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Background: This study investigated the relationship of obesity, insulin resistance, inflammation and angiogenesis with cancer progression and survival in a colorectal cancer cohort.Methods: Clinical and pathological data, along with anthropometric and follow-up data, were collected from 344 consecutive colorectal cancer patients. Serum samples at diagnosis were analysed by immunoassay for adiponectin, C-reactive protein (CRP), vascular endothelial growth factor-A (VEGF-A), angiopoietin-2 (Ang-2), insulin-like growth factor-1 (IGF-1), insulin and C-peptide.Results: Serum Ang-2 and VEGF-A levels increased with tumour T stage (P=0.007 and P=0.025, respectively) and N stage (P=0.02 and P=0.03, respectively), and correlated with CRP levels (r=0.43, P<0.001 and r=0.23, P<0.001, respectively). Angiopoietin-2 correlated with C-peptide (r=0.14, P=0.007) and VEGF-A with IGF-1 in males (r=0.25, P=0.001). Kaplan-Meier analysis showed that patients with high serum levels of CRP and Ang-2 had significantly reduced survival (both P≤0.001). After adjusting for tumour stage and age, Ang-2 remained a significant predictor of survival. The CRP levels were inversely associated with survival in American Joint Committee on Cancer stage II patients (P=0.038), suggesting that CRP could be used to support treatment decisions in this subgroup. Serum markers and anthropometric measures of obesity correlated with each other, but not with survival.Conclusion: Our study supports the concept that obesity-related inflammation, rather than obesity itself, is associated with colorectal cancer progression and survival. The study confirms serum Ang-2 as a predictive marker for outcome of colorectal cancer. [ABSTRACT FROM AUTHOR]

The in-vivo mode of action of metronidazole and the MetA protein which confers resistance to metronidazole, was investigated in Escherichia coli wild type and DNA repair-deficient strains. All the E. coli strains were moderately susceptible to metronidazole under aerobic conditions but were more susceptible under anaerobic conditions, and the amount of DNA breakage was less under anaerobic than aerobic conditions. E. coli excision (uvr) and recombination (rec) mutants were more susceptible than DNA repair wild type strains. Metronidazole did not induce cell lysis in E. coli but caused single strand DNA breaks in wild type and repair-deficient E. coli strains. The MetA protein reduced DNA breakage caused by metronidazole in E. coli wild type and DNA repair-deficient strains grown under aerobic and anaerobic conditions, and inhibited the suppressor effect of the RecE protein in E. coli recBC strains. The MetA protein did not inactivate metronidazole. [ABSTRACT FROM AUTHOR]

This paper reports a detailed analysis of the effect of low oxygen conditions (hypoxia) on the reporter green fluorescent protein (GFP). It questions the feasibility of using GFP for gene expression studies under tumor conditions. Hypoxia is a characteristic of both experimental and clinical tumors. Several important factors are pointed out which need to be considered when using GFP as reporter gene. GFP fluorescence is the final product of a long and complex pathway involving transcription, translation, and posttranslational modifications. All of these steps may be affected by the availability of oxygen. We show specifically that cellular GFP fluorescence decreased with reduced oxygenation, anoxia virtually eliminated fluorescence and protein levels, and fluorescence recovery after anoxia required 5-10 h of reoxygenation. In conclusion, GFP appears to be a good marker gene to study location or movement of proteins or cells but should be used with great caution as a reporter of gene expression under tumor conditions.

Gene therapy of cancer is a novel approach with the potential to selectively eradicate tumour cells, whilst sparing normal tissue from damage. In particular, gene-directed enzyme prodrug therapy (GDEPT) is based on the delivery of a gene that encodes an enzyme which is non-toxic per se, but is able to convert a prodrug into a potent cytotoxin. Several GDEPT systems have been investigated so far, demonstrating effectiveness in both tissue culture and animal models. Based on these encouraging results, phase I/II clinical trials have been performed and are still ongoing. The aim of this review is to summarise the progress made in the design and application of GDEPT strategies. The most widely used enzyme/prodrug combinations already in clinical trials (e.g., herpes simplex 1 virus thymidine kinase/ganciclovir and cytosine deaminase/5-fluorocytosine), as well as novel approaches (carboxypeptidase G2/CMDA, horseradish peroxidase/indole-3-acetic acid) are described, with a particular attention to translational research and early clinical results., (Copyright 2001 Wiley-Liss, Inc.)

This paper demonstrates the potential for utilizing the plant enzyme, horseradish peroxidase (HRP), in a gene-directed enzyme prodrug therapy context. Human T24 bladder carcinoma cells transfected with a mammalian expression vector containing the HRP cDNA were selectively sensitized to the nontoxic plant hormone, indole-3-acetic acid (IAA). The HRP/IAA-induced cell kill was effective in normoxic and anoxic conditions. The activated drug is a long-lived species able to cross cell membranes, and cell contact appears not to be required for a bystander effect to take place. These preliminary results suggest that the delivery of the HRP gene to human tumors followed by IAA treatment may provide a novel cancer gene-directed enzyme prodrug therapy approach, with potential to target hypoxic cells.

Low oxygen tension (hypoxia) is an important prognostic factor in cancer treatment because it affects tumor formation and malignant progression. Many genes governing these complex processes have been found to be oxygen regulated. This article reviews the present knowledge of hypoxia-inducible gene expression and how this affects angiogenesis, progression, and metastasis. Of particular importance are hypoxia-regulated transcription factors because they can modulate expression of countless different genes. Additional genes analyzed in some detail include those encoding angiogenic growth factors, factors controlling blood flow, and those involved in metastasis. Although hypoxia is generally perceived as a hindrance to cancer therapy, it is possibly exploitable because severe oxygen deficiency is tumor specific. Strategies aimed at using the presence of hypoxia in solid tumors include oxygen sensitive chemotherapy and gene therapy., (Copyright 1998 W.B. Saunders Company.)

Solid tumors with areas of low oxygen tension (hypoxia) have a poor prognosis, as cells in this environment often survive radiation and chemotherapy. In this report we describe how this hypoxic environment can be used to activate heterologous gene expression driven by a hypoxia-responsive element (HRE), which interacts with the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Our results demonstrate that the HIF-1/HRE system of gene regulation is active in hypoxic tumor cells and show the potential of exploiting tumor-specific conditions for the targeted expression of diagnostic or therapeutic genes in cancer therapy.

We report that hypoxia regulates and influences the level of the angiogenic enzyme platelet-derived endothelial cell growth factor (PD-ECGF), also called thymidine phosphorylase, in vitro and in vivo. Levels of PD-ECGF protein increased 6-fold in the breast cancer cell line MDA 231 after 16 h of growth in 0.3% oxygen. A simultaneous increase in enzyme activity was observed. Immunohistochemical staining of MDA 231 tumors grown in nu/nu mice showed increased expression of PD-ECGF in those parts of the tumor that are proximal to the areas of necrosis. In addition, increased and widespread staining for PD-ECGF protein was obtained when the tumor vascular supply was occluded for 2 h by clamping. Lowering the media pH to 6.3-6.7 in vitro also resulted in an increase in PD-ECGF protein levels. This study demonstrates that tumor microenvironmental factors can result in the specific up-regulation of an angiogenic enzyme that can also activate 5-fluorouracil prodrugs and hence is exploitable therapeutically.

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia. We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail. Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue. This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target. We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors. The list of genes that have been considered for use in the treatment of cancer is extensive. It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop. Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes). This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E. (ABSTRACT TRUNCATED)

Hypoxia-inducible factor-1 (HIF-1) has been shown to mediate the transcriptional activation of its target genes in response to oxygen concentration, most likely via a pathway involving a specific oxygen sensor. Molecular cloning of HIF-1 has shown that this widely expressed, DNA binding transcription factor is a heterodimer of two proteins, HIF-1 alpha and HIF-1 beta. A major control of HIF-1 activity by oxygen tension is achieved by changes in the level of the HIF-1 alpha subunit, which complexes with the constitutively expressed HIF-1 beta subunit. Such changes in HIF-1 alpha abundance occur via regulated stability, probably involving proteolysis, rather than at the level of transcription or translation. Further analysis has shown the existence of two separate regulatory domains in the C-terminus of the alpha subunit. Thus, a mechanism of oxygen-regulated HIF-1 activation is proposed, which involves the operation of one inducible domain being amplified by changes in protein level conferred by a second regulatory domain. Evidence for a critical role of HIF-1 in the response of diverse target genes involved in cellular growth and metabolism comes from studies on cultured, mutant mouse cells that lack a functional HIF-1 beta subunit. Furthermore, studies on tumor xenografts derived from the mutant and wild-type cells show that HIF-1 is activated in vivo, and has major effects on gene expression in response to tumor hypoxia. Thus, HIF-1 is a critical component of the oxygen-signaling pathway, and is a prime candidate regulator molecule for the role of coordinating vascular oxygen supply with cellular growth and energy metabolism.

In this review, reports of the increased expression of selected genes in response to hypoxia have been summarised. The best studied mammalian hypoxia response systems are those of the erythropoietin (Epo) and the vascular endothelial growth factor (VEGF) genes, which will be described in some detail. Other genes discussed here include those encoding growth factors, cytokines, transcription factors, metabolic enzymes and DNA repair enzymes. Short DNA sequences (hypoxia response elements) governing the increased gene expression in response to hypoxia have been discovered in the vicinity of most of these genes. The review will end by analysing the possibility of exploiting tumour hypoxia via the use of hypoxia response elements for gene therapy of cancer.