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Basic Science Investigations |
1 Department of Radiation Oncology, University of Washington School of Medicine, Seattle, Washington
2 Department of Radiology, University of Washington School of Medicine, Seattle, Washington
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: cell proliferation • fluorothymidine • 18F • thymidine kinase • tumors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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FLT was evaluated as a chemotherapeutic agent for the treatment of leukemia (14), but researchers soon discovered that this new antimetabolite had exceptional anti-HIV activity, possibly superior to that of azidothymidine (15). However, toxicity observed during phase II clinical trials with FLT (16) deterred investigations with pharmacologic doses of drug. Nonetheless, the successful labeling of FLT with 18F (17) led to a novel proposal to use 18F-FLT as a radiopharmaceutical for monitoring HIV infection. Although this application was never pursued in animal models, the concept formed the basis for imaging cell proliferation in tumors.
Several aspects of FLT biochemistry suggested that it would be a useful PET tracer for imaging cell proliferation in vivo (11,18–20). FLT is resistant to catabolism by thymidine phosphorylase in vivo and is specifically phosphorylated in cells by cytosolic thymidine kinase-1 (TK1). TK1 is a principal enzyme in the DNA-salvage pathway in cells (18), one that is strongly regulated by the cell cycle and is selectively upregulated just before and during the S phase (18–21). In contrast, FLT is a poor substrate for mitochondrial thymidine kinase-2 (TK2).
These features motivated us to investigate FLT labeling with high-specific-activity 18F for imaging tumors with PET. The availability of an in vivo tracer of the DNA-salvage pathway that is convenient, sensitive, accurate, and catabolized less than thymidine could have an important impact on the way tumors are diagnosed and assessed as responsive in clinical situations (2,11). Currently, several groups are imaging tumors with 18F-FLT (22–25), but the mechanism of its retention in cells has not been validated. Some investigators have suggested a key role for TK1, but other than preliminary work reported by our research group, this has not yet been demonstrated.
To interpret 18F-FLT images as measures of cell proliferation, we need to understand the mechanisms that account for its accumulation and retention in tissues. Our first step was to determine how well TK1 activity accounted for FLT uptake in cultured human cells under a variety of growth conditions ranging from active growth to growth arrest caused by radiation or by nutrient deprivation. Radiation was a particularly useful tool for these studies because it causes a dose-dependent delay in progression through the cell cycle of all mammalian cells, provided they retain, as do A549 lung carcinoma cells, a functional p53 tumor suppressor gene and the associated normal cell cycle checkpoints (26). However, this delaying effect of radiation can be eliminated by selected genetic manipulations that abrogate function of the p53 tumor suppressor gene (27). These manipulations of p53 function mimic changes occurring in some cells as they progress to malignancy and were a convenient additional way to investigate the link between cell growth, FLT uptake, and TK1 activity.
In this article, we report a strong positive correlation between uptake of radiolabeled FLT and levels of TK1 in A549 lung carcinoma cells in vitro. We also demonstrate that converting cells from growth arrest to a high growth rate consistently leads to elevated TK1 levels, accompanied by increased FLT uptake. Taken together, these data validate 18F-FLT as a useful imaging agent for cell proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Cell Culture Systems
A549 human lung adenocarcinoma cells were used for these studies and also as the parental cell line to construct isogenic cell lines that differ in p53 gene expression. The parent A549 cell line has wild-type p53 gene function (28). A549 cells were transduced with the retrovirus vector LXSN, which contains a selectable gene for resistance to neomycin, to introduce the human papillomavirus (HPV) type 16 E6 gene, as described previously (27), and were designated A549-E6. Controls, designated A549-LXSN, received vector only. Cells expressing E6 (A549-E6) have a functionally inactive p53 gene product and lose the G1 checkpoint control necessary for radiation-induced cell cycle arrest (27). Cells containing the vector alone (A549-LXSN) retain their p53-dependent G1 cell cycle checkpoint response. The parental cell line (A549), as well as A549-E6 and A549-LXSN, was used to examine uptake and retention of 3H-FLT and to determine thymidine kinase (TK) activity.
Cells were grown in 1:1 Dulbecco’s modified Eagle’s medium:F-12 containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin and were incubated at 37°C in an atmosphere of 95% air and 5% CO2. Cells were seeded at 2 x 105 cells in T25 flasks and grown for 6–8 d with no change of culture medium to reach an unfed plateau phase. Then, either the cells were released and reseeded into fresh medium at 2–3 x 106 cells per flask to reach exponential growth at the times specified for treatment, or else the cells were treated in this plateau phase.
For determining uptake of labeled FLT or DG under various conditions, culture dishes were removed from the incubator, growth medium was aspirated, and 1 mL of medium, containing 37 kBq/mL 3H-FLT or 18.5 kBq/mL 14C-DG, was used to rinse the culture medium from the monolayer of cells. Cells were incubated with 3 mL of medium containing 3H-FLT or 14C-DG. Alcohol carryover from 3H-FLT stock solution was negligible (0.05%). Cells were labeled 6, 10, 12, 14, 16, and 24 h after subculturing into fresh medium and were compared with cells labeled without being subcultured (referred to as 0-h samples).
At each of these times in each experiment, 3 replicate samples were taken. Each point in Figures 1–4 is an average of these 3 samples. Radiolabeled drug was taken up by incubation for 1 h at 37°C in a humidified atmosphere containing 5% CO2.
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Cell Sizing and Calculation of Radiotracer Uptake
To determine cell size, dishes identical to the treated dishes, but without added radiotracer, were harvested by trypsin treatment at 1 and 8 d. This parallel procedure was necessary because radioactive cells could not be introduced into the cell counter or, later, into the flow cytometer used to determine S phase. Cells were suspended in Isotone (Coulter Corp., Luton, U.K.) at a concentration of between 2,000 and 5,000 cells per milliliter. Cell size was determined by methods adapted from the Coulter ZM reference manual, Fine Particle Calibration, using Coulter size standards as calibration standards. Volume for cells was determined using the formula 4/3
r3 = volume.
Cell sizing allowed us to calculate tracer uptake in cells relative to activity in culture medium. We first calculated the intracellular volume of 106 cells. We then determined dpm/ 106 cells and expressed the cellular uptake as a percentage of radioactivity in the same volume of medium. Uptake equal to 100% would represent simple equilibration with the medium, and values > 100% meant that the cells concentrated radioactivity to a level greater than that in the medium. This method of calculating uptake readily allows one to determine when uptake exceeds activity levels in the medium and thus represents concentration of the tracer. This method also corrects for the unavoidable variations in dpm/mL of medium that occur in pipetting very small aliquots of high-specific-activity stock solutions of 3H-FLT. Radioactivity in aliquots of high-specific-activity stock solution varied more than did cell size. Volumes of 1-d-old A549, A549-E6, and A549-LXSN cells differed from each other by ±8%. Volumes of 8-d-old A549, A549-E6, and A549-LXSN cells differed by ±14%. On average, 8-d-old cells had 65% of the volume of 1-d-old cells.
Irradiation Conditions
For determining the effects of radiation on TK activity and the uptake of FLT in A549 cells with and without normal p53 function, A549-E6 and A549-LXSN cells were allowed to reach an unfed plateau phase of growth, were removed from the incubator, and were irradiated (81-14 cesium irradiator; Shepard, Glendale, CA). A dose of 10 Gy was delivered using a source-to-dish distance of 46 cm and a dose rate of 1 Gy/min. Cells either were immediately harvested by trypsin treatment and seeded into fresh medium to reach exponential growth at the times specified for addition of radioactive tracers or else were labeled in this plateau phase.
Flow Cytometry Methods
For determining cell cycle distributions, dishes identical to the treated dishes, but without added radioactivity, were harvested by trypsin treatment and were suspended in 10 mg/mL 4'-6-diamidino-2-phenylindole (DAPI) with 10% dimethylsulfoxide and cryopreserved at -70°C. (As noted above, radioactive cells could not be introduced into the flow cytometer.) Samples were analyzed on an Epics Elite flow cytometer (Coulter Corp.) with ultraviolet excitation at 360 nm from an Innova-90-6 water-cooled argon-ion laser (Coherent Inc., Santa Clara, CA). DAPI fluorescence was collected with a 450-nm/35-nm band-pass emission filter. A minimum of 20,000 cells was analyzed per sample. The data were collected using Elite software (version 4.01; Coulter Corp.). The Multicycle-AV software package (Phoenix Flow Systems, San Diego, CA) was used for data analysis.
TK Assay
For determining TK1 activity, dishes of cells identical to the treated dishes, but without added radioactivity, were harvested by trypsin treatment. Cells were counted on a ZM analyzer (Coulter Corp.) and lysed by a method adapted from Sherley and Kelly (21). Protein content in the 100,000g supernate of the cell lysate was determined using a detergent-compatible protein assay (Bio-Rad Laboratories Inc., Hercules, CA). The high-speed supernate was prepared to eliminate mitochondria, the source of TK2. TK activity in the high-speed supernate was determined using a method adapted from Hruby and Ball (29). Briefly, high-specific-activity TdR (2,590–3,330 GBq/mmol) was provided in a reaction mixture consisting of Na2HPO4/ NaH2PO4 buffer, pH 6.0, supplemented with disodium adenosine triphosphate and magnesium acetate. TK, provided by the cell supernate, phosphorylated the TdR to thymidine monophosphate. This phosphorylated product was captured on cellulose paper disks. Nonphosphorylated TdR was rinsed from the filters, which were then dried and counted in a 1900CA liquid scintillation spectrophotometer in 5 mL of Ultima Gold. TK1 activity was expressed as picomoles of phosphorylated TdR/30 min/mg of protein in the supernate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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A549 cells became highly growth arrested after 8 d with no medium change. The percentage of cells in S phase typically ranged from 3% to 5%, whereas 91%–94% of the cells had a G1 DNA content. Cells began to move into S phase by 12 h and reached a maximum of 54% ± 11% by 24 h after subculturing (Fig. 1A). FLT uptake also increased steadily at times later than 6 h and by 24 h reached nearly 4-fold the levels in growth-arrested cells (Fig. 1B). FLT uptake correlated well (r2 = 0.91) with the increase in the percentage of cells in S phase (Fig. 1C). For these comparisons, FLT uptake per 106 cells was expressed relative to the activity in the culture medium containing 3H-FLT, as described in the Materials and Methods.
Levels of the cell cycle-regulated enzyme TK1 were hypothesized to determine FLT retention in cells, and so we also measured TK1 activity in A549 cells as a function of time after subculturing. TK1 activity averaged 124 pmol phosphorylated TdR/30 min/mg of protein in growth-arrested cells. The first obvious increase occurred after 10 h in fresh medium and reached a maximum by 24 h, averaging 1,875 ± 235 pmol phosphorylated TdR/30 min/mg of protein, a 15-fold increase compared with growth-arrested cells (Fig. 2A). Interestingly, the increase in enzyme activity at 10 h preceded any obvious upswing in S phase fraction, which did not occur until 12–14 h. FLT uptake and TK1 activity correlated well (r2 = 0.63; Fig. 2B).
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-rays immediately before being subcultured into fresh medium, the expected delay of cells moving into S phase was seen in the vector-only (LXSN) transfected cells (Fig. 4A). However, the E6 transfectants were not delayed by radiation and entered the cell cycle, with S phase fractions increasing from 3% to 5% in the growth-arrested cells to 40%–50% within 20–24 h after subculturing (Fig. 4A). Only after 44 h did the S phase fraction began to increase in irradiated LXSN controls with normal p53 function. The time course of increase in FLT uptake (Fig. 4B) paralleled that of increase in S phase fraction in irradiated A549-E6 cells. FLT uptake showed essentially no change between 0 and 28 h in A549-LXSN cells, which remained growth arrested because of the radiation treatment. The failure of irradiated A549-LXSN cells to begin cycling or take up more FLT after subculturing into fresh medium was, as expected, accompanied by unchanged TK1 activity, which remained at the low levels characteristic of growth-arrested cells (Fig. 4C). In the radiation-resistant A549-E6 cells, the highest TK1 activities were seen 28 h after irradiation and subculturing (Fig. 4C). The difference in TK1 activity between A549-E6 and A549-LXSN cells at 28 h (9.2-fold) was even greater than predicted by the greater FLT uptake in the E6 transfectants compared with vector-only LXSN controls (4.3-fold).
Comparison of 3H-FLT and 14C-DG
We also are interested in how FLT compares with 18F-FDG for monitoring changes in cell proliferation. Labeled FLT detects changes in proliferation that are underreported by the glucose metabolism tracer 14C-DG, a surrogate for 18F-FDG (Table 1). This experiment was similar to those described above for A549 cells, except that 6-d-old rather than 8-d-old growth-arrested cultures were used. Parallel series of cultures were labeled with 14C-DG for comparison with 3H-FLT. Placing growth-arrested A549 cells into fresh medium increased DG uptake no more than 1.8 times, far less than the increase seen in FLT uptake (6.4 times) when a large portion of cells move into S phase at 20 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The imaging research group at the University of Washington has pioneered development of 18F-FLT for imaging cell proliferation. FLT, structurally similar to the anti-AIDS drug azidothymidine, is believed to be retained in virus-free cells because of specific phosphorylation by the cell cycle-regulated enzyme, cytosolic TK1 (15,18). This enzyme normally functions in the nucleoside salvage pathway to convert thymidine to thymidine monophosphate, a necessary first step before its incorporation into DNA. FLT is a poor substrate for mitochondrial TK2 (18), making it a specific marker for cells that express high levels of TK1, even though only a small amount of FLT is incorporated into DNA (15). Thus, the metabolic trapping of 18F-FLT by phosphorylation with little further processing has been hypothesized as analogous to the trapping of 18F-FDG that is due to phosphorylation by hexokinase.
The correlation of TK1 enzyme activity with cell proliferation and cell cycle stage is well established for both nonmalignant cells and tumor cells (21). TK enzyme activity is typically low in the G0/ G1 stage of the cell cycle, increases in S phase, and in some cells also remains high through mitosis. Activity drops sharply as cells reenter G1 phase after cell division (21). Serum levels of TK, presumably released by cell lysis accompanying tumor expansion, have been used as a marker of tumor burden, tumor aggressiveness, or prognosis in patients with leukemias and lymphomas and selected solid tumors including those of the breast and prostate (30–34). Correlations of prognosis with cytosolic TK1 measured in tumor samples also have been reported (35). These observations, coupled with data showing that FLT is a far better substrate for TK1 than for TK2, suggested that FLT might be a specific tracer of proliferation with a defined mechanism of trapping in cells.
The results reported in this article support the hypothesis that TK1 activity is an important determinant of FLT uptake and retention in cells. We studied A549 lung carcinoma cells because they become markedly growth arrested (only 3%–5% of cells in DNA synthesis) when they reach a high density in culture with no medium change. These growth-arrested cells move semisynchronously into the cell cycle when subcultured into fresh medium, with the maximum percentage of the cells in DNA synthesis (S phase) at 24 h (54% ± 11%). Comparing the changes in the percentage of cells in S phase and in 3H-FLT uptake as a function of time after subculturing (Fig. 1) confirmed a close association between these 2 measurements but also showed that FLT uptake increased by 10 h, before the increase in the proportion of cells in DNA synthesis. This relationship, in which FLT uptake precedes cell cycle progression, would be expected if FLT uptake depended on activation of TK1, as the activity of this enzyme begins to increase before S phase, in late G1 (21). The time course of changes in TK1 activity (Fig. 2A) also demonstrated increased activity just before cells began to move into S phase (Fig. 1A). Overall, FLT uptake correlated well with TK1 activity (r2 = 0.63), as shown in Figure 2B, and also with S phase (r2 = 0.91; Fig. 1C).
A more rigorous test of the relationship between cell cycle progression, FLT uptake, and expression of TK1 activity came from the experiments in which we prevented the normal movement of growth-arrested A549 cells into active growth by irradiating them just before subculturing. A549 cells have normal p53 function (28), a condition necessary for radiation-induced growth arrest in the G1 phase of the cell cycle. We also examined the effect of abrogating p53 function in A549 cells by stably transfecting them with HPV type 16 protein E6, a manipulation shown to eliminate postradiation G1 arrest in this lung carcinoma cell line (27). Nonirradiated, growth-arrested control (A549-LXSN) and E6-transfected (A549-E6) cells behaved similarly to parental A549 cells when subcultured into fresh medium; that is, they showed increases in S phase fraction, FLT uptake, and TK1 activity as a function of time (Fig. 3). Radiation produced the expected growth delay in vector-only control cells (A549-LXSN) having normal p53 function, that is, no increase in S phase fraction up to 24 h after subculturing (Fig. 4A). This failure to proliferate was accompanied by sustained low levels of FLT uptake and TK1 activity, characteristic of growth-arrested cells (Fig. 4). The expected increase in all 3 parameters was seen in the E6 transfectants when placed into fresh medium (Fig. 4); these cells escape the radiation-induced G1 block in the cell cycle because they lack normal p53 surveillance mechanisms.
Rapid growth is a hallmark of aggressive tumors, but high TK1 levels may be associated with other facets of tumor aggressiveness. The inherent high mutation rate in tumor cells and the complex control of TK1 activity (20,21) suggest that in more aggressive tumors, TK1 levels may not be tightly coupled with cell cycle stage. Thus TK1-dependent FLT uptake may report tumor aggressiveness as well as cell proliferation. For example, mutated tumor cells may express TK1 in the G0/ G1 stage of the cell cycle when enzyme activity normally is low (36) and overexpression of TK1 mRNA (37) eliminates cell cycle-specific regulation of TK1 activity. In addition, the postmitotic degradation of TK1 protein, implicated in the rapid decline of enzyme activity in G1 immediately after cell division, can be eliminated by mutations affecting the final 40 amino acids of the enzyme chain without eliminating enzyme function (38). Finally, TK1 activity may be up to 5 times higher in actively growing tumor cells than in exponentially growing nontransformed cells (39).
18F-FDG has been more widely used than any other PET tracer, and so it is logical that researchers would investigate how well it correlates with tumor growth or reports changes in cell proliferation. Work with animal tumors and cultured cells suggested that labeled thymidine is a better indicator of cell proliferation. Nonetheless, 18F-FDG uptake is strongly correlated with number of viable cells (40). Our comparisons of 14C-DG, a surrogate for 18F-FDG, with 3H-FLT support the hypothesis that specific tracers of the DNA synthetic pathway such as 11C-TdR, 18F-FMAU, and 3H- or 18F-FLT give a more accurate picture of the time course of changes in cell proliferation or response to cytotoxic agents than do glucose analogs.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Janet S. Rasey, PhD, Radiation Oncology, University of Washington, Box 356043, Seattle, WA 98195.
E-mail: rasey{at}u.washington.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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