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R Bicknell

Publications and source records attributed to R Bicknell.

At least 55 records · Page 3Linked to original sources
PubMed1999

Thymidine phosphorylase expression in endometrial carcinomas.

Thymidine phosphorylase (TP) is a potent angiogenic molecule shown to induce endothelial cell migration and proliferation. We investigated the expression of TP in a series of 156 endometrial carcinomas, using immunohistochemical methods. Histopathological parameters of known prognostic significance and the molecular factors of p53, bcl-2 and angiogenesis were also assessed. Thymidine phosphorylase was expressed in cancer cells, stromal fibroblasts and myometrial cells. The pattern of TP staining was nuclear or mixed nuclear/cytoplasmic, and only exceptionally was purely cytoplasmic. An exclusively cytoplasmic staining was documented for the tumour-associated foamy macrophages. Cancer cell reactivity was rather limited; only 3.2% of endometrial carcinomas expressed TP in more than 50% of the neoplastic cell population and only 12% expressed the enzyme in more than 10% of the cancer cells. By contrast, TP reactivity was frequent in the fibroblasts of the tumour supporting stroma and the fibroblasts/myometrial cells at the invading tumour front, where approximately 1/3 of the cases expressed TP in more than 50% of the respective constituent cells. A high TP reactivity in the stromal fibroblasts was significantly associated with the presence of foamy macrophages and an intense lymphocytic response. A high TP reactivity at the invading tumour front was significantly associated with an intense lymphocytic response and the adverse prognostic parameters of high tumour grade, deep myometrial invasion, advanced stage of disease and the non-endometrioid carcinomas. There was no significant association of cancer cell TP reactivity with any of the parameters studied, including nuclear p53 accumulation, cytoplasmic/perinuclear bcl-2 expression, microvessel density (MVD) and prognosis. Similarly, no relationship was established between fibroblastic or fibroblastic/myometrial TP reactivity and MVD. It is concluded that TP is not a major angiogenic factor in endometrial carcinomas. However, a prominent TP activity at the invading tumour front, which is probably induced by cytokines of histiocytic and lymphocytic origin, may promote tumour invasion and progression.

Endometrial Neoplasms↗
PubMed1999

Transcriptional profiling of human microvascular endothelial cells in the proliferative and quiescent state using cDNA arrays.

Endothelial cells are known to be a rich source of transcriptional gene expression. Recent technological advances now permit the detailed profiling of mRNA expression using arrays of known cDNAs on blots. We have used such arrays to examine expression of mRNA by primary isolates of human foreskin microvascular endothelial cells in the proliferative and quiescent state. Cells were stimulated by a combination of known growth factors for these cells including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and 'endothelial cell growth supplement (ECGS)' either alone or in combination. Analysis showed the expression of many mRNAs but of the 588 examined, only one, namely monocyte chemotactic protein-1 (MCP-1), showed a decrease on treatment with EGF. A combination of image grabbing followed by subtractive densitometry enabled identification of the mRNAs upregulated in proliferating endothelium. In consideration of the possibility of selective vascular targeting, of particular interest was the increase in expression of the mRNA for the cell surface proteins vascular endothelial (VE-) and neural (N-) cadherin and alpha5, alphav, beta1 and beta3 integrins. The alpha5 integrin offers a previously unrecognized opportunity for vascular targeting.

Journal Article↗
PubMed1999

Regulation of endometrial angiogenesis.

Bleeding problems are the most common reason for discontinuation of hormone replacement therapy. Human endometrium undergoes the unique process of benign angiogenesis under the control of ovarian steroids during reproductive life and it is presumed that similar processes occur when women take hormone replacement therapy. The key players in endometrial growth and angiogenesis are vascular endothelial growth factor, thymidine phosphorylase and adrenomedullin. Regulation of these angiogenic factors is described.

Adrenomedullin↗
PubMed1999

Urinary vascular endothelial growth factor and its correlation with bladder cancer recurrence rates.

PURPOSE: Vascular endothelial growth factor (VEGF) is a principal growth factor mediating tumor angiogenesis. The high expression of VEGF within bladder tumors is associated with a poor prognosis. We quantified urinary VEGF and determined its potential as a prognostic marker in bladder cancer. MATERIALS AND METHODS: VEGF was measured by enzyme-linked immunosorbent assay in the urine of 261 patients, including 153 undergoing cystoscopic surveillance for bladder cancer and 108 with another advanced malignancy or a benign urological condition. The source of urinary VEGF was studied through its quantification in bladder tumors and normal bladders. RESULTS: Urinary VEGF was higher in patients undergoing cystoscopic surveillance for bladder cancer than in those with an advanced nonbladder malignancy (p <0.0001) or a benign urological condition (p = 0.004). The highest levels were noted in patients with bladder cancer compared to those with clear cystoscopy (p <0.0001). In 26 cases the correlation between VEGF protein levels in bladder cancer and urine (r = 0.67, p = 0.003) suggested that the tumor is a source of urinary VEGF. Increased VEGF protein in normal urothelium in 22 patients with bladder cancer compared to that in 7 cadaveric organ donors (p = 0.002) indicates that urinary VEGF may also be derived from nonmalignant urothelium. In 61 cases we established a correlation between urinary VEGF and stage T1 or less superficial bladder tumor recurrence rates (r = 0.45, p <0.0001). CONCLUSIONS: Our study demonstrates that VEGF is high in the urine of patients with bladder cancer and it correlates with tumor recurrence rates. VEGF is implicated in the pathogenesis of bladder cancer recurrence. Its quantification may provide a valuable noninvasive marker for the early detection of bladder tumor recurrence as well as a therapy target.

Adult↗
PubMed1998

Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis.

Angiogenesis is the term used to describe the formation of new blood vessels from the existing vasculature. In order to attract new vessels, a tissue must release an endothelial-cell chemoattractant. 2-Deoxy-D-ribose is produced in vivo by the catalytic action of thymidine phosphorylase (TP) on thymidine and has recently been identified as an endothelial-cell chemoattractant and angiogenesis-inducing factor. TP, previously known only for its role in nucleotide salvage, is now known to be angiogenic. TP expression is elevated in many solid tumours and in chronically inflamed tissues, both known areas of active angiogenesis. There is evidence that TP is also involved in physiological angiogenesis such as endometrial angiogenesis during the menstrual cycle. The majority of known endothelial-cell chemoattractants are polypeptides that bind to endothelial-cell-surface receptors. In contrast, 2-deoxy-D-ribose appears to lack a cell-surface receptor. Glucose is another sugar that acts as an endothelial-cell chemoattractant. The migratory activity of glucose is blocked by ouabain. It is possible that 2-deoxy-D-ribose and glucose stimulate endothelial-cell migration via a similar mechanistic pathway.

Animals↗
PubMed1998

Role of the hypoxia sensing system, acidity and reproductive hormones in the variability of vascular endothelial growth factor induction in human breast carcinoma cell lines.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor implicated in many pathological processes. We investigated the regulation of 4 alternatively spliced isoforms (121, 165, 189 and 206 amino acids) by hypoxia, hypoglycemia, acidity, female reproductive hormones and vitamin D in breast carcinoma cell lines representing different tumor phenotypes. There was a 17-fold difference in total VEGF mRNA expression across the cell lines. The isoform expression, 121 > 165 > 189, was unchanged by different culture conditions. Hypoxia was the most potent stimulus, and the cell lines demonstrated a 1.4- to 6.9-fold range of VEGF induction, maintained when other hypoxically regulated genes (phosphoglycerate kinase 1 and glucose transporter 1) and a HIF-1-dependent reporter gene were examined. The relative inducibility of the genes was maintained in each cell line, but basal expression was independent of -fold induction. VEGF expression decreased under acidic conditions in 2 cell lines, but the hypoxia stimulus remained effective under acidic conditions. Hypoglycemia, female reproductive hormones and vitamin D exerted no effect on expression, nor did inhibitors of mutant ras. Our results show that VEGF expression varies widely between cell lines and that capacity to respond to hypoxia is also cell specific, relating mostly to the hypoxic sensing of the cell and the signal transduction mechanism. Such characteristics, if maintained in vivo, have implications for the angiogenic potential of different tumor cells under normal and hypoxic conditions.

Breast Neoplasms↗
PubMed1998

PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non estrogenic mechanism in the human endometrium.

Tamoxifen is currently the most widely used drug for the treatment of breast cancer, but there now exists considerable evidence that tamoxifen can also induce endometrial hyperplasia in pre menopausal women. We have used PCR differential display on primary human endometrial isolates in an attempt to identify genes induced by tamoxifen but not estrogen. Eight such differentially expressed bands were cloned and sequenced, one of which was found to be the peptide adrenomedullin. We have shown that adrenomedullin is a novel growth factor for endothelial cells and is angiogenic in vivo in the chick chorioallantoic membrane assay. Immunohistochemical analysis of endometrial sections have shown that while macrophages in the endometrium express adrenomedullin at a low level, endometrial macrophages of women receiving tamoxifen strongly express adrenomedullin (P=0.008). We postulate that endometrial induction of the angiogenic factor adrenomedullin by tamoxifen is part of the mechanism by which tamoxifen results in endometrial hyperplasia.

Adrenomedullin↗
PubMed1998

Validation of anti-vascular endothelial growth factor (anti-VEGF) antibodies for immunohistochemical localization of VEGF in tissue sections: expression of VEGF in the human endometrium.

Transient transfection of COS-1 cells followed by fixation, embedding in paraffin, and immunohistochemistry has identified anti-vascular endothelial growth factor (anti-VEGF) mouse monoclonal antibodies that efficiently immunostain VEGF in paraffin-embedded tissue sections. Immunohistochemical localization of VEGF in 34 specimens of normal human endometrium that had been collected at different stages of the menstrual cycle was then performed. VEGF was present at all stages of the cycle, but both the pattern and the intensity of staining varied. Thus, VEGF expression occurred predominantly in the endometrial epithelium and while weak in the proliferative phase, was strong in the secretory phase. VEGF expression in the stroma was weaker than in the proliferative phase glands and did not change throughout the cycle. These findings are in agreement with reports of VEGF mRNA expression in the endometrium, but disagree with previous immunohistochemical studies that employed an immunohistochemically unvalidated antiserum. This study has shown that the commercially available anti-VEGF monoclonal antibody M293 is excellent for the immunohistochemical localization of VEGF in paraffin sections.

Adenocarcinoma↗
PubMed1998

Expression of VEGF in routinely fixed material using a new monoclonal antibody VG1.

The aim of this study was to raise and characterize a monoclonal antibody reactive with VEGF (vascular endothelial growth factor) in routinely fixed specimens and to use it to investigate its tissue distribution in normal and pathological specimens. Recombinant VEGF 189 protein was used to raise a monoclonal antibody. The specificity of the antibody was confirmed using COS cells transfected with cDNA coding for VEGF 121, 165 and 189 protein and by western blotting studies. The resulting antibody VG1 was shown to react with the 121, 165 and 189 isoforms of VEGF protein in routinely processed material. In normal tissues, there was strong staining of endometrial and salivary glands and of the mucosa of the gastro-intestinal tract. In tumours, a proportion of the neoplastic cells in lung and breast cancer and in melanoma were labelled. In all tissues, whether normal or malignant, striking VEGF positivity was seen in plasma in vessels and stroma. This study has shown that antibody VG1 detects the 121, 165 and 189 VEGF isoforms in routinely fixed specimens. The results of the normal tissue and tumour labelling are in agreement with other studies using alternative methods of detection. This should be a useful and reliable reagent for studies of VEGF and angiogenesis in human pathological material.

Animals↗
PubMed1998

Angiogenesis in the endometrium.

Human endometrial vasculature has the unique property of undergoing benign angiogenesis during each menstrual cycle under the influence of the ovarian steroids estradiol and progesterone. However, neither has intrinsic angiogenic activity and endometrial angiogenesis involves stimulation by ovarian steroids of angiogenic factor release by the epithelium and stroma which then act on the endothelium. In vitro models using cultures of isolated endometrial epithelium, stroma and endothelium now allow mechanistic questions to be addressed. Vascular endothelial growth factor and platelet-derived endothelial cell growth factor/thymidine phosphorylase at present appear to be key players in endometrial angiogenesis.

Journal Article↗
PubMed1998

The dormant in vivo phenotype of early stage primary human melanoma: termination by overexpression of vascular endothelial growth factor.

Early stage primary human cutaneous melanoma is known to remain relatively avascular and dormant for up to a decade, after which it may give rise to more rapidly growing, vascular and metastatically- competent primary tumor. Clinical dormancy of early stage human melanomas can be recapitulated experimentally by injection of cell lines established from such tumors into nude mice. For example, WM1341B cells, which were isolated from a thin vertical growth phase (VGP) human melanoma, are non-tumorigenic in nude mice even though some of the cells remain viable for at least three weeks at the site of orthotopic injection. These cells produce little or no vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a potent stimulator of angiogenesis. In order to determine whether their in vivo dormant behaviour may therefore be related to an inability to induce tumor angiogenesis, subpopulations of WM1341B cells were engineered to constitutively overexpress the VEGF/VPF121 isoform. This apparently single modification was sufficient to induce overt and progressively growing tumors by several independent VEGF/VPF121 producing clones, which could be largely blocked by systemic treatment of mice with a monoclonal anti-VEGF neutralizing antibody (A 4.6.1). No evidence for an autocrine mechanism of growth stimulation by VEGF was found. Taken together, these results support the notion that defective angiogenesis may, at least in part, account for dormant phenotype of some early stage primary melanomas. Since the induction of an overt tumorigenic phenotype in several VEGF/VPF transfected WM1341B clones appears to depend exclusively on their expression of VEGF/VPF, such sublines should be useful for screening the activity of known or potential VEGF/VPF ligand or VEGF/VPF receptor antagonists in an in vivo context.

Journal Article↗
PubMed1998

Differential expression of vascular endothelial growth factor mRNA vs protein isoform expression in human breast cancer and relationship to eIF-4E.

Angiogenesis is the formation of new blood vessels from the existing vasculature. Vascular endothelial growth factor (VEGF) is an endothelium-specific angiogenic factor strongly implicated in pathological angiogenesis. In this study, the mRNA and protein expression of the four alternatively spliced VEGF isoforms (121, 165, 189 and 206 amino acids) were examined in normal and malignant breast tissues. Three VEGF transcripts were detected in both (121>165>189), whereas only VEGF165 protein was detected. The tumours expressed more VEGF mRNA (P = 0.02) and protein (P < 0.0001), with eight-fold more VEGF protein generated per mRNA unit (P = 0.009). To examine this further, the expression of eIF-4E, a translation initiation factor, was examined. Increased eIF-4E mRNA levels were detected in the tumours (P < 0.0001) that correlated with VEGF mRNA (P = 0.0002), implying co-regulation of these genes. VEGF mRNA expression was elevated in tumours expressing the epidermal growth factor receptor (P < 0.01), but there was no difference according to oestrogen receptor status (P = 0.9), node status (P = 0.09) or between differing histologies (P = 0.4). These data suggest that elevated VEGF protein expression, by both enhanced transcription and translation, is a potential means by which tumour angiogenesis is induced in breast carcinomas. VEGF expression is also significantly associated with factors correlating with a poor outcome, implying a role in progression of this disease.

Adult↗
PubMed1997

Endothelial cell-specific expression of tumor necrosis factor-alpha from the KDR or E-selectin promoters following retroviral delivery.

The tumor vasculature offers a target for anti-cancer gene therapy which has the advantages both of good accessibility to systemically delivered therapy and comparative homogeneity across solid tumor types. We aimed to develop retroviruses carrying endothelial-specific promoters for the delivery of genes to proliferating endothelial cells in vitro and to tumor endothelial cells in vivo. This paper reports the generation of such retroviral vectors and the level of expression of murine tumor necrosis factor-alpha (mTNF-alpha) cDNA following infection into endothelial cells and stromal fibroblasts. Retroviral vectors carrying mTNF-alpha have been generated whose expression is controlled either by the retroviral long terminal repeat or by 5' proximal promoter sequences from the endothelial-specific kinase insert domain receptor (KDR)/VEGF receptor and E-Selectin promoters within the context of a self-inactivating (SIN) vector backbone. Both KDR and E-Selectin have been shown to be upregulated on tumor endothelium. A putative polyadenylation sequence AAATAAA within the E-Selectin promoter was mutated to permit faithful transmission of retroviral vectors carrying this promoter. We demonstrate a 10- to 11-fold increase in mTNF-alpha expression from promoter elements within sEND endothelioma cells as compared to NIH-3T3 fibroblasts. Suggestions for further improvements in vector design are discussed.

3T3 Cells↗
PubMed1997

Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer.

Tumor development is angiogenesis dependent, and vascular endothelial growth factor (VEGF) is a key growth factor in this process. We demonstrate that high expression of VEGF mRNA in 55 superficial bladder cancers was associated with earlier recurrence (P = 0.001; hazard ratio, 3.09) and progression to a more invasive phenotype (P = 0.02; hazard ratio, 5.33). VEGF mRNA expression correlated with protein levels in superficial tumors (r = 0.59, P = 0.003) and normal bladder (r = 0.65, P < 0.05), although the ratio of VEGF protein to mRNA was elevated in tumors compared to normal bladder (P = 0.004), suggesting posttranscriptional regulation. In this study, VEGF is implicated as a major downstream mediator of the effects of the p53 tumor suppressor gene by the association between high p53 protein (determined immunochemically) and high VEGF protein and mRNA expression (P < 0.02), although in cases without high p53 protein expression, high VEGF mRNA also predicts a poor prognosis. The relationship between VEGF and early tumor recurrence suggests that seeding via angiogenesis may be a major mechanism in the pathogenesis of recurrence. These studies indicate that VEGF can predict the behavior of superficial bladder tumors and is a therapeutic target for intravesical therapy.

Carcinoma, Transitional Cell↗
PubMed1997

COS-1 cell expression and one-step affinity protein purification and activity of epitope-tagged human erythropoietin and of site-directed mutants.

Recombinant human erythropoietin (rhEPO) is an important glycoprotein hormone which has been successfully used in the treatment of anaemia. To facilitate the rapid evaluation of wild-type and mutant forms of rhEPO in structure-function studies, we have developed an expression system in which the recombinant hormone is tagged at the C-terminus with a c-myc peptide. One-step affinity purification of culture supernatants on an anti-myc antibody column yielded proteins which were greater than 50% pure with a specific activity of 300,000 U/mg, in agreement with the value of wild-type protein. We conclude that the additional myc-peptide does not affect receptor binding. The expression system was used to study three mutants in which the N-glycosylation sites were changed to cysteines (Asn24Cys, Asn38Cys and Asn83Cys). Specific activities of these cysteine mutants were significant, but reduced (60%, 22% and 70%, respectively), compared to wild-type. The reduction in specific activity may be due to reduced stability of the mutant proteins.

Amino Acid Sequence↗
PubMed1997

Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis.

Systemic administration of the nitric oxide (NO) synthase inhibitor Nomega-nitro--arginine methyl ester (L-NAME) to rabbits bearing a corneal implant blocked vascular endothelial growth factor (VEGF), but not basic fibroblast growth factor (bFGF)-induced angiogenesis. L-NAME completely blocked angiogenesis induced by VEGF-transfected MCF-7 breast carcinoma cells and the cells remained dormant in the cornea. Postcapillary endothelial cell migration and growth induced by VEGF were blocked by both the NO synthase inhibitor Nomega-mono-methyl--arginine and by the guanylate cyclase inhibitor LY 83583. We conclude that NO is a downstream imperative of VEGF-, but not bFGF-induced angiogenesis, and propose that the NO synthase/guanylate cyclase pathway is a potential target for controlling tumor angiogenesis in response to VEGF. Our studies support recent evidence that VEGF and bFGF induce angiogenesis by different mechanistic pathways using the alphavbeta5 and alphavbeta3 integrins, respectively.

Animals↗