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GENE THERAPY
From the Department of Bioorganic Chemistry, Centre of
Molecular and Macromolecular Studies, Polish Academy of Sciences,
Many reports indicate different nonantisense yet
sequence-specific effects of antisense phosphorothioate
oligonucleotides. Products of enzymatic degradation of the
oligonucleotides can also influence cell proliferation. The
cytotoxic effects of deoxyribonucleoside-5'-phosphates (dNMPs) and their 5'-phosphorothioate analogs,
deoxyribonucleoside-5'-monophosphorothioates (dNMPSs) on 4 human cell types (HeLa, HL-60, K-562, and endothelial cells) were examined, and the effects were correlated with the catabolism of these compounds. The results indicate that
differences in cytotoxicity of dNMPs or dNMPSs in these cells depend
upon different activity of an ecto-5'-nucleotidase. It has also been found that dNMPSs stimulate proliferation of human umbilical vein endothelial cells and HL-60 cells in a concentration-dependent manner. This stimulation might be caused by the binding of
deoxynucleoside-5'-phosphorothioates to as-yet unidentified nucleotide
receptor(s) at the cell surface.
(Blood. 2001;98:995-1002) The rapid degradation of unmodified
oligodeoxynucleotides (phosphodiester oligodeoxynucleotides [PO
oligos]) by exonucleases and endonucleases limits their application as
antisense constructs1 and requires the synthesis and use
of modified oligonucleotides. Phosphorothioate analogs of
oligonucleotides (PS oligos) containing a sulfur atom at each
internucleoside bond still constitute the most promising class of
oligonucleotide analogs for potential therapeutic purposes. Although
they are much more resistant to nucleolytic degradation than the PO
oligos, reports indicating their stereodependent hydrolysis have been
published recently.2,3 Moreover, results published by
Vaerman et al4 indicate that the cytotoxicity of PO and PS
oligos might have been, in part, caused by mononucleotides
deoxyribonucleoside-5'-phosphates (dNMPs) or their
phosphorothioate analogs, deoxyribonucleoside-5'-monophosphorothioates (dNMPSs) released during enzymatic degradation of the
oligonucleotides. The toxicity of the dNMPs to leukemia cell lines
depends on their concentration and/or the type of nucleobase. It
appeared that 2'-deoxyguanosine 5'-monophosphate (dGMP) was
more toxic to the BV 173 cell line than other dNMPs used at the same
concentration. This observation indicates that nonantisense mechanisms
of the action of oligonucleotides may be
sequence-dependent.4 However, Vaerman et al4
studied the cytotoxicity of mononucleotides and oligonucleotides using
only the leukemia BV 173 cell line bearing the Philadelphia chromosome,
and CD34+ bone marrow cells. Some earlier
data5-8 have also shown that deoxyribonucleosides
influence the growth of leukemia cell lines, but there is no evidence
that other types of cells are also sensitive to mononucleotides or
their phosphorothioate analogs. Because we have studied the synthesis
and therapeutic application of phosphorothioate oligonucleotides,9-11 we have undertaken efforts to
explain their potential nonantisense cytotoxicity.
In this study, we have analyzed the influence of mononucleotides (dNMPs
and dNMPSs) and oligonucleotides (PO and PS oligos) on 4 different
human cell types: the HeLa epitheloid carcinoma cell line, HL-60
promyelocytic leukemia cells, K-562 myelogenous leukemia cells bearing
the Philadelphia chromosome, and human umbilical vein endothelial cells
(HUVECs). We have examined the cytotoxic effects of mononucleotides,
deoxynucleosides, and free purine bases and have correlated the effects
with the catabolism of these compounds. It is known that the
extracellular metabolism of adenosine 5'-triphosphate (ATP) and
adenosine is controlled by enzymes such as ecto-ATP-ases,
5'-nucleotidases, and phosphatases, which are present on the surface of
many different cell types.12 Although the biological roles
of ATP and adenosine in the different signaling pathways have been
studied in detail, the effects of other nucleotides and nucleosides on
cell proliferation have not been characterized. Here, we demonstrate
that deoxynucleosides, their 5'-phosphates, and their
5'-phosphorothioates can also influence cell proliferation. In the cell
types investigated, the cytotoxicity of extracellular dNMPs and dNMPSs
depends on the activity of the ecto-5'-nucleotidase (ecto-5'-NT)
present at the cell surface. The membrane-bound enzyme can convert
dNMPs and dNMPSs to deoxynucleosides, which are, in turn, the
substrates for other enzymes (purine nucleoside phosphorylase,
adenosine deaminase, guanosine deaminase) or are taken up by the cells.
Because the cell lines studied demonstrate different ecto-5'-NT
activity, the cytotoxic effects of dNMPs, dNMPSs, and deoxynucleosides
are different in each of these cell lines. However, ectonucleotidase
activity does not seem to be the only mechanism responsible for the
biological activity of these compounds. In the course of our studies,
we have also observed that dNMPSs stimulate growth of HUVECs and HL-60
cells in a concentration-dependent manner. This effect might be caused
by the binding of deoxynucleoside-5'-phosphorothioates to as-yet
unidentified nucleotide receptor(s) at the cell surface.
Chemicals
Oligonucleotides
Cell lines Endothelial cells (HUVECs) were isolated from navel strings by washing of umbilical veins with 0.1% collagenase solution. The cell lines studied were maintained in RPMI 1640 medium supplemented with 10% (HeLa, K-562) or 20% (HL-60, HUVEC) thermally inactivated FBS, 100 µg/mL streptomycin, and 100 IU/mL penicillin at 37°C in a humidified atmosphere of 5% CO2. In the case of the HUVECs, the medium was additionally supplemented with ECGF (150 µg/mL) and heparin (90 µg/mL). The endothelial cells were cultured in bottles covered with 1% gelatin.Purine base, deoxynucleoside, deoxynucleotide, or oligonucleotide treatment of cell lines Cells in exponential growth phase were washed with RPMI 1640 containing FBS and resuspended in this medium at a concentration of 5 × 104 per 200 µL (K-562, HeLa, or HL-60 cells) or 7 × 104 per 200 µL (HUVECs). Guanine, xanthine, deoxynucleosides, or mononucleotides (25 or 100 µM) were added to the cell cultures as sodium salts at time zero of each experiment without any further addition of the reagents. Additionally, the concentration-dependent influence of dGMP and dGMPS on the K-562 and HL-60 cell growth was studied at a 2.5- to 100-µM concentration range. In some experiments, dNMPs or dNMPSs (100 µM concentration) were added to the cells preincubated for 1 to 2 hours with , -methylene-ADP (100 µM), or ,-methylene-ATP (100 µM). NBTI (1 µM), dipyridamole (20 µM), or dilazep (1 µM) was
dissolved in water and added to cell cultures for 1 hour before the
treatment of cells with nucleosides or mononucleotides. PO and PS
oligos were added to the cell culture at 2.5-, 10-, or 25-µM
concentration. The number of cells was determined every 24 hours by
means of the tetrazolium salt (MTT) method.14
Chromatographic analysis of extracellular catabolism of nucleotides and nucleosides Each of the studied cell lines was incubated with 200 µM substrate (deoxynucleotide, deoxynucleoside, or purine base) for 96 hours in RPMI 1640 culture medium containing 10% or 20% FBS. For the correlation of catabolism and cytotoxicity of nucleosides, nucleotides, and purine bases, these experiments were carried out under the same conditions as those done to analyze cytotoxicity. Therefore, the assay medium was not supplemented with nucleoside transport inhibitors. At given time points, 250-µL aliquots were removed from the cell cultures, denatured for 30 minutes at 95°C, and spun down. The samples were maintained at 20°C prior to HPLC analysis. The
catabolites were identified by means of reverse-phase chromatography on
5 µ BDS Hypersil (Thermo Hypersil, Cheshire, United
Kingdom). Purine bases, deoxynucleosides, dNMPs, and dNMPSs were separated with a triethylammonium bicarbonate (TEAB) and CH3CN gradient: 0 to 0.1 M TEAB from 0 to 8 minutes; then
gradient 0.1 M TEAB 24% CH3CN/0.1 M TEAB from 8 to 12 minutes; and finally isocratic conditions, 24%
CH3CN/0.1 M TEAB (12 to 18 minutes). Under these
conditions, products of the enzymatic degradation of mononucleotides
and corresponding deoxynucleosides have been readily separated, eg, the
dGMP metabolites eluted as follows: xanthine at 4.1 minutes, guanine at
6.5 minutes, deoxyguanosine at 14.6 minutes, and deoxyguanosine
5'-phosphate at 14.1 minutes. In control experiments 5'-mononucleotides
and their phosphorothioate analogs were incubated in FBS-supplemented
medium (see above) or in the medium from cell cultures from which cells
were removed after their 72-hour incubation. These experiments have
been carried out to show that ecto-5'-NT activity responsible for
mononucleotide dephosphorylation is cell-associated and that this is
not present in serum or not released from cells into medium.
Oligodeoxynucleotide degradation in cell culture media The 5'-32P-radiolabeled oligomers 1 through 4 (2.5 to 10 µM) were incubated at 37°C in RPMI 1640 medium containing 10% or 20% FBS. At various times, 10-µL aliquots were withdrawn and heated for 2 minutes at 95°C. Then, 50 µL water was added to each denatured sample. After vigorous shaking, the protein precipitates were spun down, and the aqueous solutions were dried in a Speed Vac rotary evaporator (Savant Instruments, Farmingdale, NY). The resultant samples were analyzed by 20% polyacrylamide/7 M urea gel electrophoresis. The autoradiograms were scanned by means of an LKB Ultrascan XL densitometer (Pharmacia LKB, Uppsala, Sweden).
Purine-base, nucleoside, or mononucleotide treatment of the K-562 cell line To analyze the influence of 5'-mononucleotides and their phosphorothioate analogs on the cell growth K, 562 cells were incubated with either dNMPs or dNMPSs for 96 hours. These experiments have shown that dNMPs and dNMPSs are not toxic to the K-562 cell line: after a 72-hour incubation with dNMP or dNMPS, at 100 µM concentration, cell viability was decreased only by 5% to 10%. However, deoxyguanosine and guanine, used at the same concentration, inhibited cell growth by 90% (Table 1). We hypothesized that the different effects of guanine, deoxyguanosine, and dGMP on the K-562 cell line resulted from different catabolism of these compounds, which could be catabolized not only by serum enzymes present in culture medium but also by enzymes anchored to cell membranes. To examine this hypothesis, K-562 cells were incubated with these compounds for 96 hours, and the resultant media supernatants were analyzed by HPLC. HPLC analysis of the control samples (see "Materials and methods") has shown that after a 72-hour incubation only 10% to 15% of mononucleotides or their 5'-phosphorothioate analogs were converted to the corresponding nucleosides by serum enzymes. Moreover, the cells did not release nucleotidase or phosphatase activities to the medium because dNMPs or dNMPSs were dephosphorylated in the medium taken from these cells cultures to a similar extent as the dephosphorylation in FBS-supplemented medium (Figure 1). These results have shown that an activity responsible for dephosphorylation of dNMPs and dNMPSs is cell membrane-associated. To underline cellular localization of this enzyme, we have used the term ecto-5'-NT.15 The analysis carried out for K-562 cells demonstrated that after a 72-hour incubation only 10% of the dGMP was converted to deoxyguanosine; this could result from phosphatase and/or nucleotidase activity of FBS-supplemented medium (Figure 1). This result indicates very low ecto-5'-NT activity on the K-562 cells. Therefore, dGMP is not efficiently dephosphorylated to deoxyguanosine and remains nontoxic to these cells. Probably because of the low ecto-5'-NT activity, thymidine-5'-phosphate (TMP) is also not toxic to the K-562 cells, although, after a 72-hour incubation, thymidine (100 µM concentration) decreased the number of viable cells by 40%.
Role of nucleoside transporters in cytotoxic effects of nucleosides The cytotoxic effect of 2'-deoxyguanosine to human leukemic cell lines was reported several years ago.5-8 It was postulated that this effect is caused by intracellular enzymatic phosphorylation of deoxyguanosine to its monophosphate, diphosphate, and, finally, triphosphate (dGTP), which acts as an allosteric inhibitor of ribonucleoside reductase.16-18 Therefore, the cytotoxic effects of mononucleotides on different cell lines may occur not only as the result of their dephosphorylation, but also as a consequence of their effective transport across cell membranes. Some nucleoside carriers responsible for cellular uptake of nucleosides can be inhibited by NBTI, dilazep, or dipyridamole.19 Using these inhibitors, we determined their influence on the toxicity of the deoxyguanosine to the K-562 cells. We separately added NBTI, dipyridamole, or dilazep to the K-562 cell cultures, and after 1 hour of incubation, the cells were treated with deoxyguanosine as described above. It appeared that deoxyguanosine was less toxic to the cells preincubated with the inhibitors than to the cells without them (Figure 2). After 48-hour incubation with deoxyguanosine, the number of K-562 cells was decreased by 60% as compared with the control, while dipyridamole, dilazep, or NBTI partially protected the K-562 cells from deoxyguanosine toxicity: in the presence of the inhibitors, the cell numbers were decreased by 30% to 40%. These results indicate that the antiproliferative effect of deoxyguanosine on the K-562 cell line is at least partially dependent on the nucleoside transport mechanism. However, some cytotoxic effects of deoxyguanosine have been observed even in the presence of the inhibitors; they decreased the antiproliferative effect of deoxyguanosine by 20% to 30%. The low effectiveness of the inhibitors can be, at least partially, caused by the activity of a purine nucleoside phosphorylase and generation of guanine. Guanine transport across cell membranes cannot be inhibited by NBTI, dilazep, or dipyridamole, and therefore, the decrease of K-562 cell numbers has been observed despite the presence of these inhibitors. Similar experiments using dipyridamole have also been carried out for other cells (HUVECs, HeLa, and HL-60), and their partial protection against deoxyguanosine cytotoxicity has also been observed (data not shown).
Purine base, nucleoside, and mononucleotide treatment of the HL-60 cells In contrast to the K-562 cells, HL-60 cells do not possess the Philadelphia chromosome. In general, dNMPs (used at 25 or 100 µM concentration) were not toxic to the HL-60 cell line. On the other hand, exposure of a cell suspension to 100 µM deoxyguanosine resulted in a 40% to 50% decrease in cell numbers. HL-60 cells were also sensitive to guanine: it caused a 60% inhibition of cell growth after a 72-hour incubation. HPLC analysis has shown that during a 96-hour incubation, dNMPs (200 µM) were only slightly dephosphorylated to the corresponding deoxynucleosides. TMPs and dGMPs were dephosphorylated by 10%, while none of the dNMPSs were converted to the corresponding deoxynucleosides. These observations indicate that the HL-60 cells are protected from the cytotoxic effect of dGMP owing to the low ecto-5'-NT activity. We have not observed any cytotoxicity of deoxycytidine, deoxyadenosine, or thymidine to the HL-60 cells. Also, none of the dNMPSs inhibited cell growth (Table 1). Interestingly, we have observed that each dNMPS stimulated HL-60 cell growth. After a 72-hour incubation with dNMPS (100 µM, the number of viable cells was increased by 20% to 40%, as compared with the control experiments. The greatest proliferative effects were observed for thymidine-5'-phosphorothioate (TMPS) and dGMPS (40% to 50%) (Table 2). The essential role of the phosphorothioate function in the stimulation of the HL-60 cell growth was confirmed by the addition of a phosphatase-pretreated dGMPS sample to the cell culture. Phosphorothioate anions, released from the dGMPS molecules by the enzyme, did not stimulate cell growth. Additional experiments performed with thymidine 3'-phosphorothioate and thymidine 5',3'-bisphosphorothioate have shown that the compounds did not influence HL-60 cell growth (data not shown). These results indicate that a target molecule binding 5'-dNMPS possesses significant specificity to phosphorothioate derivatives of deoxyribonucleosides.
Mononucleotide treatment of HUVECs HUVECs were used in these studies as an example of normal nontumor human cells. Because these cells form an endothelium of blood vessels, they may be influenced by mononucleotides and nucleosides released during nucleolytic degradation of antisense oligonucleotides intravenously administered to blood. Among dNMPs and dNMPSs added to the cell culture at a 100 µM concentration for 72 hours, only TMP and dGMP decreased the number of viable cells, by 10% and 30%, respectively. Under the same conditions, deoxyribonucleoside 5'-phosphorothioates (deoxyadenosine-5'-phosphorothioate [dAMPS], TMPS, and dGMPS) stimulated growth of the HUVECs by 20% to 60%. The highest proliferative effect was caused by TMPS after a 24-hour incubation (70% to 90%), but after longer incubation times (48 or 72 hours), the number of viable cells was increased by 50% to 60%. Further increase of dNMPS concentration up to 500 µM caused 300% increase in cell proliferation (data not shown). We supposed that the time dependence of the stimulatory effect was caused by ecto-5'-NT-catalyzed dephosphorylation of TMPS and the subsequent decrease of its concentration in cell culture. Detailed analysis of metabolites from the HUVEC culture revealed significant ecto-5'-NT activity, which is able to dephosphorylate both dNMP and dNMPS (Table 3, Figure 3). To our best knowledge, this is the first observation that ecto-5'-NT is able to recognize and hydrolyze 5'-phosphorothioate analogs of dNMP. It has been earlier demonstrated that ectonucleotidases in pig smooth-muscle cells dephosphorylated adenosine 5'-monophosphorothioate (AMPS) to adenosine, but ectonucleotidases from endothelial cells could not catabolize adenine nucleotides when sulfur was substituted on the terminal phosphate (ie, AMPS, ADPS, ATP S).20,21 In contrast
to the ecto-5'-NT from HeLa cells (see below), the HUVEC enzyme
dephosphorylates not only TMPS, but also other dNMPSs. In our attempts
to inhibit ecto-5'-NT activity and to increase proliferation of HUVECs
by dNMPS, we have used ,-methylene-ADP, which is known to
selectively inhibit the enzyme.22 HPLC analysis has
confirmed that in the presence of ,-methylene-ADP,
dephosphorylation of dNMP or dNMPS was effectively inhibited (Table 3).
However, the inhibition of ecto-5'-NT activity did not increase the
proliferative effect of TMPS or other
deoxyribonucleoside-5'-phosphorothioates, but rather abolished
dNMPS-stimulated proliferation of the HUVECs (Figure
4). This effect might be caused by a
competition of ,-methylene-ADP with dNMPS for binding to the
same nucleotide receptor. It is known that ,-methylene-ATP binds
to ionotropic (P2X) and metabotropic (P2Y) nucleotide
receptors and acts as an agonist.23 It cannot be excluded
that ,-methylene-ADP also possesses some affinity for P2
receptors. It has been found that membranes of WEC cells (endothelial-derived cell line) contain binding sites of high affinity
toward ,-methylene-ATP and
,-methylene-ADP.24 Although ,-methylene-ADP
has been recommended as a selective inhibitor of ecto-5'-NT
activity,22 and ,-methylene-ATP has been used as a
selective P2X agonist,23 some authors suggest that these ADP and ATP analogs can bind to the nucleotide-binding sites of both
ectonucleotidases and nucleotide receptors.25 Taking into account the above suggestions,22-25we tested the influence
of ,-methylene-ATP on the dNMPS-stimulated proliferation of
HUVECs. If the cells were first treated with 100 µM
,-methylene-ATP, an addition of TMPS did not stimulate HUVEC
growth (Figure 4). Even a 10-µM concentration of
,-methylene-ATP significantly diminished dNMPS-induced
stimulation of HUVEC growth.
Mononucleotide treatment of HeLa cells Among dNMPs used in the experiments, only TMP and dGMP were toxic to the HeLa cells: TMP caused a 30% inhibition of cell growth after 72-hour incubation (Table 1). Under similar conditions, TMPS appeared to be nontoxic because it decreased the cell number by only 10%. It should be noted that thymidine alone caused a 30% to 40% inhibition of the HeLa cell growth. Deoxyguanosine decreased the number of viable cells by 40%, while dGMP caused a 20% inhibition of cell growth. These observations suggest that the sensitivity of HeLa cells to extracellular TMP and dGMP could result from the activity of the ecto-5'-NT, releasing the corresponding deoxynucleosides. HPLC analysis has confirmed this hypothesis: after a 48-hour incubation with the HeLa cells, dGMP was completely hydrolyzed to deoxyguanosine, which was, in turn, converted to guanine (15%) and xanthine (85%). Other deoxynucleoside-5'-phosphates (dAMP, dCMP, and TMP) were hydrolyzed by the ecto-5'-NT at rates similar to that of dGMP. Among the deoxyribonucleoside-5'-phosphorothioates, only TMPS was effectively dephosphorylated by the enzyme: 50% conversion of TMPS (200 µM) to thymidine was observed after a 48-hour incubation. However, under the same conditions, dAMPS was not dephosphorylated. It should be noticed that the enzyme from HeLa cell membranes was less sensitive to,-methylene-ADP than the ecto-5'-NT from endothelial cells:
addition of ,-methylene-ADP to HeLa cell cultures decreased the
rate of dNMP dephosphorylation only slightly (Table 3).
Oligonucleotides (PO and PS oligos) and their antiproliferative effects To compare biological effects caused by mononucleotides and oligonucleotides, we constructed PO and PS oligomers of the sequences given in Table 4. Preliminary experiments performed with mononucleotides have shown that the cells were more sensitive to dGMP and TMP than to other dNMPs. Therefore, to test the nonantisense effects of oligonucleotide sequences on cell viability, we synthesized oligomers 1 and 2 containing different tetranucleotides: namely, d[TGGT] and d[ACCA] at their 3'-ends. The homo-oligonucleotides d[A]16 and d[T]16 (oligomers 3 and 4) were synthesized as additional control oligomers. The stability of phosphate and phosphorothioate oligonucleotides toward serum 3'-exonuclease activity was determined by polyacrylamide gel electrophoresis. We found that the PO oligos were degraded by 50% after a 4-hour incubation in cell culture media, while PS oligos incubated under the same conditions appeared to be much more stable. Even after 24-hour incubation of the PS-d[T]16 in RPMI medium containing 10% heat-inactivated FBS, we did not observe any bands corresponding to shorter oligomers. However, the use of nonthermally inactivated FBS caused partial degradation of the fully phosphorothioated oligonucleotides for which half-life time was approximately 8 hours. In similar experiments carried out by Vaerman et al,4 with the use of nonthermally inactivated 10% fetal calf serum or 2.5% normal human serum, PO oligos were degraded with half-life times of about several minutes. The half-life time for the PO/PS oligo containing a single internucleotide phosphorothioate bond at the 3'-end was about 15 hours. Such experimental conditions generated a significant amount of dNMP and, as interpreted by the authors, dNMPS.4 The oligonucleotides 1 through 4 (used at 10 µM) did not cause any toxic effects to the HL-60 or K-562 cells. Although deoxyguanosine-5'-phosphate was released from oligomer 1a by plasma 3'-exonuclease activity, concentration of the resulting dGMP after a 24-hour incubation was not higher than 15 to 20 µM. Because of the low activity of ecto-5'-NT, dGMP was not dephosphorylated to deoxyguanosine, and therefore, no cytotoxic effect could be observed. For the same reason, the PS oligos 1 through 4b did not influenced the growth of K-562 and HL-60 cells. Only HeLa cells appeared to be sensitive to one of the oligonucleotides used in our experiments. After a 72-hour incubation, the unmodified oligonucleotide d[T]16 (4a) caused 15% to 30% inhibition of cell growth. This effect was clearly concentration-dependent: d[T]16 used at 2.5 µM concentration caused only 15% inhibition, while 10 µM of the same oligomer gave 30% inhibition of the cell growth. The inhibitory effect results from exonucleolytic degradation of the oligonucleotide 4a and TMP release. In our estimation, the concentration of TMP released after 72-hour incubation was approximately 100 µM. Therefore, the inhibitory effects observed for the oligomer 4a used at a concentration of 10 µM and for the free TMP used at 100 µM were almost identical. A further increase of the oligonucleotide concentration (up to 25 µM) did not cause significant enhancement of the antiproliferative effect from what might result from the limited activities of the 3'-exonuclease and/or the ecto-5'-NT. The homo-oligonucleotide d[A]16 (3a) was degraded by the exonuclease with the same efficacy as d[T]16 but dAMP resulting from the cleavage was not toxic to the cells. The phosphorothioate homo-oligonucleotide 4b did not inhibit HeLa cell growth, probably because of its higher resistance to serum 3'-exonuclease activity as compared with the unmodified oligonucleotide d[T]16.
Vaerman et al4 postulated that dNMP and dNMPS
released from PO and PS oligos by plasma exonucleases might inhibit
growth of different cell lines. Deoxyribonucleoside-5'-phosphates
(especially dGMP and TMP) added to cell cultures caused significant
cytotoxic effects to the leukemic BV 173 cell line and to
CD34+ bone marrow cells.4 Vaerman et
al4 did not examine the toxicity of dNMPS after direct
addition to the BV 173 cell cultures. Our experiments, carried out with
the use of chemically synthesized dNMPS, have shown that the influence
of mononucleotides and their 5'-phosphorothioate analogs on the
proliferation of different cell lines is more complex than has been
presented so far. Deoxyribonucleoside-5'-phosphates (dGMP and TMP) are
toxic to some types of cells, but under the same conditions, their
5'-phosphorothioate analogs do not decrease cell viability. Moreover,
in some cases, dNMPSs stimulate cell proliferation independently of the
type of nucleobase. A detailed HPLC analysis of media supernatants
allowed us to identify ecto-5'-NT activity responsible for
dephosphorylation of extracellular mononucleotides to the corresponding
nucleosides. The cell lines studied demonstrate different ecto-5'-NT
activities. The enzyme from K-562 cells showed very low activity, which
resulted in only a 5% yield of dNMP dephosphorylation after a 72-hour
incubation, independently of the type of nucleobase. HL-60 cells also
degraded the extracellular mononucleotides at a very slow rate. This
observation is in agreement with earlier data, because low activity of
the ecto-5'-NT from HL-60 cells has been reported by Spychala et
al26 and Clifford et al.27 The lack of the
ecto-5'-NT activity in K-562 and HL-60 cells results in the high
stability of extracellular mononucleotides. They are not
dephosphorylated to the corresponding deoxynucleosides and, therefore,
are not toxic to these cells. In contrast to the HL-60 and K-562 cells,
HUVECs and HeLa cells possess elevated ecto-5'-NT activity, which can
dephosphorylate both dNMP and dNMPS. Thymidine 5'-phosphate is slightly
toxic to the HeLa cells, because this mononucleotide has been
hydrolyzed to thymidine, which can be taken up by the cells and which
can inhibit intracellular thymidine kinase.28,29
Deoxyguanosine 5'-phosphorothioate is toxic to HUVECs because of its
dephosphorylation to deoxyguanosine. To our knowledge, there are no
data available on stimulation of mammalian cell growth by
2'-deoxyribonucleoside-5'-monophosphorothioates. However, it is
possible that dNMPSs influence HL-60 and HUVEC proliferation by acting
on a cell-surface receptor belonging to a P2 nucleotide receptor class
activated by nucleoside triphosphates and/or diphosphates. Recent
studies reveal much higher diversity among the P2 receptors than was
previously thought.30,31 In comparison with ATP or uridine
5'-triphosphate, 5'-mononucleotides usually have much lower agonist
activity for the nucleotide receptors.32,33 However, it
has been found that AMPS is a weak agonist of a novel P2 receptor on
undifferentiated HL-60 cells. Its pharmacological profile is very
similar to that of the P2Y11 receptor identified in 2 different cell
lines: the 1321N1 astrocytoma and the Chinese hamster over K1 cell
line.34-36 One cannot exclude the possibility that HL-60
cells and HUVECs express a similar, as-yet unidentified nucleotide
receptor possessing an affinity for 2'-deoxyribonucleoside 5'-phosphorothioates but not for 3'-phosphorothioates or
5',3'-bisphosphorothioates. The influence of
We thank Dr P. Guga, M. Olesiak, and D. Krajewska for synthesis of mononucleotide 5'-phosphorothioates; Daria Szymanowicz for synthesis of thymidine 3'-phosphorothioate and thymidine 5',3'-bisphosphorothioate; and an anonymous referee for valuable comments.
Submitted November 20, 2000; accepted April 13, 2001.
Supported by the State Committee for Scientific Research, grant 6 PO4B 014 15 (M.K.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maria Koziolkiewicz, Department of Bioorganic
Chemistry, Centre of Molecular and Macromolecular Studies, Polish
Academy of Sciences, Sienkiewicza 112, 90-363
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  N. Dias and C. A. Stein Antisense Oligonucleotides: Basic Concepts and Mechanisms Mol. Cancer Ther., March 1, 2002; 1(5): 347 - 355. [Full Text] [PDF]
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, Poland; and the Columbia University, College of
Physicians & Surgeons, New York, NY.
Top
Abstract
Introduction
indicates dGuo; 
, dGuo and NBTI (1 mM); 
,
dGuo and dilazep (1 mM); 
, dGuo and dipyridamole (20 mM); 
,
dipyridamole (20 mM).
