Rate Of DNA Synthesis Research Articles

An examination of the inhibitory effects of three antibiotics in combination on ribosome biosynthesis in Staphylococcus aureus

Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. The use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects on ribosomal subunit synthesis rates. In this work, combination antibiotic treatments gave a significant decrease in cell numbers following growth in the presence of ciprofloxacin or rifampicin with azithromycin compared to those grown with azithromycin or rifampicin alone. DNA, RNA and protein synthesis rates were reduced with single antibiotic treatments and showed further decreases when drug combinations were used. 70S ribosome levels were reduced with every antibiotic treatment. DNA gyrase subunits A and B showed significant decreases for double and triple antibiotic-treated samples. Ribosomal subunit synthesis rates were diminished for each different antibiotic combination. Turnover of 16S and 23S rRNA was also observed in each case and was stimulated by antibiotic combinations. The frequency of spontaneous resistance was reduced in all double selections, and no triply resistant mutants were found.

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.

The Erythropoietin Receptor Regulates The Number Of Cell Divisions and The Duration Of Erythroblast Terminal Differentiation By Regulating Erythroblast Iron

Signaling by the erythropoietin receptor (EpoR) is essential for the survival of definitive colony-forming unit-erythroid (CFU-e) progenitors and their erythroblast progeny. Here we used EpoR-null embryos to ask whether EpoR signaling might also exert essential non-survival functions in erythropoiesis.To address this, we rescued EpoR-null fetal liver cells from death by transducing them in vitro with either the anti-apoptotic protein Bcl-xL, or, as control, with the wild-type EpoR. The Bcl-xL-transduced EpoR-null cells survived, expressed hemoglobin and underwent morphological erythroid maturation and enucleation. However, unlike exogenous EpoR, exogenous Bcl-xL was unable to support the formation of EpoR-null CFU-e colonies in methylcellulose. The absence of colonies was explained by the finding that the Bcl-xL-transduced progenitors underwent fewer cell divisions than equivalent EpoR-transduced cells (1.1 vs. 2.9 in 24 hr, respectively) and had a slower rate of intra-S phase DNA synthesis, suggesting longer S phase duration. Multispectral imaging showed that the Bcl-xL-transduced cells matured prematurely, attaining smaller cell and nuclear size and a lower nuclear/cytoplasmic ratio at earlier time points than EpoR-transduced cells. Premature maturation was also evident by flow cytometric analysis. Thus, EpoR-null fetal liver cells in vivo arrest in their differentiation at the transition from subset S0 (Ter119-neg CD71-low) to S1 (Ter119-neg CD71-high) (Pop et al, PLoS Biology 2010). Rescue with EpoR in vitro allows EpoR-null progenitors to resume differentiation, sequentially upregulating CD71 and Ter119. By contrast, rescue of EpoR-null cells with Bcl-xL results in their premature upregulation of Ter119 and failure to upregulate CD71 to high levels. The cell cycle and differentiation deficits in Bcl-xL-supported, EpoR-null erythropoiesis were associated with a slower loss of DNA methylation from the erythroid genome, and with slower erythroid gene transcription.CD71 (the transferrin receptor) is a known target of EpoR and Stat5 signaling. We asked whether the deficits of EpoR-null erythropoiesis might be the result of low cell surface CD71 and the consequent reduced iron transport. In support of this hypothesis, we found that EpoR-null fetal liver cells that are transduced with both CD71 and Bcl-xL resume the normal maturation rate characteristic of EpoR-supported differentiation, as judged by multispectral imaging measurements of cell size and nuclear/cytoplasmic ratio. Further, we were able to restore rapid S phase to Bcl-xL-transduced EpoR-/- erythroblasts by culturing them in the presence of the cell-permeant iron chelator Fe-SIH (salicylaldehyde isonicotinoyl hydrazone), which is able to supply the cell interior with iron even in the absence of CD71 (Figure 1). We suggest that EpoR-mediated upregulation of CD71 at the onset of erythroid terminal differentiation determines the number and duration of erythroblast cell divisions by regulating iron homeostasis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Systems Biology and Epigenetic Mechanisms in Erythropoiesis

Irreversible rapid cellular decisions are often controlled by network motifs known as bistable switches. We identified a cell-cycle regulated bistable switch that controls activation of the erythroid transcriptional program during early S phase of the last generation of erythroid colony-forming-unit progenitors (CFUe). This switch drives a rapid, multi-layered commitment event that activates GATA-1 transcription, renders the cells dependent on erythropoietin, and brings about chromatin reconfiguration at erythroid gene loci. In addition, it triggers an unusual process of genome-wide DNA demethylation, the first known example of such a process in somatic cell development. Approximately 25 to 30 percent of all methylation marks are lost from essentially all genomic elements during erythroid terminal differentiation. The bistable switch activating erythroid transcription consists of two linked double-negative feedback interactions of the erythroid transcriptional repressor PU.1, which antagonizes both S phase progression, and the erythroid master transcriptional regulator GATA-1. During operation of the switch, a rapid S phase-dependent decline in PU.1 activates GATA-1 transcription. The dependence of this switch on S phase progression coincides with a dramatic change in the nature of S phase itself, which becomes shorter and 50 percent faster. The accelerated intra-S phase DNA synthesis rate is essential for the loss of genome-wide DNA methylation, which in turn is required for the rapid induction of erythroid genes. Disclosures:No relevant conflicts of interest to declare.

Autophagy stimulates apoptosis in HER2‐overexpressing breast cancers treated by lapatinib

HER2-overexpressing breast cancers often show hyperactivation of the HER2/AKT/mTOR signaling pathway. Lapatinib is an oral dual tyrosine kinase inhibitor (TKI) that targets both EGFR and HER2 to inhibit the proliferation of breast cancer cells. However, it is obscure whether and how lapatinib could induce autophagy in breast cancer cells, an important cell response with drug treatment. In this study, we investigated the apoptosis and the autophagy in the HER2-overexpressing breast cancer cells BT474 and AU565 treated with lapatinib, and further examined their relationship. Lapatinib inhibited the proliferation and the rate of DNA synthesis in HER2-positive cells, as observed by MTT, colony formation and EDU assays. Lapatinib not only induced apoptosis accompanied by an increased expression of cleaved Caspase-3 and cleaved PARP, but it also induced autophagy in vitro, as confirmed by electron microscopy (EM), acridine orange (AO) staining and LC3-II expression. Meanwhile, lapatinib inhibited the phosphorylation of HER2, AKT, mTOR, and p70S6K, whereas that of AMPK was activated. When the cells were pre-incubated with 3-Methyladenine (3-MA), the specific autophagy inhibitor, the growth inhibitory ratio and apoptosis rate were frustrated, whereas colony formation and DNA synthesis ability were encouraged. In addition, 3-MA application could up-regulate Caspase-3 and PARP expression, compared with the treatment with lapatinib alone. The addition of 3-MA could attenuate the inhibitory role on HER2/AKT/mTOR pathway and the active role on AMPK that was raised by lapatinib. Therefore, lapatinib simultaneously induced both apoptosis and autophagy in the BT474 and AU565 cells, and in these settings, autophagy facilitates apoptosis.

Effects of leptin and adiponectin on the growth of porcine myoblasts are associated with changes in p44/42 MAPK signaling

We hypothesized that both adiponectin and leptin affect the growth of porcine skeletal muscle cells, with fatty acids acting as modifiers in adipokine action and that both adipokines influence the gene expression of their receptors. Therefore, the objective of this study was to investigate the effects of recombinant adiponectin and leptin on cell number (DNA) and DNA synthesis rate with and without oleic acid supplementation, on cell death, and on key intracellular signaling molecules of proliferating porcine myoblasts in vitro. Moreover, the mRNA expression of genes encoding for the leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) as affected by leptin or adiponectin was examined. Recombinant porcine adiponectin (40 μg/mL) and leptin (20 ng/mL) increased DNA synthesis rate, measured as [3H]-thymidine incorporation (P < 0.01), reduced cell viability in terms of lactate dehydrogenase release (P < 0.05), or lowered DNA content after 24 h (P < 0.05). In adiponectin-treated cultures, oleic acid supplementation increased DNA synthesis rate and reduced cell number in a dose-dependent manner (P < 0.05). Both adiponectin (P = 0.07) and leptin (P < 0.05) induced a transient activation of p44/42 mitogen-activated protein kinase (MAPK) after 15 min, followed by decreases after 60 and 180 min (P < 0.05). Adiponectin tended to increase c-fos activation (P = 0.08) and decreased p53 activation at 180 min (P = 0.03). Both adiponectin and leptin down-regulated the abundance of ADIPOR2 mRNA and, transiently, of LEPR mRNA (P < 0.05). In conclusion, adiponectin and leptin may adversely affect the growth of porcine myoblasts, which is related to p44/42 MAPK signaling and associated with changes in ligand receptor gene expression.

The DNA methyl transferase inhibitor, 5′-aza-2-deoxycitidine, enhances the apoptotic effect of Mevastatin in human leukemia HL-60 cells

Statins induce antiproliferative effects and apoptotic response in various cancer cell types. Moreover, they also sensitize tumor cell lines from different origins to many agents. We aimed to investigate possible effects of Mevastatin (Mev) alone and sequential treatment of 5'-aza-2-deoxycitidine (DAC) and Mev on HL-60 cell line using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay, lactate dehydrogenase release assay, flourescence microscopy, DNA fragmentation analysis, determination of DNA synthesis rate, and active caspase-3 assay. Messenger RNA (mRNA) expression of apoptotic and antiapoptotic genes were also evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for BAX, BCL2, and XIAP genes and quantitative Real-time PCR for CASP3, CASP8, and CASP9 genes. We showed that treatment with Mev alone and DAC followed by Mev resulted in apoptotic response in a time- and dose-dependent manner. We also found that pretreatment with DAC sensitized HL-60 cells to Mev and caused more apoptotic cell death than Mev-alone treatment via caspase-3 activation and DNA fragmentation. Moreover, sequential addition of Mev after DAC diminished DNA synthesis rate more effectively than Mev-alone treatment. Furthermore, DAC pretreatment significantly increased CASP3 and CASP9 mRNA expression even with lower doses of Mev. BAX, BCL2, and XIAP gene mRNA levels were also found to be changed in the presence of DAC and Mev. Determination of the exact molecular effects of statins and DAC would allow us to identify new molecular targets to develop more effective treatment regimens for cancer.

Magnetic control of the DNA synthesis

By using polymerases β loaded with isotopic ions 24Mg2+, 25Mg2+ and 26Mg2+ magnetic isotope effect was detected: 25Mg2+ ions with magnetic nuclei 25Mg suppress enzymatic activity by 2–3 times with respect to that of polymerases β loaded by 24Mg2+ and 26Mg2+ ions. No difference in enzymatic activity of polymerases β with 24Mg2+ and 26Mg2+ ions exists. The rate of DNA synthesis strongly depends on the magnetic field. The polymerase chain reaction is also suppressed by 25Mg2+ ions with respect to the ions with nonmagnetic nuclei. Magnetic control of the DNA synthesis may be used for medical purposes.

Magnetic isotope and magnetic field effects on the DNA synthesis

Magnetic isotope and magnetic field effects on the rate of DNA synthesis catalysed by polymerases β with isotopic ions 24Mg2+, 25Mg2+ and 26Mg2+ in the catalytic sites were detected. No difference in enzymatic activity was found between polymerases β carrying 24Mg2+ and 26Mg2+ ions with spinless, non-magnetic nuclei 24Mg and 26Mg. However, 25Mg2+ ions with magnetic nucleus 25Mg were shown to suppress enzymatic activity by two to three times with respect to the enzymatic activity of polymerases β with 24Mg2+ and 26Mg2+ ions. Such an isotopic dependence directly indicates that in the DNA synthesis magnetic mass-independent isotope effect functions. Similar effect is exhibited by polymerases β with Zn2+ ions carrying magnetic 67Zn and non-magnetic 64Zn nuclei, respectively. A new, ion–radical mechanism of the DNA synthesis is suggested to explain these effects. Magnetic field dependence of the magnesium-catalysed DNA synthesis is in a perfect agreement with the proposed ion–radical mechanism. It is pointed out that the magnetic isotope and magnetic field effects may be used for medicinal purposes (trans-cranial magnetic treatment of cognitive deceases, cell proliferation, control of the cancer cells, etc).

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.

Fewer forks make faster progress

Just as a large number of forks can inhibit someone unaccustomed to formal dining, an excess of replication forks can slow the rate of DNA synthesis, [Zhong et al.][1] reveal. Cdc7 is the catalytic subunit of the Dbf4-dependent kinase, which initiates DNA replication by activating the DNA helicase

Abstract 3944: Serine phosphorylation of vasodilator-stimulated phosphoprotein regulates the tumorigenic potential of colon cancer cells.

Abstract Colorectal cancer is one of the most common and deadly cancers in developed nations. Molecular mechanisms precisely defining colorectal tumorigenesis and cancer progression remain unclear, an obstacle for innovative research applications. The vasodilator-stimulated phosphoprotein (VASP) is an actin-binding protein which critically regulates the cytoskeleton and its dependent functions, including cell shape, adhesion and migration. Of relevance, VASP Ser phosphorylation becomes deregulated in colorectal transformation and promotes invasive protrusion dynamics. Here, VASP Ser phosphorylation was selectively inhibited in colon carcinoma cells employing dominant negative mutants. Effects on cell tumorigenic potential were examined with two mouse models of colorectal cancer. Compared to control conditions, suppression of VASP Ser239 phosphorylation resulted in an enhanced cancer cell capability to establish tumor colonies in both the subcutaneous and peritoneal xenograft model. Increased tumorigenicity reflected, in part, hyper proliferative kinetics of cancer cells bearing VASP Ser239 mutants, which exhibited accelerated growth and DNA synthesis rates. In contrast, selective inhibition of VASP Ser157 slowed cell proliferation and reduced the in vivo tumorigenic potential of colon carcinoma cells, compared to respective controls. These observations support the suggestion that VASP Ser phosphorylation represents a fine signaling mechanism to control intestinal tumorigenesis and invasion, a novel paradigm for targeted anticancer strategies in patients with colorectal cancer. Citation Format: Mehboob Ali, Inna Chervoneva, Giovanni M. Pitari. Serine phosphorylation of vasodilator-stimulated phosphoprotein regulates the tumorigenic potential of colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2013-3944

Histone Acetyl Transferase (HAT) HBO1 and PHD Finger Protein 17 (PHF17/JADE1) in Epithelial Cell Regeneration

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of chromatinal histones. We characterized the role of JADE1‐HBO1 complexes in vitro and in vivo during epithelial cell replication. Downregulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury (AKI) to examine expression of HBO1‐JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels which returned to the baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum while JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysine 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S‐HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicated JADE1‐HBO1 complex in AKI and suggested distinct roles for JADE1 isoforms during epithelial cells recovery.NIH RO1 DK087910, and ACS IRG‐72–001‐32 (to M. V. P.). NIH 1KO8DK090143 (to A.H.).

REV7 is required for anaphase-promoting complex-dependent ubiquitination and degradation of translesion DNA polymerase REV1

REV1 is a Y-family polymerase specialized for replicating across DNA lesions at the stalled replication folk. Due to the high error rate of REV1-dependent translesion DNA synthesis (TLS), tight regulation of REV1 activity is essential. Here, we show that human REV1 undergoes proteosomal degradation mediated by the E3 ubiquitin ligase known as anaphase-promoting complex (APC). REV1 associates with APC. Overexpression of APC coactivator CDH1 or CDC20 promotes polyubiquitination and proteosomal degradation of REV1. Surprisingly, polyubiquitination of REV1 also requires REV7, a TLS accessory protein that interacts with REV1 and other TLS polymerases. The N-terminal region of REV1 contains both the APC degron and an additional REV7-binding domain. Depletion of REV7 by RNA interference stabilizes REV1 by preventing polyubiquitination, whereas overexpression of REV7 augments REV1 degradation. Taken together, our findings suggest a role of REV7 in governing REV1 stability and interplay between TLS and APC-dependent proteolysis.

Equivocal Differential Effect of NDRG1 in Human Ovarian Cancer Cells

Inactivation of tumor suppressor genes is a key factor in cancer regulation. N-myc downstream regulated gene 1 (NDRG1) is a tumor suppressor gene well known to be involved in carcinogenesis of numerous cancer types. The present study was designed to investigate the role of NDRG1 in human ovarian cancer, using SKOV-3 and SW626 (moderately and well differentiated cancer cells, respectively). Our results revealed that over-expressed NDRG1 significantly up-regulated the differentiation marker p21, in the ovarian cancer cell lines. This regulation led to decrease in cell viability and DNA synthesis rates in SW626 cells (83% and 89.5%, respectively). However, no effect on viability or on DNA synthesis was observed in SKOV-3 NDRG1-transfected cells. These findings prove that NDRG1 is indubitably functional in human ovarian cancer cells, as it up-regulated p21 expression. Nevertheless, this regulation showed differential effect on cell viability and DNA formation thus promoting the perception that downstream regulation of p21 could be inefficient in some cancer cells, a concept that needs to be further explored in order to understand its disability to play as regulator of cell cycle progression.

Histone Acetyl Transferase (HAT) HBO1 and JADE1 in Epithelial Cell Regeneration

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of histones in chromatin context. We characterized the role of JADE1-HBO1 complexes invitro and invivo during epithelial cell replication. Down-regulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury to examine expression of HBO1-JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels, which returned to baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum, whereas JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysines 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S-HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicate JADE1-HBO1 complex in acute kidney injury and suggest distinct roles for JADE1 isoforms during epithelial cell recovery.

Growth kinetics of CD133‐positive prostate cancer cells

In the adult human prostate CD133 expression is thought to mark rare prostate epithelial stem cells and malignant tumor stem/initiating cells. Such putative stem cell populations are thought to proliferate slowly, but possess unlimited proliferative potential. Based on this, we hypothesized that CD133(pos) prostate cancer cells proliferate slower than CD133(neg) cells. Human prostate cancer cell lines were analyzed for CD133 expression and DNA content using flow cytometry. Rates of cell division and DNA synthesis were determined using CFSE cell tracing and BrdU uptake, respectively. Changes in cell cycle distribution and the percentage of CD133(pos) cells were assayed under conditions of different cell density and AR-pathway modulation. Lastly, we over-expressed lentiviral CD133 to measure whether CD133 regulates the cell cycle. The cell cycle distribution differs between CD133(pos) and CD133(neg) cells in all three human prostate cancer cell lines studied. CD133(pos) cells have a greater proportion of cells in G2 and proliferate faster than CD133(neg) cells. High cell density increases the percentage of CD133(pos) cells without changing CD133(pos) cell cycle progression. Treatment with the AR agonist R1881, or the anti-androgen MDV3100, significantly changed the percentage and proliferation of CD133(pos) cells. Finally, ectopic over-expression of CD133 had no effect on cell cycle progression. Contrary to our hypothesis, we demonstrate that CD133(pos) cells proliferate faster than CD133(neg) cells. This association of CD133 expression with increased cell proliferation is not directly mediated by CD133, suggesting that surface CD133 is a downstream target gene of an undefined pathway controlling cell proliferation.

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines

The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser’s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.

Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication

Bacteriophage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7% of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.

Molecular Mechanism of Antagonism between the Y181C and E138K Mutations in HIV-1 Reverse Transcriptase

Etravirine (ETR) is an expanded-spectrum nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use as an antiretroviral agent in treatment-experienced patients. Y181C and E138K in HIV-1 RT are among 20 different drug resistance mutations associated with ETR. However, E138K can be consistently selected by ETR when wild-type viruses but not viruses containing Y181C are grown in tissue culture. This study was carried out to evaluate any possible mechanisms that might explain antagonism between the Y181C and E138K mutations. Accordingly, we performed tissue culture studies to investigate the evolutionary dynamics of E138K in both a wild-type (WT) and a Y181C background. We also generated recombinant enzymes containing Y181C and E138K alone or in combination in order to study enzyme processivity, rates of processive DNA synthesis, enzyme kinetics, and susceptibility to ETR. We now show that the presence of the Y181C mutation prevented the emergence of E138K in cell culture and that the simultaneous presence of E138K and Y181C impaired each of enzyme activity, processivity, rate of processive DNA synthesis, and deoxynucleoside triphosphate (dNTP) affinity. The addition of E138K to Y181C also decreased the level of resistance to ETR compared to that obtained with Y181C alone.