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Delayed Addition of Deoxycytidine Protects Normal CD34⁺ Cells against Cytotoxicity of Gemcitabine without Compromising Its Activity against Human Leukemic Cells

^a Department of Nephrology,
^b Department of Internal Medicine, Faculty of Medicine, University of Innsbruck, Innsbruck, Austria;
^c Eli Lilly Area Medical Center, Vienna, Austria

Key Words. 2',2'–Difluorodeoxycytidine • Gemcitabine • 2-Deoxycytidine • Delayed addition • CD34⁺ • HL-60 cells • Coculture

Prof. G. Konwalinka, M.D., Department of Internal Medicine, University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria. Telephone: 43-512-504-3415; Fax: 43-512-504-3415; e-mail: Guenther.Konwalinka{at}uibk.ac.at

	ABSTRACT

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In phase I and II clinical trials, the deoxycytidine analogue 2',2' difluorodeoxycytidine (dFdC, gemcitabine) has shown promising antitumor activity in leukemia as well as in solid tumors. Preclinical and clinical studies of gemcitabine suggested that myelosuppression was the dose-limiting toxicity. The present investigations were designed to test the effect of continuously administered gemcitabine on the in vitro clonal growth of normal CD34⁺ cells isolated from peripheral blood and the promyelocytic cell line, HL-60. For this purpose, CD34⁺ and HL-60 cells were cultured in methylcellulose in the continuous presence of 0.1–16 nM of gemcitabine. The results show a dose-dependent inhibition of colony growth of normal as well as leukemic cells. However, HL-60 cells were up to 12-fold more sensitive towards gemcitabine than normal progenitors. For rescue experiments, the natural pyrimidine deoxycytidine (dCyd) was added to CD34⁺ and HL-60 cells simultaneously or with delay. Coadministration of 1mM dCyd to separate cultures resulted in complete restoration of colony formation capacity of CD34⁺ and HL-60 cells. Delayed addition of 1 mM dCyd after 48 and 72 hours recovered up to 90% and 40%, respectively, of stem cell proliferation, whereas HL-60 cells remained substantially inhibited (4.5% ± 3.5% versus 0%). Delayed addition after 48 and 72 hours protected about 80% and 50%, respectively, of myelopoietic and erythropoetic colony formation, whereas colony formation obtained from HL-60 cells remained significantly inhibited (9.6% ± 4.17% versus 0%). These in vitro data suggest that there is a marked difference in the susceptibility of leukemic and normal CD34⁺ cells to gemcitabine and that delayed administration of dCyd may further reduce the bone marrow cytotoxicity of gemcitabine without impairing its antitumor effect.

	INTRODUCTION

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Gemcitabine (2',2' difluorodeoxycytidine [dFdC]) is a deoxycytidine analogue with proven in vitro and in vivo antitumor activity [1–6]. For activation, gemcitabine requires phosphorylation by deoxycytidine kinase (dCK) in a stepwise fashion to the corresponding monophosphate, diphosphate, and ultimately, triphosphate (2',2-'difluorodeoxycytidine triphosphate [dFdCTP]) [2]. dCK activity is the rate-limiting step for the phosphorylation of gemcitabine. Incorporation of the active triphosphate into DNA causes inhibition of DNA synthesis and cell death [7]. Pharmacokinetic studies of mononuclear and leukemic cells, both in vitro and from patients during phase I trials, revealed a plateau of dFdCTP when plasma levels of gemcitabine exceeded 15–20 µM, indicating saturation of the critical enzyme dCK. This was achieved after 30-minute infusions at conventional doses of 790 mg/m²/week [8, 9].

However, Grunewald et al. observed a fourfold higher accumulation of the active form of gemcitabine, dFdCTP, when the infusion time was prolonged [8]. Recently, it was shown that a protracted infusion of a reduced dose of gemcitabine (300–500 mg/m²) over 3 hours achieved remissions in refractory, heavily pretreated advanced solid tumors [10]. The same antitumor effect could be achieved despite a major dose reduction, when gemcitabine was applied for a prolonged period.

Preclinical and clinical studies of gemcitabine have shown that myelosuppression is the main and dose-limiting toxicity [11]. Deoxycytidine (dCyd) was found to antagonize the antitumor activity of nucleoside analogues such as gemcitabine and cytosine arabinoside (Ara-C) by preventing their phosphorylation [4]. A previous in vitro study of normal and ovarian cancer cells showed that dCyd can reduce the host toxicity of otherwise lethal doses of gemcitabine and Ara-C, leading to a net gain in therapeutic index for the combination [12].

Results in murine colon tumors suggest that the application of prolonged infusion schedules with low doses of gemcitabine might be warranted [13]. In this study, continuous intravenous infusion or subcutaneous application of low-dose (2 mg/kg/day) gemcitabine for up to 7 days proved equally effective as bolus administration of high-dose gemcitabine in tumor-bearing mice, as far as antitumor activity is concerned.

The aim of this study was A) to investigate the effect of continuously administered low-dose gemcitabine on the proliferation of normal CD34⁺ cells and leukemic cells, and B) to determine the effect of coadministered or delayed addition of dCyd on HL-60 and CD34⁺ cells in the presence of gemcitabine. Our results suggest that there are differences between normal and malignant cell sensitivities to gemcitabine. Coadministration of high-dose dCyd protects both normal and malignant cells, whereas its delayed addition after 48 and 72 hours rescues up to 90% and 40%, respectively, of normal CD34⁺ cells, without compromising the cytotoxic effect of gemcitabine on HL-60 cells.

	MATERIALS AND METHODS

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Drugs, Chemicals, and Cell Lines
Gemcitabine was purchased from Eli Lilly (Indianapolis, IN; http://www.lilly.com). DCyd and other chemicals were purchased from Sigma Chemicals (St. Louis, MO; http://www.sigmaaldrich.com). Drugs were stored as dry powder at –20°C and reconstituted in 0.9% sterile sodium chloride before use.

The human promyelocytic cell line HL-60 was maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS), L-glutamine (5 mM), penicillin (50 U/ml), and streptomycin (50 g/l) at 37°C with 5% CO₂. Experiments were performed on cells growing in logarithmic phase.

Preparation of CD34⁺ Cells and Clonal Assays
The methods of collection and selection of CD34⁺ cells have recently been described [14]. Briefly, peripheral blood mononuclear cells (PBMCs) were separated from the whole blood of healthy volunteers (with informed consent), who did not show any hematological disorder, using a CS 3000 Plus cell separator (Baxter Healthcare, Fenwal Division; Deerfield, IL; http://www.baxter.com). Three leukaphereses were performed on each of the subjects. The PBMCs of the first two leukapheresis products were pooled, and CD34⁺ cells were positively selected on a Ceprate SC Stem Cell Concentration System loaded with a CD34⁺-specific biotinylated monoclonal antibody (CellPro; Bothell, WA). This resulted in a 91% median enrichment for CD34⁺ cells (range, 86%–95%). Before use, the cells were cryopreserved in 10% dimethylsulfoxide by controlled rate freezing and stored in liquid nitrogen.

After suspension in Iscove’s medium containing 10% FCS, CD34⁺ and HL-60 were each plated at a final concentration of 2,000/dish and 400/dish, respectively (1 ml culture medium per dish) in 0.8% methylcellulose in Iscove’s medium containing 30% FCS, 10% bovine serum albumin, 1% 2-mercaptoethanol (1 x 10^-4 M) and 200 mM L-glutamine, using 3 U/ml recombinant erythropoetin (Cilag; Vienna, Austria) and 10% agar-stimulated human leukocyte-conditioned medium as stimulants, along with the designated concentrations of gemcitabine. The assays were performed in duplicate. Plates were then incubated in a fully humidified atmosphere in 5% CO₂ at 37°C.

Using an inverted microscope, bursts derived from primitive erythroid progenitors (BFU-E) and colonies derived from granulocytic/macrophagic progenitor cells (CFU-GM) containing more than 50 cells were scored after 18 days of incubation. Colonies derived from HL-60 cells containing more than 50 cells were scored after 12 days of incubation. The inhibitory effect of gemcitabine (with or without dCyd) on progenitor cell growth was expressed as the percentage of colony formation by treated cells relative to untreated controls.

Rescue of HL-60 and CD34⁺ Cells from Cytotoxic Effect of Gemcitabine by Delayed Addition of dCyd
Experiments in which application of dCyd was delayed were performed as described above with the following modification: cultures of HL-60 and human progenitor cells were plated at a final concentration of 2 x 10³/dish and 4 x 10²/dish, respectively, along with lethal concentrations of gemcitabine (16 nM). Zero, 24, 48, 72, or 96 hours after cytostatic treatment, cultures were overlayered with 1 x 1mM dCyd and scored as described above after 18 days.

For the study of the reversal of gemcitabine-mediated cytotoxicity on normal and malignant progenitor cells in coculture, a mixture of CD34⁺ and HL60 cells were seeded out in methylcellulose at a final concentration of 2 x 10³/dish and 3 x 10²/dish, respectively, along with the designated concentrations of gemcitabine as described above.

Again, 0, 48, and 72 hours after cytostatic treatment, cultures were overlayered with 1 x 1mM dCyd. On day 16, all colonies were enumerated using an inverted microscope. The BFU-E colonies were distinguished from CFU-GM and HL-60 colonies by hemoglobinization. CFU-GM colonies consisted of smaller, transparent cells that formed dispersed colonies. Morphologically, HL-60 colonies were clearly different from CFU-GM colonies, and consisted of larger, opaque cells that gave rise to only clump-like colonies. Cell aggregation of more than 50 cells was defined as a colony for both CFU-GM and HL-60. In order to make correct identification of the cell types, however, a morphological differentiation between CFU-GM and HL-60 colonies was made in most of the dishes after Wright-Giemsa staining as previously described [15].

Statistical Analysis
The half-maximal inhibition constant (IC₅₀), determined as the concentration required for 50% of inhibition, was determined by a hyperbolic fit. Unless indicated otherwise, the data are given as the standard error (SE) of at least six independent determinations. Significant differences between experiment groups were assessed using the Student’s t-test with p values of less than 0.05 being considered as significant. All calculations were performed on Hewlett Packard computers using Prism Version 2.01.

	RESULTS

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Effect of Gemcitabine on Colony Formation of HL-60 and CD34⁺ Cells
For a better insight into the toxicity profile of gemcitabine, CD34⁺ and HL-60 cells were cultured in methylcellulose along with the designated concentrations of gemcitabine (0.1–16 nM). As illustrated in Figure 1, growth factor-induced colony formation derived from CFU-GM and BFU-E, and colony formation from HL-60 were inhibited by gemcitabine in a dose-dependent manner. However, HL-60 cells proved more sensitive than CD34⁺ cells. Thus, if the inhibitory effect of gemcitabine was expressed as the IC₅₀ value, it could be seen that for a 50% inhibition of CFU-GM, a 12.4-times-higher concentration (8.61 ± 1.5 nM) was required compared with that of HL-60 cells (0.69 ± 0.18 nM; p = 0.0011). For a comparable BFU-E inhibition, a 3.1-times-higher concentration was necessary (2.15 ± 0.39; p = 0.0088).

View larger version (16K): [in this window] [in a new window]   Figure 1. Inhibitory effect of varying doses of gemcitabine on colony formation derived from CFU-GM (), BFU-E (), and HL-60 cells (). The results for BFU-E, CFU-GM, and HL-60 cells are expressed as the mean ± SE of percent inhibition for gemcitabine compared with control cultures.

Similar differences in the sensitivity of normal and malignant cells for gemcitabine were found when the dose necessary for 100% inhibition of colony formation by CD34⁺ and HL-60 cells was compared. Again, it could be seen that the HL-60 cells (2 nM) were significantly more sensitive than normal progenitors (BFU-E, CFU-GM) (8 nM versus 16 nM; p < 0.0001).

Effect of Immediate versus Delayed Addition of dCyd on Gemcitabine-Mediated Growth Inhibition of CD34⁺ and HL-60 Cells
Channeling of gemcitabine into the cellular trinucleotide pool is achieved by the pyrimidine salvage enzyme deoxycytidine kinase (dCK) [2]. According to this proposed mechanism, it has been suggested that accumulation of gemcitabine nucleotides by cultured malignant cells can be prevented by dCyd, the naturally occurring substrate of dCK [4].

In order to find out whether dCyd is able to protect colony formation of HL-60 and normal myeloid and erythroid progenitor cells from the growth-inhibitory effect of gemcitabine, a concentration of 1mM of dCyd was coadministered to cultures incubated with 16 nM of gemcitabine. This completely inhibited colony formation of HL-60 and CD34⁺ cells. As shown in Table 1, coadministration of 1mM of dCyd was sufficient for restoring HL-60 proliferation as well as colony formation of CD34⁺ cells. In order to find out whether a delayed addition of dCyd to cytostatic-treated cultures results in different restoration of normal and malignant cells, dCyd was added after a 24- to 96-hour delay to gemcitabine-treated HL-60 and CD34⁺ cells. As can be seen from Table 1, delayed addition of dCyd significantly protected normal but not malignant cells. Thus, addition of dCyd to HL-60 cells after a delay of 24 and 48 hours after treatment with gemcitabine restored 40% ± 11% and 4.5% ± 3.5%, respectively, of the colony formation capacity of HL-60 cells, whereas up to 95% of the colony formation capacity of erythropoetic and myeloid progenitor cells could be rescued. If dCyd was added to gemcitabine-treated HL-60 and CD34⁺ cells after 72 hours, there was a significant proliferation of myeloid and erythroid progenitor cells (45.5% ± 2.5% and 39.5% ± 19.5%), whereas no HL-60 colony formation occurred at all.

Figure 2 illustrates the effect of delayed addition of dCyd on gemcitabine-treated HL-60 cells mixed with CD34⁺ cells. As found in separate experiments, coadministration restored the colony formation capacity of both CD34⁺ and HL-60 cells. A delayed addition of dCyd after 48 hours was followed by a significant restoration of myeloid and erythroid progenitor cells (94% ± 17% versus 83% ± 17%), whereas HL-60 cells were substantially inhibited (9.6% ± 4.17%). Administration of dCyd after 72 hours did not compromise the lethally toxic effect of gemcitabine on HL-60 cells, whereas colonies obtained from normal myeloid and erythroid progenitor cells were protected up to 63.5% ± 1.5% and 43% ± 15%, respectively.

View larger version (15K): [in this window] [in a new window]   Figures 2. Reversal of gemcitabine-mediated growth inhibition of normal CD34+ cells and HL-60 cells by immediate or delayed addition of 1mM dCyd. CD34+ and HL-60 were grown in coculture in the presence of 16 nM gemcitabine. At 0, 48, and 72 hours, cytostatically treated cultures were overlayered with dCyd and were scored for colonies after an incubation period of 16 days. Dark-shaded bar = CFU-GM; medium-shaded bar = BFU-E; unshaded bar = colonies derived from HL-60. The values represent mean ± SE of control colony formation determined from three experiments. Signed p values indicate statistically significant differences in the protective effect of dCyd on CFU-GM or BFU-E compared with HL-60 cells (Student’s t-test).

	DISCUSSION

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It has previously been shown that in vitro coadministration of high-dose dCyd (1 x 1mM) to bone marrow and malignant cells treated with 1 x 10^-5 M of Ara-C preferentially restored about 90% of CFU-GM, whereas HL-60 growth remained substantially inhibited (10%) [16]. This preferential rescue of normal cells by dCyd was suggested to be due to differences in the metabolism of dCyd and Ara-C between normal and malignant cells. Thus, higher intracellular ratios of dCTP/Ara-CTP were reached in normal than in leukemic cells, which were followed by a reduction of Ara-C DNA incorporation in normal cells.

However, our study shows that coadministration of 1mM dCyd to CD34⁺ and HL-60 cells treated with relatively low doses of gemcitabine (16 nM) did not confer a growth advantage on normal cells over malignant elements but completely restored colony growth of normal and malignant progenitors.

This discrepancy can be explained by the fact that phosphorylation of dCyd analogues is a time- and dose-dependent process where higher concentrations of dCyd analogues lead to an earlier peak value of the corresponding triphosphate than do lower concentrations [17]. Therefore, if low doses of the nucleoside analogue gemcitabine (16 nM) are used, peak values of dFdCTP are delayed in normal and malignant cells.

A delayed addition of dCyd (1mM), however, resulted in a significant protection of normal cells without compromising the antitumor effect on HL-60 cells. High doses of dCyd (100 µM) added after 24 and 48 hours rescued up to 90% of gemcitabine-treated normal stem cells, whereas only 40% at 24 hours and 4.5% at 48 hours of HL-60 cells could be rescued (Table 1). If dCyd was added after 72 hours, up to 40% of normal stem cells could still be rescued, but HL-60 cells were completely inhibited.

The significant growth advantage of normal CD34⁺ cells over leukemic HL-60 cells was also seen in experiments in which dCyd (1mM) was added after a delay to a mixture of gemcitabine-treated HL-60 and CD34⁺ cells. As illustrated in Figure 2, the delayed addition of dCyd after 72 hours to gemcitabine-treated cocultured HL-60 and CD34⁺ cells resulted in the colony growth of normal myeloid (63.5 ± 1.5) and erythroid progenitor cells (43 ± 15) only, whereas HL-60 proliferation was suppressed.

This differential rescue of CD34⁺ over HL-60 cells by a delayed dCyd addition might be explained by the fact that gemcitabine is phosphorylated to a higher extent in malignant cells than dCyd. Consequently, increasing the delay of dCyd results in an increasing ratio of dFdCTP/dCTP in malignant cells compared with that in normal CD34⁺ cells. Therefore, no colony formation of HL-60 was found, whereas colony formation of CD34⁺ cells could still be obtained with application of dCyd after a prolonged delay.

This hypothesis is in accordance with previous studies of Ara-C in which it was shown that phosphorylation of Ara-C by dCK in myeloid leukemic cells was double that of dCyd, whereas in normal cells the maximal velocity of the two substrates was equal [18, 19]. Different activities of dCK in normal and malignant cells were suggested to be responsible for this phenomenon. In another study, it was found that the v_max for Ara-C was double that for dCyd in leukemic cells (5.3 ± 1.0 nmol/h/mg versus 2.5 ± 0.5 nmol/h/mg), whereas in normal cells the v_max for both substrates remained equal (1.4 ± 0.2 nmol/h/mg versus 1.1 ± 0.1 nmol/h/mg) [20].

In conclusion, gemcitabine has an inhibitory effect on CD34⁺ cells and HL-60 cells with increased sensitivity of leukemic compared with normal cells. The results of the present in vitro study further indicate that continuously administered low concentrations of gemcitabine combined with delayed administration of high but nontoxic and clinically achievable levels of dCyd result in enhanced chemotherapeutic selectivity of gemcitabine. Although caution is required for extrapolating in vitro data to the in vivo situation, these findings have implications for designing future drug regimens incorporating gemcitabine and dCyd, with the potential for an improved therapeutic index.

	ACKNOWLEDGMENT

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We are indebted to Hanni Linert, Michalea Karches-Böhm, and Renate Sperk for excellent technical assistance. This work was supported by a grant from the Verein zur Förderung der hämatologischen Forschung.

	REFERENCES

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