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Stem Cells, Vol. 14, No. 3, 351-362, May 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

2',2'-Difluorodeoxycytidine (Gemcitabine) Induces Apoptosis in Myeloma Cell Lines Resistant to Steroids and 2-Chlorodeoxyadenosine (2-CdA)

Johann Grubera, Françoise Geisenb, Roswitha Sgoncc, Alexander Eglea, Andreas Villungera, Guenther Boeckc, Günther Konwalinkab, Richard Greila

a Laboratory of Molecular Cytology, Department of Internal Medicine, University of Innsbruck, Innsbruck, Austria;
b Stem Cell Laboratory, Department of Internal Medicine, University of Innsbruck, Innsbruck, Austria;
c Institute for General and Experimental Pathology, Innsbruck, Austria

Key Words. Multiple myeloma • Apoptosis • Steroid resistance • Gemcitabine • 2-chlorodeoxyadenosine • CFU-GM colony growth

Dr. Richard Greil, Department of Internal Medicine, Laboratory of Molecular Cytology, University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10–6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 µM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 µM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells.

The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Steroids are the mainstay in the treatment of multiple myeloma both in the induction phase of the treatment and in salvage protocols like VAD (vincristine, adriamycin, dexamethasone) for relapsing or refractory patients. They act in a dose-dependent manner [1] and induce programmed cell death [2]. Steroids have been shown to make the major contribution to anti-tumor effects in refractory myeloma when applied together with anthracyclines and/or vincristine (e.g., in the VAD protocol) [3].

Although high-dose chemotherapy followed by stem cell support may be effective and even superior to standard-dose chemotherapy [4], the majority of myeloma patients will either not qualify for these cytotoxic protocols or not be definitely cured by this procedure [5]. In addition, failure to induce remission by conventional treatment is the single most important adverse predictor of outcome in such a scenario [6]. For these reasons, there is an urgent need for the development of new efficient cytotoxic substances to combat this disease.

We, therefore, studied four myeloma cell lines (U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia line (HS Sultan), and a myeloid control cell line (HL 60), all of which are definitely resistant to the treatment by steroids as a model to test the efficacy of two new substances, i.e., the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine). 2-CdA has previously been shown to be highly effective in slowly proliferating and terminally differentiated B cell neoplasias like B-chronic lymphocytic leukemia (B-CLL) [7] and hairy cell leukemia [8]. 2-CdA was also used on a small scale in myeloma, but no significant clinical benefit could be demonstrated in 10 patients [9] on whom it was tested. Because the result of this trial has been questioned [10] and also for the purpose of making comparisons, the substance has been included in our analysis. Gemcitabine has been shown to be promising in the treatment of some solid tumors like ovarian cancer [11] and non-small cell lung cancer [12], but it has not yet been tested in multiple myeloma. Our in vitro results, which demonstrate for the first time that gemcitabine is effective in multiple myeloma cells resistant to steroids and 2-CdA at concentrations below those suppressing growth of bone marrow myeloid progenitor cells, suggest that this substance could be used in phase I clinical trials on patients with multiple myeloma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
U 266, OPM 2, IM 9, and HS Sultan cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Biol. Inc.; Beth Hamek, Israel). RPMI 8226 and HL 60 cells were cultured in 15% FCS in a humidified atmosphere with 5% CO2. Immunophenotype, proliferative activity, interleukin 6 (IL-6) receptor expression and growth factor production of these cell lines have been assessed in detail [13].

DNA Preparation and Agarose Gel Electrophoresis
DNA was extracted from 1 x 107 cells, using phenol/chloroform and electrophoretically separated on an agarose gel, as described previously [14]. Cell pellets were resuspended in 100 µl ice-cold phosphate-buffered saline (PBS)/EDTA, to which 900 µl of lysis buffer (0.3 M sucrose, 10 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1% Triton X-100, [Sigma; Deisenhofen, Germany]) were added. The suspension was agitated horizontally for 10 min on ice and centrifuged at 400 g for 10 min at 4°C. The supernatant was then discarded and the pellet resuspended in 900 µl lysis buffer, centrifuged again as described above, and resuspended in 450 µl nuclei-resuspension buffer (75 mM NaCl, 24 mM EDTA, pH 8.0), to which 10 µl proteinase K (10 mg/ml; Boehringer Mannheim; Mannheim, Germany), 12.5 µl 10% SDS, 1.5 µl bidistilled water were added, and the solution was incubated for two hours at 55°C. The sample was then extracted three times with an equal volume of phenol/chloroform [24:1 (v/v), Tris-HCl (pH 8.0), 1 mM EDTA] followed by an extraction with 1 volume of chloroform/isoamylalcohol [24:1 (v/v)]. Finally, DNA was precipitated with 0.1 volume 3 M Na-acetate (pH 8.0) and 2 volumes ethylalcohol. Eight µg DNA per lane were electrophoretically separated on 1.8% agarose gels containing ethidium bromide. DNA was visualized by a UV (302 nm) transilluminator, and gels were photographed with a Polaroid DS 34 camera system.

Morphological Analysis and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL)
Morphological analysis and determination of apoptosis on a single cell level was done with a TUNEL technique in combination with confocal laser scan microscopy as described by Sgonc et al. [15]. Briefly, 1 x 106 cells were fixed in 200 µl freshly prepared 2% paraformaldehyde dissolved in PBS for 30 min at room temperature on a horizontal shaker, washed once in PBS/1% bovine serum albumin (BSA), permeabilized with 100 µl 0.1% Triton X-100/0.1% Na-citrate for 2 min on ice, washed twice and then incubated for the in situ end-labeling with 0.03 nmol fluorescein isothiocyanate (FITC)-12-dUTP (Boehringer Mannheim), 3 nmol dATP, 2 µl 25 mM CoCl2, 25 units terminal deoxynucleotidyl transferase (TdT) (Boehringer Mannheim) and TdT buffer (30 mM Tris pH 7.2, 140 mM sodium cacodylate) in a total reaction volume of 50 µl in a moist chamber for 60 min at 37°C. The TUNEL reaction was stopped by adding 2 µl 0.5 M EDTA. Then the cells were washed twice and analyzed on a FACScan (Becton Dickinson, San Jose, CA) for quantitative analysis of DNA fragmentation and on a laser scanning microscope LSM 10 (Zeiss; Oberkochen, Germany) using a 488 nm argon laser, filter setting BP 530/30 for morphological analysis of TUNEL positive cells.

Quantitative Analysis of Apoptotic Cells
Quantitation of apoptotic cells was performed with a propidium iodide [(PI), Serva, Heidelberg, Germany] assay [16]. After centrifugation (300 g) cell pellets were resuspended in 500 µl hypotonic fluorochrome solution (50 µg/ml PI in 0.1% sodium citrate/0.1% Triton X-100) (Sigma) for permeabilization and DNA staining. Analysis of cell size and fluorescence intensity was performed in the forward/side scatter program on a FACScan (Becton Dickinson) using an argon laser (488 nm). After exclusion of necrotic debris, apoptotic cells were defined by a decrease in DNA content as compared to G1 phase cells.

Treatment of Myeloma and Plasma Leukemia Cells with Steroids, Gemcitabine and 2-CdA
Cells were treated with dexamethasone (Sigma) in concentrations of 10–6 M and 10–5 M, respectively. Gemcitabine (Eli Lilly; Indianapolis, IN) was used in concentrations of 10 nM, 100 nM, 1 µM and 10 µM, respectively, for 30 min, after which the cells were centrifuged (300 g), and fresh culture medium was added for the indicated times. Clinically achievable concentrations for gemcitabine are in the range of 10-20 µM [17,18]. In clinical trials it is normally given as a 30 min infusion [19,20]. Therefore, an incubation time of 30 min was used in our experiments. In addition, long-term treatment of up to 72 h was carried out. 2-CdA, synthesized as described by Carson et al. [21] and Cardinaud [22], was kindly provided by Prof. E. Beutler, Scripps Research Institute, La Jolla, CA. The lyophilized substance with the molecular mass of 271 Da was dissolved in 0.9% sodium chloride and was used in the concentrations of 30 or 300 nM for the indicated periods of time.

Treatment with Gemcitabine and 2-CdA of Colony Growth of Normal Myeloid Progenitor Cells
Human bone marrow mononuclear cells (MNC) were obtained from healthy volunteers after informed consent. Ten to 15 ml of marrow was aspirated from the iliac crest and collected in syringes containing preservative-free heparin.

For clonogenic assay of marrow MNC, we used Iscove's methylcellulose culture system [23]. In brief, bone marrow MNC were isolated according to the method of Boyum [24], washed twice in Hank's balanced salt solution (HBSS) and suspended in Iscove's medium containing 20% FCS. They were plated at a final concentration of 1 x 105 cells/dish (1.1 ml culture medium per dish) in 0.8% methylcellulose in Iscove's medium containing 30% FCS, 10% BSA, 1% 2-mercaptoethanol (1 x 10–4 M) and 200 mM L-glutamine. In addition, 3 U/ml recombinant human erythropoietin (Epo; Cilag, Vienna, Austria) and 10% agar stimulated leukocyte medium were used as stimulants in the presence of the designated concentrations of 2-CdA (5, 10, 20, 40, 80, 160 nM final concentration) and gemcitabine (1, 2, 4, 8, 32, 100, 500 nM, 1µM, 10 µM final concentration), respectively. Each assay was set up in duplicate. Plates were incubated in a fully humidified atmosphere with 5% CO2 at 37°C.

After 18 days of incubation, colonies derived from granulocytic macrocytic progenitor cells containing more than 50 cells were scored, using an inverted microscope.

Time Course Studies
Marrow MNC (1 x 106/ml), suspended in Iscove's medium containing 20% FCS, were incubated with increasing doses of 2-CdA (5 to 160 nM) and gemcitabine (1 to 32 nM), respectively, in a fully humidified atmosphere with 5% CO2 at 37°C for 24 or 48 h. For short time exposure (30 min) marrow MNC were incubated with increasing doses of gemcitabine (10 nM, 100 nM, 500 nM, 1 µM, 10 µM). After incubation, cells were washed three times in HBSS and resuspended in Iscove's medium containing 20% FCS. Cell viability was determined by dye exclusion test; the viability of MNC with and without 2-CdA or gemcitabine was over 90%. Cells were then grown without 2-CdA and gemcitabine in methylcellulose cultures and examined for their capability of colony formation as described above.

Statistical Analysis
Statistical analysis was performed with an unpaired Student's t-test using Stat View® software, 4.1 (Abacus Concepts; Berkeley, CA) on a Macintosh IIci computer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of Apoptosis with Gemcitabine
For investigating the mechanisms of action of gemcitabine, we used three different assays for apoptosis detection: the classical DNA ladder analysis by agarose gel electrophoresis of the extracted DNA, a modified TUNEL technique combined with laser scanning microscopy for simultaneous detection of DNA fragmentation and morphological changes on a single cell level [15], and, third, flow cytometry analysis of permeabilized and PI stained cells to show DNA loss [16 ]. In all the cell lines examined, gemcitabine (10 µM) induced the typical ladder pattern reflecting the presence of an activated endonuclease (Fig. 1Go). Furthermore, the morphological features of apoptosis, like cell shrinkage, chromatin condensation and membrane blebbing [25] were observed in treated cells which simultaneously incorporated FITC-labeled nucleotides in an in situ nick end-labeling assay (Fig. 2Go). The DNA loss, also a typical feature during the apoptotic process, resulted in a sub-G1 peak in the flow cytometric analysis of PI stained cells [16]. Since all three assays showed comparable results, PI-staining was used for all further studies, for it is a very reliable method for the quantification of apoptotic cells [26].



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  		Figure 1. Gemcitabine-induced DNA degradation. Agarose gel electrophoresis of DNA from the HL 60 control cell line, one plasma cell leukemia and four myeloma cell lines after treatment with 10 µM gemcitabine for 24 h. Gemcitabine induces the typical ladder pattern of DNA characteristic of apoptosis. IM 9 (lane 2), RPMI 8226 (lane 4), OPM 2 (lane 6), and U 266 (lane 8) cells were cultured in RPMI 1640 medium supplemented with 10% FCS; L 363 (lane 10), HL 60 (lane 12), and HS Sultan (lane 14) were cultured in 15% FCS. Lanes 1, 3, 5, 7, 9, 11, and 13 represent the respective controls.

 


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  		Figure 2. Gemcitabine-induced morphological changes. Confocal laser scan microscopic images of myeloma cell lines treated with 10 µM gemcitabine for 48 h showing overlays of transmission scans and fluorescence scans. Apoptotic cells reveal nuclei stained green after 3' OH labeling by the TUNEL technique. A) OPM 2, B) IM 9, C) RPMI 8226, D) U 266 (original magnification x63, oil, x60 zoom).

 
Sensitivity of Cell Lines to Dexamethasone and 2-CdA
There was no statistically significant difference in the degree of apoptosis between myeloma and plasma cell leukemia cells treated with 10–6 M dexamethasone and those of the control group (Figs. 3A-FGo).



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  		Figure 3. Effect of dexamethasone, 2-CdA and gemcitabine on apoptosis of tumor cells. The percentage of apoptotic cells after treatment with the relevant substances is indicated in a box plot and whiskers model, in which boxes contain the data of the interquartile range and whiskers depict the upper and lower quartile of the data. Median values are indicated by horizontal lines. Asterisks characterize effects statistically different from control values (** p < 0.001. * p < 0.05). Data, pooled from four experiments, were analyzed after 48 h of continuous incubation of cells with the relevant substances. The following concentrations were applied: dexamethasone [10–6 M, (Dex)]; 2-CdA [300 nM (C1); 30 nM (C2)]; gemcitabine [10 µM (G1); 100 nM (G2); 10 nM (G3)] for 48 h. Results are shown for (A) the HL 60, (B) the U 266, (C) the RPMI 8226, (D) the OPM 2, (E) the IM 9 and (F) the HS Sultan cell line. Statistical analysis was performed using Student's t-test and Stat View® software.

 
All cell lines were treated with 30 or 300 nM 2-CdA for 30 min, 24 and 48 h, respectively. Forty-eight-hour values are shown in Figure 3Go. The median numbers of apoptotic cells in the myeloma cell lines and plasma leukemia cell line, as measured by the PI assay, were not statistically different from those in the control group (Fig. 3Go).

Effects of Gemcitabine on Tumor Cell Kill
Myeloma and plasma cell leukemia cells were treated with 10 nM, 100 nM, 1 µM, 10 µM and 100 µM of the pyrimidine antagonist gemcitabine for 30 min, 24 and 48 h, respectively. Treatment with the extremely low concentration of 10 nM was sufficient to induce a significant degree of apoptosis in the U 266 and IM 9 myeloma cell lines, and the HS Sultan plasma cell leukemia line (Fig. 3Go). A 30-min treatment with 1 µM gemcitabine followed by a 48-h incubation in a medium without this substance induced apoptosis in the RPMI 8226 cell line (p < 0.05 compared to controls, data not shown), but failed to induce apoptosis in the OPM 2 cell line. Significant tumor cell kill was observed in all of the cell lines, at concentrations of 10 µM (Figs. 3A-FGo). Increasing the dose of gemcitabine beyond the optimal in vitro dose [27] to 100 µM did not alter the percentage of apoptotic cells (Table 1Go). This is consistent with the findings of Grunewald et al. [17] in human acute myeloid and acute and chronic lymphoid leukemias. In the time course of gemcitabine effects, the maximal degree of apoptosis was seen when cells were analyzed after 48 h of continuous treatment (Fig. 4Go).


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  		Table 1. Percentage of apoptotic cells after incubation with different concentrations of gemcitabinea
 


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  		Figure 4. Time-course of gemcitabine-induced apoptosis in the RPMI 8226 cell line. RPMI 8226 cells were treated with gemcitabine, 2-CdA and Dex, for the times and with the concentrations indicated in the figure. The percentage of apoptotic cells was determined by the PI-incorporation assay at the times indicated, as described in Materials and Methods. The figure depicts the data from one out of three experiments. All three experiments gave similar results.

 
Bolus Versus Continuous Treatment
We also investigated the possibility whether a continuous treatment for 48 h and a "bolus" treatment for 30 min with gemcitabine might have different apoptotic efficacies. For the bolus treatment, gemcitabine was added for 30 min after which the cells were centrifuged and resuspended in a fresh culture medium. After further incubation of 48 h in this fresh medium, cells were analyzed for apoptosis. For continuous treatment, the culture medium was not changed and cells were also analyzed after 48 h. As shown in Figure 5Go, there was no significant difference between the two modes of treatment.



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  		Figure 5. Short-term (30 min) versus continuous (48 h) treatment of tumor cells with gemcitabine. Black bars indicate the number of apoptotic cells resulting from a 30-min treatment with gemcitabine (10 µM), subsequent washout of the substance, reseeding of cells in fresh culture medium and analysis for apoptosis by the PI assay after 48 h of culture. White bars represent the results of tests in which cells were incubated with gemcitabine continuously (10 µM). One out of three representative experiments is shown. Differences between bolus and continuous application of gemcitabine were statistically insignificant.

 
Effect of Short-Time and Continuous Exposure to 2-CdA and Gemcitabine on CFU-GM Progenitor-Derived Colony Formation
To determine the exposure time required for a significant inhibition of colony formation, MNC were seeded in cultures, treated with gemcitabine or 2-CdA for 24 h, 48 h or continuously for up to 18 days. After 18 days, colony formation of CFU-GM was determined as described in Materials and Methods. When the substances were present throughout the experiment, there was a dose-dependent growth inhibition of CFU-GM as compared to controls with a maximal inhibition occurring at 32 nM gemcitabine and at 80 nM 2-CdA, respectively (Figs. 6A, BGo).



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  		Figure 6. Effect of short-time (24 and 48 h) and continuous exposure of 2-CdA and gemcitabine on CFU-GM-derived colony formation. For short-time incubation, marrow MNC were incubated with varying doses of 2-CdA or gemcitabine for 24 and 48 h, then washed and plated without 2-CdA or gemcitabine in methylcellulose at a concentration of 1 x 105 cells/dish (1.1 ml). For continuous exposure, cells were directly plated with varying doses of 2-CdA or gemcitabine in methylcellulose at a concentration of 1 x 105 cells/dish (1.1 ml). Results represent the mean ± SE of percent of growth at 24 and 48 h obtained from nine and five experiments, respectively.

 
Compared to untreated controls, however, a 48-h incubation with 2-CdA resulted in an 82% inhibition of colony growth (Fig. 6BGo), whereas in gemcitabine treated cells only 15% inhibition was observed, even at the highest concentration used (32 nmol; Fig. 6AGo). These results indicate that a prolonged exposure time is required for gemcitabine to exert an inhibitory effect on the growth of CFU-GM.

Effect of Short-Time Incubation of Gemcitabine on CFU-GM
In order to analyze the toxicity of a short-time incubation of gemcitabine, MNC were incubated for 30 min in a concentration range of 10 nM up to 10 µM. The results showed that inhibition was only found at the highest dose, i.e., 10 µM (Fig. 7Go).



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  		Figure 7. Effect of short-time incubation of gemcitabine on CFU-GM. Bone marrow MNC were incubated with varying doses of gemcitabine (10 nM, 100 nM, 500 nM, 1 µM, 10 µM) for 30 min, then washed and plated without gemcitabine in methylcellulose at a concentration of 1 x 105 cells/dish (1.1 ml). Results represent the mean percentage ± SE of colony formation obtained from six experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Standard substances for the treatment of multiple myeloma comprise alkylating agents, vincristine, adriamycin, methylprednisolone and dexamethasone. When these substances were tested in an in vitro system on RPMI 8226, ARH, U 266 and HS Sultan cell lines, vincristine (2 nM) was clearly superior to the other substances [28]. Adriamycin (26 nM) was less effective on growth inhibition in RPMI 8226 and U 266 cells. Methylprednisolone (2 µM) and dexamethasone (0.5 µM) showed only a moderate effect. In order to compare the efficacy and toxicity of new therapeutic agents, in our study they were tested in three of the above-mentioned and well-characterized cell lines i.e., RPMI 8226, U 266, HS Sultan, in addition to IM 9, OPM 2 cell lines, and compared with the HL 60 control cell line.

Gemcitabine is already being used in clinical trials for the treatment of slowly proliferating tumors like head and neck cancer [29], colorectal cancer [30] and non-small cell lung cancer [31]. So far, however, neither in vitro nor in vivo data have been published as to the efficacy of this substance in multiple myeloma. In this article we report for the first time that gemcitabine definitely induced apoptosis in all of the cell lines, namely four myeloma cell lines, a plasma cell leukemia line and myeloid control cell line, all of which are resistant to steroids. The evidence for this is as follows: (A) a DNA degradation typical of apoptosis was shown on agarose gels (Fig. 1Go), (B) gemcitabine induced the well-known cell morphology characteristic of this process [32], (C) these morphologically apoptotic cells simultaneously integrated FITC-labeled nucleotides into their DNA as demonstrated by an in situ nick end-labeling technique (Fig. 2Go), and (D) apoptotic cells were characterized by a decrease in cell size and simultaneous loss of DNA content.

Gemcitabine has previously been shown to induce apoptosis in the HL 60 myeloid cell line in concentrations as low as 1 µM and with incubation times as short as two h [33]. In this investigation Bouffard et al. reported that the degree of apoptosis clearly proved to be dose- and time-dependent. Under longer exposure times, gemcitabine caused programmed cell death at concentrations even 100-fold lower than Ara-C [33]. In two out of four myeloma cell lines as well as in one plasma cell leukemia line, we demonstrate a sensitivity to this drug at a concentration of 10 nM which is 100 times below that reported in the literature for the HL 60 cell line [33], and still 10 times below the concentration at which gemcitabine is active in the HL 60 cell line in our study. Although higher concentrations (10 µM) of gemcitabine were necessary for the induction of apoptosis in the other two myeloma cell lines (i.e., the RPMI 8226 and the OPM 2 cell lines, Figs. 3C, DGo), these plasma cells were responsive after a short exposure of only 30 min (Fig. 5Go). In our test system, dose escalation from 10 µM up to 100 µM failed to further increase the percentage of apoptotic cells (Table 1Go). The relatively high sensitivity of myeloma and plasma cell leukemia lines to gemcitabine is underlined by the fact that concentrations of this substance up to 238 µM were necessary to induce in vitro response in tumor cells from patients suffering from other hematologic diseases [34], such as acute myelocytic and lymphocytic leukemia. Using this high dose of gemcitabine, an in vitro response of freshly isolated leukemic cells to gemcitabine was observed in 45% of patients with acute myelocytic leukemia and in only 27% of patients with acute lymphocytic leukemia [34].

In order to assess the value of gemcitabine for the in vivo situation, we also analyzed the dose- and time-dependent effect of gemcitabine on the clonal growth of normal bone marrow CFU-GM progenitor cells. A short-time incubation with gemcitabine at concentrations up to 1 µM did not result in significant inhibition of CFU-GM (Fig. 7Go), whereas under these conditions apoptosis was induced in three out of four myeloma and one plasma cell leukemia cell line.

In an in vitro assay, 2-CdA at the concentration of 238 µM induced a response in all samples isolated from patients with chronic myelocytic leukemia, whereas only 70% of cells from patients with acute lymphocytic leukemia showed a response [34]. The effects of 2-CdA were heterogeneous when tested in mature B cell neoplasias. It induced up to 90% response rates in previously untreated patients suffering from Waldenström's disease and up to 50% reduction in macroglobulinemia in patients resistant to standard treatment [35]. In contrast, treatment with 0.1 mg/kg 2-CdA, i.e., the standard dose used in clinical trials [36], had no effect on myeloma protein levels and on clinical response [9]. In the present in vitro study, 2-CdA was used in concentrations of up to 300 nM, which although an achievable plasma concentration in vivo [37], is nevertheless beyond the plasma levels usually measured in clinical studies (i.e., 20-50 nM [38]) using standard doses of the substance. However, in our hands even a concentration of 300 nM of 2-CdA failed to induce apoptosis, whereas a 48-h exposure at concentrations of up to 160 nM already inhibited 80% of colony growth of CFU-GM (Fig. 6BGo).

As an explanation for this marked difference between the efficacy of a pyrimidine and that of a purine antagonist in causing programmed cell death in myeloma cells, we propose the following working hypothesis: In order to become active, both 2',2'-difluorodeoxycytidine, an antimetabolite of deoxycytidine, and 2-CdA, an adenosine deaminase-resistant analog of deoxyadenosine, require phosphorylation to the monophosphate form by deoxycytidine kinase (dCytK), conversion into their triphosphate forms by other kinases and incorporation into DNA. In contrast to gemcitabine, the net accumulation of 2-CdA phosphates depends on the balance between phosphorylation of the nucleoside by the dCytK and the dephosphorylation of its nucleotide product by cytoplasmic 5-nucleotidase (5' NT). Thus, cells in which 5' NT levels exceed those of dCytK are resistant to 2-CdA [39]. The main mechanism for acquired resistance to gemcitabine in cell lines has been shown to be the impaired phosphorylation attributed to decreased activity or altered substrate specificity of dCytK [40]. A gemcitabine-resistant ovarian cell line, deficient in dCytK activity has been shown to be cross-resistant to 2-CdA [41]. Therefore, based on our observation that myeloma cell lines and a plasma cell leukemia line are sensitive to gemcitabine but resistant to 2-CdA, we assume that these cells probably contain not only high levels of dCytK but even higher levels of 5' NT.

In summary, gemcitabine consistently induced apoptosis in four myeloma cell lines and one plasma cell line resistant to both steroids and 2-CdA. The higher sensitivity of myeloma cells to gemcitabine compared to that of normal bone marrow progenitor cells in a clinically achievable dose range looks encouraging enough to initiate phase I clinical trials.


   Acknowledgments
 
We thank Heidrun Recheis for preparing the confocal images. This work was supported by Austrian Research Council (FWF) Grants P 8947 med. (R.G.) and P 10132 med. (G.K.).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received December 8, 1995; accepted for publication March 8, 1996.




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