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R Bicknell

Publications and source records attributed to R Bicknell.

At least 91 records · Page 5Linked to original sources
PubMed1995

Platelet-derived endothelial cell growth factor/thymidine phosphorylase expression in normal tissues: an immunohistochemical study.

Angiogenesis is the formation of new blood vessels from the existing vascular bed. It is a complex multi-step process controlled by a number of angiogenic factors. One such factor is platelet-derived endothelial cell growth factor (PD-ECGF), recently shown to be thymidine phosphorylase (TP), which is angiogenic in several in vivo assays and tumour systems. PD-ECGF/TP catalyses the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine. Since PD-ECGF/TP has an important role in cellular metabolism and in angiogenesis and its expression has been only partially characterized, we raised a monoclonal antibody against recombinant PD-ECGF/TP and used an immunohistochemical approach to examine the expression of PD-ECGF/TP in a comprehensive range of normal human tissues. The clone P-GF44.C, which recognizes recombinant PD-ECGF/TP and cell lysates transfected with a plasmid expressing PD-ECGF/TP cDNA on Western blotting, was selected for its ability to stain routinely processed tissue. Staining was observed in both the cytoplasm and/or the nucleus. Immunoreactivity was strongly expressed by macrophages, stromal cells, glial cells, and some epithelia. Gastrointestinal epithelium, smooth muscle, adrenal, lung, and testis were negative. Although endothelial cell expression was observed, there was no correlation with sites of new vessel growth. This pattern of expression suggests tight PD-ECGF/TP regulation and that cellular thymidine pools may serve to control its different functions. Thus, in the nucleus it might modulate the pool for DNA synthesis, whilst in the cytoplasm it could control other effects through different enzyme systems. The high expression present in macrophages and skin might be important for total body thymidine homeostasis.

Blotting, Western↗
PubMed1995

Controlling the vasculature: angiogenesis, anti-angiogenesis and vascular targeting of gene therapy.

Angiogenesis is the development of new blood vessels from an existing vascular bed. Normal vascular proliferation occurs only during embryonic development, the female reproductive cycle and wound repair. By contrast, many pathological conditions (for example, cancer, atherosclerosis and diabetic retinopathy), are characterized by persistent, unregulated angiogenesis. Conversely, inadequate angiogenesis can lead to failure of ulcers to heal and myocardial infarction. Control of vascular development could permit new therapeutic approaches to these disorders. For example, several anti-angiogenic drugs are currently undergoing clinical trials for the treatment of cancer, whereas enhancement of angiogenesis by exogenous growth factors can prevent or limit the damage in chronic wounds and duodenal ulcers. Here Tai-Ping Fan, Rhys Jaggar and Roy Bicknell highlight recent achievements and discuss the prospects of receptor antagonists, enzyme inhibitors, tumour suppressor genes and vascular targeted approaches, especially that of gene therapy, in the future development of angiotherapy.

Animals↗
PubMed1995

Increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine and modulation of 5-fluoro-2'-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase.

Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line (1 microM dThd increased the IC50 value 1000-fold), but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 (1 microM dThd raised the IC50 value 3-fold). We observed a substantial 'bystander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.

Antineoplastic Agents↗
PubMed1995

The isolation, characterization and culture of human decidual endothelium.

A novel method has been developed for the isolation of microvascular endothelium from first trimester decidua. Enzymatic dispersion and Percoll gradient centrifugation, followed by positive selection using Ulex europaeus coated immunomagnetic beads, yields pure isolates of endothelium. Typical 'cobblestone' monolayers form within 3-5 days; cells express the classical endothelial markers CD31 and Von Willebrand factor and the inducible cell adhesion molecules endothelial-leukocyte adhesion molecule and vascular cell adhesion molecule. Cell growth is dependent on both high concentrations of magnesium ions and human serum. Of the endothelial growth factors examined, only vascular endothelial growth factor stimulated growth, suggesting an important role for this factor in uterine angiogenesis.

Cell Division↗
PubMed1995

Urinary basic fibroblast growth factor in patients with bladder cancer and benign prostatic hypertrophy.

OBJECTIVE: To investigate the role of urinary measurements of an angiogenic factor, basic fibroblast growth factor (bFGF), in the assessment of patients with bladder cancer. PATIENTS AND METHODS: Urine from 83 patients was assayed using a commercially available ELISA for bFGF. Thirty-eight patients had a bladder tumour and 21 had a history of bladder cancer but no disease at the time of testing. Twenty-four patients acted as controls, 16 of whom were about to undergo transurethral resection of the prostate (TURP) for benign prostatic hypertrophy (BPH) and eight who had no urological disease. RESULTS: Median urinary bFGF was higher in patients with active bladder cancer than in those with a clear cystoscopy (5.20 and 2.13 ng/g creatinine, respectively; P < 0.005). Median urinary bFGF was also elevated in patients about to undergo TURP (4.52 ng/g creatinine). Using a threshold value of 6.0 ng/g creatinine, the sensitivity of the test for detecting cancer was 42% and specificity was 88%. At a threshold value of 4.0 ng/g the sensitivity was 62% and the specificity 70%. CONCLUSION: The relationship between urinary basic FGF and the presence of bladder cancer was significant. The test is not sufficiently sensitive or specific to use as a screening test for bladder cancer but may be very useful in monitoring the effectiveness of systemic therapies in bladder cancer. Elevated levels of bFGF in the urine of patients about to undergo TURP suggests a role for bFGF in the pathogenesis of BPH.

Aged↗
PubMed1995

The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges.

A highly reproducible and technically straightforward technique for the isolation and long-term culture of normal human endometrial epithelial cells is described. The essential conditions for long-term culture are that the cells be seeded onto a gelatin matrix and that 'endothelial cell growth supplement' be present in the culture medium. Normal endometrial epithelial cells express cytokeratins and oestrogen receptors. They may be passaged five to six times without change in properties. Growth of normal endometrial epithelial cells was stimulated by 17-beta-oestradiol and epidermal growth factor. Expression of the mRNA coding for seven polypeptide angiogenic factors, by normal endometrial epithelial, stromal and three endometrial carcinoma lines, was examined. The endometrial epithelial and stromal cells express mRNA for the polypeptide angiogenic factors, basic fibroblast growth factor, vascular endothelial cell growth factor, transforming growth factor-beta 1 and pleiotrophin, as well as the cytokine midkine. Expression of the mRNA for both vascular endothelial growth factor and midkine by normal endometrial epithelial cells showed a 2-fold increase on treatment with a physiological dose of 17-beta-oestradiol (10(-10) M) while, in contrast, the mRNA of transforming growth factor-beta 1 decreased 4-fold on treatment with 17-beta-oestradiol (10(-10) M) and was abolished by exposure to progesterone (5 x 10(-9) M). Expression of the mRNAs for angiogenic polypeptides by the endometrial carcinoma lines was more restricted.

Adenocarcinoma↗
PubMed1994

Association of ovarian malignancy with expression of platelet-derived endothelial cell growth factor.

BACKGROUND: At the present time, the pathogenesis of ovarian cancer remains poorly understood, with invasive diagnosis and ineffective treatment for women with the disease. Despite scientific and medical advances in oncology, the overall 5-year survival rate of 30% for ovarian cancer patients has not changed in 20 years. An understanding of the angiogenic process as it occurs in ovarian cancer would not only increase our knowledge of the pathogenesis of this cancer but also might offer novel opportunities for therapeutic intervention. PURPOSE: Our aim was to study the expression of messenger RNA (mRNA) coding for four putative angiogenic factors in normal ovaries and benign and malignant ovarian tumors: platelet-derived endothelial cell growth factor (thymidine phosphorylase), vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1. METHODS: Four normal ovaries and 25 tumors (seven benign, one of borderline malignancy, and 17 malignant) were collected from 29 patients during elective oophorectomy. The site of sampling (areas of high-velocity blood flow) was directed by transvaginal color Doppler imaging performed within 24 hours of the surgery. Increased blood flow within the tissues was demonstrated by the presence of color (i.e., the velocity was > 7 cm/s) and, together with a pulsatile index of less than 1.0, constituted a positive scanning result. In scan-positive tissues, the area of maximum blood flow was chosen. In scan-negative tissues, a solid area was chosen in complex lesions, or the cyst wall was chosen in simple lesions. Ovarian RNA was subsequently extracted from areas of high-velocity flow (i.e., tissues with a positive scanning result) or from solid areas or septa in tissues with a negative scanning result. A ribonuclease protection assay was used to assess the expression of mRNA coding for the four angiogenic factors. RESULTS: Two normal ovaries (containing a corpus luteum) and one benign and 17 malignant tumors (plus the borderline) gave a positive scanning result. There was a significant difference between the expression of mRNA for platelet-derived endothelial cell growth factor between scan-positive and scan-negative tissues (P < .001) and between benign and malignant tumors (P < .001). CONCLUSIONS: Areas of high blood velocity in ovarian tumors are associated with increased expression of platelet-derived endothelial cell growth factor. IMPLICATIONS: Drugs that affect the angiogenic activity of platelet-derived endothelial cell growth factor offer a potential route for therapeutic intervention.

Autoradiography↗
PubMed1994

Gene therapy through signal transduction pathways and angiogenic growth factors as therapeutic targets in breast cancer.

Angiogenesis is a major new prognostic factor in breast cancer. Small vessels quantitatively assessed by staining with anti-CD31 antibodies correlate with lymph node involvement and are a better independent predictor of survival. There are many vascular growth factors, but predominant in primary tumors assessed by nuclease protection assays are vascular endothelial growth factor and platelet-derived endothelial cell growth factor. Acidic and basic fibroblast growth factor are also detectable. A common feature of these angiogenic factors is heparin binding, so novel analogues of suramin that can compete for heparin binding have been developed. These are more potent in vitro against endothelial cells and are less toxic in vivo, thereby giving a much better therapeutic ratio. Protein kinase C is also important in endothelial growth, as it is in carcinoma growth. Thus, a novel agent inhibiting this pathway, and inducing transforming growth factor-beta production has been assessed in a Phase I trial; this agent is bryostatin. It does not cause marrow suppression and has stimulatory effects of tumor necrosis factor-alpha and interleukin (IL)-6 production. High expression of epidermal growth factor (EGF) receptors and erbB-2 has been related to poor prognosis. EGF receptors are mainly regulated by transcription, as are some cases of high erbB-2 expression. Thus, a novel approach to gene therapy is being developed using direct tumor injection of cDNA, with a tumor specific promoter ligated to the IL-2 gene. This avoids many problems associated with targeting. Because IL-2 stimulation of cytotoxic T-cells will depend on appropriate antigen presentation, human lymphocyte antigen Class I expression was studied, as was the peptide transporter system RING4 (TAP1). Losses were found in 50% of cases, and in some cases only in lymph nodes but not primary cancers, thereby providing evidence for a role in suppressing metastasis. Thus, many new approaches to therapy are possible as a result of understanding growth factors and intracellular signaling pathways.

Angiogenesis Inducing Agents↗
PubMed1994

Differential expression of human Wnt genes 2, 3, 4, and 7B in human breast cell lines and normal and disease states of human breast tissue.

Wnt gene expression was investigated by ribonuclease protection analysis in human breast cancer, nontumorous breast tissue, and a variety of human breast cell lines. We report the expression of Wnt3, Wnt4, and Wnt7b in human breast cell lines and Wnt2, Wnt3, Wnt4, and Wnt7b in human breast tissues. Wnt3a and Wnt7a were absent in the cell lines and tissues tested. The level of expression of Wnt2 and Wnt4 was 10- to 20-fold higher in fibroadenomas than it was in normal or malignant breast tissue, and in 10% of tumors Wnt7b expression was 30-fold higher than in normal or benign breast tissues. In contrast to the mouse, in which Wnt1 and Wnt3 are involved in tumorigenesis, our results suggest that Wnt2, Wnt4, and Wnt7b may be associated with abnormal proliferation in human breast tissue.

Adult↗
PubMed1994

Activin-A binds to a heterotrimeric receptor complex on the vascular endothelial cell surface. Evidence for a type 3 activin receptor.

The effect of transfection of the type 2 activin receptor, ACTR2, on binding of 125I-activin-A to the surface of bovine aortic endothelial cells (BAEC) was investigated. BAEC transfected either with full-length ACTR2 or with a truncated form of ACTR2 lacking the intracellular kinase domain (ACTR2T) displayed two classes of 125I-activin-A binding sites, one of high affinity (Kd = 250-254 pM) and one of low affinity (Kd = 6.5-16 nM). Affinity labeling of ACTR2-transfected BAEC with 125I-activin-A revealed labeled species of 55, 95, 100, and 160 kDa, all four of which were immunoprecipitated by an anti-ACTR2 monoclonal antibody. Only the 95- and 100-kDa species, however, were immunoprecipitated following denaturation of the affinity-labeled cell lysate with SDS. BAEC transfected with an epitope-tagged form of ACTR2T (ACTR2TMyc) displayed intense 55- and 70-kDa affinity-labeled forms of the truncated receptor, together with a 160-kDa species. As with the full-length receptor, the 160-kDa species associated non-covalently with ACTR2TMyc. These data indicate that, in vascular endothelial cells, ACTR2 forms a high affinity heterotrimeric receptor complex with activin-binding proteins characteristic of type 1 and type 3 activin receptors, and that formation of the complex does not require the kinase domain of ACTR2.

Activin Receptors↗
PubMed1994

A structure-activity analysis of antagonism of the growth factor and angiogenic activity of basic fibroblast growth factor by suramin and related polyanions.

The ability of a series of polysulphonated naphthylureas structurally related to suramin to inhibit basic fibroblast growth factor (bFGF) or serum-stimulated growth of endothelial cells [either large vessel, human umbilical vein endothelial cells (HUVEC) or microvascular, bovine adrenal capillary endothelial (BACE) cells] and angiogenesis in vivo has been examined. The polyanions encompassed two main structural variations, namely the number of aromatic amide groups intervening between two terminal naphthyl rings and/or variation in the substitution pattern of the naphthyl rings. The polyanions were either inactive (group I) or inhibited (group II) bFGF-stimulated uptake of [3H]methylthymidine by BACE cells. Group I compounds shared a common structural feature in that they were simple binaphthyl-substituted ureas. In contrast, group II compounds all had an extended multiple ring structure with at least two aromatic groups intervening between the two terminal naphthyl rings. Compounds with either two or four intervening groups were equipotent in blocking bFGF in vitro. However, compounds with two bridging aromatic groups were 5- to 10-fold less toxic than suramin in mice, suggesting a potential for an improved therapeutic ratio. The ability of the polyanions to block bFGF-driven endothelial cell proliferation in vitro correlated with antiangiogenic activity in vivo as shown by use of the rat sponge angiogenesis model. These observations could substantially widen the anti-tumour therapeutic opportunities for this class of compound.

Animals↗
PubMed1994

Monoclonal antibodies that recognize the type-2 activin receptor, ACTR2.

Monoclonal antibodies (MAbs) were raised in mice against a bacterial fusion protein composed of the intracellular serine/threonine kinase domain of the type-2 activin receptor, ACTR2, fused to glutathione S-transferase. Three MAbs with high affinity toward the ACTR2 kinase domain were isolated, one of which recognized specifically ACTR2 expressed transiently in vascular endothelial cells. These reagents should be of use in the elucidation of mechanisms of transmembrane signaling by this member of the emerging receptor serine threonine kinase family.

Activin Receptors↗
PubMed1994

Vascular targeting and the inhibition of angiogenesis.

Recent advances in our understanding of the process by which tumours elicit an angiogenic response, and of differences between normal and tumour vasculature, are revealing new strategies for anticancer therapy. The current status of anti-angiogenic therapy, together with approaches to targeting tumour vasculature, is reviewed. In particular, the advantages of using tumour vasculature as a target for anticancer gene therapy are outlined.

Angiogenesis Inducing Agents↗
PubMed1994

Antibody for detecting p53 protein by immunohistochemistry in normal tissues.

AIMS: To establish whether PAb248 recognises human p53 as well as murine p53 and if so, to determine its distribution in normal tissues. METHODS: The ability of PAb248 to recognise human p53 was established by analysis of the human osteosarcoma derived Saos-2 cell line, which lacks the p53 gene, before and after transfection with p53 cDNA, using western blotting and immunoprecipitation. Immunostaining on normal tissues and cell lines was carried out using an immunoperoxidase technique. The two anti-p53 antibodies PAb 240 and DO-7 were used as controls. RESULTS: The anti-p53 PAb248 monoclonal antibody stained the Saos-2 cell line after, but not before, transfection with p53 cDNA. Both western blots and immunoprecipitations performed with this antibody revealed a 53,000 molecular weight band. With immunostaining, this antibody detects p53 protein in most lymphoid and human epithelial cells in a cytoplasmic-perinuclear localisation that has not been described before. In the same tissues nuclear staining could be seen in a few scattered cells using the PAb240 antibody. The topographical distribution of wild type p53 was not related to proliferating areas but, rather, to short-lived populations of cells. CONCLUSIONS: Immunostaining of wild type p53 is demonstrable not only in its nuclear form using antibody PAb240 but also in it common cytoplasmic-perinuclear localisation in normal tissues using the PAb248 monoclonal antibody. This opens up new possibilities for its study in both physiological and pathological conditions.

Antibodies, Monoclonal↗
PubMed1993

Inhibition of vascular endothelial cell growth by activin-A.

The demonstration of type 2 activin receptor expression in human umbilical vein endothelial cells prompted an investigation of the effects of the activin/inhibin family of hormones on vascular endothelial cell growth. Recombinant activin-A inhibited [3H]methylthymidine uptake and growth of a panel of endothelial cell types; recombinant inhibin-A was without effect. Affinity cross-linking studies demonstrated the presence of type 1 and type 2 activin receptors on the surface of bovine aortic endothelial cells, while detailed analysis of type 2 activin receptor expression revealed both type 2 and type 2B activin receptor mRNA in all endothelial cell types analyzed. In addition, capillary endothelial cells were found to express activin-beta A subunit mRNA and protein, the levels of which were increased in response to transforming growth factor (TGF)-beta. Furthermore, activin-A and TGF-beta caused additive inhibition of capillary endothelial cell [3H]methylthymidine uptake. These findings implicate the activins in the regulation of endothelial cell function, and suggest that TGF-beta and activin may interact to inhibit capillary endothelial cell growth.

Activin Receptors↗
PubMed1993

Relationship of endothelial cell proliferation to tumor vascularity in human breast cancer.

Current studies of tumor angiogenesis rely on the concept that endothelium proliferates 30-40 times faster in tumors than in normal tissues. This evidence is based on histological autoradiographic data largely from animal studies. To assess endothelial cell proliferation in human cancer we used the more sensitive and specific technique of immunohistochemistry. We measured the frequency and distribution of endothelial cell proliferation and examined their relationship to tumor cell proliferation. For the first time, we also correlated endothelial and tumor cell proliferation with tumor vascularity. Twenty breast carcinomas from patients exposed to bromodeoxyuridine 3-8 h prior to surgery were double immunostained using antibodies to CD31 (as a marker of endothelium) and bromodeoxyuridine (as a marker of proliferation). The labeling index (LI) for both tumor and endothelial cells was determined and tumor vascularity was assessed by counting the number of CD31 positive vessels. Endothelial cell proliferation was predominantly at the tumor periphery while tumor cell proliferation occurred throughout the lesion. The mean LIs for endothelium and tumor were 2.2% (range, 0.8-5.3) and 7.3% (range, 1.3-17.1), respectively. There was no correlation between tumor and endothelial cell LI (P = 0.414) or between the tumor LI or endothelial cell LI and tumor vascularity (P = 0.08 and P = 0.39, respectively). These findings suggest that previous studies in animal tumors have significantly overestimated endothelial cell proliferation and that its importance in tumor angiogenesis may be related more to continual remodeling and migration of vessels than to proliferation alone.

Adult↗