression of ac-FoxO1 protein in prelabor fetal membranes overlying the cervix: possible role in human fetal membrane rupture.

Forkhead box O proteins have critical roles in a number of cellular processes, including apoptosis. Acetylation and phosphorylation of forkhead box O proteins are posttranslational modifications that attenuate their transcriptional activity. As supracervical fetal membranes are characterized by increased cell death, the aim of this study was to compare the expression of forkhead box O1, acetylated-forkhead box O1, and Ser-256 phosphorylated forkhead box O1 at supracervical and distal site fetal membrane. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 7) was used to analyze the protein expression of forkhead box O1, acetylated-forkhead box O1, and Ser-256 phosphorylated forkhead box O1. There was no difference in forkhead box O1 and Ser-256 phosphorylated forkhead box O1 protein expression between the 2 sites. However, when compared with distal site, the intensity and extent of staining of acetylated-forkhead box O1 were greater in amnion and chorion obtained from the supracervical site. In summary, supracervical fetal membranes are characterized by increased acetylated-forkhead box O1 protein expression. Although the precise role and contribution of acetylated-forkhead box O1 in the process of human fetal membrane rupture are unknown, it has been implicated in apoptosis and/or cell cycle regulation.

Preterm and infection-driven preterm labor: the role of peroxisome proliferator-activated receptors and retinoid X receptor

Approximately 8% of births are complicated by preterm delivery. To improve neonatal outcomes, a greater understanding of the mechanisms surrounding preterm parturition is required. Peroxisome proliferator-activated receptors (PPARs) have been implicated in the regulation of labor at term where they exhibit anti-inflammatory properties. Thus, we hypothesize that dysregulation of PPAR expression and activity may be associated with preterm labor and infection-associated preterm labor. The aim of this study was to compare the expression and activity of PPARs and the expression of retinoid X-receptor alpha (RXRA) in gestational tissues from term and preterm deliveries, and from infection-associated preterm deliveries. Quantitative RT-PCR, western blotting and activity ELISA were used to study expression and DNA binding profiles. Compared with term, preterm parturition was associated with an increased expression of PPAR delta (PPARD; mRNA and protein), PPAR gamma (PPARG; protein) and RXRA (protein) in the placenta and PPARD (mRNA and protein) and RXRA (mRNA) in the choriodecidua. There was, however, no change in preterm PPAR DNA binding activity compared with term. Preterm chorioamnionitis (CAM) demonstrated protein degradation in the choriodecidua and was associated with a decline in the mRNA expression of PPAR alpha (PPARA) and RXRA compared with uninfected preterm cases. PPAR DNA binding activity increased in the placenta (PPARD and PPARG) and decreased in the amnion (PPARA and PPARG) in association with preterm CAM. In conclusion, idiopathic preterm deliveries were associated with an increase in PPAR:RXR expression and preterm CAM was associated with a decrease in PPAR:RXR expression and tissue-specific alterations in transcriptional activity. The reasons for such dysregulation remain to be determined; however, the data are consistent with the hypothesis that PPARs may play a role in preterm labor and infection-complicated preterm deliveries.

A Randomized Controlled Trial of a Smoking Cessation Intervention During Pregnancy

This study was a randomized controlled trial of a smoking cessation intervention for pregnant smokers. Women who reported smoking at their first antenatal visit and satisfied the inclusion criteria were asked to participate in the trial. Analysis was restricted to 393 evaluable women in the control group (received usual antenatal care) and 339 women to the study group (received usual antenatal care plus the intervention). The primary hypotheses were that the intervention would result in a higher proportion of quitters and that the mean birth‐weight of babies born to women receiving the intervention would be greater than that of babies born to women in the control group. The outcome measures were smoking status based on self‐report combined with a urinary cotinine level of <115 ng/mL, and birth‐weight. There was no significant difference in quit rate between women receiving the intervention and women in the control group (11.9% versus 9.8% p=0.41). Babies born to women receiving the intervention were on average 84 g heavier than babies born to controls (p=0.04). The factors that contribute to the lack of a significant increase in smoking cessation in the intervention group and the possible explanation for the changes in birth‐weight are discussed.

Identification of differentially regulated proteins in a patient with Leber's Congenital Amaurosis – a proteomic study

BackgroundTo identify the pattern of protein expression in the retina from a patient with Lebers Congenital Amaurosis (LCA) secondary to a mutation in the AIPL1 gene. The retina from one eye of a patient with LCA and 7 control eyes were studied. The tissue was subjected to high resolution two-dimensional gel electrophoresis, image analysis and mass spectrometry, in an effort to identify differentially regulated proteins.ResultsIn the LCA retina seven protein spots were differentially expressed. Six proteins were significantly up-regulated of which three could be identified as: αA-crystallin, triosephophate isomerase, and an N-terminal fragment of the β-chain of ATP synthase. One protein spot that was down-regulated in the LCA retina was identified as a C-terminal fragment of β-tubulin.ConclusionRetinal tissue in LCA is characterised by an up-regulation of αA-crystallin, triosephosphate isomerase, and ATP synthase (β-chain fragment) and down-regulation of a fragment of β-tubulin. These proteins/protein fragments may play a crucial role for the retinal degeneration processes in LCA and other retinal dystrophies.

REDUCTION OF HUMAN RECOMBINANT TYPE II PHOSPHOLIPASE A2 AND PROSTAGLANDIN F2ALPHA RELEASE BY MICROTUBULE DEPOLYMERIZING AGENTS

1. The present study examines the effects of the microtubule depolarizing agent colchicine on secretory type II phospholipase A2 (PLA2) function in Chinese hamster ovary (CHO) cells that specifically overexpress human type II PLA2 and the effect of both colchicine and tubulazole on the release of type II PLA2 and prostaglandin (PG) F2α from human placental explants.