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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2127-2135
Copyright © 2004 by The Endocrine Society

Imatinib Mesylate (Gleevec; STI571) Monotherapy Is Ineffective in Suppressing Human Anaplastic Thyroid Carcinoma Cell Growth in Vitro

Joshua M. Dziba and Kenneth B. Ain

Thyroid Cancer Research Laboratory, Medical Service, Veterans Affairs Medical Center, Lexington, Kentucky 40511; and Department of Internal Medicine, University of Kentucky Medical Center, Lexington, Kentucky 40536-0298

Address all correspondence and requests for reprints to: Kenneth B. Ain, M.D., Thyroid Oncology Section, Division of Hematology and Oncology, Department of Internal Medicine, Room MN524, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536-0298. E-mail: kbain1{at}uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Imatinib mesylate is remarkably effective in treating chronic myeloid leukemia and metastatic gastrointestinal stromal tumors. Meanwhile, anaplastic thyroid carcinoma (ATC) remains a fatal malignancy for which there are currently no effective curative interventions. In chronic myeloid leukemia and gastrointestinal stromal tumors, imatinib inhibits the constitutive tyrosine kinase activity of BCR-ABL and c-KIT, respectively. Reports suggest that imatinib may also be effective against ABL and platelet-derived growth factor receptor kinase-dependent pathological conditions. These mechanisms provide a wide scope of possible clinical applications for the drug. Potentially, diseases instigated by constitutive kinase activity that can be inhibited with imatinib should be treatable with this drug. We evaluated the effects of imatinib on the viability, cycling, and tyrosine phosphorylation of ATC cells in vitro. Our data indicate that imatinib has negligible antineoplastic activity against ATC cell lines within established therapeutically useful concentrations. No constitutive kinase activity was detected in these cell lines that could be exploited as a therapeutic target by imatinib. We conclude that imatinib mesylate monotherapy would not be effective in ATC patients. Current preclinical data do not warrant future clinical studies of imatinib monotherapy for ATC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMATINIB MESYLATE (GLEEVEC; STI571) was first approved by the Food and Drug Administration (FDA) authority for treatment of the chronic phase of chronic myeloid leukemia (CML) and is currently being used as a first line of treatment for this disease (1). Imatinib is also approved for the treatment of gastrointestinal stromal tumors (GISTs) (2, 3, 4). Imatinib monotherapy has been highly effective in treating CML patients, with complete remission seen in most patients in the early stages of the disease (5), whereas those in the later stage of the disease respond but eventually relapse. The success of imatinib derives both from the complete remission of CML in a large percentage of the patients treated and also from the remarkably limited side effects in the patients (6, 7, 8).

The successful treatment of CML and GISTs as well as lymphoblastic leukemia (6) with imatinib suggests that this drug may have a wide application in treating other cancers such as anaplastic thyroid carcinoma (ATC) that remain incurable. Indeed, at least one study has suggested that imatinib inhibits the growth of anaplastic thyroid cancer cells in vitro, as well as in a mouse xenograft model (9). Positive findings from preclinical in vitro and xenograft studies provide validation for the pursuit of riskier and more costly clinical studies in humans and would be particularly welcome in ATC, because this is a rapidly fatal malignancy for which there are no effective curative modalities (10). To evaluate the possible role of imatinib as a therapy for ATC, we initiated in vitro studies on its effect on human ATC cell lines early in 2002. Data obtained since the initiation of our studies suggested that imatinib had no toxic effects on ATC cell lines within clinically relevant dosages. The publication of a study suggesting that the drug inhibited the growth of ATC cell lines (including one cell line we had tested) prompted us to further analyze the effects of imatinib mesylate against a large panel of ATC cell lines (9). We investigated the antineoplastic properties of imatinib monotherapy against nine such cell lines (ARO-81, BHT-101, C643, DRO-90, KAT-4, KAT-18, SW1736, HTh-7, and HTh-74) using standard effective methodologies (11). The effects of imatinib against the ATC cell lines were compared with its activity against the K562 cell line, which naturally expresses BCR-ABL, and provided a positive control as an imatinib-responsive cell line (12, 13, 14, 15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Imatinib was provided by Novartis Pharmaceuticals Corp. (East Hanover, NJ) through the Cancer Therapy Evaluation Program of the National Cancer Institute, National Institutes of Health (Bethesda, MD). The drug was dissolved in sterile distilled water at 10 mM stock. Reagents for traditional PCR were obtained from Invitrogen Life Technologies (Chicago, IL), whereas TaqMan real-time PCR primers were obtained from Applied Biosystems (Foster City, CA). Actin (I-19), p-Tyr (PY99) antiphosphotyrosine, and the horseradish peroxidase (HRP)-conjugated goat antimouse (sc-2005) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti c-Abl and anti-phospho-c-Abl (Tyr245) antibodies came from Cell Signaling Technology (Beverly, MA). The HRP-conjugated antirabbit IgG was from Pierce Biotechnology (Rockford, IL). RNase A and protease inhibitor cocktail tablets (Complete Mini, EDTA free) were obtained from Roche Molecular Biochemicals (Roche Applied Science, Indianapolis, IN). All other analytical grade reagents were purchased from Sigma Chemical Co. (St. Louis, MO) with the exception of fetal bovine serum, PBS, Hanks’ buffered salt solution, RPMI 1640 medium, nonessential amino acids, sodium pyruvate, and trypsin 0.25% EDTA, which were purchased from Life Technologies, Inc./Life Technologies (Grand Island, NY).

Cell lines

Dr. G. F. J. Juillard (Department of Radiation Oncology, University of California-Los Angeles School of Medicine, Los Angeles, CA) provided ATC cell lines ARO-81 and DRO-90. ATC cell line BHT-101 was provided by Dr. I. Pályi (National Institute of Oncology, Budapest, Hungary) (16), whereas SW1736 was developed by Leibowitz and McCombs III at the Scott and White Memorial Hospital (Temple, TX) in 1977 and provided by Dr. Nils-Erik Heldin (Uppsala University, Uppsala, Sweden). ATC cell lines C643, HTh-7, and HTh-74 were also provided by Dr. Heldin (17). K562, a BCR-ABL-expressing cell line, was derived from a CML patient in blast crisis by Dr. Lozzio (13) and obtained from Dr. E. A. Hirschowitz at the University of Kentucky (Lexington, KY). U-1242 MG is a human astrocytoma cell line, used as a positive control for platelet-derived growth factor-ß receptor (PDGFR-ß) expression (18) and obtained from Dr. Hany Saqr of Ohio State University (Columbus, OH). KAT-4 and KAT-18 are ATC cell lines, which were developed by our laboratory. KAT-210 tissue was obtained from normal human thyroid derived from a 58-yr-old white female under an Institutional Review Board-approved protocol.

Cell culture conditions

Cultured cells were maintained in 75-cm2 culture flasks from Sarstedt (Newton, NC) in an atmosphere of 5% CO2 and 95% humidity and at 37 C. All cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate. Cytotoxicity assays were conducted in 24-well culture plates, phosphorylation assays in six-well plates, and cell cycle assays in 100-mm cell culture dishes (Corning Inc., Corning, NY).

Dose-response experiments

Nine ATC cell lines (ARO-81, BHT-101, C643, DRO-90, HTh-7, HTh-74, KAT-4, KAT-18, and SW1736) as well as a CML cell line, K562, were tested for their response to a range of doses (0.1, 0.316, 1.0, 3.16, and 10 µM) of imatinib mesylate. The drug was added to cultures at 5–10% confluence with control cultures receiving equal volumes of vehicle (water). Each experimental condition was studied with six replicates and then repeated in duplicate. Cell quantification was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (19, 20). Viability for the K562 suspension cell line as well as for ARO-81 cells was determined using hemocytometry with the trypan blue exclusion assay (21). Cells were exposed to drugs for 72 h before quantification.

Phosphorylation assay and Western blot analysis

Cell lines were grown to 50% confluence (ARO-81 cells) and then exposed in triplicate to either 10 µM imatinib or vehicle alone for different intervals, washed with 10 ml of cold PBS, and lysed in buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 5% ß-mercaptoethanol, protease inhibitor cocktail tablets, and sodium orthovanadate (Sigma)]. Samples were sonicated, using a Kontes KT50 ultrasonic cell disruptor (settings 60, 50) for 30 sec each and then incubated at 37 C for 30 min before loading on a 4–15% linear gradient Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA) for electrophoresis at 100 V. After electrophoresis, proteins were wet-transferred to nitrocellulose membranes (Bio-Rad) at 225 mA for 75 min and then immunoblotted using antiphosphotyrosine (p-Tyr/PY99), anti-c-ABL, or anti-phospho-c-ABL (Tyr245) antibodies at dilutions of 1:1000. Immunoblots were standardized with antiactin (I-19) antibody at a dilution of 1:240. The HRP-conjugated antimouse and antirabbit IgGs were used at a dilution of 1:2000 each. Membranes were developed in enhanced chemiluminescence reagent from Amersham Biosciences (Piscataway, NJ).

RNA extraction and RT-PCR

Total RNA was extracted from cultured cells, using the Trizol method with minor modifications to the protocol recommended by the supplier of the reagent (Life Technologies). Tissue or cultured cells were homogenized in 1 ml Trizol with 0.1 ml of 1-bromo-3-chloropropane replacing 0.2 ml of chloroform. The isolated RNA was dissolved in 0.1% diethyl pyrocarbonate water (Sigma) and then treated using the DNA-free DNase treatment and removal reagents from Ambion, Inc. (Austin, TX) according to the manufacturer’s protocol. A GENESYS 5 spectrophotometer from Thermo Spectronic (Madison, WI) was used in quantifying the RNA.

We evaluated the expression of the BCR-ABL oncogene, as well as the ABL, KIT, and PDGFR-ß genes in the anaplastic thyroid cancer cell lines, with the U-1242 MG cells used as a positive control for the expression of the PDGFR-ß gene (18) and the K562 cells as a positive control for the expression of the BCR-ABL fusion oncogene. Traditional PCR was performed on a PTC-200 thermal cycler manufactured by MJ Research, Inc. (Waltham, MA) using 1 µg of mRNA from each cell line to synthesize cDNA by RT using the Advantage RT-for-PCR kit (Clontech), according to the manufacturer’s protocol (Palo Alto, CA) with random hexamer primers, 50 mM Tris-HCl (pH 8.3), 75 mM KCL, 3 mM MgCl2, 0.5 mM each dNTP, 1 U/µl RNase inhibitor, and >=200 U/µg RNA of the Maloney murine leukemia virus reverse transcriptase enzyme. Each reaction mix was incubated at 42 C for 1 h and then heated at 94 C for 5 min. The cDNA was diluted in 80 µl of cDNA dilution buffer [10 mM Tris/6 mM EDTA (pH 8.0) in 0.1% diethyl pyrocarbonate water].

Primers were designed to yield a final BCR-ABL product of either 305 or 234 bp, depending on the expression of exon 3 on the BCR gene (Table 1Go) (22, 23). Three microliters of template were added to a 50-µl PCR mix of 25 pmol of the oligonucleotides CMLNA and CMLNB, 1x Advantage 2 DNA polymerase enzyme manufactured by Clontech, 0.2 mM each of the dNTPs, 0.3 mM Tris-HCL (pH 8.0), 1.5 mM KCl, 3.5 mM Mg, and water. Each reaction mixture was preheated for 5 min at 94 C followed by 45 cycles of amplification at 94 C for 30 sec, 66 C for 1 min, and 72 C for 1 min with an elongation step of 72 C for 7 min.


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  		TABLE 1. Sequences of oligonucleotides used to amplify BCR-ABL and real-time PCR standards

 
Dilutions of the original K562 RNA stock (1:30; 1:1,000; 1:30,000; and 1:1,000,000) were used to synthesize cDNA standards to determine the relative levels of BCR-ABL gene expression. The PCR data were normalized to human ß-actin PCR product (661 bp) (Stratagene, La Jolla, CA). The ß-actin PCR mixture was preheated for 10 min at 94 C followed by 25 cycles of amplification at 94 C for 20 sec, 62 C for 30 sec, and 72 C for 1 min with an elongation step of 72 C for 5 min. PCR products were resolved by agarose gel electrophoresis with ethidium bromide and analyzed with a Kodak-1D imaging system (New Haven, CT).

Real-time quantitative PCR (Q-PCR) analysis

Expression of the ABL, KIT, and PDGFR-ß genes in the ATC cell lines was determined using Q-PCR analysis performed on an Opticon 2 DNA Engine from MJ Research. Primers were purchased from Applied Biosystems’ Assays-on-Demand program using their unique assay IDs (Hs00245445_m1, Hs00174029_m1, and Hs00182163_m1 for ABL, KIT, and PDGFR-ß, respectively), recognizing sequences available on the GenBank database (accession numbers NM_005157, NM_007313 for ABL, NM_000222 for KIT, and NM_002609 for PDGFR-ß) with two primers for amplifying the sequence of interest, as well as one TaqMan MGB probe (6-FAM dye-labeled) for quantification. Standard curves were generated for c-ABL, c-KIT, and PDGFR-ß products and the results normalized to eukaryotic 18S ribosomal RNA (18S rRNA) transcript amplified using the relevant primers (assay ID Hs99999901_s1; GenBank accession number X03205) from Applied Biosystems.

Preparation of Q-PCR standards

cDNAs derived from U-1242 MG and K562 cells as well as KAT-210 tissue were used to prepare Q-PCR standards for PDGFR-ß, ABL-1, and KIT, respectively, using traditional primers (Table 1Go). The primers complemented sequences approximately 200 bp upstream [ABL1(F), KIT(F), and PDGFR-ß(F)] and downstream [ABL1(R), KIT(R), and PDGFR-ß(R)] of the sequences recognized by the AB TaqMan MGB probes (Table 1Go). PCR products were resolved on a 1% agarose gel containing ethidium bromide, and bands of interest were cut out under illumination from a hand-held Mineralight lamp, model UVSL-25 (UVP, Inc., San Gabriel, CA). DNA was extracted from the gels using a Qiaex II Gel extraction kit according to the manufacturer’s instructions (QIAGEN Inc., Valencia, CA) and used for further amplification of target genes with final PCR products precipitated in ethanol and resuspended in 50 µl of 10 mM Tris (pH 8.0)/1 mM EDTA.

The PDGFR-ß gene was similarly amplified except that the reaction mixture was preheated for 5 min at 94 C, followed by 35 cycles of amplification at 94 C for 20 sec, 55 C for 30 sec, and 72 C for 1 min with an elongation step of 72 C for 5 min. Amplification of the c-ABL and c-KIT genes was similar except for the use of 2.5 U of Pfu Turbo Hotstart DNA polymerase enzyme from Stratagene and conditions of preheating for 2 min at 94 C followed by 30 cycles of amplification at 94 C for 30 sec, 65 C for 30 sec, and 72 C for 45 sec with an elongation step of 72 C for 10 min. Five standard dilutions were prepared from the PCR products of each of the three genes (c-ABL, c-KIT, and PDGFR-ß) at concentrations of 1 x 105, 1 x 104, 1 x 103, 1 x 102, and 5 x 101 copies/µl. These were used for standard curves for each gene and to determine the number of copies of each target gene in the ATC cell lines.

The 18S rRNA standard curves for Q-PCR were prepared from six serial dilutions of plasmid DNA, ranging in concentration from 6.4 x 108 to 6.4 x 103 copies/µl, with a 10-fold difference between sequential dilutions. A 472-bp DNA fragment starting from sequence position 391 and ending at position 863 was prepared by regular PCR amplification of cDNA from a thyroid cancer cell line using the primers 18s(F) and 18s(R) (Table 1Go). The PCR product was resolved on a 1% agarose gel, and the correct DNA fragment (472 bp) was extracted from the gel and purified and inserted into a pCR 2.1 vector using the TA Cloning Kit from Invitrogen Life Technologies. The resulting plasmid was sent to Elim Biopharmaceuticals, Inc. (Hayward, CA) for sequencing.

Cell cycle analysis

Cell cycle analysis was performed after cells were given imatinib at 5–10% confluence and incubated for 48 h in either 10 or 3.16 µM imatinib or an equal volume of vehicle and then harvested and fixed in 70% ethanol as described below.

Cells were detached from the culture plates using trypsin-EDTA, precipitated by centrifugation at 500 x g for 5 min, and resuspended in 0.5 ml of chilled PBS. The process was repeated once more to wash the cells. The cells were fixed in 70% ethanol (added dropwise) for at least 15 min and then washed in 1 ml of chilled PBS before precipitating and resuspending in 1 ml of freshly prepared propidium iodide/RNase-A solution (0.01 mg/ml and 0.25 mg/ml, respectively). Monodispersed cells were transferred to prelabeled (12 x 75 mm) polystyrene tubes, protecting the samples from light all the time. The samples were incubated at 37 C for at least 15 min and then taken for FACS analysis at the University of Kentucky Medical Center’s Flow Cytometry Service Facility.

Data analysis

Antineoplastic growth parameters, total growth inhibition (TGI), 50% growth inhibition (GI50), and concentration resulting in 50% lethality (LC50), were evaluated for each of the cell lines from the survival curves obtained using data from the dose-response assays (Fig. 1Go) (24). The TGI value is the concentration of drug at which a complete cytostatic effect is observed. The LC50 is the concentration of drug that causes a 50% reduction in the number of cells, compared with the number of cells initially present at the beginning of the experiment. In the dose-response experiments, the growth response of the cells treated with drugs was normalized to the control groups, exposed to the diluents alone. Percentage survival values of 100 indicate no drug effect, whereas values approaching 0 indicate increasing cytostatic drug effects. Values below 0 indicate increasing cytotoxicity.



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  		FIG. 1. Imatinib cytotoxicity determined from dose-response experiments. Cells were exposed to 0.1, 0.316, 1, 3.16, 10, or 31.6 µM imatinib mesylate for 72 h before measurement of cell viability. The viability of ARO-81 and K562 cells was determined by hemocytometry, whereas the viability of the other cell lines was determined using the MTT assay. Data shown are for up to 10 µM imatinib. Each experiment was repeated at least twice. The arrow along the x-axis indicates the maximal achievable concentration of imatinib mesylate in humans.

 
The detection limits for the real-time PCR (Q-PCR) assays were set to the highest tenth whole value above the number of copies detected in the no-template control and were thus 90 copies per reaction for c-ABL, 170 copies per reaction for c-KIT, and 210 copies per reaction for PDGFR-ß. Q-PCR data are presented as the number of copies of the target gene per million copies of the 18S rRNA transcript.

Flow cytometry data from two replicates of the cell cycle assay was modeled using ModFit LT version 2.0, from Verity Software House (Topsham, ME). Data with a reduced {chi}2 value over 5 were considered inconclusive and were not used. At least 10,000 events (excluding debris and aggregates) were modeled for respective control cells. The coefficient of variation in the untreated cells was less than 8%, and the background aggregates and debris were less than 20% (25, 26, 27).

Statistical analysis of data was performed with GraphPad Prism 4 for Macintosh (GraphPad Software, Inc., San Diego, Ca). Dose-response data were analyzed using two-way ANOVA and the Bonferroni posttests were used to determine statistical significance of the data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent effects of imatinib mesylate

The dose-dependent effects of imatinib mesylate on cultured ATC cell lines were compared with the effects of the drug on K562 cells. The cells were exposed to 0.1, 0.316, 1.0, 3.16, or 10 µM imatinib mesylate for 72 h (Fig. 1Go). Cell survival was evaluated using the MTT assay and expressed relative to the control cells exposed to an equal volume of the vehicle (nanopure water) alone.

Growth of K562 cells was inhibited in a dose-dependent manner by imatinib mesylate (Fig. 1Go). The GI50, TGI, and LC50 values for all the nine ATC cell lines except ARO-81 and BHT-101 cells were beyond the range of dosages studied. None of the ATC cell lines had any significant antineoplastic (GI50, TGI, or LC50) response below the maximal achievable dose of 6.78 µM in humans (Fig. 1Go, arrow) (28). ARO-81 cells had a GI50 of 9.24 µM, whereas BHT-101 cells had a GI50 of 7.17 µM, concentrations that were greater than 25 times the GI50 concentration for the K562 cells. The TGI and LC50 concentrations for K562, ARO-81, and BHT-101 were beyond the range studied (Fig. 1Go and Table 2Go).


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  		TABLE 2. Antineoplastic parameters determined from dose-response experiments

 
Tyrosine phosphorylation assay

The effects of imatinib on the general tyrosine phosphorylation of proteins in ARO-81 cells was investigated and compared with the effects in K562 cells. K562 cells exposed to 10 µM imatinib for as short as 5 min or as long as 24 h were similarly dephosphorylated at various tyrosine targets (Fig. 2AGo). The tyrosine phosphorylation of cells treated with the diluent alone was not attenuated and was the same in all control cases. The tyrosine phosphorylation profile for ARO-81 cells exposed to imatinib mesylate was identical with the profile of the control cells not exposed to imatinib (Fig. 2BGo). There was no attenuation of tyrosine phosphorylation with prolonged exposure to imatinib over the time interval studied.



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  		FIG. 2. Effects of imatinib mesylate on tyrosine phosphorylation in ARO-81 cells. A, Results of a Western blot assay using the antiphosphotyrosine antibody (PY99) on K562 cells exposed either to 10 µM imatinib mesylate or to an equal volume of diluent for different time periods (5, 30, or 60 min or 24 h); B, results for ARO-81 cells treated similarly. The observations are standardized to ß-actin (sc-1616) shown in the bottom panels of each data set.

 
Effects of imatinib mesylate on ABL phosphorylation

The effect of imatinib mesylate on the phosphorylation of the c-ABL protein in ARO-81 cells was determined by Western blot. The results were compared with the K562 cells (Fig. 3Go) as well as with the effect of the drug on ABL expression in both cell lines. Observations were standardized to actin expression using the antiactin antibody (sc-1616). ABL protein was expressed to significant levels in both cell lines. The expression of c-ABL was not affected by exposure of either cell line to 10 µM imatinib for up to 24 h (Fig. 3Go, A and B). The levels of ABL protein expression in drug-treated cells were comparable to levels in the control cells exposed to diluent alone. Phosphorylated c-ABL was not detected under the conditions of the current study in either the ARO-81 cell line or the CML cell line K562 (Fig. 3Go, C and D). The light band seen in the 24-h positive control lane in Fig. 3CGo is probably nonspecific, as it runs closer to 100 kDa compared with the expected 120 kDa for phosphorylated c-ABL.



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  		FIG. 3. Effects of imatinib mesylate on the phosphorylation of c-ABL protein. A, Western blot staining for c-ABL protein in K562 cells treated with either 10 µM imatinib mesylate or with an equal volume of diluent for up to 24 h; B, ARO-81 cells treated similarly; C and D, K562 and ARO-81 cells respectively, representing the results of immunostaining with an antibody specific to phosphorylated c-ABL. The results are standardized to ß-actin protein.

 
BCR-ABL, ABL, KIT, and PDGFR-ß gene expression

The expression of the BCR-ABL fusion oncogene was investigated by RT-PCR. The BCR-ABL fusion oncogene was not detected in any of the nine ATC cell lines, but this oncogene was successfully detected in all but the 1:1,000,000 dilution of the K562 positive control (Fig. 4Go). The results were standardized to ß-actin expression that was detectable in all cases except the no-template control and the 1:1,000; 1:30,000; and 1:1,000,000 dilutions of the K562 control.



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  		FIG. 4. Expression of BCR-ABL fusion oncogene in ATC and K562 CML cell lines. The expression of the BCR-ABL fusion oncogene in ATC cell lines, as well as in the CML cell line (K562), was analyzed by RT-PCR. The BCR-ABL expression profile was compared with ß-actin expression. Diluted K562 mRNA was used to synthesize cDNA, which was then used to provide a semiquantitative comparison for the level of expression of the gene in ATC cell lines. K562 mRNA was used at dilutions of 1:30; 1:1,000; 1:30,000; and 1:1,000,000 of the original mRNA stock concentration.

 
The expression of the c-ABL, c-KIT, and PDGFR-ß genes was investigated using real-time PCR (Q-PCR). All the ATC cell lines expressed significant levels of the c-ABL transcript, with the highest expressions relative to the 18S rRNA transcript seen in KAT-18 and K562 cells (Table 3Go). K562 cells expressed the highest ratio of c-ABL copies, which was approximately five times higher than in the KAT-18 cells and approximately 10 times higher than the rest of the ATC cell lines. The detection of the c-ABL transcripts in all the cells tested was confirmed by the detection of the c-ABL protein (Fig. 3Go, A and B). The c-KIT gene was detected in normal thyroid tissue (KAT-210) and at lower levels in KAT-18 cells (Table 3Go). The normal thyroid tissue had a higher ratio of c-KIT copies at over 130 copies per million of the 18S rRNA compared with 0.1 copies in KAT-18 cells (Table 3Go). No c-KIT was detected in the K562 cell line. The PDGFR-ß gene was detected in four (C643, HTh-7, HTh-74, and KAT-18) of the nine ATC cell lines tested as well as in normal thyroid tissue. Normal thyroid tissue and C643 cells had comparable expression ratios of the PDGFR-ß gene, at approximately 16 copies per million of 18S rRNA. No PDGFR-ß was detected in the K562 CML cell line.


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  		TABLE 3. Detection of c-ABL, c-KIT, and PDGFR-ß genes by real-time RT-PCR

 
Cell cycle analysis

The effects of imatinib mesylate on the cycling of ATC cells were compared with the effects of the drug on the cycling of K562 cells. Cells were exposed to either 3.16 or 10 µM imatinib mesylate or an equal volume of the diluent for 48 h. The percentages of cells in the G0-G1 phase, the G2-M phase, and the total S phase were compared across concentrations within each cell line. The percentages of cells in each cell cycle after treatment with 3.16 and 10 µM imatinib mesylate were compared with that of the untreated control cells (Table 4Go). The percentage of cellular debris (representing dead cells) was also determined for each cell line on exposure to 3.16 or 10 µM imatinib and compared with the control condition with the diluent alone. Bonferroni posttests revealed statistically significant increases in cellular debris in K562 cells treated with 3.16 µM imatinib (50% increase) and with 10 µM imatinib (53.9% increase) compared with the control condition (P < 0.001; Table 4Go). No statistically significant increases in cellular debris were detected in any of the ATC cell lines. The percentage of cellular debris in all ATC cell lines remained less than 7% on exposure to either 3.16 or 10 µM imatinib mesylate.


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  		TABLE 4. Dose-dependent effects of imatinib mesylate on cell cycling

 
A statistically significant increase in the percentage of K562 cells in the G0-G1 phase was detected after treatment with 10 µM imatinib mesylate (P < 0.05; Table 4Go). There was also a decrease in the percentages of cells in the G2-M and S phases with increasing concentrations of imatinib, although these changes were not statistically significant by the Bonferroni posttests (Table 4Go). The only statistically significant change detected in any of the ATC cell lines was in the KAT-18 cell line where an increase in the percentage of cells in the G0-G1 phase was detected on treatment with both 3.16 and 10 µM imatinib (P < 0.01 and P < 0.05, respectively; Table 4Go). These changes were accompanied by decreases in the percentage of cells in the G2-M phase and total S phase, which were, however, not determined to be statistically significant by the posttests.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The success of imatinib mesylate in treating CML patients is attributed to its inhibition of the constitutive kinase activity of a product of the BCR-ABL fusion oncogene (12, 14, 15, 29). We looked for the expression of the BCR-ABL fusion oncogene by RT-PCR in the nine ATC cell lines. Because other studies have indicated that imatinib can also target the kinase activity of the ABL, c-KIT, and PDGF-ß receptors (30, 31, 32, 33), we used Q-PCR to determine the expression of these genes in the ATC cell lines. The effects of imatinib on cell growth as well as on cell cycling were investigated with findings compared with the effects of the drug on the CML cell line K562. The expression of c-ABL as well as phosphorylated c-ABL proteins was also determined by Western blot analysis.

K562 cells express the fusion oncogene BCR-ABL, which encodes the oncogenic protein target of imatinib. BCR-ABL is a fusion gene on the Philadelphia chromosome generated by a specific translocation involving chromosomes 9 and 22. The human c-ABL gene is located on chromosome 9 and is a member of the non-receptor tyrosine kinases (34). The gene appears to be ubiquitously expressed, and the c-ABL protein appears to have a major role in regulating cell cycle progression for normal growth and development. The BCR gene is located on chromosome 22 and is ubiquitously expressed. In quiescent cells, the BCR protein is localized in the cytoplasm but translocates to a perichromosomal location during mitosis, suggesting a possible role in cell regulation. The BCR-ABL oncogene translates into a 210-kDa protein, exhibiting deregulated, constitutively active tyrosine kinase activity and is found exclusively in the cytoplasm of the cell, inducing a leukemic phenotype. The constitutive tyrosine kinase activity of the BCR-ABL protein activates signaling cascades resulting in profound effects on cell growth and differentiation. Unlike K562 cells, none of the ATC cell lines express BCR-ABL protein, thus lacking this well characterized imatinib target.

The possibility that imatinib may affect ATC cell growth by directly regulating c-ABL independent of the BCR-ABL pathway was considered. Despite the demonstration of comparable protein expression levels in ARO-81 and K562 cells (Fig. 3Go, A and B), the cell lines responded dramatically differently from each other to imatinib treatment. Furthermore, the response of the ARO-81 cells to imatinib within the established physiologically relevant dosages of imatinib was fairly characteristic of the response seen in all the other anaplastic cell lines and different across the board from the response by K562 cells to similar treatment. For all practical purposes then, it seems improbable that the level of c-ABL protein expression alone, in the ARO-81 cells in particular and perhaps in anaplastic cells in general, would be a significant determinant of the cytotoxic efficacy of imatinib on these cells. Imatinib inhibited general tyrosine phosphorylation in the K562 cells (Fig. 2AGo) grown in 10% serum, but there was no such corresponding response in the ARO-81 cells. The absence of an effect on general tyrosine phosphorylation in the ATC cell line was mirrored by a similar observation by other investigators in a serum-starved follicular thyroid cancer cell line, WRO (35). Wild-type c-ABL protein is auto-inhibited under basal conditions and so is nonphosphorylated unless it is bound to its ligand or is deregulated by a structural modification (36, 37). Thus, we did not expect to find hyperphosphorylated c-ABL in either the CML cell line or the ATC cells. The effects of imatinib on the K562 positive control cell line are mediated via the BCR-ABL fusion oncogene protein and not through c-ABL. Our findings on the effects of imatinib on the tyrosine phosphorylation status of c-ABL were inconclusive because of the lack of an appropriate positive control for the Western blot assay (Fig. 3Go, C and D). However, we were unable to detect any phosphorylated c-ABL that could serve as a target for regulation by imatinib. To ascribe a BCR-ABL oncogene-independent role for c-ABL in any imatinib-induced modulation of ATC cell growth, a constitutively active or a mutated, deregulated form of c-ABL kinase would need to be identified (38). The clinical range of imatinib concentrations used in our studies did not induce significant reductions in ATC cell growth, indicating lack of a significant effect by the drug in modulating cell growth and thus invalidating any need to pursue investigations into the role of c-ABL in any such modulation.

The effect of imatinib on the survival of ATC cell lines was investigated in vitro between 0.1 and 10 µM. Imatinib binds highly to albumin and {alpha}1-acid glycoprotein and less than 5% to lipoproteins and {gamma}-globulins (28). Imatinib also binds to blood cells in a concentration-dependent manner. The maximal achievable plasma levels of imatinib in patients are no higher than 6.78 µM at maximal administered doses of 600 mg/d (28, 39). The effectiveness of imatinib in CML patients is corroborated by the low GI50 concentration (0.24 µM) seen in cultured K562 cells. This is at least 25-fold lower than the lowest GI50 concentration observed for ATC cells (7.17 µM in BHT-101 cells). The typical clinical levels of imatinib are under 2.6 µM, and at the upper limit of this range, the free form of imatinib ranges from 4–5% (28). These numbers are consistent with the effective concentrations of imatinib on K562 cells in vitro, corresponding to clinical activity of this agent in CML. One recent report suggested remarkable growth inhibition in ATC cells after exposure to 10 µM imatinib mesylate (STI571) for more than 72 h (9). Our studies, in conjunction with the protein-binding data for imatinib (28), suggest that this concentration is clinically irrelevant and corresponds to unobtainable serum levels in patients that would be at least 25-fold higher than concentrations effective in responsive malignancies (39). Such levels of imatinib, if achieved by higher dosing in patients, would result in unacceptable dose-limiting toxicity.

C643, HTh-7, HTh-74, and KAT-18 were the only ATC cells in which PDGFR-ß was detectable. The expression of PDGFR-ß in C643 and HTh-74 cells and the loss of c-KIT in the thyroid cancer cells is consistent with earlier findings by other investigators (40, 41). Previous studies have indicated that normal thyroid epithelial cells, including thyroid follicle cells, do not express PDGFRs (41). We detected PDGFR-ß in the KAT-210 normal thyroid control tissue (Table 3Go). Others have reported detecting PDGFR-ß in homogenates of KAT-4 tumors (42), although we were not able to detect mRNA for PDGFR-ß in the KAT-4 cell line using real-time PCR (Table 3Go). This apparent discrepancy is simply explained by the fact that PDGFRs are expressed in cells of mesenchymal and glial origin, which include connective tissue cells such as fibroblasts (43, 44).

The fact that PDGF plays an important role in oncogenesis is well accepted and appears to be one way by which tumor cells mediate stromal reactions (41, 45). Although the tumor cells may not respond to PDGF directly, tumor growth can still be regulated by changes to the stromal environment mediated via PDGFR pathways. So even in the absence of direct effects on the thyroid cancer cells, imatinib can lower interstitial fluid pressure by inhibiting stromal PDGFR-ß to facilitate delivery of cytotoxic drugs to the tumors (42, 46).

The dose-response assays in this study were conducted on cells cultured in 10% serum, conditions that more closely mimic the physiological setting. These conditions are also the established optimal culture conditions for the growth of all the cell lines used in this study, including the ARO-81 cells (47, 48, 49). The use of suboptimal culture conditions (9) to grow the cells while investigating the cytotoxic effects of an agent may compromise subsequent conclusions. ATC cell lines do not express the BCR-ABL oncogene, which is the most potent imatinib target. Additionally, ATC cells do not appear to express other possible targets for imatinib (c-KIT, deregulated c-ABL, or the PDGFR-ß) in a manner that correlates with any effects of this drug on cell viability. KAT-18 cells expressed minimal levels of c-KIT (Table 3Go) and were the only ATC cells in which there was a significant change in the percentage of cells entering the G0-G1 phase (Table 4Go). However, this observation did not correlate to a reduction in cell survival. The cell cycle studies showed a significant imatinib-dependent effect in only one ATC cell line, KAT-18. In comparison with the K562 cells in which a significant shift of cells into the G0-G1 phase was accompanied by noticeable reductions in the population of cells in the G2-M and total S phases, with 48 h of drug exposure, there were no such responses observed in any of the ATC cell lines (Table 4Go). The shift into the G0-G1 phase by KAT-18 cells was not accompanied by any change in the amount of cellular debris, indicating absence of any effect on cell survival.

Clinically relevant doses, at which imatinib is reported to induce potent antineoplastic effects in patients with CML and GISTs, are far below the minimally active doses necessary for ATC cell lines in vitro in this and other studies (9, 42, 46). In fact, one study reports that imatinib may promote tumor metastasis by regulating the Met receptor, which is overexpressed in thyroid carcinomas (35). Responses by the ATC cells were attained only by using far higher dosages of imatinib than those tolerable by patients. The popularity of imatinib derives from its effectiveness in treating CML at relatively low concentrations with minimal toxicity to the patient. Current data suggest that this advantage of imatinib would be greatly attenuated if not lost before deriving any theoretical benefits in treating ATC patients. The data from our investigations as well as other studies noted in this paper do not warrant additional studies on monotherapeutic use of this agent in a clinical setting for the treatment of ATC patients.


   Footnotes
 
Abbreviations: ATC, Anaplastic thyroid carcinoma; CML, chronic myeloid leukemia; GI50, 50% growth inhibition; GIST, gastrointestinal stromal tumor; LC50, concentration resulting in 50% lethality; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PDGFR-ß, platelet-derived growth factor-ß receptor; TGI, total growth inhibition.

Received October 6, 2003.

Accepted January 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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