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Biomedical subjects

A L Harris

Publications and source records attributed to A L Harris.

At least 289 records · Page 16Linked to original sources
PubMed1995

Horizontal 'solvent-free' lipid bimolecular membranes with two-sided access can be formed and facilitate ion channel reconstitution.

We present here an easily used method and apparatus for formation of horizontal 'solvent-free' lipid bilayer membranes affording two-sided access. These horizontal bilayers allow direct delivery of submicroliter volumes of samples onto the membrane upper surface increasing the efficacy of reconstitution by several orders of magnitude, as demonstrated using Staphylococcus aureus alpha-toxin. Also, they permit creation of locally high and transient transbilayer osmotic gradients to initiate fusion of ion-channel containing liposomes with planar membrane, which, following fusion, leaves the membrane and channel in essentially symmetric bathing solutions. This method is especially advantageous for cases where thickness of the membrane, absence of hydrocarbon solvent, or presence of differing lipid compositions in the two monolayers is critical.

Bacterial Toxins↗
PubMed1995

Regulation of Wnt5a mRNA expression in human mammary epithelial cells by cell shape, confluence, and hepatocyte growth factor.

The Wnts are a family of genes with a role in cell fate and morphological development in numerous embryonic and adult tissues. In mouse mammary tissue a subset of the Wnts have a function in the normal development of the gland, and aberrant expression of Wnts normally silent in this tissue causes mammary carcinomas. We have previously shown that Wnt5a expression is elevated in the epithelial component of proliferative lesions of human breast and have therefore examined the regulation of Wnt5a mRNA expression in the human mammary epithelial cell line HB2, which has a luminal phenotype and thus represents the most commonly transformed cell type in human breast cancer. Wnt5a was up-regulated 30-fold at confluence. This up-regulation was induced specifically by confluence and not by the growth arrest that accompanied it. In addition, Wnt5a was down-regulated 3-fold by changes in cell shape associated with the transition from growth on a two-dimensional surface (flat cell morphology) to growth in three-dimensional gels (spherical cell morphology). Cytoskeletal disruption with non-toxic doses of colchicine also induced a spherical morphology and brought about a dose-dependent down-regulation of Wnt5a. Wnt5a was also down-regulated 10-fold during the hepatocyte growth factor-induced branching of HB2 cell aggregates in collagen gels. The down-regulation of Wnt5a preceded the branching process. A similar result was obtained with primary human breast epithelial populations and the breast cancer cell line MDA468. We conclude that regulation of Wnt5a expression is a down-stream effect of signaling by hepatocyte growth factor. These results are consistent with a role for Wnt5a in mammary epithelial cell motility and are in accord with Xwnt5a's function in embryonal cell migration. If Wnt5a's function in human mammary epithelial cells is similar to that of Xwnt5a, its up-regulation at confluence may be a mechanism for inhibition of cell migration beyond confluence.

Breast↗
PubMed1995

Thymidine phosphorylase is angiogenic and promotes tumor growth.

Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies.

Animals↗
PubMed1995

Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a secreted endothelial-specific growth factor that is angiogenic in vivo. It is commonly expressed in a range of carcinomas. PURPOSE: The study was designed to investigate the effect of constitutive expression of VEGF on tumor formation by estrogen-dependent human MCF-7 breast carcinoma cells. METHODS: A full-length complementary DNA encoding the shortest isoform of VEGF (VEGF121) was stably transfected into MCF-7 cells. Transfected clones were screened for VEGF121 messenger RNA (mRNA) expression by ribonuclease protection analysis and for secretion of VEGF121 protein by Western blot analysis. Secretion of biologically active VEGF121 by transfectants was confirmed by 1) a competitive radioreceptor-binding assay, 2) stimulation of the growth of microvascular endothelial cells in vitro, and 3) potent angiogenic activity in the rabbit corneal assay. Tumor models were then established by subcutaneously implanting wild-type or VEGF121-transfected MCF-7 cells, together with either mouse BALB/3T3 clone A31 fibroblasts or human MDA-435S breast carcinoma cells, into ovariectomized nude mice either with or without a separately implanted slow-release estrogen pellet. Tumor vascularity was quantitatively assessed by capillary vessel counting after staining with the pan-endothelial marker CD31. RESULTS: Stable VEGF121-overexpressing MCF-7 cells were isolated and designated V12 cells. When implanted into the rabbit cornea, V12 cells elicited a strong directional outgrowth of capillaries. The growth rate of V12 cells in vitro was indistinguishable from that of MCF-7 wild-type cells. V12 cells formed faster growing tumors than did wild-type cells (P < .01) when xenografted subcutaneously into nude mice with either 3T3 fibroblasts or MDA-435S cells. Tumors formed from V12 cells were more vascular (P < .01) and showed a heterogeneous distribution of vessels when compared with the homogeneous distribution seen in tumors formed from wild-type cells. VEGF121 overexpression had no effect on hormone dependence or tamoxifen sensitivity of tumor formation by MCF-7 cells in mice. No macroscopic evidence for metastasis from subcutaneous implants was obtained. CONCLUSIONS: VEGF121 expression by breast carcinoma cells confers a growth advantage in vivo but not in vitro. Tumors formed by V12 transfectants were more vascular than those formed by wild-type MCF-7 cells, and we surmise that the growth advantage arises from increased tumor vascularization induced by VEGF121. IMPLICATIONS: Tumor formation by V12 cells could provide a useful model for the assessment of anti-angiogenic drugs.

3T3 Cells↗
PubMed1995

Different angiogenic pathways characterize superficial and invasive bladder cancer.

We have investigated by RNase protection analysis the expression of 2 angiogenic factors in 45 primary bladder tumors and 8 normal bladders. Expression of vascular endothelial growth factor (VEGF) was 3-fold higher and that of platelet-derived endothelial cell growth factor was 40-fold higher in tumors compared to normal bladder. However, the factors were differentially expressed in different stages of the cancer. Expression of VEGF in superficial tumors was 4-fold higher than in invasive tumors and 10-fold higher than in normal bladder (superficial versus invasive, P < 0.0006; superficial versus normal, P < 0.0002). Expression of platelet-derived endothelial cell growth factor in invasive tumors was 33-fold higher than in superficial tumors and 260-fold higher than in normal bladder (invasive versus superficial, P < 0.0001; invasive versus normal, P < 0.0003). In well differentiated or moderately differentiated superficial tumors which had invaded the lamina propria (pT1G1/2) VEGF expression was 4-fold higher in tumors which subsequently recurred at 3 months compared to those which did not recur (P < 0.002). Thus there are two distinct angiogenic pathways involved in different stages of bladder cancer, which is in keeping with the evidence for two different genetic pathways. Elevated VEGF expression predicts early recurrence of pT1G1/2 tumors.

Antisense Elements (Genetics)↗
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

Microvessel count predicts metastatic disease and survival in non-small cell lung cancer.

The growth of newly formed vessels, or neoangiogenesis, represents an important step in both physiological and pathological situations: in particular, tumour growth and metastasis require angiogenesis. Microvessel count (MC), which represents a measure of tumour angiogenesis, has been associated with metastatic spread in cutaneous, mammary, prostatic, head and neck, and early-stage lung cancer. In this study, the role of tumour angiogenesis as a prognostic indicator was examined in 253 primary non-small lung cancer (NSCLC) patients. Microvessels were counted by highlighting endothelial cells with anti-Factor VIII monoclonal antibody (Mab) in methacarn-fixed tumour samples. In univariat analysis, MC (P< 0.000001), sex (P=0.0036), histotype (P < 0.014), tumour status (P <0.007), and vessel invasion (P < 0.019) were significantly related to hilar and/or mediastinal nodal involvement. However, in the stepwise logistic regression analysis, MC (P<0.000003) retained the most important influence on nodal metastasis. The overall survival analysis calculated by the Kaplan-Meier method revealed that tumours with high MC ( > 25 vessels/field) were significantly associated with increased death risk (log-rank test P = 0.00067; Cox's test P = 0.00046; Gehan's Wilcoxon test P = 0.00108). In 94 patients, the development of metastatic disease during follow-up was significantly related to MC. Indeed, patients who developed metastasis during follow-up showed a higher MC, either as a dichotomous (P = 0.01) or as a continuous (P = 0.003) variable, than patients who had developed no metastasis at the time of the analysis. Moreover, in the stepwise logistic regression analysis, MC retained the most important influence on distant metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis.

In some studies of breast cancer, quantitation of immunohistochemically highlighted microvessel 'hot spots' has been shown to be a powerful prognostic tool. However, the antibody used, the number and size of the 'hot spots' assessed, and the stratification of patients into high and low vascular groups vary between studies. Furthermore, little is known about the relationship between microvessel density and other vascular parameters. These uncertainties and the laborious nature of the technique make it unsuitable for diagnostic practice. Both manual and computerized image analysis techniques were used in this study to examine the relationship between microvessel density and the vascular parameters in different sized microscopic fields in a pilot series of 30 invasive breast carcinomas. Automated pixel analysis of immunohistochemical staining, Chalkley point counting, and observer subjective vascular grading were also assessed as more rapid methods of measuring tumour vascularity. A Chalkley count was also performed on a further 211 invasive breast carcinomas. Significant correlations were observed between manual microvessel density and luminal perimeter (r = 0.6, P = 0.0004), luminal area (r = 0.56, P = 0.002), and microvessel number (r = 0.57, P = 0.0009) by computerized analysis. There were also significant correlations between the microscopic hot spots of 0.155 mm2 and 0.848 mm2 for microvessel number (r = 0.81, P < 0.00005), luminal perimeter (r = 0.78, P < 0.00005), and luminal area (r = 0.65, P = 0.0001). In addition, a significant correlation was observed between microvessel density and both subjective vascular grade (P = 0.002) and Chalkley count (P = 0.0001). A significant reduction in overall survival was observed between patients stratified by Chalkley count in both a univariate (P = 0.02) and a multivariate (P = 0.05) analysis in the 211 invasive breast carcinomas. These findings show that Chalkley counting is a rapid method of quantifying tumour angiogenesis and gives independent prognostic information which might be useful in diagnostic practice.

Adult↗
PubMed1995

The increased expression of adhesion molecules ICAM-3, E- and P-selectins on breast cancer endothelium.

Sequential interaction of neoplastic cells with the endothelium of tumour neovasculature is believed to be a significant step in tumour metastasis. Increasing evidence suggests that inducible endothelial adhesion molecules are intimately involved in this process. An immunohistochemical approach was used to examine the expression of adhesion molecules in 14 normal controls and a series of 64 invasive breast carcinomas. Endothelium in normal breast showed constitutive expression of PECAM (100 per cent), ICAM-2 (100 per cent), and P-selectin (64 per cent); variable and focal expression of ICAM-1 (71 per cent); and only weak staining for E-selectin (21 per cent). No ICAM-3 or VCAM-1 expression was observed. Similarly to normal breast endothelium, widespread and intense immunoreactivity on the endothelium of tumour-associated vessels was seen for PECAM (100 per cent), ICAM-1 (69 per cent), and ICAM-2 (95 per cent). In contrast to normal tissues, E- and P-selectins showed increased intensity of staining (52 and 67 per cent of cases, respectively) and expression of E- and P-selectins was more prominent at the tumour periphery. ICAM-3 expression was increased on tumour endothelium (15 per cent of cases), but in common with VCAM-1 (10 per cent) expression was focal. A previously unreported finding was the immunoreactivity of the neoplastic epithelial cells for the non-epithelial lineage markers ICAM-1 (34 per cent), ICAM-3 (10.9 per cent), PECAM (1.6 per cent), and E- and P-selectins (7 and 37 per cent of cases, respectively). These findings show that tumour endothelium displays significant heterogeneity and can assume a pro-inflammatory phenotype, probably as a result of cytokine stimulation. Upregulation of adhesion molecules might contribute to changes in invasive phenotype by promoting endothelial cell adhesion and angiogenesis, as well as forming a substratum for tumour cells to assemble and attract macrophages.

Adult↗
PubMed1995

Expression of messenger RNA for amphiregulin, heregulin, and cripto-1, three new members of the epidermal growth factor family, in human breast carcinomas.

The expression of amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1) mRNA transcripts was assessed in 60 human primary breast carcinoma. AR and HRG transcripts were expressed respectively in 58% and 25% of the carcinomas as measured by Northern blot analysis. CR-1 mRNA was found in 77% of the carcinomas using Reverse Transcriptase-PCR analysis. Coexpression of two or three of these peptides was observed in several specimens. There was no significant association between AR, HRG, and CR-1 expression and nodal status, EGF receptor, or c-erbB-2 protooncogene expression in these tumors. However, a significant association between AR expression and estrogen receptor positivity was observed.

Amphiregulin↗
PubMed1995

The prognostic value of quantitative angiogenesis in breast cancer and role of adhesion molecule expression in tumor endothelium.

Angiogenesis is the formation of new capillaries from the existing vascular network and is essential for tumor growth and metastases. Increased microvessel density in breast cancer is associated with lymph node metastasis and reduced survival. We have assessed tumor vascularity in 211 breast carcinomas using a more rapid technique based on a Chalkley point eyepiece graticule. We confirmed using this method a significant reduction in overall survival between patients stratified by Chalkley count in both a univariate (p = 0.02) and multivariate (p = 0.05) analysis. Since studies have suggested that cell adhesion molecules (CAMs) might be important in the angiogenic process, and interaction of neoplastic cells with this neovasculature is a significant step in tumor metastasis, we have also examined the expression of CAMs in a subset of these tumors (n = 64). Using immunohistochemistry we observed widespread and intense staining on the endothelium of tumor-associated vessels for PECAM (100%), ICAM 1 (69%), and E- and P-selectins (52% and 59% of cases respectively). Endothelial expression of the selectins was more prominent at the tumor periphery. Immunoreactivity of ICAM-1 (34%), PECAM (1.6%), and E- and P-selectins (7% and 37% of cases respectively) was also observed on the neoplastic element of the tumors.

Analysis of Variance↗
PubMed1995

An open study to assess the safety, tolerance and pharmacokinetics of an intravenous infusion of granisetron given at 3 mg over 30 s in patients receiving chemotherapy for malignant disease.

Granisetron is a highly potent and selective 5-hydroxytryptamine3 (5-HT3) receptor antagonist indicated for the prevention of cytotoxic-induced nausea and vomiting. Clinical trials have demonstrated granisetron to be effective and well tolerated at a standard dose of 40 micrograms/kg or 3 mg given i.v. as a 5-min infusion. In this study, the efficacy and safety of granisetron given as a 30-s infusion was assessed. A total of 21 patients, scheduled to undergo chemotherapy, received a single 3-mg i.v. dose of granisetron over 30 s, completed at 1 h before chemotherapy administration. Patients were allowed two further i.v. doses of granisetron at 3 mg within the 24-h assessment period. Changes from baseline values in vital signs were analysed prior to granisetron administration and at 30 s as well as 1, 10, 15, 30 and 60 min after granisetron administration. Holter ECG recordings were taken for 6 h prior to and 1 h after administration. No significant change was found in vital signs at 30 s or 1 min after granisetron infusion. There was a small but statistically significant fall in diastolic blood pressure as compared with baseline and a non-significant trend in favour of a reduction in heart rate at 10 and 15 min. No ECG abnormality was recorded post-infusion that had not been present pre-infusion. None of these changes was considered to be clinically relevant. The treatment was well tolerated. The most frequently reported adverse events were constipation (n = 6) and headache (n = 5). Maximal plasma levels of granisetron were within the range of 44.57-410 ng/ml except in one patient. The median values recorded for peak concentration (Cmax) and area under the curve (AUC) were 195 ng/ml and 71.2 ng h ml-1, respectively. In conclusion, granisetron at 3 mg was shown to be safe and well tolerated when given as a 30-s i.v. infusion to patients receiving chemotherapy for malignant disease.

Adult↗
PubMed1995

Immunohistochemical assay for epidermal growth factor receptor on paraffin-embedded sections: validation against ligand-binding assay and clinical relevance in breast cancer.

Epidermal growth factor receptor (EGFR) has been the subject of much research since it was first described as a prognostic factor in breast cancer. The assay methods used and results obtained vary widely between studies. In this study 88 primary breast cancers were assayed for EGFR using a novel immunohistochemical assay performed on paraffin-embedded sections. The monoclonal antibody used was raised against purified, denatured EGFR, reacts with an epitope on the external domain and does not interfere with ligand binding. Twenty-two per cent of the tumours were EGFR positive using this assay. The results obtained were significantly correlated with those obtained by ligand-binding assay (r = 0.621, P = 0.011). The concordance rate was 82% (P < 0.001). The majority of discordant results could be explained by the presence of benign breast tissue and other non-malignant elements which could be seen to express EGFR on the immunohistochemical assay and were excluded from the score for this, but would be incorporated into ligand-binding assay results. The well-established inverse relationship between EGFR (as measured by this assay) and oestrogen receptor (ER) was seen (chi 2 = 24.9, P < 0.0001). In addition, in this exploratory study on a limited tumour set, EGFR was a significant adverse prognostic factor (on univariate but not multivariate analysis) for both relapse-free survival (P = 0.02) and overall survival (P = 0.03) when measured by this immunohistochemical assay, but was not significant when measured by ligand-binding assay.

Adult↗
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

Bryostatin 1, a novel antineoplastic agent and protein kinase C activator, induces human myalgia and muscle metabolic defects: a 31P magnetic resonance spectroscopic study.

Bryostatin 1, a novel antineoplastic agent and protein kinase C (PKC) activator, has been found to induce myalgia (muscle pain) 48 h after administration in clinical trials. This is the dose-limiting toxicity and has restricted the duration of therapy in phase I trials. To investigate the mechanisms and try to increase toleration of the drug, we studied calf muscle metabolism of 14 patients at rest and during exercise and subsequent recovery using 31P magnetic resonance spectroscopy (MRS) before and 4 h, 48-72 h and 1-2 weeks following bryostatin therapy. In resting muscle there was a significant (P < 0.001) increase in the phosphodiester/adenosine 5'-triphosphate (PDE/ATP) ratio 48 h post bryostatin and in patients with myalgia compared with pre-bryostatin control studies. Following exercise, patients with myalgia showed significantly slower phosphocreatine (PCr) and ADP recovery half-time (P < or = 0.05) suggesting impaired mitochondrial (oxidative) energy production, possibly due to a direct effect on the mitochondria or secondary to reduced blood flow. The apparent proton efflux rate following exercise was significantly reduced 4 h after bryostatin (P < or = 0.05), suggesting reduced blood flow. The rate of post-exercise reoxygenation was studied in four patients by near-infrared spectroscopy 4 h post bryostatin. In three of these the rate was reduced, consistent with reduced muscle blood flow. Bryostatin 1 appeared to cause a long-lasting impairment of oxidative metabolism and proton washout from muscle, consistent with a vasoconstrictive action. Thus these studies provide evidence for two mechanisms of the dose-limiting toxicity for bryostatin. Prospective studies on the use of vasodilators to improve the tolerance of the drug should be carried out.

Adenosine Triphosphate↗