Received date: January 05, 2013; Accepted date: January 18, 2013; Published date: January 21, 2013
Citation: Bukhvostov AA, Shatalov OA, Orlov AP, Kuznetsov DA (2013) An Atypical DNA Polymerase Beta Overexpressed in Human Aml/Hl-60 Malignant Cells. J Cancer Sci Ther 5:094-099. doi: 10.4172/1948-5956.1000191
Copyright: © 2013 Bukhvostov AA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Human acute myeloid leukemia cells overexpresses a beta–like DNA polymerase (EC 22.214.171.124) which is found
to be a chromatin associated single subunit protein (66.5 kDa) purified by original extraction/gel filtration procedure
allowing to gain the 122,000-fold purification degree as corrected to a total cell protein. The enzyme possesses
some key DNA pol β-specific catalytic properties such as the processing of short (200n-250n) single strand DNA
sequences, activation in the presence of 200 mM KCl, resistance to N-ethyl-melamide and Aphidicolin, lack of
3’,5’-exonuclease activity, and low dTTP utilization rates (KM=0.016 mM, Kcat=0.622 (μM dTTP/min)/mg protein). A
possible significance of the unique enzyme studied as a target for its pharmaceutical inhibitors is under discussion.
This work is a full–length version of a study presented as a Poster at the OMICS managed 2nd World Congress on
Science Cancer and Therapy, Sept 10–12, 2012, San Antonio, TX.
Acute myeloid leukemia; DNA polymerase beta; Target enzymes in cancer therapies
DNA polymerases beta, EC 126.96.36.199 (DNA pol β), represent the special subpopulation of the rich-n-variable DNA polymerases superfamily. A remarkable peculiarity of DNA pol β relates to its participation in the DNA base–excision repair [1-4]. Being the chromatin-associated proteins [4,5], most DNA pol β species were found to be overexpressed in many malignant tumors [6-12]. It makes these enzymes the legitimate targets for inhibitors or, to be exact, for a chemotherapeutic attack provided by DNA pol β -recognizing high affinity suppressors playing a role of pharmaceutical agents [13-17].
The latter circumstance attracts an attention of not only enzymologists but of oncologists and pharmacologists as well [14,18,19]. However, a broad structural diversity of DNA pol β species isolated from normal and cancer cells dictates a necessity of detail structural and functional (catalytic) characterization of each one, usually tumor– specific, enzyme of this group.
Thus, most frequently, DNA pol β examples are Mg2+-coordinating proteins having pI within 8.3–8.7 and a molecular mass within 35 kDa– 55 kDa ranges, respectively [19-21]. Normally, these enzymes are too slowly produce the single strand DNA chain consisting of no more than 300 nucleotides (DNA repair requirement) showing a high resistance to such common DNA polymerase (alpha, gamma, epsilon, etc) specific inhibitors as Aphidicolin and N-ethyl-malemide [19,22]. A total lack of 3’,5’-exonuclease activity is also a marking sign of DNA pol β [20-24].
On other hand, there are some remarkable instances of an exceptionally high, up to 260 kDa, molecular mass values estimated for several chromatin affiliated enzymes with the above specified catalytic activity (β-like DNA polymerases) [7,23,24]. Moreover, a DNA pol β molecular size itself might be a rather critical parameter in chromatin structural organization making an impact on genome expression control which seems to be particularly essential to the high molecular weight β-like enzymes [5,24].
Although the DNA pol β research counts nearly 20 years of its history, a list of tumors subjected to purification–characterization of their specific DNA pol β is still far of being completed. Noteworthy, one of the abundant human blood cancers, an acute myeloblast leukemia or AML, has not been engaged yet to isolate and investigate its specific DNA pol β . So this work is a first report to describe an original procedure of purification and characterization of such enzyme from chromatin of HL60 cells (AML).
The HL-60 human myeloid leukemia cell line has been purchased from the Hungarian Cell Bank, Pasteur Institute of Hungary, Szeged, NCBI Code C427. Cells were maintained in suspension culture at +37°C under 5% CO2/air in RPMI 1640 (Gibco, UK) supplemented with 10% FCS and antibiotics: 100 U/mL Penicillin and 100 μg/mL Streptomycin. The cells were subcultured three times weekly, ATRA (Sigma, USA). This procedure has been originally adopted by Olins et al.  and then modified by Roy et al. .
The cells were precipitated by centrifugation at 12,000 rpm for 20 min (+4°C). The pellets were then suspended and homogenized in 5 volumes of 10 mM Tris-HCl (pH 7.80)/200 mM sucrose/1.5 mM EDTA/20 mM MgCl2/0.5% (v/v) Triton X100 using the glass-teflon Potter-Elvehjem homogenizer with a snug-fitting pestle (1,800 rpm).
Homogenates were filtered through the 5-layer cotton graze and then the filtrates were centrifuged at 800 g for 30 min (+4°C). The pellets were collected and re-suspended in 10 volumes of 15 mM Tris-HCl (pH 8.0)/1.5 mM EDTA/10 mM MgCl2/1.5 mM KCl/25 mM sucrose/Trypsin 20 μg/mL (w/v) and incubated at +37°C for 1 hr with a following centrifugation at 800 g, 40 min (+4°C). The resulted pellets were treated as a crude nuclei subcellular fraction, P0.8. To obtain nucleoplasm samples, separate portions of homogenate were utilized for isolation of P.08 fractions with their following Triton X100 treatment (2.0%, v/v)/15 mM Tris-HCl (pH 8.0) and a subsequent centrifugation at 150,000 g, 2 hrs (+4°C).
For isolation of chromatin, the crude nuclei fractions (P0.8) were subjected to procedure described by Voss et al.  and modified by Lerman et al.  with a subsequent phenol-chloroform extraction of total protein . The resulting protein fractions were mixed with 10 volumes of an ice-cold acetone and kept at +4°C overnight. An acetone–insoluble material was precipitated at 20,000 rpm, 20 min, +4°C. The pellets were extensively re-washed with acetone using the same procedure and then dissolved in 5–6 volumes (w/v) of 25 mM potassium–phosphate (pH 6.30)/0.5% NaCl/1.5 mM EDTA/0.01% glutathione/0.05% heparin/1.0% 2-mercaptoethanol/80–100 U/mL nuclease S followed by 40 min incubation at +37°C. All post–incubation mixtures were treated by sonication at 80 KHz, 30 min, +60°C, under a non-stop extensive shaking. Then these mixtures were submitted to a scalar fractionation path reaching the 30%–70% ammonium sulfate saturation, consequently. The precipitates obtained were collected at 10,000 rpm, 20 min, and dissolved in 15 mM potassium phosphate buffer (pH 6.0)/0.2% NaCl (10 vols, w/v). The solutions were subjected to dialysis against 20 mM potassium phosphate buffer (pH 6.0) and lyophilized.
The lyophilized powders were first dissolved in 15 mM potassium phosphate (pH 6.30)/5.0 mM MgCl2/1.5 mM EDTA/0.0001% sodium azide and passed through the fiberglass filters with 0.3-0.4 μ pore diameter (Millipore 5R, Millipore, France). The transparent solutions were subjected to ultrafiltration on membranes with the molecular size exclusion limit of 5.0 kDa at 800 p.s.i. (Diaflo Y5.0 25 mm membranes, Amicon BV, The Netherlands). The membrane–retained material was then extracted with 10 mM Tris-HCl (pH 8.0)/1.0% 2-mercaptoethanol (v/v), 5.0 mL per a razor–disintegrated membrane, +30°C, 12 hrs, with a following concentration in a rotor evaporizer.
The 1.5-2.5 mL samples were then applied onto a 1.5 × 50 cm (V=98 mL) column packed with the TOYOPEARL HW 55F gel and equilibrated by the eluent buffer consisting of 15 mM potassium phosphate (pH 6.30)/5.0mM MgCl2/ 0.0001% sodium azide. Elution rate: 0.8 mL/min (room temperature). In each one of the consequently eluted 1.5 mL fractions, the DNA polymerase activity has been measured according to Matsumoto and Kim  and modified by Piersen et al. . To get the enzyme specific activity measure, the amounts of protein were estimated by the Bradford colometric method . A column was calibrated with the protein markers set (Serva Heidelberg, Germany) to cover the following molecular mass values range: 12.5 kDa (cytochrome C)–24.0 kDa (trypsine)–45 kDa (ovalbumin)–70 kDa (HSA)–145.0 kDa (L-asparaginase).
A slightly modified Laemmli method  has been employed to estimate both molecular mass and purification extent of the DNA polymerase isolated. The peculiar fractionation parameters: 10% PAAG separation system based on a standard Tris-glycine (pH 8.30)/0.5 % SDS, 0.1 × 100 × 100 mm slab gels, 180 V/gel (4.5 mA/cm). Calibration of gels was performed with a standard QR 460/5 protein marker kit covering the 12.5-120.0 kDa range (Miles Laboratories, USA).
A routine 2.0%-agarose gel electrophoretic technique has been employed . For the nascent single strand DNA chain length estimation, the 150n–300n ranged poly (dT) markers were applied (Calbiochem-Novabiochem International, USA).
The descendent linear 10.0–3.0 pH gradient 1.0×80×80 mm PAAG slabs with a reported pI performance range of 9.6–3.5 (KomaBiotech, Korea) were used as originally described by Walker  and modified by Katoh . Additionally, a cellophane–attached ultrathin 0.12 × 260 × 125 mm PAAG slabs with the 10.0–3.0 pH gradients (Clean Gel–IEF plates, GE HealthCare Europe GmbH, Germany) were used according to Görg et al. . In a latter case, the processed and unfixed gels were also used to trace-or-exclude a 3´,5´-exonuclease activity in DNApolβ-fraction . These IEF procedures were also employed to get the protein composition profiles of chromatin and nucleoplasm obtained from HL60 cells and from myelocytes of the adult human male donors [27,28].
DNA polymerase (ec 188.8.131.52) activity measurements
The enzyme catalytic activity was measured by method  adopted for 0.15 mL incubation mixtures consisting of 50 mM Tris-HCl (pH 8.0)/8.0 mM dithiothreitol/15 mM MgCl2/15% glycerol (v/v)/27 μg act DNA, calf thymus/50 μg each of dATP, dCTP, dTTP, dGTP/0.25 μmole [Methyl-1,2-3H]dTTP (90–120 Ci/mmol, NET520A, NEN)/150 mM NaCl. The tritium-labeled nucleotide was purchased from New England Nuclear, USA. These compound concentration values were first pre-optimized within both pH 6.0–9.0 and 5.0 mM–50.0 mM MgCl2 ranges. These mixture samples were first pre-incubated at +37°C for 60 min. Then 5.0–7.5 μg of pure enzyme was added to each one of these running samples and they were incubated at +37°C for 60 min longer. The ice cold incubation samples (0°C, 60°C after pre-incubation) as well as the trypsin treated samples (20 μg/mL trypsin, Merck GmbH, Germany, +37°C, 60°C) were taken for controls.
The post-incubation mixtures were subjected to a quantitative extraction of the DNA ultramicro–amounts using an AccuPrep Genomic DNA Extraction Kit (Bioneer Corp., Korea) as described by Mikami et al.  and modified by Haratian et al. . The extracted DNA aliquots were used for electrophoretic determination of the DNApolβ-processed DNA chain sizes  and for [3H]-radioactivity measurements in Wallac 2200LX LS Counter (Wallac OY, Finland). The DNA pol β -specific catalytic activity values were expressed in [3H] cpmDNA/mg enzyme. The protein ultramicro amounts were estimated according to Fukami et al. . The DNA ultramicro amounts measurements were performed in diluted water solutions as described by Müller et al. .
The kinetic constants, KM (mM) and Kcat ([μM dTTP/min]/mg enzyme), were estimated by the free dTTP pool depletion rates  measured using the HPLC analysis of acetone–soluble fractions of pre- and post-incubation mixtures: Altex 1800E (18×220 mm) column/ODS-S5CN stationary phase/mobile phase, 10-60% linear pyridine gradient based on 10% water-methanol/2,000 p.s.i./+22°C / Waters DL600 HPLC Analytical System [42,43].
The UV-spectrophotometry (Lambda 1050 Scanning Spectrophotometer, Perkin Elmer, Inc., USA) and a circular dichroism spectrometry (J815 CD Spectrometer, JASCO, Inc., USA) were employed using the Spectra Manager II cross-platform software (JASCO) for automated data treatment. To dissolve the lyophilized enzyme samples, 10 mM potassium phosphate adjusted to pH 5.20/pH 6.20/pH 8.30 (UV absorption tested in 190 nm–400 nm range) and to pH 8.00 (CD spectrometry, 190 nm-255 nm) has been employed.
As seen from the data presented in Figure 1, the procedure we proposed allows to isolate the perfectly purified 66.5 kDa monomer protein (Figure 1d and Figure 1 gel filtration profile) with a marked DNA polymerase activity limited to produce DNA chains within 200n–250n size range (Figure 1a). The enzyme purified is found to possess the following properties: pI=8.45 (Figures 1B, C and E); pH 8.0/15 mM MgCl2 optimal incubation parameters (Materials and methods); kinetic constants estimated by dTTP utilization mode, KM=0.016 mM and Kcat=0.622 (μM dTTP/min)/mg protein.
The enzyme is overexpressed in HL60 cells compared to normal myelocytes being affiliated with chromatin not nucleoplasm (Figure 1e). In the same IEF tests, a total lack of 3’,5’-exonuclease activity was found for purified enzyme employing the technique described by Görg et al. and Rule [36,37]–see Materials and methods. UV absorption spectra of the enzyme isolated shows not only a lack of impurities such as poly- and oligonucleotide contaminants but a nativity of the protein as well  (Figure 2). A high nativity extent along with a predominantly alpha-helix contribution to a secondary structure of enzyme  has been detected by CD-spectrometry (Figure 3).
The enzyme is sensitive to ddTTP inhibitor being resistant to Aphidicolin and N-ethyl-melamide, a high concentration of KCl (200 mM) leads to a sharp increase of catalytic activity (Table 1). A progressive increase of the enzyme beta-specific catalytic activity occurred in the consequent steps of purification procedure is seen clearly from the data listed in Table 2. As corrected to a total cell protein, the final yield (purification extent) of the enzyme isolated was found equal to 1:122,000 (w/w). To make sure that the DNA polymerase activity measured (Table 2) was indeed related to a beta-type enzyme, it has always been determined in the presence of Aphidicolin, 5.0 μg/mL [19,22,24].
The reason why the enzyme studied considered a β -like DNA polymerase not just a DNA pol β is a relatively large molecular size (66.5 kDa) (Figure 1) of this single-subunit protein. This alone makes this enzyme a sort of “non canonical” to fit a conventional definition [1,2,19–21] of DNA pol beta. For such rare and peculiar cases, a β-like DNA polymerase notion has been deliberately proposed [45-47]. A comparison between the molecular size determination data obtained by
(a) gel filtration column chromatography (native conditions allowing enzyme to work perfectly), and by
(b) SDS–PAGE analysis (tough denaturing conditions) show no difference in molecular mass of the enzyme purified (66.5 kDa, both gel filtration and SDS–PAGE) (Figure 1). This proves a monomer, i. e. single-subunit, nature of the enzyme which itself is an unusual, attention catching, DNA pol β-characterizing pattern. On other hand, a numerous known DNA pol β-specific marker signs (“taxonomic criteria”) like a unique capability to get overactivated in the presence of 200 mM KCl , high resistance to Aphidicolin and N-ethyl-melamide [19,22,24], short (smaller than 300 n) single strand DNA chains processed [1,3,4,24], a total lack of 3’,5’- exonuclease activity [2,5,20,45], slow rate of the nascent DNA chain growth (low processivity) indicates that the enzyme belongs namely to a DNA pol β group. All these criteria listed were experimentally found and measured for the enzyme studied (Figure 1 and Table 1, KM and Kcat-see Results). So we’re indeed dealing with a special type of DNA pol β or, better to say, β -like DNA polymerase. Both kinetic constants measured (Results) shows a rather slow reaction processing rate which is in a favor to precise and accurate, error-free, DNA repair known for most of the DNA pol β species [1,10,12,18,47]. As per the pI value estimated (8.45), Mg2+ dependence and sensitivity to ddTTP (Figure 1 and Table 1, Materials and methods), these patterns are common for all kinds of DNA polymerase subpopulations including a DNA pol β group as well [13,19,22]. A mere fact that the chromatin associated enzyme we purified is overexpressed in malignant cells compared to the normal ones (Figure 1 e) is in a good accordance with the variable data claiming a critical role of DNA pol β in carcinogenesis [6-17]. These data are about to offer a concept of cytostatic effect caused by a selective turning off the DNA pol β function in tumor as a part of the enzyme targeted cancer therapies [14,17-19]. Considering this point, a detail study on pure DNA pol β species isolated from cancer cells (Results) seems practically the same task as a study on the tumor specific molecular target for new drugs or pharmacophores [13,16,17].
|Agent tested||DNA pol β activity, [3H]DNA cpm/mg protein (M ± SEM, n=6)|
|Aphidicolin, 5.0 µg/mL||30,789 ± 398|
|N-ethyl-melamide, 0.5 mM||27,632 ± 437|
|ddTTP, 2.5 µM||1,370 ± 186|
|Trypsin, 20 µg/mL||207 ± 16|
|KCl, 200 mM||74,613 ± 441|
|No effectors added (optimized incubation mixture)||29,838 ± 322|
Table 1: Catalytic activity of the beta-like DNA polymerase purified from HL60 chromatin: effects of inhibitors and potassium chloride.
Noteworthy, our purification procedure allows not only to separate some non-specific chromatin structural “ballast” proteins but to remove the DNA pol β peptide inhibitors [6,13,29] as well (Figure 1 and Table 2).
|Enzyme purification step||DNA pol β–specific catalytic activity, [3H]DNA c.p.m./mg protein (M ± SEM, n=6)|
|Total cell homogenate||588 ± 63|
|Crude nuclei (P0.8)||1,864 ± 87|
|Crude chromatin||2,476 ± 118|
|Phenol-chloroform extraction||6,081 ± 202|
|Ammonium sulfate 30%-70% saturation gradient||10,112 ± 233|
|Diaflo Y5.0 ultrafiltration||19,423 ± 270|
|TOYOPEARL HW 55F Gel Chromatography||32,687 ± 331|
Table 2: An increase of DNA pol β activity in a multi–step purification procedure
A known DNA polymerase beta - specific resistance to Aphidicolin [19,22,24] helps to trace an increase of its catalytic activity in crude biological extracts and subcellular fractions in a course of the multi-step enzyme purification procedure (Results, Table 2). This is an advantage of the inhibitor chase sufficient to turn off all DNA polymerases except for their beta subpopulation [22,24,29]. The UV-absorption spectra show some peculiarities (Figure 2). Thus, there is no marked absorption in a region close to 210 nm (peptide bond) at pH 5.20 while the maximum absorption was registered over there at pH 8.30. Since pH-optimum (8.0–see Materials and methods) and pI 8.45 (Figures 1b,1c and 1e) makes enzyme active, this protein is expected to show a high level of nativity in UV-absorption spectra registered at pH 8.30. So the lack of 210 nm–absorbance at pH 5.20 reflects a mild misfolding occurred under acidic conditions . Having a moderate-low UV-absorption peak nearby 280 nm (aromatic amino acids residues), we should pay attention to 225 nm–high absorption revealed at all pH tested (Figure 2) which is, most likely, relates to tryptophan contribution typical for some alpha helix–rich chromatin proteins . UV-spectrophotometry as well as the CD-spectrometry data (Figure 3) reveals a nativity of enzyme at pH close to 8.0 and a high degree of the protein purity.
The ellipticity of the CD spectrum obtained is typical for the “far” UV (190–240 nm) and show the native structure of the alpha-protein with the secondary structure comprising insignificant quantity of the beta-fold elements [44,48]. So the enzyme studied is a predominantly alpha-helical monomer with a common for chromatin proteins alkaline pI point (Figures 1b,1c,1e,2 and 3). A superficial, UV-accessible, location of the Trp residues usually means a rigid globular structure enriched with the alpha helical elements [44,49] which corresponds to our results (Figure 2).
Human acute myeloblast leukemia cells, HL60, are found to be overexpressing a chromatin associated DNA polymerase beta–like enzyme that manifests the EC 184.108.40.206–specific activity being different from a vast majority of DNA pol β species by molecular mass (66.5 kDa) of the catalytic autonomous monomer.
For a complete purification of this enzyme, an original multi-extraction/gel chromatography procedure has been proposed.