THE PURINE NUCLEOTIDE CONTENT IN HUMAN LEUKEMIA CELL LINES

Abstract: The HPLC method was used to determine the purine nucleotide (ATP, ADP, AMP, GTP, GDP, GMP, NAD+) contents and the values of the adenylate energy charge (AEC) and guanylate energy charge (GEC) for three human acute myelogenous leukemia (AML) cell lines: HL60 (M3 subtype of AML), THP1 (M5 subtype of AML), and HEL (M6 subtype of AML) in French-American-British classification (FAB) and for one chronic myelogenous leukemia (CML) cell line: K562. The results showed that the examined leukemic cells had some significant changes in their purine nucleotide concentrations relative to healthy cells. On the basis of the obtained results, it seems that two of the tested acute myelogenous leukemia cell lines, HL60 and HEL, have similar purine nucleotide metabolisms, while the third AML cell line, THP1, has a purine nucleotide metabolism like that of the chronic myelogenous leukemia cell line, K562.

Key Words: Acute Myelogenous Leukemia, Chronic Myelogenous Leukemia,Purine Nucleotides, HPLC Technique

INTRODUCTION

There is a good deal of data on the increase in purine nucleotide contents in tumour cells relative to healthy cells [1-3]. Purine nucleotide metabolism is based on three fundamental pathways: de novo biosynthesis, the salvage pathway and catabolic conversions. Purine nucleotides are responsible for the biosynthesis of the building blocks for nucleic acids. Nucleotides also serve as co-substrates in the activation reactions of many metabolites, as phosphate donors for kinases, and as allosteric regulators of the activity of many enzymes[1,2]. With regard to cancer chemotherapy, it is very important that the intracellular ATP level determines a cell's ability to undergo apoptosis [4]. GTP mainly participates in the pathways of intracellular signalling involving Gproteins as well as in protein synthesis [1]. NAD+ is an important substrate for the formation of ADP-ribosylated proteins giving an ATP-ribose moiety to form long and branched polymers attached to nuclear proteins. It is postulated that the ADP-ribos lation of nuclear proteins could play an important role in the maturation, growth and proliferation of cells [5, 6]. Recent studies showed that poly(ADP-ribose) polymerase is involved in the regulation of gene expression through the modification of transcription factors by poly(ADP-ribosyl)ation orits direct binding to gene-regulating DNA sequences [7]. It was also demonstrated that NAD+ modulates p53 DNA binding specificity and function via interactions with p53 tetramers leading to a conformational change in the p53 tetramers [8]. Additionally, it was demonstrated that NAD+ participates in histone deacetylation, which can attenuate p53 transcriptional activity [9].

Thus, there is no doubt that precise and detailed knowledge on purine metabolism in normal and tumour cells would be very important for a good understanding of purine function and for identifying the main changes between normal and tumour cells with an aim to finding a method of selectively killing the latter [10]. Indeed, purine antimetabolites are often used in the treatment of leukemia diseases. Purine antimetabolites can be divided into three groups: i) structural analogues of normal purines; ii) inhibitors of de novo purine biosynthesis; and iii) inhibitors of the purine salvage pathway [11]. It was reported, for example, that the purine nucleoside analogues fludarabine and 2-chlorodeoxyadenosine are very active in chronic lymphocytic leukaemia (CLL) both in salvage therapy and in newly diagnosed patients. These drugs have revolutionized therapeutic strategies for patients with CLL, which used to be treated with alkylating agents that have no potential for long-term remission and cure. Purine nucleoside analogues have also been successfully combined with other drugs e.g. mitoxantrone, cyclophosphamide, corticosteroids and monoclonal antibodies [12]. However, recent papers reported on the appearance of resistance to purine and pyrimidine nucleoside and nucleobase analogues in multidrug resistant tumour cells (MDR). It is proposed that the overexpression of MDR exporting pumps (e.g. P-glycoprotein) may be involved in the resistance of these cells to purine and pyrimidine analogues [13]. Taking all of these findings into account, it seems that a determination of purine nucleotide levels in leukemia cells may be helpful for the design of effective chemotheraputic methods.

The aim of this study was to examine the purine nucleotide contents, specifically ATP, ADP, AMP, GTP, GDP, GMP and NAD+, for three human acute myelogenous leukemia (AML) cell lines, HL60, THP1, HEL [14-17], and one chronic myelogenous leukemia (CML) cell line, K562 [18]. All the measurements were made using the HPLC method [19].

MATERIALS AND METHODS

Cell lines and cultures

Human leukemia cells HL60, THP1, HEL and K562 were obtained from ATCC, Manassas, VA, USA. They were grown in RPMI medium supplemented with 2mM L-glutamine and 10% foetal calf serum in a humidified atmosphere of 95% air and 5% CO2. Cultures initiated at a density of 105 cells ml-1 grew exponentially to about 106 cells ml-1 in 72 hours. They were counted with a hemocytometer, immediately before use. Cell viability was assessed by trypan blue exclusion.

Cell cycle distribution by flow cytometry

The cells (106 cells ml-1) were pelleted by centrifugation and washed twice with PBS. The cell pellets were suspended in 0.5 ml PBS and fixed in 5 ml ice-cold 70% ethanol at 4°C. The fixed cells were centrifuged at 300×g for 10 minutes and the pellets were washed with PBS. After resuspension with 1 ml PBS, the cells were incubated with 10 ml of RNase I (10 mg/ml) and 100 ml of propidium iodide PI (400 mg/ml; Sigma) and shaken for 1 hour at 37ºC in the dark [20]. Samples were analysed by flow cytometry (FACScan, Becton Dickinson). The red fluorescence (F2) of single events was recorded using a laser beam at 610nm (lex = 488 nm) to measure the DNA content. The percentage of cells in each phase of the cell cycle was calculated with the Cell Fit Software (Becton Dickinson).

Preparation of samples for HPLC analysis

The cell sample containing 106 cells in culture medium was centrifuged at 300×g for 5 minutes at 4ºC. After removal of the supernatant, the cells were washed with 1 ml of PBS and the sample was centrifuged again at 300×g for 5 minutes at 4ºC. The washed cells were suspended in 200 ml of PBS and maintained at -28ºC for 20 minutes. After this time, the sample was unfrozen and 200 μl of 1.3M perchloric acid (HCLO4) was added. The obtained mixture was centrifuged at 19000×g for 10 minutes at 4ºC. The obtained supernatant (300 μl) was neutralised with 2 M KOH solution and centrifuged at 19000×g for 10 minutes at 4ºC.

HPLC separation of purine nucleotides

Twenty microlitre aliquots of the obtained samples were injected onto the chromatograph column, and purine nucleotides were separated using a linear phosphate buffer gradient elution system (buffer A, 150 mM KH2PO4, 150 mM KCl adjusted to pH 6.0 with K2HPO4; buffer B, 15% (v/v) solution of acetonitrile in buffer A) at a flow rate of 0.666 ml/min. Peaks were detected by absorption measurements at 254 nm. The composition of the mobile phase was controlled by a low-pressure gradient mixing device. The cycle time was 12.8 minutes between injections. The analytical column was maintained at a constant temperature of 20.5°C. The Hewlett Packard series 1110 chromatographic system consisted of a quaternary pump with a degasser and continuous seal wash option (G1311A), a variable-wavelength detector (G1314A) and a thermostatted column compartment (G1316A). The analytical column (100´4.6 mm L C) was packed with 18.3 mm Hypersil BDS-C (Hewlett Packard). Samples were introduced using a Rheodyne 7725 injection valve equipped with a 20 ml loop.Sample peaks were integrated, calibrated and quantified using a HPLC 2D chromatography data system operating on Chemstation Software for Windows 98 (Hewlett Packard).

Statistical analysis

Results are presented as the mean + S.D. of 4-5 independent experiments.Statistical analysis of the significance level of differences observed between cell lines was performed using the Kruskal-Wallis test.

RESULTS AND DISCUSSION

In our experiments, we used three human acute myelogenous leukemia (AML)cell lines: HL60, M3 subtype of AML; THP1, M5 subtype of AML; and HEL, M6 subtype of AML, in French-American-British classification (FAB) and one chronic myelogenous leukemia (CML) cell line, K562. For each cell line, the cell cycle distibution was analysed by flow cytometry using the PI staining method [20]. Representative histograms of PI fluorescence (F2) for all the examined cell lines are presented in Fig. 1. The distribution of cells in each phase of the cell cycle is shown in Tab. 1. As can be seen, the cell cycle distribution of HL60 and K562 cells were similar, whereas in the case of THP1 cells, the cell cycle distribution resembles that for the HEL cells. It was found that the percentages of HL60 and K562 cells in the growth phase of the cell cycle (S-G2/M) were significantly higher (50.7 ± 2.4% and 46.6 ± 3.0% for HL60 and K562, respectively) than in the case of THP1 and HEL cells (39.8 ±2.6% and 35.9 ± 2.6%, respectively).

Tab. 1. The cell cycle distribution for acute (HL60, THP1, HEL) and chronic myelogenous (K562) human leukemia cell lines.
                         Leukemia cell line
Phase of cell              % of cells
 cycle         HL60      THP1       HEL       K562
                              
 G1          41.3±1.4  50.0±1.4  44.7±1.0 42.0±4.8
 S           36.7±1.4  23.9±1.0  21.0±2.1 37.3±1.9
 G2/M        14.0±1.0  15.9±1.6  14.9±0.8  9.3±1.1

The values are the mean ± S.D. of four independent experiments.

The concentrations of purine nucleotides (ATP, ADP, AMP, GTP, GDP, GMP,NAD+) were determined using the HPLC method. This technique is widely used for the determination of nucleotides and related metabolite concentrations. The HPLC method used in this study permits the separation of a wide spectrum of metabolites present at low concentrations in a small sample volume of 20 ml[19].

For each cell sample, the adenylate energy charge value (AEC) and the guanylate energy charge value (GEC) [21] were calculated according to the following formulas:

Figs.2 and 3 present the contents of adenine nucleotides (ATP, ADP, AMP),guanine nucleotides (GTP, GDP, GMP) and NAD+, and the values of the adenylate energy charge (AEC) and guanylate energy charge (GEC) determined for the examined cell lines. For comparative purposes, all the obtained data are presented in diagram form. The control values reported for human blood lymphocytes from healthy subjects are also included in Tab. 2 [2].
Concerning adenine nucleotides, all the examined leukemic cell lines had high levels of ATP lying in the range 8.03-9.61 nmol/106 cells. However, it was observed that THP1 and K562 cell lines had slightly elevated ATP contents relative to HL60 and HEL cells. These differences were statistically significant(p<0.0001). Important differences were observed in ADP and AMP contents between the tested leukemia cell lines. THP1 and K562 cells had significantly higher (p < 0.0001) levels of ADP than HL60 and HEL (about 2-fold higher). It was also found that THP1 had about a 6- to 8-fold higher content of AMP than the other tested leukemia cells. Consequently, the AEC value determined for THP1 cell line was significantly lower (p < 0.0001) (0.803 ± 0.027) than that for the other cell lines: HL60, HEL and K562 (0.896-0.918).
Concerning guanine nucleotides, THP1 cells had the highest content of GTP(3.55 ± 0.35 nmol/106 cells) among the examined leukemia cell lines (p<0.001). The GDP contents were comparable for all the tested cells (about 1.5nmol/106 cells), although GDP content was slightly lower (1.25 ± 0.24 nmol/106 cells) in the case of HL60 cells (p < 0.001). Important differences were observed in GMP contents between the tested leukemia cells. It was found that THP1 and K562 cells had significantly higher (p < 0.0001) GMP levels than HL60 and HEL cells (about 3- to 5-fold differences). K562 cells had the lowest value of GEC (0.713 ± 0.021) among the examined leukemia lines. For the three other tested cell lines, HL60, THP1 and HEL, the values of GEC were in the range 0.765-0.797.

Tab. 2. The content of purine nucleotides (ATP, ADP, AMP, GTP, GDP, GMP, NAD+) and adenylate/guanylate energy charge values (AEC/GEC) in human lymphocytes of healthy subjects (data published by Carlucci et al. [2]).
Nucleotide    Human lymphocytes
               pmol/106cells
 ATP            1128±411
 ADP             648±166
 AMP             89±23
 GTP             345±73
 GDP             162±36
 GMP             67±10
 NAD+            347±109
   Value of energy charge
 AEC            0,739
 GEC            0,742

Important differences were also observed in NAD+ level between the tested leukemia cell lines (Fig. 3). THP1 and K562 cells had significantly higher (p<0.0001) NAD+ contents than HL60 and HEL cells (about 3.5-fold differences).

The obtained data showed that the purine nucleotide contents in the examined leukemia cell lines were 2- to 10-fold higher than in lymphocytes of healthy human subjects (Tab. 2) [2]. This is in agreement with literature data reporting the significant increase in nucleotide levels in many actively dividing tumour cells in comparison to normal cells [1]. AEC values found for all the examined leukemia cell lines (0.803-0.918) were also very high relative to the values for human blood lymphocytes from healthy subjects (0.739).

In summary, the presented data showed that the examined leukemic cells had important changes in purine nucleotide metabolism in comparison to normal lymphocyte cells. Some significant differences in the concentrations of purine nucleotides were also observed between the leukemic cell lines studied. Taking into account the recent data concerning the biological role of NAD+, (see Introduction section), the great differences in NAD+ levels observed for the examined leukemic cell lines could suggest important differences in their maturation, growth and proliferation [5, 6, 22, 23]. On the basis of the obtained results, it seems that two tested human acute myelogenous leukemia (AML) cell lines, HL60 (M3 subtype of AML) and HEL (M6 subtype of AML), have similar purine nucleotide metabolism while the third acute myelogenous leukemia (AML) cell line: THP1 (M5 subtype of AML) has purine nucleotide metabolism like that of the chronic myelogenous leukemia (CML) cell line K562. Data obtained in the study showed that there was no similarity between the nucleotide levels and percentage of cells in the growth phase of the cell cycle (S-G2/M) among the cell lines examined. There is no doubt that for the better understanding of the reported data more research is needed on purine nucleotide metabolism in acute myelogenous leukemia and chronic myelogenous leukemia cells at the level of enzymes participating in the conversion of purines and their analogues, as well as at the level of transcription regulation of genes encoding the purine interconversion enzymes.

Because purine metabolism is an important target for leukemia chemotherapy[11-13], it seems that the presented data could be helpful for future efforts to design more effective antileukemic drugs. Nevertheless, further studies are needed to prove the potential importance of the presented data in leukemia therapy.

Acknowledgements. This study was supported by the Faculty of Natural Science of the University of Szczecin. The authors acknowledge Maria Ignaczak and Magdalena Rutkowska for their technical assistance.

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