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Biomedical subjects

A L Harris

Publications and source records attributed to A L Harris.

At least 109 records · Page 6Linked to original sources
PubMed2001

Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia-inducible factor-2 alpha in human bladder tumors and cell lines.

Hypoxia is an important regulator of vascular endothelial growth factor (VEGF) expression, and VEGF is associated with poor prognosis in bladder cancer. To investigate further the mechanisms of VEGF regulation, we examined VEGF expression by mRNA and protein analysis in four human bladder cancer cell lines, showing a progression from well to poorly differentiated phenotypes under varying conditions of confluence and hypoxia (0.1% O(2)) and with chemical mimics of hypoxia. Hypoxia significantly increased VEGF protein expression in all cell lines, although this effect was dependent on the degree of confluence. The superficial bladder cancer cell line RT4 lost hypoxia inducibility at confluence, whereas inducibility was maintained in the invasive cell lines 253J and EJ28. This pattern of VEGF expression in the invasive cell lines correlated with the expression of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) and with hypoxia-inducible factor-2 alpha (HIF-2 alpha) and in RT4 correlated with a marked reduction in HIF-1 alpha inducibility at confluence. Using the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-inducible pathway regulated by HIF-1 alpha is distinct from a PI 3-kinase-dependent pathway, which regulates basal amounts of VEGF, but does not affect inducibility. Both HIF-1 alpha and HIF-2 alpha protein and mRNA were up-regulated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results suggest that components of the hypoxia response pathway, including HIF-1 alpha and HIF-2 alpha, are important cofactors in the regulation of VEGF in bladder cancer and are therapeutic targets in this disease.

Basic Helix-Loop-Helix Proteins↗
PubMed2001

Phase I and pharmacological study of the oral matrix metalloproteinase inhibitor, MMI270 (CGS27023A), in patients with advanced solid cancer.

This Phase I study of MMI270, an p.o. administered matrix metalloproteinase inhibitor, assessed toxicity, pharmacokinetics, and tumor response data and investigated markers of biological activity to recommend a dose for Phase II studies. MMI270 was administered continuously at seven dose levels (50 mg once daily to 600 mg three times/day). Patients were evaluated for toxicity and tumor response, and blood and urine samples were taken for pharmacokinetics, bone resorption markers, direct targets of the inhibitor [matrix metalloproteinase-2 (MMP-2), MMP-8, and MMP-9], indirect targets [tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, basic fibroblast growth factor, vascular endothelial growth factor, vascular cell adhesion molecule-1, soluble urokinase plasminogen activator receptor, and cathepsins B and H] and for a tumor necrosis factor-alpha cytokine release assay. Ninety-two patients were entered. There was no myelotoxicity. Eighteen patients developed a widespread maculopapular rash, which increased in frequency and severity at doses > or = 300 mg bid. Thirty nine patients developed musculoskeletal side effects, which were related to duration of treatment, not to dose level. Pharmacokinetics were linear, and MMI270 was rapidly absorbed and eliminated with minimal accumulation on chronic dosing. Sustained plasma concentrations in excess of 4 x mean IC(50) for the target enzymes were observed at dose levels > or = 150 mg bid. There were no tumor regressions; however, 19 patients had stable disease for > or = 90 days. There was a dose-response increase of MMP-2 and TIMP-1 with MMI270. Transient effects on the bone resorption markers were detected. MMI270 was generally well tolerated, with adequate plasma levels for target enzyme inhibition. The two main toxicities were rash, resulting in a maximum tolerated dose of 300 mg bid and musculoskeletal side effects. Biological marker data indicate drug effects. The rise in TIMP-1 suggests that a reflex rise in inhibitors could modify the effects of MMI270. The recommended Phase II dose is 300 mg bid.

Administration, Oral↗
PubMed2001

Soluble Tie2 and Flt1 extracellular domains in serum of patients with renal cancer and response to antiangiogenic therapy.

Antiangiogenesis drugs can be difficult to evaluate because they produce disease stabilization rather than tumor regression. Markers of endothelial mass in tumors may be of value to monitor therapy and evaluate such drugs. Soluble domains of the endothelial receptor tyrosine kinases, sTie2 (angiopoietin receptor) and sFlt1 (vascular endothelial growth factor receptor-1) were analyzed by sandwich ELISA in serum samples from 43 patients with advanced renal cancer before and 1 month after antiangiogenic therapy with razoxane. Pretreatment sFlt1 levels were 0.77 ng/ml +/- 0.48 (SD) and sTie2 74.3 ng/ml +/- 15 (SD). Pretreatment sFlt1 levels above the median were associated with a lesser chance of stable disease (P = 0.04) and poorer survival (P = 0.01). Fall of sTie2 on treatment was associated with stable disease (P = 0.05) and improved survival (P = 0.04). The soluble receptors measured weeks before response were assessed and correlated with response and survival, showing they may be useful to monitor and develop antiangiogenic therapy.

Antineoplastic Agents↗
PubMed2001

Thioredoxin expression is associated with lymph node status and prognosis in early operable non-small cell lung cancer.

PURPOSE: Thioredoxin (TRX), a low molecular weight protein, exerts reduction-oxidation control over a number of transcription factors involved in cell activation and proliferation. High TRX mRNA levels have been found in lung carcinomas, a trait associated with a growth and survival advantage. EXPERIMENTAL DESIGN: In this study, we examined the immunohistochemical expression of human TRX in normal lung and in 102 primary non-small cell lung carcinomas. RESULTS: In normal lung, the staining for TRX was cytoplasmic in the respiratory bronchial epithelium, alveolar epithelium, and alveolar macrophages. Bronchial glandular cells demonstrated a mixed nuclear and cytoplasmic staining. In lung carcinomas, the pattern of expression for TRX was predominantly cytoplasmic and only occasionally nuclear. A strong association between absence of TRX expression and regional lymph node negativity was observed (P = 0.004). High proliferation index, as detected with Ki-67 antibody, was associated with high TRX expression (P = 0.02). A significant correlation between high cytoplasmic p53 reactivity and low TRX expression was observed (P = 0.04). No association with grade, tumor stage, histology, or bcl-2 was noted. A significant coexpression of TRX with human activator protein endonuclease 1 was recorded (P = 0.04). Absence of TRX expression was associated with a better outcome (P < 0.05). CONCLUSIONS: We conclude that overexpression of TRX in non-small cell lung carcinomas is indicative of a more aggressive tumor phenotype and is associated with bad prognostic features and possibly with a poorer outcome.

Aged↗
PubMed2001

Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy.

PURPOSE: Carbonic anhydrases are proteins involved in the catalytic hydration of carbon dioxide to carbonic acid. Recent studies show that carbonic anhydrase 9 (CA9) is up-regulated by hypoxia and that its immunohistochemical tissue distribution follows the distribution of the radiosensitizer pimonidazole (C. C. Wykoff et al., Cancer Res. 60: 7075-7083, 2001). Therefore, CA9 expression may show hypoxia levels of clinical importance. EXPERIMENTAL DESIGN: We assessed the expression of CA9 and the microvessel density (MVD; CD31-positive) in 75 locally advanced squamous cell head and neck cancers treated with concurrent chemoradiotherapy with carboplatin. RESULTS: Strong membrane/cytoplasmic CA9 expression, noted in 20/75 (26.6%) tumors, mainly occurred in tumors with very poor vascularization (expression in 63% versus 14%; P < 0.0001), was located around areas of focal necrosis, and was related to poor complete response rate (40% versus 70%; P = 0.02). These observations suggested that CA9 might be a marker of clinically important hypoxia. Combining the CA9 staining and the tumor angiogenicity (MVD), we identified three groups of patients: (a) hypoxic tumors; (b) euoxic highly angiogenic tumors; and (c) euoxic non-highly angiogenic tumors. Groups (a) and (b) had a very poor local relapse-free survival (P < 0.0001). CONCLUSIONS: Stratification of patients undergoing radical radiotherapy using the CA9/MVD model may be useful for the individualization of therapeutic strategies combining antiangiogenesis and hypoxia targeting with radiotherapy.

Antigens, Neoplasm↗
PubMed2000

Hypoxia-inducible expression of tumor-associated carbonic anhydrases.

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Blotting, Western↗
PubMed2000

Relationship of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression to vascular endothelial growth factor induction and hypoxia survival in human breast cancer cell lines.

Hypoxia-inducible factors (HIF-1 and HIF-2) are two closely related protein complexes that activate transcription of target genes in response to hypoxia. Expression of HIF-1alpha and HIF-2alpha and their effects on survival under hypoxia were studied in six human breast cancer cell lines. We also evaluated the basal and inducible expression of two hypoxically regulated genes, vascular endothelial growth factor (VEGF) and lactate dehydrogenase-A (LDH-A). All of the cell lines studied expressed HIF-1alpha at various levels, but HIF-2alpha was low or absent from the more aggressive cell lines. There was an inverse correlation between HIF-1alpha and HIF-2alpha induction and clonogenic survival under hypoxia. Thus, cell lines with reduced induction of HIF-1alpha or HIF-2alpha showed high basal levels of VEGF and improved survival under hypoxia. A reduction in HIF expression was also associated with a more aggressive phenotype in vivo. To confirm these results, we carried out stable transfection of the MDA 435 cell line with human HIF-2alpha cDNA. There was no change in the growth rate in monolayer culture. However, in vitro growth as colonies and in vivo tumor growth of the HIF-2alpha overexpressing cells were significantly impaired compared with the control transfectants. Thus, despite the fact that HIF proteins are necessary for optimal tumor growth and angiogenesis in vivo, overexpression of these molecules seems detrimental to tumor growth. A balance between the angiogenic and tumor-inhibiting levels of HIF proteins may, therefore, be necessary for optimal tumor growth.

Animals↗
PubMed2000

Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling.

The von Hippel-Lindau tumour suppressor gene (VHL) targets hypoxia inducible factor (HIF)-alpha subunits for ubiquitin dependent proteolysis. To better understand the role of this and other putative pathways of gene regulation in VHL function we subjected mRNA from VHL defective renal carcinoma cells and transfectants re-expressing a wild type VHL allele to differential expression profiling, and analysed VHL target genes for oxygen regulated expression. Among a group of newly identified VHL target genes the majority but not all were regulated by oxygen, indicating that whilst dysregulation of the HIF system makes a dominant contribution to alterations in transcription, VHL has other influences on patterns of gene expression. Genes newly defined as targets of the VHL/hypoxia pathway (conditionally downregulated by VHL in normoxic cells) include aminopeptidase A, collagen type V, alpha 1, cyclin G2, DEC1/Stra13, endothelin 1, low density lipoprotein receptor-related protein 1, MIC2/CD99, and transglutaminase 2. These genes have a variety of functions relevant to tumour biology. However, not all are connected with the promotion of tumour growth, some being pro-apoptotic or growth inhibitory. We postulate that co-ordinate regulation as part of the HIF pathway may explain this paradox, and that evolution of anti-apoptotic pathways may be required for tumour growth under VHL-dysregulation. Our results indicate that it will be necessary to consider the effects of abnormal activity in integral regulatory pathways, as well as the effects of individual genes to understand the role of abnormal patterns of gene expression in cancer.

Carcinoma, Renal Cell↗
PubMed2000

A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response.

In a significant proportion of melanoma patients, CTL specific for the melan-A(26/7-35) epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer(+) cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer(+) cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7(+) CD45R0(-) CD45RA(+) phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer(+) cells were shifted toward a CCR7(-) CD45R0(+) CD45RA(-) phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

Adult↗
PubMed2000

Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors.

Thymidine phosphorylase (TP) (E.C. 2.4.2.4), also known as platelet-derived endothelial cell growth factor, is a potent angiogenic factor. The expression of TP correlates with poor prognosis in a range of tumor types. 2-Deoxy-D-ribose-1-phosphate, a product of thymidine catabolism by TP, is a strongly reducing sugar that generates oxygen radical species during the early stages of protein glycation. We show that thymidine induces oxidative stress in TP-overexpressing carcinoma cells, promoting secretion of the stress-induced angiogenic factors vascular endothelial growth factor and interleukin-8, and inducing matrix metalloproteinase-1. Our findings outline a putative mechanism for TP-induced angiogenesis and identify novel targets for intervention.

Carcinoma↗
PubMed2000

A variant within the DNA repair gene XRCC3 is associated with the development of melanoma skin cancer.

Exposure to UV radiation is a major risk factor for the development of malignant melanoma. DNA damage caused by UV radiation is thought to play a major role in carcinogenesis induction. Multiprotein pathways involved in repairing UV-DNA damage are the base excision, the nucleotide excision, and the homologous double-stranded DNA repair pathways. This study used a sequence-specific primer PCR (PCR-SSP) genotyping method to investigate the association between polymorphisms in DNA repair genes from these pathways with the development of malignant melanoma. The patient cohort was comprised of 125 individuals with malignant melanoma with lesions or staging suggesting a high risk of relapse or metastatic disease. The control population consisted of 211 individuals. We found the presence of a T allele in exon 7 (position 18067) of the XRCC3 gene was significantly associated with melanoma development (P = 0.004; odds ratio, 2.36; relative risk, 1.74). This gene codes for a protein involved in the homologous pathway of double-stranded DNA repair, thought to repair chromosomal fragmentation, translocations, and deletions. These results may provide further insights into the pathogenesis and the mechanism of UV-radiation induced carcinogenesis as well as having a role in prevention.

Adult↗
PubMed2000

Vascular endothelial growth factor/KDR activated microvessel density versus CD31 standard microvessel density in non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) is an important angiogenic factor, linked to poor outcome in human malignancies including non-small cell lung carcinoma (NSCLC). We used the 11B5 monoclonal antibody recognizing the VEGF/KDR complex (R. A. Brekken et al., Cancer Res., 58: 1952-1959, 1998) to assess the VEGF expression in cancer cells and the VEGF/KDR activated microvessel density (aMVD) in early operable NSCLC. The JC70 anti-CD31 monoclonal antibody was used to assess the standard MVD (sMVD). The aMVD was significantly higher in the invading front of the tumors and in the normal lung adjacent to the tumors as compared with normal lung distant to the tumor or to inner tumor areas (P < 0.0002). The sMVD was higher in the normal lung and decreased from the invading front to inner tumor areas (P < 0.0001). However, the vascular activation (aMVD:sMVD) was 4-6 times higher in the tumor areas as compared with lung from normal individuals (36-58% versus 9%; P < 0.0001). Fibroblast 11B5 reactivity, noted in 25% of cases, correlated with high aMVD and sMVD in the inner tumor areas. Multivariate analysis showed that aMVD was the most potent and independent prognostic factor (P = 0.001; t-ratio, 3.28). It is concluded that intense VEGF/KDR angiogenic pathway activation is a tumor-specific feature in more than 50% of NSCLC cases and is associated with poor postoperative outcome. Clinical trials involving targeting of the VEGF/KDR-positive vasculature with specific antibodies, such as 11B5, are, therefore, encouraged.

Adenocarcinoma↗
PubMed2000

The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions.

Analogues of EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole-4-7-dione]prop-2-e n-1-ol) which lack functionality at either the C-2 or C-3 position were synthesised. The aim was to establish the importance of each group towards toxicity and to give an indication as to whether substitution at either position altered activation and toxicity after metabolism by cellular NADPH: cytochrome c (P450) reductase (P450R). MDA231 breast cancer cells were transfected with the cDNA for human P450R and stable clones were isolated. These high P450R-expressing clones were used to determine the aerobic and hypoxic toxicity of EO9 and the two analogues that lacked functionality at either C-2 or C-3. The results showed that P450R was strongly implicated in the bioactivation of EO9 and its analogues under both of these conditions. This data also showed that the C-3 functionality was primarily implicated in hypoxic toxicity.

Antineoplastic Agents↗
PubMed2000

Macrophage infiltration is associated with VEGF and EGFR expression in breast cancer.

Angiogenesis is esential for tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and is an important component of the angiogenic stimulus in a range of human neoplasias. In addition to its mitogenic activities, VEGF has also been found to stimulate migration in macrophages via the flt-1 VEGF receptor. It has previously been shown that increased focal tumour macrophage infiltration is associated with increased angiogenesis and worsened relapse-free and overall survival in breast cancer. Macrophages are able to stimulate angiogenesis by their production of a range of factors including VEGF, tumour necrosis factor-alpha (TNF-alpha), and thymidine phosphorylase (TP). Thus, in breast cancer, VEGF could have a dual role in the regulation of angiogenesis, by direct mitogenic stimulation of endothelial cells, and also indirectly by attracting macrophages into avascular tumours. The purpose of this study was to localize VEGF protein in a series of 96 consecutive primary breast carcinomas and to determine its relationship to focal macrophage infiltration (macrophage index). These two variables were also compared with the pathological features of the tumours, as well as oestrogen receptor (ER), epidermal growth factor receptor (EGFR), microvessel density, macrophage index, and survival. An inverse relationship (p=0.0006) was noted between VEGF and EGFR, with high VEGF expression correlating with low EGFR levels. In the EGFR-negative group of cases (n=56), positive associations were observed between VEGF expression and macrophage index (p=0.005), ER (p=0.05), p53 (p=0. 006), tumour grade (p=0.02), and tumour necrosis (p=0.03). Macrophage counts were higher in EGFR-positive tumours (p=0.0006) and no associations were found between VEGF expression and increased microvessel density. These results show that in breast cancers there are two types of macrophage infiltrates, one associated with the presence of EGFR and low VEGF expression in tumours and the other with high VEGF expression in EGFR-negative tumours. VEGF expression may be an important factor in the recruitment of tumour-associated macrophages into breast carcinomas and may thus have an additional, indirect, pathway of angiogenic stimulation in this type of tumour.

Adult↗
PubMed2000

Expression of vascular endothelial growth factor by macrophages is up-regulated in poorly vascularized areas of breast carcinomas.

Angiogenesis is essential to the growth and metastasis of solid tumours. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic cytokine that is overexpressed in malignant tumours such as invasive carcinoma of the breast. The low oxygen tensions (hypoxia) present in these tumours are known to up-regulate the expression of VEGF by tumour cells. Human macrophages also respond to hypoxia by increasing their release of VEGF in vitro, although the effect of hypoxia on VEGF expression by macrophages in vivo has yet to be demonstrated. The present study compared the expression of VEGF by macrophages in areas of low and high vascularity in 24 invasive breast carcinomas (12 lobular, 12 ductal). The cellular distributions of VEGF protein, CD31 (vessels), and CD68 (macrophages) were compared in sequential sections for each tumour. In ten tumours, both tumour cells and macrophages were immunoreactive for VEGF protein. Use of non-isotopic in situ hybridization to localize VEGF mRNA showed that these cell types also expressed VEGF mRNA. No significant differences in the cellular distribution of VEGF protein were found between lobular and ductal carcinomas. In all tumours, macrophages accumulated in higher numbers in poorly vascularized than in highly vascularized areas. In VEGF-positive tumours, macrophages were immunoreactive for VEGF only in avascular areas where tumour cells also expressed VEGF. This suggests that VEGF expression by these two cell types may be regulated by the same microenvironmental stimuli in breast carcinomas. In addition, significantly more macrophages were present in poorly vascularized areas of VEGF-positive than VEGF-negative tumours. This suggests that VEGF may exert a chemotactic action on macrophages in vivo and guide their migration into avascular tumour sites.

Adult↗
PubMed2000

'Invading edge vs. inner' (edvin) patterns of vascularization: an interplay between angiogenic and vascular survival factors defines the clinical behaviour of non-small cell lung cancer.

Neo-angiogenesis during neoplastic growth involves endothelial mitogenic and migration stimuli produced by cancer or tumour stromal cells. Although this active angiogenesis takes place in the tumour periphery, the process of vessel growth and survival in inner areas and its clinical role remain largely unexplored. The present study compared the microvessel score (MS) as well as the single endothelial cell score (ECS) in the invading edge and in inner areas of non-small cell lung carcinomas (NSCLCs). Three different patterns of vascular growth were distinguished: the edvin (edge vs. inner) type 1, where a low MS was observed in both peripheral and inner tumour areas; the edvin type 2, where a high MS was noted in the invading front but a low MS in inner areas; and the edvin type 3, where both peripheral and inner tumour areas had a high MS. The ECS was high in the invading edge in edvin type 2 and 3 cases and was sharply decreased in both types in inner areas, suggesting that endothelial cell migration is unlikely to contribute to the angiogenic process in areas away from the tumour front. Expression of the vascular endothelial growth factor (VEGF) and of thymidine phosphorylase (TP) was associated with a high MS in the invading edge. VEGF was associated with a high MS in inner areas (edvin 3), while TP expression was associated with edvin type 2, showing that VEGF (and not TP) contributes to the preservation of the inner vasculature. Both edvin type 2 and 3 cases showed an increased incidence of node metastasis, but edvin type 3 cases had a poorer prognosis, even in the N1-stage group. The present study suggests that tumour factors regulating angiogenesis and vascular survival are not identical. A possible method is reported to quantify these two parameters by comparing the MS in the invading edge and inner areas (edvin types). This observation may contribute to the evaluation of the effectiveness of different therapeutic approaches, namely vascular targeting vs. anti-angiogenesis.

Carcinoma, Non-Small-Cell Lung↗