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Biomedical subjects

R Bicknell

Publications and source records attributed to R Bicknell.

At least 109 records · Page 6Linked to original sources
PubMed1993

cDNA transfection followed by the isolation of a MCF-7 breast cell line resistant to tamoxifen in vitro and in vivo.

A tamoxifen resistant cell line (clone 9) has been isolated from the tamoxifen sensitive, hormone responsive MCF-7 breast carcinoma cell line after transfection with mixed cDNA libraries, followed by tamoxifen selection in the presence of oestrogens. Transfection was confirmed by Southern analysis with vector probes. Clone 9 in several-fold more resistant to tamoxifen and other anti-oestrogens than wild type cells when cultured either as a monolayer or as colonies in soft agar but retains oestrogen receptors. Clone 9 was less responsive to 17-beta-oestradiol than were wild type MCF-7. In addition to showing in vitro tamoxifen resistance, clone 9 was also tamoxifen resistant in vivo when xenografted into the nude mouse. Culture medium conditioned by clone 9 cells stimulated quiescent cells of the same clone as well as wild type cells, whereas medium conditioned by wild type MCF-7 was inhibitory to both, suggesting that clone 9 may be secreting an autocrine growth factor. Clone 9 provides a novel model for further investigation of the mechanism of anti-oestrogen resistance that occurs without loss of oestrogen receptors. Preliminary results suggest that an autocrine growth stimulatory mechanism may be one pathway of such resistance.

Animals↗
PubMed1993

Immunohistochemical properties of the endothelial cells in the human uterus during the menstrual cycle.

Expression of vascular cell adhesion molecule (VCAM) and endothelial leukocyte adhesion molecule (ELAM)-1 on human uterine endothelium was examined by immunohistochemistry. Endomyometrial blocks were obtained from 26 hysterectomy specimens (n = 6, menstrual; n = 6, follicular; n = 14, luteal). VCAM and ELAM-1 expression was compared to that of the classic endothelial markers Factor VIIIRA, CD31, CD34 and CD36, which were found to be present in samples taken from all stages of the cycle. Factor VIIIRA, CD31 and CD34 were expressed on most endothelium whereas CD36 was expressed primarily on that of small vessels. CD36 was also found to be strongly expressed on endometrial epithelium and no changes were apparent during the menstrual cycle. ELAM-1 expression was not detected in any of the tissues examined. VCAM expression varied during the cycle, appearing on endometrial endothelium in the mid- and late luteal phases, stages at which large granular lymphocytes migrate into endometrium. VCAM or a VCAM-like molecule may be involved in migration of large granular lymphocytes into endometrium.

Adult↗
PubMed1993

Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors.

BACKGROUND: Endothelial cells are important in initiating adhesive processes between circulating cells and extracellular structures and changes in their distribution are believed to be important in many pathologic conditions. Since little is known about the detailed distribution of adhesion molecules in human endothelium in different sites and circumstances, the present study has undertaken a detailed analysis of 5 of the putative most important adhesion molecules on a wide range of normal tissue endothelium. We have compared this reactivity with that seen in a comprehensive range of vascular tumors both benign and malignant. EXPERIMENTAL DESIGN: Fresh samples of a wide range of normal tissues and vascular tumors were stained by antibodies against the following adhesion molecules; intercellular adhesion molecule-1 (CD54), E-selectin (endothelial cell adhesion molecule-1), vascular cell adhesion molecule-MUC-1, P-selectin (platelet activation-dependent granule to external membrane protein, CD62) and MUC-18 using either the APAAP immuno-alkaline phosphatase or an immunoperoxidase method. RESULTS: Labeling of the endothelial cells in different normal tissues with intercellular adhesion molecule-1, P-selectin, and MUC-18 was heterogeneous both in terms of vessel size and strength of staining. Vascular cell adhesion molecule-1 and E-selectin were largely absent. The vascular tumors were likewise variable in their staining patterns which frequently differed from the immunophenotype of the reactive vessels surrounding the tumor. CONCLUSIONS: This study demonstrates that the expression of adhesion molecules of the immunoglobulin and selectin family on normal tissue endothelium, and vascular tumors is much less predictable than that obtained with other vascular markers such as F8 RA, CD31, CD34, and CD36. Adhesion molecules show considerable heterogeneity of expression on vascular endothelium which presumably reflects their varied functions on different types of vessel. In general their expression is markedly reduced on vascular tumors.

Antibodies, Monoclonal↗
PubMed1992

Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity.

Platelet-derived endothelial cell growth factor (PD-ECGF) has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The fusion protein was purified by one-step affinity chromatography on glutathione-agarose beads, and recombinant PD-ECGF was proteolytically cleaved with thrombin from its GST leader peptide to yield pure protein. Recombinant PD-ECGF stimulated [3H]methylthymidine uptake by endothelial cells in vitro; however, we were unable to detect stimulation of cell proliferation under a wide variety of conditions. We confirm that in accord with the recent report that PD-ECGF and human thymidine phosphorylase are products of the same gene [Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., & Akiyama, S. I. (1992) Nature 356, 668] recombinant PD-ECGF has thymidine phosphorylase activity comparable to that of E. coli thymidine phosphorylase. Further, E. coli thymidine phosphorylase was able to mimic the activity of recombinant PD-ECGF in the [3H]methylthymidine uptake assay, and it appears that recombinant PD-ECGF's effect on the uptake of thymidine by endothelial cells may be due to modulation of cellular thymidine pools. The mechanism by which PD-ECGF stimulates angiogenesis remains to be elucidated.

Amino Acid Sequence↗
PubMed1992

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.

Animals↗
PubMed1992

Inhibition of colon and breast carcinoma cell growth by interleukin-4.

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.

Breast Neoplasms↗
PubMed1992

Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours.

AIMS: To determine the distribution of factor VIII related antigen, CD31, CD34 and CD36 in normal and malignant human vascular tissues using a panel of well characterised monoclonal antibodies. METHODS: Frozen and fixed material from a wide range of normal tissues and routinely processed material from 43 benign and malignant vascular tumours were examined. Single immunocytochemical labelling was performed using the APAAP technique. Double staining involved the sequential use of APAAP with the peroxidase method. RESULTS: Human vascular endothelium was antigenically heterogeneous. One of the most restricted markers was factor VIII related antigen, despite its having been widely used in diagnostic pathology as a marker of vascular endothelium and of the tumours which arise from it. Three antibodies against factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10) were identified as immunostaining routinely processed, formalin fixed, paraffin wax sections. Each antibody gave different staining when tested on a range of vascular tumours, both benign and malignant. CONCLUSIONS: A small panel of three reagents (factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10)) should be used by diagnostic pathologists who want to show the presence of cells of endothelial origin in routine material.

Antigens, CD↗
PubMed1991

Interleukin-4 is a potent mitogen for capillary endothelium.

Interleukin-4 (IL-4) is a mitogen for both microvascular (human adrenal capillary, HACE) and large vessel (human umbilical vein, HUVEC) endothelial cells. Comparison of growth promotion by IL-4 to that by the potent endothelial mitogen fibroblast growth factor (FGF) showed the activity of IL-4 on HACE cells to be strong (50% of that with FGF) but on HUVEC's weak (12% of that with FGF). Growth stimulation was characterised by both 3H-thymidine incorporation and by cell number, and was maximal at 1 nM IL-4. The presence of IL-4 receptors on HACE cells and HUVEC's was confirmed by specific binding of radioiodinated IL-4. Scatchard analysis confirmed a single high affinity binding receptor on both HACE cells (Kd = 80 pM, 358 receptors/cell) and HUVEC's (Kd = 88 pM, 2,580 receptors/cell). Potent activity on capillary as opposed to large vessel endothelium places IL-4 in a unique position amongst endothelial mitogens.

Capillaries↗
PubMed1991

Isolation and properties in culture of human adrenal capillary endothelial cells.

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.

Adrenal Glands↗
PubMed1991

Heterogeneity of the endothelial cell and its role in organ preference of tumour metastasis.

The vascular endothelium is a remarkably heterogeneous organ. In addition to well-characterized anatomical diversity in situ, specific differences are increasingly being recognized between surface antigens on endothelial cells from different tissues, including absence of the classic endothelial marker factor VIII-related antigen (von Willebrand factor) from many endothelial cells. Microvascular heterogeneity extends to properties of endothelial cells thought to be involved in tumour angiogenesis and metastasis, such as growth factor responsiveness and expression of cell adhesion molecules. These findings are not only of relevance to the unambiguous identification and characterization of cultured endothelial cells, but, as Roy Bicknell and colleagues discuss, may explain the phenomenon of preferential organ tumour metastasis and provide novel opportunities for antitumour therapy.

Cell Adhesion↗
PubMed1990

Long-term follow-up of the Indiana conservative resurfacing hip arthroplasty.

Sixty-four Indiana conservative (Depuy, Warsaw, IN) hip arthroplasties performed in 61 patients were reviewed for loosening, mechanisms of loosening, and revision rate. Mean follow-up time was 6.8 years. There were revisions in 26 hips (40.6%) with 4 hips awaiting revision, yielding a total of 30 hips (47%) defined as failures. Acetabular failure occurred in 20 hips, femoral failure in 18, and combined failure in 13. Failure of this prosthesis persists over time. There seems to be little or no place for this design in contemporary hip joint arthroplasty.

Adult↗
PubMed1989

Angiogenin depresses aortic smooth muscle cell cAMP by a pertussis toxin sensitive mechanism.

Angiogenin transiently depresses the cAMP level of rat aortic smooth muscle cells. The dose response is similar to angiogenin activation of the inositol-specific phospholipase C in this cell line [Moore, F. & Riordan, J.F. (1989) Biochemistry. Submitted]. The time course showed a maximal depression (28%) in cAMP at 2 min, followed by a return to that of unstimulated cells by 3.5 min. Angiogenin also inhibited isoproterenol stimulated cAMP formation, but the percentage depression in cAMP (9%) was less than that in cells treated with angiogenin alone (28%). In contrast angiogenin enhanced forskolin stimulation of adenylate cyclase, an effect previously linked with agonist activation of protein kinase C. The effect of angiogenin on cellular cAMP was abolished by pre-incubation with pertussis toxin. Angiogenin had no effect on cellular cGMP. These results are consistent with activation of adenylate cyclase Gi following exposure of the cells to angiogenin and provide further evidence for interaction between cellular signalling pathways.

Adenylate Cyclase Toxin↗
PubMed1989

Angiogenin stimulates endothelial cell prostacyclin secretion by activation of phospholipase A2.

Angiogenin stimulates capillary and umbilical vein endothelial cell prostacyclin secretion but not that of prostaglandins of the E series. The response was quantitated by radioimmunoassay and by [3H]arachidonate labeling followed by analysis of the secreted prostaglandins. The stimulated secretion lasts for several minutes and is optimal at 2-4 min. The dose-response (peak at 1-10 ng/ml) is similar to that previously observed for activation of endothelial cell phospholipase C. Stimulated secretion was blocked by pretreatment with the inhibitors of prostacyclin synthesis, indomethacin and tranylcypromine, and also the specific inhibitor of phospholipase A2, quinacrine, as well as pertussis toxin and the diglyceryl and monoglyceryl lipase inhibitor RHC 80267. Stimulated secretion was also abolished in cells that were either pretreated for 48 hr with phorbol ester to down-regulate protein kinase C or incubated with the protein kinase inhibitor H7. Hydrolysis of phosphatidylinositol by phospholipase A2 appears to be the source of angiogenin-mobilized arachidonate; angiogenin-induced hydrolysis of phosphatidylcholine was not detected. Activation of phospholipase A2 occurs in the absence of an angiogenin-induced calcium flux. The results are discussed in terms of mechanisms of agonist-induced intracellular arachidonate mobilization and relevance to angiogenesis.

Angiogenesis Inducing Agents↗
PubMed1989

Modulation of mitogenic stimuli by angiogenin correlates with in vitro phosphatidylinositol bisphosphate synthesis.

125I-labeled angiogenin binds rapidly to the plasma membrane of several cell lines at 37 degrees C (t1/2 less than 1 min) but in comparatively small amounts. Competition with unlabeled angiogenin varies markedly with different cell lines, being most effective in vascular smooth muscle and fibroblast cells. Angiogenin modulates mitogenic stimuli in bovine adrenal capillary endothelial (BACE), rat aortic smooth muscle (RASM), and fibroblast (3T3) cells. Thus, it enhances the mitogenic effect of certain conditioned media on RASM and 3T3 cells, but it inhibits the mitogenic effect on BACE cells. In RASM and 3T3 cells, mitogenesis is increased at low (less than 5 ng/ml) and high (greater than 100 ng/ml) but not at intermediate concentrations of angiogenin. Plasma membranes from RASM and 3T3 cells that have been treated with angiogenin show an enhanced in vitro synthesis of phosphatidylinositol bisphosphate (PtdInsP2) with an angiogenin concentration dependence similar to that of enhanced mitogenesis. PtdInsP2 synthesis arises by activation of a fatty acid (arachidonyl) coenzyme A synthetase and either a plasma membrane fatty acid acyltransferase or phosphatidylinositol kinase(s), or both. Increased PtdInsP2 or the derived second messengers (e.g., diacylglycerol) may mediate modulation of the mitogenic stimulus. The differential mitogenic interaction of angiogenin with several cell types, either stimulation or inhibition, probably reflects the multistep nature of angiogenesis.

Animals↗