Presence Of DNA Synthesis Inhibitor Research Articles

Epigenetic Targeting of Adipocytes Inhibits High-Grade Serous Ovarian Cancer Cell Migration and Invasion.

Ovarian cancer (OC) cells frequently metastasize to the omentum, and adipocytes play a significant role in ovarian tumor progression. Therapeutic interventions targeting aberrant DNA methylation in ovarian tumors have shown promise in the clinic, but the effects of epigenetic therapy on the tumor microenvironment are understudied. Here, we examined the effect of adipocytes on OC cell behavior in culture and impact of targeting DNA methylation in adipocytes on OC metastasis. The presence of adipocytes increased OC cell migration and invasion, and proximal and direct coculture of adipocytes increased OC proliferation alone or after treatment with carboplatin. Treatment of adipocytes with hypomethylating agent guadecitabine decreased migration and invasion of OC cells toward adipocytes. Subcellular protein fractionation of adipocytes treated with guadecitabine revealed decreased DNA methyltransferase 1 (DNMT1) levels even in the presence of DNA synthesis inhibitor, aphidicolin. Methyl-Capture- and RNA-sequencing analysis of guadecitabine-treated adipocytes revealed derepression of tumor-suppressor genes and epithelial-mesenchymal transition inhibitors. SUSD2, a secreted tumor suppressor downregulated by promoter CpG island methylation in adipocytes, was upregulated after guadecitabine treatment, and recombinant SUSD2 decreased OC cell migration and invasion. Integrated analysis of the methylomic and transcriptomic data identified pathways associated with inhibition of matrix metalloproteases and fatty acid α-oxidation, suggesting a possible mechanism of how epigenetic therapy of adipocytes decreases metastasis. In conclusion, the effect of DNMT inhibitor on fully differentiated adipocytes suggests that hypomethylating agents may affect the tumor microenvironment to decrease cancer cell metastasis.Implications: Epigenetic targeting of tumor microenvironment can affect metastatic behavior of ovarian cancer cells. Mol Cancer Res; 16(8); 1226-40. ©2018 AACR.

Enhanced genetic rescue of negative-strand RNA viruses: use of an MVA-T7 RNA polymerase vector and DNA replication inhibitors

A modified cDNA rescue system that improves recovery of recombinant nonsegmented, negative-strand RNA viruses from cloned DNAs is described. Rescue systems based on vaccinia virus-T7 RNA polymerase vectors have been used to derive many negative-strand viruses; however, some strains can be recalcitrant to rescue possibly because of the simultaneous replication of the vaccinia virus-T7 vector. Our goal was to engineer a system where replication of the vaccinia virus-T7 vector could be blocked, yet allow for sufficient T7 RNA polymerase expression to enable genetic rescue. To that end, a recombinant modified vaccinia virus Ankara (MVA) was engineered that contained the bacteriophage T7 gene-1 under the control of a strong early promoter that would enable T7 RNA polymerase expression in the absence of MVA DNA replication. The new T7 helper, MVAGKT7, was then utilized successfully for the genetic rescue of a measles virus minigenome and full-length cDNAs, in the presence of DNA synthesis inhibitors. In addition to blocking completely MVAGKT7 replication, AraC treatment was found to enhance minigenome-encoded gene expression and the efficiency of measles virus rescue.

Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays.

Early during the herpes simplex virus (HSV) lytic cycle or in the presence of DNA synthesis inhibitors, core viral replication machinery proteins accumulate in intranuclear speckled punctate prereplicative foci, some of which colocalize with numerous sites of host cellular DNA synthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replication compartments are formed; these compartments also contain progeny viral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair. In this study, we demonstrate that several forms of both prereplication foci and active viral replication compartments that display an appearance similar to that of the compartments in HSV-infected cells can be successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork proteins together with oriS target plasmid DNA. Furthermore, bromodeoxyuridine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized with the HSV single-stranded DNA-binding protein (SSB) in these replication compartments, implying that active viral DNA replication may be occurring. The assembly of complete HSV replication compartments and incorporation of BrdU were both abolished by treatment with phosphonoacetic acid (PAA) and by omission of any one of the seven viral replication proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essential for viral DNA replication. Consistent with the fact that both HSV IE175 and IE63(ICP27) localize within replication compartments in HSV-infected cells, the assembled HSV replication compartments were also able to recruit both of these essential regulatory proteins. Blocking viral DNA synthesis with PAA, but not omission of oriS, prevented the association of IE175 with prereplication structures. The assembled HSV replication compartments also redistributed cotransfected cellular p53 into the viral replication compartments. However, the other two HSV immediate-early nuclear proteins IE110(ICP0) and IE68(ICP22) did not enter the replication compartments in either infected or transfected cells.

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n-Butyrate has been shown to induce Epstein-Barr virus (EBV) antigen synthesis in certain EBV genome-carrying lymphoblastoid cell lines. We have studied the effect of cis-dichloro-diamine-platinum (CDDP) on induction of EBV antigen synthesis not only by n-butyrate treatment of the EBV producer, P3HR-1 cells, but also by superinfection of EBV to the EBV non-producer, Raji cells. Simultaneous treatment of the cells with n-butyrate and CDDP or MMC blocked VCA and EA induction, but no suppressive effect was observed by the treatment of the cells with n-butyrate and Ara-C. The induction of EBV antigens was also observed in EBV-superinfected Raji cells, even in the presence of DNA synthesis inhibitor, CDDP or MMC. From these results, we discussed the mechanisms of the EBV antigens synthesis in EBV latently infected cells.

Kinetic analysis of UV-induced incision discriminates between fibroblasts from different xeroderma pigmentosum complementation groups, XPA heterozygotes and normal individuals

The capacity of a variety of human fibroblasts to incise DNA following exposure to far ultraviolet-light is determined from the rate of single-strand DNA break accumulation in the presence of DNA synthesis inhibitors. We have quantitated incision, one of the early steps in the UV excision repair pathway, in cells form normal, xeroderma pigmentosum groups C, D, G, H and variant individuals, and in the parents of one XPA patient. On the basis of the estimated initial rates of incision the different XP cells examined in this work can be marked as follows: XP variant ⋙ XPH > XPD > XPC > XPG > XPA . In each cell strain breaks accumulate immediately after irradiation over a range of 0.5–20 Jm −2 with the exception of the XPC strain examined, where there is an initial delay of 15 min. The rate of incision in XPA heterozygote cells is roughly half that of normal fibroblasts. Analysis of the kinetics of break accumulation over short intervals after irradiation permits estimation of the apparent enzymatic parameters, K m and V max, for the incision step. The approximate values of K m and V max for normal and XP variant are similar while for the heterozygotes of an XPA individual K m values are normal (around1 Jm −2), but there is only half the amount of normal enzyme activity. XPD and H cells express low levels of active enzyme, between 5 and 15% of that of the normal, but while the K m of KPH is very similar to that of normal cells, that of two XPD strains examined is between 2- and 3-fold higher.

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state.

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.

Early enhanced glucose uptake in human cytomegalovirus-infected cells.

The uptake rate of some sugars was demonstrated in human embryo fibroblasts following infection by human cytomegalovirus (HCMV) and a significant increase in glucose uptake was demonstrated. Quantitative and sequential analysis of glucose uptake during the HCMV replication cycle showed that the enhanced uptake began during the first 20 h after infection and occurred even when a high glucose level was constantly present in the medium. The increase in sugar uptake requires an active viral genome, de novo protein synthesis and seems to be dependent only on the early transcription of the viral genome, as it did occur when replication of the viral genome was limited (non-permissive cells or cells infected in the presence of DNA synthesis inhibitors).

Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA.

In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.