Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Self assembling cluster crystals from DNA based dendritic nanostructures

Cluster crystals are periodic structures with lattice sites occupied by several, overlapping building blocks, featuring fluctuating site occupancy, whose expectation value depends on thermodynamic conditions. Their assembly from atomic or mesoscopic units is long-sought-after, but its experimental realization still remains elusive. Here, we show the existence of well-controlled soft matter cluster crystals. We fabricate dendritic-linear-dendritic triblock composed of a thermosensitive water-soluble polymer and nanometer-scale all-DNA dendrons of the first and second generation. Conclusive small-angle X-ray scattering (SAXS) evidence reveals that solutions of these triblock at sufficiently high concentrations undergo a reversible phase transition from a cluster fluid to a body-centered cubic (BCC) cluster crystal with density-independent lattice spacing, through alteration of temperature. Moreover, a rich concentration-temperature phase diagram demonstrates the emergence of various ordered nanostructures, including BCC cluster crystals, birefringent cluster crystals, as well as hexagonal phases and cluster glass-like kinetically arrested states at high densities

A subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics

A subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics

Effects of recombinant proteins on cell viability as determined by MTT assay.

<p>(A) The effects of barnase and scFv 4D5-dibarnase on the viability of human cancer and normal cells. SKOV-3 cells were treated for 72 h with barnase (long dashed line) or scFv 4D5-dibarnase (solid line), and hPBMCs were treated with barnase (short dashed line) or scFv 4D5-dibarnase (dashed-dotted line). (B) The competitive inhibition of scFv 4D5-dibarnase cytotoxicity by scFv 4D5. SKOV-3 cells were treated for 72 h with scFv 4D5-dibarnase in the absence (black circles) or presence (white triangles) of 300 nM scFv 4D5 or with scFv 4D5 alone (white squares). (C) The inhibition of barnase cytotoxicity and scFv 4D5-dibarnase cytotoxicity by barstar. SKOV-3 cells were treated for 72 h with barnase (white circles), barnase and equimolar amounts of barstar (white triangles), scFv 4D5-dibarnase (black circles), scFv 4D5-dibarnase with three-fold molar excess of barstar (black triangles), or barstar alone (black squares). (D) The effects of hRI on the cytotoxicity of scFv 4D5-dibarnase. SKOV-3 cells were treated for 72 h with either scFv 4D5-dibarnase in the absence of hRI (black circles), scFv 4D5-dibarnase in the presence of hRI (white diamonds), or hRI alone (black diamonds). Cell viability is expressed as the percentage of the metabolic activity of treated cells with respect to untreated cells (crosshair). Each regression curve in panel A (with 95% confidence intervals indicated by dotted lines) represents at least three independent experiments. Sigmoid regression was performed with SigmaPlot software. Curves in B–D represent typical experiments. Error bars (B–D) were obtained from triplicate measurements.</p

Ribonuclease activity assay.

<p>(A) The ribonuclease activities of barnase (dashed line and diamonds) and scFv 4D5-dibarnase (dotted line and circles) were determined according to the method of Rushizky et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#pone.0002434-Rushizky1" target="_blank">[58]</a>. The x-axis represents the concentration of barnase alone or the half-concentration of scFv 4D5-dibarnase. The absorbance of 0.5 AU₂₆₀ corresponds to the activity of 2 nM native barnase as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#pone.0002434-Hartley2" target="_blank">[27]</a>. (B) Susceptibility of barnase to hRI (solid line and circles) and of scFv 4D5-dibarnase to barstar (dashed line and triangles). Data are means±SD of triplicate determinations; the curves are the results of sigmoid regression performed with SigmaPlot software.</p

Cellular RNA undergoes degradation in SKOV-3 cells treated with barnase.

<p>SKOV-3 cells were exposed to 50 µM barnase for 24 h (lane 3) or 48 h (lane 4). Total RNA was isolated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a> and analyzed on a 9% polyacrylamide gel containing 7.5 M urea. Each sample lane was loaded with RNA from 2×10⁵ treated (+) or untreated (−) cells. Lane 2 corresponds to mock-treated control. The positions of the RNA molecular weight standards (lane 1) are shown as the number of bases to the left of panel. Asterisks indicate the most prominent bands that appear as a result of enzymatic cleavage of high molecular weight rRNA by barnase (lane 3).</p

Binding and internalization of scFv 4D5-dibarnase in BT-474 cells visualized by confocal microscopy.

<p>(A) Cells were incubated with scFv 4D5-dibarnase at 4°C or (B) at 37°C. The scFv 4D5-dibarnase was detected with rabbit anti-barnase antiserum followed by GAR-PE. Fluorescence was observed predominantly on the surface of cells incubated at 4°C and inside the cells incubated at 37°C. This difference in the localization of the fluorescent label suggests internalization of scFv 4D5-dibarnase at 37°C in BT-474 cells.</p

Barnase and scFv 4D5-dibarnase cause DNA fragmentation in SKOV-3 cells.

<p>(A) Flow cytometric analysis of the cell cycle distribution was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a>. Histograms represent the differences in the percentages of cells between barnase- or scFv 4D5-dibarnase-treated and untreated cells for each cell cycle stage (sub-G1, G1, S, and G2/M) measured after 24 h (black bars), 48 h (blue bars), and 72 h (green bars) of treatment. Error bars show the standard deviation. Positive controls for DNA fragmentation were SKOV-3 cells cultured for 7 days in serum-free medium (orange bars). (B) DNA electrophoresis assay. Cells were treated with either 50 µM barnase or 50 nM scFv 4D5-dibarnase. Seventy-two hours later, genomic DNA of both treated (+) and untreated (−) cells was isolated and DNA from equal numbers of cells was resolved in non-denaturing 1.5% agarose gels. The DNA was visualized by ethidium bromide staining. Chromatin fragments resulting from internucleosomal cleavage were present in samples of DNA from cells treated with barnase (lane 2) and scFv 4D5-dibarnase (lane 6). DNA of serum-starved (ss) cells were cleaved irregularly (lane 7). Lanes 3 and 5 represent untreated controls. Lanes 1 and 4 are molecular weight markers ((M) HyperLadder I, Bioline). (C) Cells were exposed to 50 nM scFv 4D5-dibarnase for 72 h and then stained with acridine orange, analyzed by fluorescence microscopy, and photographed. A representative case of nuclear pyknosis and fragmentation (karyorrhexis) is shown (inset). Magnification, 400× (1200×, inset).</p

Binding of barnase and scFv 4D5-dibarnase to cells.

<p>The cell-binding ability of the recombinant proteins demonstrated by fluorescent microscopy. Cells were incubated at 4°C for 1 h with either 20 nM scFv 4D5-dibarnase (A, B and D), or a mixture of 20 nM scFv 4D5-dibarnase and 20 nM scFv 4D5 (C), or 20 µM barnase (E). Unbound proteins were removed, and then living (A–D) or fixed (E) cells were stained with rabbit anti-barnase antiserum and GAR-TR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a>. The scFv 4D5-dibarnase bound to HER2-positive SKOV-3 cells (A and B), this specific binding was inhibited by scFv 4D5 (C). The scFv 4D5-dibarnase did not bind to HER2-negative CTLL-2 cells (D). Cytoplasmic staining of SKOV-3 cells with 20 µM barnase was observed (E). Magnification, 400×.</p