THP1核酸代谢

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].