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Limitations of the Reporter Green Fluorescent Protein Under Simulated Tumor Conditions1

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Limitations of the Reporter Green Fluorescent Protein Under Simulated Tumor Conditions1 [CANCER RESEARCH 61, 4784–4790, June 15, 2001] Limitations of the Reporter Green Fluorescent Protein under Simulated Tumor Conditions1 Claudia Coralli, Maja Cemazar, Chryso Kanthou, Gillian M. Tozer, and Gabi U. Dachs2 Tumour Microcirculation Group, Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom ABSTRACT normal cellular activities and is easily detectable by fluorescence microscopy and quantifiable by FACS analysis (13). This paper reports a detailed analysis of the effect of low oxygen A major advantage in using GFP as an in vivo marker protein is the conditions (hypoxia) on the reporter green fluorescent protein (GFP). It lack of requirement for exogenous substrates or cofactors to produce questions the feasibility of using GFP for gene expression studies under tumor conditions. Hypoxia is a characteristic of both experimental and the active fluorescent molecule (7). However, GFP requires molecular clinical tumors. Several important factors are pointed out which need to oxygen to catalyze the posttranslational cyclization to form the pro- be considered when using GFP as reporter gene. GFP fluorescence is the tein’s fluorophore (14). This requirement may be a problem when final product of a long and complex pathway involving transcription, GFP is used as a reporter in biological systems where oxygen is translation, and posttranslational modifications. All of these steps may be limiting. affected by the availability of oxygen. We show specifically that cellular Low oxygen tension (hypoxia) is a common feature of both exper- GFP fluorescence decreased with reduced oxygenation, anoxia virtually imental and clinical tumors. Tumor hypoxia arises from insufficient eliminated fluorescence and protein levels, and fluorescence recovery after and abnormal blood supply and is the result of an imbalance in oxygen anoxia required 5–10 h of reoxygenation. In conclusion, GFP appears to delivery and consumption (15). Originally, diffusion-limited hypoxia, be a good marker gene to study location or movement of proteins or cells resulting from large intercapillary distances, was perceived as the sole but should be used with great caution as a reporter of gene expression under tumor conditions. cause of tumor hypoxia. However, hypoxic cells can also arise from perfusion-driven changes in oxygen supply. Such cells are subjected to rapid and reversible changes in oxygenation (16, 17). INTRODUCTION This tumor environment provides some unique opportunities for The progressive development of reporter gene technology has therapy, especially gene therapy. The expression of several genes greatly contributed to the study and understanding of cellular events important for tumor growth and spread, including those encoding associated with signal transduction and gene expression. Several growth factors (e.g., vascular endothelial growth factor), oncopro- genes, with easily measurable phenotypes distinguishable above a teins, and transcription factors, has been shown to be induced by background of endogenous proteins, are used commonly as reporters hypoxia (18, 19). The cellular response to hypoxia consists of two in a broad range of applications, including gene transfer and expres- main components, namely, the HIF-1-dependent transcriptional reg- sion studies (1, 2). The most widely used reporter genes encode: (a) ulation and a hypoxia-dependent stabilization of certain mRNAs. the bacterial enzyme ␤-galactosidase (3); (b) the bacterial enzyme HIF-1 is a heterodimeric nuclear transcription factor consisting of chloramphenicol acetyltransferase (4); (c) the bioluminescent protein HIF-1␣, the oxygen-sensitive subunit, and HIF-1␤. The transcription luciferase (also known as aequorin or monooxygenase) from firefly factor HIF-1 binds to its recognition sequence, the hypoxia regulatory (Photinus pyralis) or the sea pansy (Renilla reniformis; Ref. 5); and element (HRE), in the vicinity of oxygen-sensitive genes. It is com- (d) the GFP3 from jellyfish (6). Because the first three reporters mon to all mammalian cells, tissues, and organs tested to date with require exogenously added substrates and/or cofactors, they are of high abundance in human tumors (20–22). The use of this oxygen- limited use in living organisms. GFP, however, has no such require- sensitive gene regulation system has been proposed for targeted ments (7). gene therapy (23). The GFP from the jellyfish Aequorea victoria is a 238 amino acid Vascular endothelial growth factor-promoter-regulated GFP fluo- polypeptide, which is highly fluorescent and stable in many assay rescence in wound healing and in tumor formation has been demon- conditions (6). Reports on its sequence (8) and studies on its expres- strated in vivo (24). However, little is known about how GFP fluo- sion in heterologous systems (7) made it a unique reporter gene. rescence is affected by tumor conditions. The aim of the present study Applications for which GFP has been used successfully include mon- was to determine the feasibility of using GFP under low and variable itoring the transfer and expression of genes in living cells and tissues, oxygenation conditions, which are prevalent in solid tumors. Specif- subcellular location and protein movement within living cells by ically, we have determined the effects of simulated tumor conditions fusion to genes of interest (9), and location and fate of labeled cells on GFP fluorescence, GFP protein levels, and GFP mRNA levels in within whole organisms, to trace, e.g., metastasis (10). Mutagenized vitro and carried out a preliminary analysis of GFP fluorescence in GFP variants with improved fluorescence intensity and spectral qual- solid s.c. tumors. ities (11) and with reduced half-life for studies of transient gene expression (12) have increased the use of GFP in a variety of biolog- MATERIALS AND METHODS ical applications. GFP shows low toxicity and no interference with Cell Line and Growth Media. The human bladder carcinoma cell line T24 Received 8/16/00; accepted 4/30/01. (European Collection of Cell Cultures, Salisbury, UK; Refs. 25 and 26) and its The costs of publication of this article were defrayed in part by the payment of page transfected derivatives were used. The cells were maintained in DMEM (Life charges. This article must therefore be hereby marked advertisement in accordance with Technologies, Inc., Paisley, UK) supplemented with 10% FCS (Sigma Chem- 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Cancer Research Campaign Grant SP2292/0102. ical Co., Gillingham, UK), 2 mML-glutamine (Life Technologies, Inc.), 100 2 To whom requests for reprints should be addressed, at Gray Laboratory Cancer units/ml penicillin, and 100 ␮g/ml streptomycin (Sigma Chemical Co.). Cy- Research Trust, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United King- cloheximide (Sigma Chemical Co.) at 0.3 mM was used to inhibit protein dom. Phone: (44)-1923-828611; Fax: (44)-1923-835210; E-mail: [email protected]. synthesis. 3 The abbreviations used are: GFP, green fluorescent protein; d2EGFP, destabilised enhanced GFP; RT-PCR, reverse transcription-PCR; FACS, fluorescence-activated cell Cell proliferation and cell viability were monitored by cell counting using sorting; HIF-1, hypoxia-inducible factor 1; CMV, cytomegalovirus. a hemocytometer and trypan blue (Sigma Chemical Co.) exclusion staining. 4784 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. GFP AS A REPORTER IN HYPOXIA Oxygenation Conditions. To mimic the heterogeneous oxygenation of (Micron Separations, Inc., Westborough, MA) using a Pharmacia-Biotech solid tumors, cells were plated in 6-cm oxygen-impermeable dishes (Per- semidry blotter. Immunoblotting was performed with primary monoclonal manox; Nalge Nunc International) and maintained at 37°C under various anti-GFP antibodies, which detect all GFP variants (1:1000; Clontech Labo- oxygen conditions: (a) incubator: humidified air, 5% CO2;(b) anaerobic glove ratories), and secondary peroxidase-conjugated goat antimouse immunoglobu- cabinet (DON Whitley Scientific, Ltd., Shipley, UK): 90% N2,5%H2,5% lins (1:2000; Dako, Ely, UK), according to manufacturer’s instructions. De- CO2 with palladium catalyst; and (c) air-tight Perspex boxes flushed contin- tection of immunoreactive bands was performed using the enhanced uously with a humidified gas mixture containing Ͻ0.0005, 0.02, 0.1, 0.3, 1, 2, chemiluminescence technique (ECL kit; Amersham Pharmacia Biotech, 5, or 95% O2 and 5% CO2, balance N2 (BOC Gases, London, UK). Amersham, UK). The bands were analyzed by densitometry using Visilog In the experiments involving anoxia, the samples were not exposed to software (Noesis, Leshlis, Coutaboeuf, France). oxygen until their final analysis by performing manipulations in the anaerobic To confirm equal loading, the blots, probed previously for GFP, were glove cabinet and keeping tubes tightly closed outside the cabinet. briefly washed, re-blocked, and probed with anti-actin monoclonal antibodies The media pH was measured in air (21% O2) and in anoxia (0% O2) and (1:1000; Sigma Chemical Co.). found to be the same (pH 7.5). Competitive RT-PCR. Total RNA was extracted from cells using RNAzol DNA Constructs. All DNA manipulations were performed according to B (Biogenesis, Ltd, Poole, Dorset, UK), according to manufacturer’s instruc- standard procedures (27) using restriction enzymes, T4 DNA ligase, Mung tions. A glycogen solution (Rosche) added
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