An Atypical DNA Polymerase Beta Overexpressed in Human Aml/Hl-60 Malignant Cells

Abstract

Materials and Methods Cell cultureThe 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% CO 2 /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. [25] and then modified by Roy et al. [26]. Chromatin fractionationThe 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 MgCl 2 /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

Retinoblastoma Case: Shall we Get A Paramagnetic Trend in Chemotherapy?

“…Thus, a preclinical test step for 25Mg 2+ -MIE has been confirmed lately to complete the AML related arsenal of medicinal isotopes with the Magnesium-25 enriched, so to say "easy-to-permit", Mg-containing officinal drug forms [9]. In this case, a mode of the isotope anti-tumor action, caused by deprivation of the AML-specific DNA repair capabilities, has unambiguously determined by a number of circumstances including an MIE-allowing structural uniqueness of the DNA Polymerase Beta [4,5], hyperexpression of this target enzyme in AML [4] and by the ion-radical manner of the 25Mg 2+ -induced dysfunction of the tartget [2][3][4][5].…”

“…6 hrs after, the nascent DNA were labeled with [Methyl-1,2-3H] dTTP, 180-220 Ci/ mmol, 100 µCi per 1.0 mL culture. The resulting NEN-resistant ([3H]DNA cpm/mg protein) values were considered as the in situ DNApolB specific activity patterns according to Bukhvostov AA et al [5,9]. All protein measurements, DNA extraction, DNApolB catalytic activity determination, standard DNA 1.8%-agarose gel electrophoresis followed by the DNA chain size The results listed in Table 1 shows that the RB cells are extremely sensitive to all three MIE-promoters ( 25 Mg 2+ , 43 Ca 2+ , 67 Zn 2+ ) leading to a sharp increase of the cell mortality values which is likely relates to a marked DNApolB functioning breakdown.…”

“…Noteworthy, a significant MIE-provoked shortening of the DNA fragments processed, by at least 20% to 25% in length, is also occurred (Table 1). This itself is about to deprive the cell of its usual DNA repair capabilities [1,2,8] which is a reason to expect the tumor growth to get limited [3,9]. Thus, the MIE-dependent formation of these "size The applied medicinal significance of these data looks promising due to the followings: (a) the MIE ion-radical mode [4][5][6][7] does not allow the most members of DNA Polymerases diversity (α, γ, δ) to get operated this way since the electron transfer distances in their catalytic sites are too long to make a "magnet" manifesting its MIE [1,3,5]; while (b) DNApolB is a perfect target for MIE-ions considering this enzyme active site 3D-structure [4,7].…”

“…Particularly, retinoblastoma (RB) is an appropriate object for such an impact due to the DNA repair engaging paths of epigenetic control revealed in this peculiar cancer [8]. To find out the in situ degree and validity of an impact like that, all cellular DNA polymerase species except for the beta ones (i.e., alpha, gamma and delta) might be selectively suppressed by N-ethyl-melamide (NEM) [9]. Investigating a potential of the [nMe 2+ ] MIE-Chemotherapeutic approach.…”

Retinoblastoma: Magnetic Isotope Effects Might Make a Difference in the Current Anti-Cancer Research Strategy

“…Chromatin-associated DNApolβ fraction was isolated from the cell lysate samples under the conditions preserving native proteins using our slightly modified technique consisting in a subsequent phenol-chloroform/ammonium sulfate treatment followed by the gel filtration on TOYOPEARL HW 55F column (5). For the DNApolβ catalytic activity measurements, an incorporation of (Methyl-1,2-3 H)dTTP (90-120 Ci/mmol, NE-T520A, NEN) into the nascent enzyme-produced DNA sequences was detected to be expressed then as ( 3 H)cpmDNA/ mg protein.…”