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Increased Sensitivity of Hydroxyurea-resistant Leukemic Cells to Gemcitabine¹

Division of Hematology/Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ABSTRACT

Tumor cell resistance to certain chemotherapeutic agents may result in cross-resistance to related antineoplastic agents. To study cross-resistance among inhibitors of ribonucleotide reductase, we developed hydroxyurea-resistant (HU-R) CCRF-CEM cells. These cells were 6-fold more resistant to hydroxyurea than the parent hydroxyurea-sensitive (HU-S) cell line and displayed an increase in the mRNA and protein of the R2 subunit of ribonucleotide reductase. We examined whether HU-R cells were cross-resistant to gemcitabine, a drug that blocks cell proliferation by inhibiting ribonucleotide reductase and incorporating itself into DNA. Contrary to our expectation, HU-R cells had an increased sensitivity to gemcitabine. The IC₅₀ of gemcitabine was 0.061 ± 0.03 µM for HU-R cells versus 0.16 ± 0.02 µM for HU-S cells (P = 0.005). The cellular uptake of [³H]gemcitabine and its incorporation into DNA were increased in HU-R cells. Over an 18-h incubation with radiolabeled gemcitabine (0.25 µM), gemcitabine uptake was 286 ± 37.3 fmol/10⁶ cells for HU-R cells and 128 ± 8.8 fmol/10⁶ cells for HU-S cells (P = 0.03). The incorporation of gemcitabine into DNA was 75 ± 6.7 fmol/10⁶ cells for HU-R cells versus 22 ± 0.6 fmol/10⁶ cells for HU-S cells (P < 0.02). Our studies suggest that the increased sensitivity of HU-R cells to gemcitabine results from increased drug uptake by these cells. This, in turn, favors the incorporation of gemcitabine into DNA, resulting in enhanced cytotoxicity. The increased sensitivity of malignant cells to gemcitabine after the development of hydroxyurea resistance may be relevant to the design of chemotherapeutic trials with these drugs.

INTRODUCTION

Tumor cell resistance to chemotherapy is one of the major obstacles to the successful treatment of cancer. Hence, knowledge of cross-resistance among different antineoplastic agents is important because it provides direction for the design of clinical trials.

Because of its key role in the synthesis of deoxyribonucleotides, ribonucleotide reductase is an important target for antineoplastic drugs (1 , 2) . Mammalian ribonucleotide reductase is composed of two subunits termed R1 and R2 (or M1 and M2; Refs. 3, 4, 5 ). The R1 subunit has substrate and effector binding sites, whereas the R2 subunit contains a nonheme iron center and a tyrosyl free radical, both of which are essential for enzyme activity (3, 4, 5, 6) . Of the various ribonucleotide reductase inhibitors available, hydroxyurea has been in clinical use for over two decades, and its action on malignant cells in vitro and in vivo has been studied extensively (7) . It is known that hydroxyurea inhibits ribonucleotide reductase activity by action on the R2 subunit (6 , 8) and that resistance to cell growth inhibition by hydroxyurea results from overexpression of the R2 subunit (9, 10, 11, 12) .

Gemcitabine (2',2'-difluorodeoxycytidine) is a new deoxynucleoside analogue that has shown clinical antitumor activity against a variety of malignancies (13 , 14) . This drug exerts its cytotoxicity by action on ribonucleotide reductase and incorporation into DNA, the latter of which results in a block in DNA replication (15) .

In the present investigation, we have examined whether lymphoid leukemic cells that are resistant to hydroxyurea by virtue of overexpression of the R2 subunit of ribonucleotide reductase are cross-resistant to gemcitabine. Contrary to our expectation, we found that HU-R³ CCRF-CEM cells take up and incorporate a greater amount of gemcitabine into DNA than HU-S cells and display an increased sensitivity to growth inhibition by gemcitabine.

MATERIALS AND METHODS

Gemcitabine and [³H]gemcitabine (specific activity, 20 Ci/mmol) were obtained from Lilly Research Laboratories (Indianapolis, IN). Hydroxyurea was purchased from Calbiochem, whereas MTT was obtained from Sigma Chemical Co. (St. Louis, MO). [³²P]dCTP was obtained from New England Nuclear Life Sciences (Boston, MA). The cDNA for the R2 subunit of human ribonucleotide reductase cloned into vector pCRII (Invitrogen, Carlsbad CA) was kindly provided by Dr. Yun Yen (City of Hope, Duarte, CA) and has been described previously (16) . The R2 cDNA insert was excised from the plasmid using BamHI and labeled with ³²P by the random primer method using the RadPrime DNA Labeling System (Life Technologies, Inc., Gaithersburg, MD). Mouse monoclonal antibody AD203 against human R1 was obtained from Accurate Chemical and Scientific Corp (Westbury, NY), whereas rabbit antiserum against human R2 was generously provided by Dr. Timothy Kinsella (Case Western Reserve University, Cleveland, OH) and has been described previously (17) .

Cells and Cell Growth Conditions.
Human T lymphoblastic leukemic CCRF-CEM cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 supplemented with 10% FCS in an atmosphere of 6% CO₂. A subclone of these cells that was relatively resistant to growth inhibition by hydroxyurea (HU-R cells) was developed by incubation with incremental concentrations of hydroxyurea over several months. HU-R cells were routinely maintained in culture medium containing 90 µM hydroxyurea. The effects of hydroxyurea and gemcitabine on the proliferation of HU-S and HU-R CCRF-CEM cells were measured by MTT assay as described by Mosmann (18) , with modifications as reported previously by us (19) . In this assay, cells were plated at a density of 2 x 10⁵ cells/ml in 96-well microwell plates in the presence of increasing concentrations of hydroxyurea or gemcitabine (shown in the figures) and analyzed for growth after 72 h of incubation.

RNA Isolation and Northern Blotting.
Total cellular RNA was isolated from cells using RNAzol (Tel-Test, Inc., Friendswood, TX) as recommended by the manufacturer, and the integrity of the RNA was verified by agarose gel electrophoresis before use. RNA (20 µg) was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde and transferred from the gel to a Nytran membrane (Schleicher & Schuell, Keene, NH) by capillary blotting. Equal loading of RNA on the gel was confirmed visually by ethidium bromide staining of the RNA bands. R2 mRNA was detected by hybridization of the membrane to ³²P-labeled cDNA probe (1.2 x 10⁶ cpm/ml) using QuickHyb Hybridization Solution (Stratagene, La Jolla, CA) as recommended by the manufacturer. The membrane was autoradiographed by exposing it to Kodak XAR-5 film (Eastman Kodak, Co., Rochester, NY) with intensifying screens at -70°C for 24–48 h.

Western Blotting.
R1 and R2 protein levels in HU-S and HU-R cells were detected by Western blotting using an enhanced chemiluminescence Western blotting detection system (Amersham, Arlington Heights, IL). Cells in exponential growth phase were harvested and lysed in 200 µl of radioimmunoprecipitation assay buffer (1% Triton X-100, 1% sodium deoxycholate, 50 mM Tris, and 150 mM NaCl) containing 100 µg/ml phenylmethylsulfonyl fluoride, 75 µg/ml aprotinin, and 1 mM sodium orthovanadate. The protein content of the clarified lysate was measured by the BCA protein assay (Pierce, Rockford ILL). SDS-PAGE of the samples was performed as described by Laemmli (20) , and proteins were transferred from the gel onto a nitrocellulose membrane as described by Towbin et al. (21) , using a Transblot system (Bio-Rad, Richmond, CA). Membranes were incubated with specific primary antibodies against R1 or R2, followed by secondary antibodies conjugated to horseradish peroxidase. For detection of protein bands, membranes were immersed in enhanced chemiluminescence detection solution and autoradiographed using Kodak XAR-5 film as recommended by the manufacturer.

Cellular Uptake of [³H]Gemcitabine and Incorporation of [³H]Gemcitabine into DNA.
HU-S and HU-R cells were plated in fresh medium (10⁶ cells/ml) containing 0.25 µM [³H]gemcitabine. After an 18-h incubation, cells were harvested and washed with ice-cold 10 mM KPO₄ (pH 7.5)/150 mM NaCl buffer. The total cellular uptake of [³H]gemcitabine was measured by lysing an aliquot of the cell suspension in de-ionized water and counting the total cell-associated radioactivity in a scintillation counter. The incorporation of [³H]gemcitabine into DNA was determined using a modification of the method described by Plunkett and Cohen (22) . Washed cells that had incorporated [³H]gemcitabine over 18 h were resuspended in 3 ml of 0.4 N PCA on ice for 30 min and then centrifuged at 40,000 x g for 5 min. The pellet was resuspended in 6 ml of 0.4 N PCA, and the suspension was centrifuged again at 40,000 x g for 5 min. The pellet was then digested overnight at 37°C in 3 ml of 0.3 N KOH. DNA in the sample was precipitated by the addition of an equal volume of 0.8 N PCA to the sample, and the sample was chilled on ice for 10 min. The DNA pellet was washed twice by centrifugation with ice-cold 0.4 N PCA and then extracted twice by incubation at 70°C for 30 min. The radioactivity in the pooled extracts was counted using a scintillation counter, and the amount of dpm incorporated into DNA/10⁶ was cells determined.

RESULTS

HU-R Cells Overproduce R2 mRNA and Protein.
The growth of HU-S and HU-R CCRF-CEM cells in the presence of hydroxyurea is shown in Fig. 1 . HU-R cells were approximately 6–7-fold more resistant to growth inhibition by hydroxyurea than HU-S cells. To confirm that our HU-R cells had an increase in the expression of the R2 subunit of ribonucleotide reductase similar to that reported by others (9, 10, 11, 12) , steady-state R2 mRNA levels were measured by Northern blotting. These studies showed that R2 mRNA was indeed increased in HU-R cells (Fig. 2) . Consistent with prior reports, the cDNA probe for R2 detected two transcripts of 3.4 and 1.6 kb (16) . Western blot analysis revealed that HU-R cells had a corresponding increase in their content of R2 subunit protein but no increase in R1 subunit protein (Fig. 3) .

View larger version (22K): [in this window] [in a new window]   Fig. 1. Effect of hydroxyurea on the proliferation of HU-S and HU-R CCRF-CEM cells. Cells were plated at 2 x 105 cells/ml in the presence of increasing concentrations of hydroxyurea. Cell growth was determined by MTT assay after a 72-h incubation. Control (100%) for each cell type represents growth in the absence of hydroxyurea. Values shown represent the means ± SE of two separate experiments, each of which was performed in quadruplicate.

View larger version (80K): [in this window] [in a new window]   Fig. 2. Northern blot of R2 subunit mRNA. Steady-state mRNA levels for the R2 subunit were determined in HU-S and HU-R cells after 24 h of growth in culture, as described in "Materials and Methods." S, HU-S cells; R, HU-R cells. Equal loading of RNA in each lane was confirmed by ethidium bromide staining of the gel (data not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Western blot analysis of R1 and R2 protein. HU-S and HU-R cells were analyzed after 24 and 48 h of growth in culture. Protein (30 µg) from the cell lysates was loaded in each lane. S, HU-S cells; R, HU-R cells.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effect of gemcitabine on the proliferation of HU-S and HU-R cells. Cells were plated at 2 x 105 cells/ml in the presence of increasing concentrations of gemcitabine. Cell growth was determined by the MTT assay after a 72-h incubation. Control (100%) for each cell type represents growth in the absence of hydroxyurea. Values shown represent the means ± SE of three separate experiments, each of which was performed in quadruplicate.

View larger version (27K): [in this window] [in a new window]   Fig. 5. A, cellular uptake of [3H]gemcitabine. HU-S and HU-R cells were incubated with 0.25 µM [3H]gemcitabine for 18 h, and the total cellular uptake of radiolabeled gemcitabine was measured as described in "Materials and Methods." B, [3H]gemcitabine incorporation into DNA. Incubation conditions were similar to those described in A. Radiolabed gemcitabine incorporation into DNA was determined as described in "Materials and Methods." Values shown represent the means ± SE (n = 3).

In the present investigation, we sought to determine whether cross-resistance exists between hydroxyurea and gemcitabine. We developed HU-R CCRF-CEM cells, and, consistent with other reports (9, 10, 11, 12) , these cells displayed an increase in the expression of mRNA and protein of the R2 subunit. However, our studies show that rather than being cross-resistant to gemcitabine, HU-R cells displayed a significant increase in sensitivity to gemcitabine. Further examination of the mechanism involved revealed that both the cellular uptake of gemcitabine and its incorporation into DNA were significantly increased in HU-R cells. It is known that in addition to inhibiting ribonucleotide reductase, gemcitabine exerts its cytotoxicity by incorporating into DNA and terminating DNA chain elongation (15) . Our results strongly suggest that the increased sensitivity of HU-R cells to gemcitabine is likely to be due to the increased cellular uptake of gemcitabine, resulting in an expanded intracellular gemcitabine pool. The latter, in turn, would favor gemcitabine incorporation into DNA and lead to enhanced cytotoxicity.

Little information exists regarding the effect of hydroxyurea resistance on the cellular uptake of gemcitabine. Recently, however, Mackey et al. (23) have shown that the influx of gemcitabine into different cells, including CCRF-CEM cells, requires specific cell membrane nucleoside transporters, and that the cytotoxicity of gemcitabine varies among cell lines with different nucleoside transporters. Although HU-S and HU-R cells were derived from the same parent cell line, it is possible that the development of hydroxyurea resistance somehow alters the expression of nucleoside transporters responsible for gemcitabine uptake. Increased expression and/or function of a gemcitabine transporter in these cells could render them more sensitive to this drug. Additional studies are planned to elucidate this potential mechanism of action.

The cross-resistance pattern of a variety of ribonucleotide reductase inhibitors has previously been examined by others. However, in most of these studies, hydroxyurea resistance has been associated with parallel resistance to other drugs. Carter and Cory (24) have shown that R2-overproducing L1210 murine leukemic cells display cross-resistance to 2,3-dihydrox-1H-pyrazolo[2,3-]imidazole, an inhibitor of the R2 subunit, but remain sensitive to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone and 1-isoquionolylmethylene-N-hydroxy-N'-aminoguanidine tosylate, agents that also inhibit R2. Along the same lines, Huang et al. (25) recently showed that HU-R cells with elevated R2 expression exhibit decreased sensitivity to methotrexate and N-(phosphonacetyl)-L-aspartate, agents that act on targets other than ribonucleotide reductase. Interestingly, Yen et al. (16) found that HU-R KB cells with increased ribonucleotide reductase activity displayed increased sensitivity to 6-thioguanine. The underlying mechanism of this supersensitivity was felt to be increased conversion of 6-thioguanine to the deoxynucleotide, leading to enhanced incorporation into DNA and subsequent cell growth arrest (16) .

The results of our studies may be relevant for the use of gemcitabine and hydroxyurea in the clinical setting. Hydroxyurea has activity in hematological malignancies and, to a lesser extent, in certain solid cancers. Gemcitabine is being intensively investigated in Phase II trials in a variety of malignancies and has shown activity against pancreatic cancer, non-small cell lung cancer, head and neck cancer, and urothelial and gynecological malignancies (13 , 14) . Both hydroxyurea and gemcitabine also appear to be potent radiosensitizing agents (7 , 26) .

Our studies suggest that a therapeutic strategy in which tumor cells are exposed to hydroxyurea and gemcitabine in an alternating fashion may be more efficacious than treatment with either drug alone. With such treatment, clones of cells that develop resistance to hydroxyurea after exposure to hydroxyurea may be effectively eradicated with subsequent exposure to gemcitabine. Studies are planned to evaluate the effect of gemcitabine in an animal model implanted with HU-R cells and to confirm our in vitro observations. The result of such studies will be highly relevant to the design of clinical trials and the use of gemcitabine in tumors that may be resistant to hydroxyurea.

FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by USPHS Grant RO1 CA68028 and funds from the Sampson family. This work was presented in abstract form at the 89^th Annual Meeting of the American Association for Cancer Research held March 28-April 1, 1998 in New Orleans, Louisiana.

² To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, WI 53226. Phone: (414) 805-4604; Fax: (414) 805-4606; E-mail: chitambr{at}mcw.edu

³ The abbreviations used are: HU-R, hydroxyurea-resistant; HU-S, hydroxyurea-sensitive; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCA, perchloric acid.

Received 10/ 7/98; revised 10/29/98; accepted 11/ 8/98.

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