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

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 to the samples at the concentration Bean nuclease, and buffers according to manufacturer’s instructions (Life of 20 ␮g/ml was used to enhance the precipitation of RNA in isopropanol. Technologies, Inc., Rosche and New England BioLabs, Herts, UK). Possible residual genomic DNA contamination was removed by RNase-free A modified version of the plasmid pd2EGFP-N1 (Clontech Laboratories, DNaseI treatment (Sigma Chemical Co.), whereas proteins were removed by Basingstoke, UK), named pCONGFP, containing the 9-27 gene promoter (a phenol: chloroform: isoamyl alcohol extraction (Life Technologies, Inc.). RNA low expressing, physiologically relevant promoter; Ref. 28) instead of the concentration and purity were estimated by reading the absorbance at 260 and CMV promoter was constructed as follows. The CMV promoter was removed 280 nm; RNA integrity was demonstrated by 1% agarose gel electrophoresis. from pd2EGFP-N1 by AsnI-NheI restriction, followed by Mung Bean nuclease First-strand cDNAs were synthesized from total RNA preparations using ⌬ – digestion and ligation to produce p CMVd2EGFP-N1. The plasmid pDW9- SuperScript II RNase H reverse transcriptase and Oligo (dT)12–18 Primer (Life 27CD2 (kindly provided by Dr. G. Stark) was linearized with HindIII and Technologies, Inc.), according to the manufacturer’s instructions. A reaction blunted, and the 9-27 gene promoter was excised by EcoRI digestion. This without reverse transcriptase was performed to control for genomic DNA fragment was ligated to the p⌬CMVd2EGFP-N1, which had been restricted contamination. All PCR amplifications were performed using Reddy-Load with XhoI, blunted, and EcoRI digested to produce pCONGFP. PCR Mix (Advanced Biotechnologies, Ltd., Epsom, UK) in a Mastercycler A competitor DNA template, to be used in measurements of steady-state Gradient Thermo cycler (Eppendorf, Cambridge, UK). Primers were custom levels of d2EGFP mRNA by competitive RT-PCR (see description below) was made by Life Technologies, Inc. For competitive PCR, a fixed amount of constructed by excision of a 32-bp fragment from pd2EGFP-N1 by BcgI cDNA was mixed with serial dilutions of competitor DNA and subjected to digestion, followed by Mung Bean nuclease treatment and religation. PCR amplifications using the following primers for d2EGFP: forward, 5Ј CGA Cell Transfection. Transfection was performed according to the method CGT AAA CGG CCA CAA GTT CAG 3Ј and reverse, 5Ј GTC CTC CTT GAA Ј described by Hart et al. (29). Briefly, T24 cells at 5 ϫ 104 cells/well were GTC GAT GCC CTT 3 at 0.5 ␮M each. transfected in air in 24-well plates for 5 h with complexes containing, at a ratio The expected product sizes were 339 bp for the endogenous d2EGFP and of 0.75:4:1 by weight, lipofectin reagent (Life Technologies, Inc.), integrin- 303 bp for the competitor DNA. The following thermocycling conditions were binding peptide (Institute of Child Health, London), and plasmid DNA (1 ␮g used: 94°C for 5 min, followed by 30 cycles of amplification consisting of of DNA/well final concentration of pd2EGFP-N1, p⌬CMVd2EGFP-N1, or 94°C for 1 min, 48°C for 1 min, 72°C for 1 min, and a final 72°C extension pCONGFP) in OptiMEM (Life Technologies, Inc.). for 10 min. Aliquots of PCR reactions were electrophoresed through an Transfected cells were maintained in complete DMEM containing Geneticin ethidium bromide-stained 4% MetaPhor Agarose gel (FMC Bioproduct, Rock- (G-418 sulfate; Life Technologies, Inc.) at the concentration of 0.5 mg/ml land, ME) in 1 ϫ Tris-Acetate-EDTA. The bands were analyzed by densitom- active drug. After 30–60 days from transfection, G-418 resistant clones were etry using Visilog software (Noesis). tested for the expression of the reporter protein d2EGFP (excitation maximum The “house-keeping gene” ␤-actin was amplified as a control using the 488 nm, emission maximum 507 nm) by FACS analysis (FACScan; Becton following primers: forward, 5Ј TCA TCA CCA TTG GCA ATG AG 3Ј and Dickinson, Cowley, Oxfordshire, UK). Cells were scored positive if they reverse,5Ј CAC TGT GTT GGC GTA CAG GT 3Ј. The thermocycling and showed fluorescence above controls transfected with p⌬CMVd2EGFP-N1. In electrophoresis conditions used were the same as those described for the particular, a clone, stably transfected with pCONGFP and expressing amplification of d2EGFP. The expected product sizes for ␤-actin of 250 bp d2EGFP, was selected and named C17. (template: genomic DNA) and 155 bp (template: cDNA) were detected (results Experimental Conditions in Vitro. C17 stable transfectants and T24 un- not shown). transfected controls were plated in 6-cm Permanox dishes at 105 or 2.5 ϫ 105 Several technical challenges were encountered when optimizing the elec- cells/dish, allowed to attach for 24 h in air, and then, after replacement of the trophoretic separation of competitive PCR products. Good resolution and oxygenated media with preconditioned anoxic one, either: (a) exposed for 16 h separation between bands is essential to analyze band intensity by densitom- to different oxygen conditions (Figs. 1 and 2); (b) exposed for 0–24 h to etry. Separation of the competitor band (303 bp) from the endogenous d2EGFP normoxia or anoxia and then reoxygenated for up to 24 h (Fig. 3); or (c) treated test band (339 bp) on an agarose gel, even at the high percentages of 2.5–3%, with cycloheximide in normoxia, anoxia, or during reoxygenation (Fig. 4). was found to be insufficient. The use of 4% MetaPhor gel improved separation Green fluorescence was monitored by FACS analysis, protein levels were and resolution of the bands but also revealed a third band situated between the monitored by Western blot analysis, and d2EGFP mRNA levels were moni- test and the competitor band. This band has been demonstrated to be a hybrid tored by competitive RT-PCR. consisting of a mixture of test and competitor DNA by several means: (a) FACS Analysis. Green fluorescence was monitored by FACS analysis, and alkaline denaturing 4% MetaPhor gel electrophoresis eliminated the hybrid the results were recorded as means of the main GFP peak. Fluorescence, (but reduced resolution compared with a native gel, results not shown) and (b) expressed in arbitrary units, was recorded as the ratio of the fluorescent signal when PCRs were carried out separately on the test and on the competitor produced by transfected cells compared with untransfected cells. On the samples, they produced the expected bands of 339 and 303 bp; when these fluorescence scale, untransfected T24 cells have a fluorescence of 10 units, were mixed, subjected to a total protein phenol extraction (to remove Taq whereas T24 cells transfected with promoter-less constructs showed a range polymerase from previous PCR) followed by a final PCR cycle, a hybrid band between 9 and 20 units. of the predicted size was detected on 4% MetaPhor gel (results not shown). It Western Blot Analysis. Western blot analysis was performed essentially is therefore clear that in competitive PCRs, where PCR products of very as described previously (30). Cell monolayers were lysed in Triton X-100 lysis similar sequences are generated, the appearance of a hybrid band on high buffer, and equal quantities of proteins from total cell lysates were separated separation matrice gel electrophoresis cannot be eliminated. Because the extra by SDS-PAGE (12% polyacrylamide gels; Invitrogen-Novex, Groningen, band is necessarily a 1:1 hybrid of test and competitor DNA, which removes Netherlands). Proteins were then transferred to nitrocellulose membranes an equal amount of each pool (test and competitor), the two bands representing 4785

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. GFP AS A REPORTER IN HYPOXIA these two species can be compared with each other (quantified) while ignoring the hybrid. Formula used for quantification: Number of copies of d2EGFP mRNA/cell ϭ [␮g total RNA/␮g total RNA used for RT] ϫ 20*ϫ [number of copies of competitor equal to cDNA**/ number of cells used in extraction] * 1/20th of total cDNA was used for competitive PCR ** determined from MetaPhor gel Mice and Tumors. Female severely combined immunodeficient mice from our pathogen-free colony were used. T24 tumors were initiated by s.c. injection of 106 viable and stably transfected T24 cells, highly expressing GFP (under the transcriptional control of the strong CMV promoter, pEGFP-N1; Clontech Laboratories), in 0.1 ml DMEM under the skin on the left flank. Mice were used for experiments at 30–40 days after inoculation, when tumors reached ϳ40 mm3 in volume, as described below. Treatment protocols were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and with approval from the Ethical Review Committee of the Gray Laboratory Cancer Research Trust. Experimental Protocol in Vivo. Immunohistochemical detection of hypoxia in the tumors was performed using the hypoxia marker pimonidazole (31). Pimonidazole (Hypoxyprobe-1; Natural Pharmacia International, Inc., Belmont, MA) was injected i.p. (60 mg/kg in 0.2 ml PBS) into tumor-bearing mice. Tumors were excised 90 min thereafter, frozen, and stored at Ϫ80°C for cryosectioning. Sections (10 ␮m) were cut at three different levels throughout the tumor. An epifluorescence microscope (Eclipse TE200; Nikon United Kingdom, Ltd., Kingston Upon Thames, UK) with a narrow band filter (500–510 nm) and equipped with a custom made imaging system was used to visualize GFP fluorescence. Tumor sections were subsequently fixed in cold acetone for 10 min, stained for pimonidazole according to manufacturer’s instructions, and viewed under transmitted light. Fig. 1. a, effect of different oxygen tensions on GFP fluorescence produced in stably Statistical Analysis. Significance tests were carried out on the data groups transfected cells (clone C17). The means of at least five independent experiments Ϯ SE using ANOVA followed by the Student t test for individual pairwise compar- are shown. Fluorescence of cells under catalyst-induced anoxia is significantly reduced ءء Ͻ isons, with values of P Ͻ 0.01 considered as significant. (P 0.01) compared with all other conditions ( ); under physiological O2 tension and in hyperoxia it is significantly increased (P Ͻ 0.01) compared with normoxic condition .On this fluorescence scale, untransfected T24 cells have a fluorescence of 10 units .(ء) RESULTS ----, the level of fluorescence in normoxia. In b, different protein levels of d2EGFP (31 kDa) were detected under a range of oxygenation conditions by Western blot analysis. N, Effect of Different Oxygen Tensions on GFP Fluorescence, P, H, and A, normoxia (21% O2), physiological O2 (2% O2), hypoxia (0.1% O2), and anoxia (0% O2), respectively. Immunoblotting for actin (42 kDa) was used to confirm Protein, and mRNA Levels. GFP fluorescence in human bladder equal loading of total proteins. carcinoma cells, T24 stably transfected with pCONGFP (clone C17), was analyzed under a variety of oxygen tensions, ranging from anoxia

(catalyst-induced anoxia and gassing with 95% N2 and 5% CO2) blot analysis (Fig. 1b). A band of about 31 kDa corresponding to the through hypoxia (0.02, 0.1, 0.3, and 1% O2) to physiological oxygen d2EGFP was detected. A band of 42 kDa, corresponding to the conditions (2 and 5% O2), normoxia (21% O2), and finally, hyperoxia internal control actin, was present at similar intensity in all conditions (95% O2; Fig. 1a). Green fluorescence was quantified by FACS analyzed, confirming that equal amounts of total proteins had been analysis, and results were recorded as fluorescence units compared loaded. Quantification of chemiluminescent signal by scanning den- with untransfected controls (which have been demonstrated to have a sitometry showed similar GFP protein levels under physiological similar background fluorescence as cells stably transfected with the oxygenation compared with air (88% of normoxic value). GFP was promoter-less construct, p⌬CMVd2EGFP-N1). Cells under physio- reduced under hypoxia (41% of normoxic value), and little or no logical oxygen tensions and hyperoxic conditions showed signifi- protein was detected under anoxia. cantly increased fluorescence up to 161–174% of normoxia. This To analyze if the reduced protein levels were attributable to a result was unexpected and suggests that several competing factors reduction in the GFP mRNA pool, competitive RT-PCR studies were determine net fluorescence through this oxygen range (see “Discus- carried out in parallel to the protein assays. No significant differences sion”). Cells under hypoxia showed fluorescence similar to that of in the steady-state levels of d2EGFP mRNAs were found under the cells under normoxia, except for 0.02% O2 where significantly re- four oxygen conditions analyzed (Fig. 2). duced fluorescence was observed (P Ͻ 0.01). Severe catalyst-induced Growth characteristics recorded after 16 h incubation under the four anoxia reduced GFP fluorescence significantly (33% of normoxia) different oxygen conditions showed a reduction of viable cells under compared with all other oxygenation conditions (P Ͻ 0.01). Nor- hypoxia (73%) and anoxia (52%) compared with air (defined as moxic fluorescence was less than both physiological and hyperoxic 100%), with no difference under physiological conditions. The reduc- conditions. tion in cell numbers was attributable to a reduction in growth rather To assess whether the variations in fluorescence observed under than an increase in cell death. C17 cells showed a total cessation of different oxygen tensions were because of changes in protein expres- growth under anoxia, with anoxia-related cell death up to 13% after sion or fluorophore formation, Western blot analysis was performed. 31 h under anoxia (results not shown). Four different oxygen conditions were investigated: catalyst-induced GFP Fluorescence Over Time in Normoxia and Anoxia. The anoxia (0% O2), hypoxia (0.1% O2), physiological O2 (2% O2), and effect of oxygen deprivation and reoxygenation on GFP fluorescence normoxia (21% O2). C17 cells were exposed for 16 h to the different was analyzed over time. Preplated C17 cells were exposed to nor- oxygenation conditions, and whole cell lysates were used for Western moxic or anoxic conditions, and the green fluorescent signal was 4786

reoxygenated in the presence of cycloheximide, they showed a rapid but transient increase in fluorescence (Fig. 4b). It is unlikely that this is because of a new protein synthesis and may therefore be indicative of a small pool of noncyclised GFP. Cells under long-term anoxia (after 16 h of anoxia) treated with cycloheximide showed no further reduction in fluorescence (Fig. 4b). GFP Fluorescence and Hypoxia in Solid Tumors. To determine the correlation between GFP fluorescence and hypoxia in solid tumors, seven s.c. T24 tumors initiated by injection of stably transfected T24 cells expressing GFP were analyzed for green fluorescence and assessed for hypoxia using the hypoxia marker pimonidazole. Distri-

Fig. 2. Competitive RT-PCR was used to quantify mRNA levels of d2EGFP in C17 cells under different oxygen conditions. In a, a representative ethidium bromide-stained equal number of copies ,ء MetaPhor gel of a competitive PCR reaction is shown. White of competitor molecules (303 bp) and d2EGFP cDNA molecules (339 bp). Lanes 1–9 of each normoxia (N), physiological O2 (P), hypoxia (H), and anoxia (A) correspond to the following numbers of copies of competitor molecules used in the competition: 5 ϫ 105; 4 ϫ 105;3ϫ 105; 2.5 ϫ 105;2ϫ 105; 1.5 ϫ 105;105; 7.5 ϫ 104; and 5 ϫ 104, respectively. M, the marker lane (100 bp ladder). In b, the number of copies of d2EGFP mRNA per cell were calculated from the competitive RT-PCR reactions. No significant differences in the steady-state levels of d2EGFP mRNA were detected under the oxygen conditions analyzed. Means of at least three independent experiments Ϯ SE are shown. monitored by FACS analysis every 2 h for up to 12 h of incubation and at 24 h (Fig. 3a). No significant fluctuations in fluorescence were detected in normoxia over the period analyzed. However, cells incubated in anoxia showed, after an initial 2-h delay, a significant reduction of fluorescence over the first8htobackground levels. It is known that in a cell-free system maintained in air at room temperature, a synthetic GFP precursor molecule rearranges to form an active fluorophore able to emit green fluorescence under near-UV light in ϳ5 h (32). To observe the rate of fluorescence recovery, C17 cells were moved from long-term anoxia to air, and fluorescence was monitored by FACS analysis (Fig. 3, a and b). Results showed that 5–10 h of reoxygenation were necessary for cells to recover to the levels of fluorescence present in cells constantly maintained in normoxia. Parallel Western blot analysis showed an increase in GFP Fig. 3. GFP fluorescence over time in stable transfectants (C17) in normoxia, anoxia, protein associated with reoxygenation (Fig. 3c). and during reoxygenation. After 4–8 h of incubation under severe catalyst-induced C17 cells incubated in the presence of cycloheximide to inhibit anoxia, the green fluorescent signal reduced to background levels (hatched area) and Ϯ protein synthesis in normoxia showed a loss of fluorescence over recovered within 5–10 h (a). Means of at least three independent experiments SE are shown. A representative FACS assay of reoxygenation is shown in b. Fluorescence of C17 time, which paralleled the loss of fluorescence in cells under anoxia cells in air (solid histogram) and C17 during reoxygenation (open histogram) is shown. (Fig. 4a). Cells placed under anoxic conditions while being treated The recovery of fluorescence during reoxygenation is mirrored at the protein level as detected by Western blot analysis (c). Actin stain was used as a loading control. Lanes 1–5 with cycloheximide showed a similar rate of loss of fluorescence, correspond to proteins extracted at different times during reoxygenation (0, 2.5, 5, 8, and showing that the treatments were not additive. When anoxic cells were 24 h) and Lanes 6–9 during normoxia (0, 2.5, 8, and 24 h). 4787

and posttranslational modifications, which all contribute to the resulting emission of green fluorescence. For gene regulation studies, conclusions about the rate of transcription are drawn by analyzing the strength of the fluorescence signal. Only by the careful analysis presented here could one possibly approach this difficult approx- imation. It is known that the formation of the GFP final fluorophore, a posttranslational modification resulting from spontaneous cyclization

and oxidation of the protein’s amino acids -Ser65 (or Thr65)-Tyr66- Gly67-, requires molecular oxygen (14). Our data now provides in- formation on the amount of oxygen required to visualize the fluorophore in live cells. A general dose response to oxygenation was found, where higher oxygen concentrations resulted in higher fluorescence and lower concentrations resulted in reduced fluorescence (Fig. 1a), but statistical analysis showed that only catalyst-induced anoxia resulted in a significant reduction of fluorescence signal compared with

all other oxygenation conditions. However, normoxia (21% O2)isnot a realistic oxygen concentration under physiological conditions,

where median pO2 values range from 24–66 mmHg (3.1–8.7% O2; Ref. 33). Importantly for in vivo studies, fluorescence under radiobi- Ͻ ological hypoxic conditions ( 0.3% O2) was significantly lower (by more than 50%) compared with physiological conditions (2 and 5%

O2). Normoxic fluorescence is lower than the signal detected under both physiological and hyperoxic conditions, the reason for which remains unclear. It may be possible that the reasons for increased fluorescence compared with normoxia may be different, in that physiological oxygen concentrations may be optimal for fluorescence, whereas hyperoxia may induce a stress response resulting in increased fluorescence. Additionally, although Western analysis showed that hypoxia and anoxia reduced GFP protein levels, physiological conditions did not increase the levels compared with normoxia, which does not correlate with the fluorescence data (Fig. 1b). The variation in protein level with oxygenation is likely to depend on both the rate of translation (which is known to be reduced under severe hypoxia, Ref. 34), as well as protein half-life. The destabilized GFP used in the in vitro experiments described here has the mouse ornithine decarbox- ylase amino acid residues 422–461, which contain a Proline, Glu-

Fig. 4. Cycloheximide treatment of stable transfectants (C17) in normoxia, anoxia, and tamic Acid, Serine, Threonine (PEST) amino acid sequence targeting during reoxygenation. In a, cycloheximide treatment inhibited GFP de novo synthesis proteins for degradation (12) fused to the COOH terminus. The equally in normoxia and anoxia (solid symbols); in b, cycloheximide treatment of cycloheximide results presented here (Fig. 4a) showed that a similar long-term anoxic cells did not further reduce fluorescence, but reoxygenation in the presence of cycloheximide resulted in a transient increase in fluorescence (solid symbols). rate of protein degradation occurred in air and anoxia, indicating that The fluorescence in air, anoxia, and reoxygenation is represented as reference (open reduced protein half-life may not be the main reason for the loss of symbols). Background fluorescence levels are indicated as a hatched area. The means of GFP under anoxia. The variation in fluorescence and protein levels three independent experiments Ϯ SE are shown. appears not to be attributable to variations in the GFP mRNA pool. Using competitive RT-PCRs, no significant difference in the GFP bution and intensity of GFP fluorescence differed both between tu- species (14–21 copies per cell) between different oxygenation con- mors and within a tumor, suggesting different levels of GFP expres- ditions was detected (Fig. 2). sion in different tumor areas (Fig. 5, a and c). Subsequent staining of Interestingly, a transient increase in fluorescence was detected the same tumor sections for pimonidazole showed that staining for when cells were treated with cycloheximide during reoxygenation pimonidazole was also heterogenous (Fig. 5, b and d). Although some (Fig. 4b). This may indicate the build-up of a small pool of nonfluo- tumors showed diminished fluorescence in areas of pimonidazole rescent protein under anoxia, which only requires oxygen to form the staining (Fig. 5, a and b), others showed GFP fluorescence overlap- fluorophore. However, nonfluorescent GFP appears not to be the main ping into areas of hypoxia (Fig. 5, c and d). reason for a lack of fluorescence under anoxia, because Western analysis showed a significant loss of protein as well (Figs. 1b and 3c). DISCUSSION It should be noted that the limit of detection for GFP using Western analysis might differ from the level of detection by FACS analysis. An Our study reports the detailed studies of the effect of simulated alternative explanation for the transient increase in fluorescence dur- tumor conditions on the reporter GFP. It questions the feasibility of ing cycloheximide-treated reoxygenation may be a delay in the inhi- using GFP for gene regulation studies in an in vivo system. Our bition of protein synthesis. This appears unlikely judging from the findings identify several important factors that need to be considered immediate effect seen in air and anoxia (Fig. 4a). when analyzing gene expression by monitoring GFP fluorescence. For this in vitro analysis, a physiologically relevant promoter was Final fluorescence is used as a surrogate marker for a long and chosen rather than a strong viral promoter such as CMV, which may complex pathway starting with the rate of transcription, translation, have saturated the fluorescence signal. The 9-27 promoter was used 4788

Fig. 5. Representative images of T24 tumors, stably expressing GFP (a and c) and pimonidazole staining (brown areas) of the same tumor sections (b and d). Dis- tribution and intensity of GFP fluorescence were different between tumors and also heterogeneous within a tumor, indicating different levels of GFP expression in various tumor areas. Some tumors showed diminished fluorescence in areas of pimonidazole staining (a and b), whereas others showed GFP fluorescence overlapping into areas of hypoxia (c and d). Scale bar: 200 ␮m.

previously as the backbone for the insertion of hypoxia-regulated In addition, our preliminary in vivo results demonstrate that distri- elements in a feasibility study of transcriptional regulation for gene bution of GFP is heterogeneous throughout the tumor, suggesting therapy (23). In the previous study, as well as the current one, the 9-27 different levels of GFP expression in different tumor areas. The promoter demonstrated low transcriptional activity and did not re- heterogeneity of GFP expression appeared to correlate with pimonida- spond to hypoxic conditions. These qualities make it a good candidate zole staining in some tumors, but not in others, implying that GFP can for further optimization of gene therapy constructs and for gene still be detected in oxygen conditions that are sufficient to bioreduc- expression studies under physiological conditions. Fluorescence mi- tively activate pimonidazole. On the other hand, GFP expression was croscopy requires a much higher level of expression of GFP than the absent in areas that were not positive for pimonidazole staining. 9-27 promoter permitted. Hence, in the in vivo work described here, Therefore, factors other than hypoxia may influence GFP expression, we have used a CMV promoter to drive GFP synthesis in the T24 such as tumor energy status, tumor infiltration by normal cells, and tumor. Using this promoter, GFP fluorescence was visible in all genetic instability of some stably transfected cells, resulting in loss of analyzed tumors. However, the distribution and intensity of fluores- functional incorporation of the GFP gene. In addition, tumor cells will cence differed between tumors and within a tumor, suggesting differ- be in different cell cycle phases and may therefore exhibit different ent levels of GFP expression in different tumor areas. levels of GFP. All these issues further support our concern that the use Reporter genes other than GFP were considered for this study. of GFP as a reporter gene for real-time in vivo studies of gene ␤ Other genetic reporters in general use, such as luciferase, -galacto- regulation is limited. sidase, and chloramphenicol transferase, are enzymes and thereby In conclusion, it appears doubtful that GFP could be used effec- ␤ require substrates, such as luciferin, O-nitrophenyl- -D-galactopyr- tively as a reporter gene for real-time in vivo studies of gene regula- anoside, and chloramphenicol, respectively, to produce detectable tion in tumors. Specifically: (a) cellular GFP fluorescence reduces products (27). This makes their use as real-time reporters for gene with reduced oxygenation; (b) fluorophore formation, depending on expression in vivo more difficult. However, apart from GFP, lucifer- the variant used, was shown to take from 1–5 h (14, 40), and GFP ase is currently the only other marker gene used for real-time live fluorescence in our system was regained over 5–10 h of reoxygen- imaging (35, 36). Luciferase was therefore considered carefully. ation; (c) even the PEST-destabilized version of GFP has a half-life Luciferase is a monomeric protein that does not require posttrans- which is unsuitable to detect rapid changes in gene expression; and (d) lational modification, and therefore, its activity can be assessed im- low oxygen reduced GFP protein levels significantly. Therefore, GFP mediately after translation (37). Although luciferase has successfully should be used with great care as a reporter of gene expression under been used in vivo (36), it has added complications for use under tumor tumor conditions. Still, GFP is a clearly good marker gene to study conditions. Luciferase requires molecular oxygen and ATP at the time location and movement of proteins or cells and can be used effectively of activation of luciferin to produce luminescence, both of which are in normal tissues under physiological oxygenation conditions. in limited supply in most solid tumors (38). The use of luciferase for gene regulation studies involving cyclic AMP-modulating agents has been questioned in the past (39). It was shown that cyclic AMP- ACKNOWLEDGMENTS elevating agents could lead to a significant change in luciferase activity independently of a transcriptional activation of promoter We thank Mick Woodcock for help with FACS, Kevin Prise for assistance elements. It appears, therefore, that the modified GFP used in this with Visilog software, George Wilson and Frances Daley for help with the study, with its shortcomings, is still superior to other reporter genes pimonidazole experiments, Gemma Lewis for contribution to the in vivo currently available. experiments, and Richard Foxon for critical discussions. 4789

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Claudia Coralli, Maja Cemazar, Chryso Kanthou, et al.

Cancer Res 2001;61:4784-4790.

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