Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.

Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. IMPLICATIONS: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.
©2019 American Association for Cancer Research.

The epigenomes of mammalian sperm and oocytes, characterized by gamete-specific 5-methylcytosine (5mC) patterns, are reprogrammed during early embryogenesis to establish full developmental potential. Previous studies have suggested that the paternal genome is actively demethylated in the zygote while the maternal genome undergoes subsequent passive demethylation via DNA replication during cleavage. Active demethylation is known to depend on 5mC oxidation by Tet dioxygenases and excision of oxidized bases by thymine DNA glycosylase (TDG). Here we show that both maternal and paternal genomes undergo widespread active and passive demethylation in zygotes before the first mitotic division. Passive demethylation was blocked by the replication inhibitor aphidicolin, and active demethylation was abrogated by deletion of Tet3 in both pronuclei. At actively demethylated loci, 5mCs were processed to unmodified cytosines. Surprisingly, the demethylation process was unaffected by the deletion of TDG from the zygote, suggesting the existence of other demethylation mechanisms downstream of Tet3-mediated oxidation.
Copyright © 2014 Elsevier Inc. All rights reserved.

A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a little-understood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.

We investigated the effect of aphidicolin, an inhibitor of DNA polymerase alpha and delta, on the induction of apoptosis by arabinosyl nucleosides in a human promyelocytic leukemia cell line, HL-60. Pretreatment of HL-60 cells with aphidicolin (2 microM) significantly increased the number of morphologically apoptotic cells induced by 1-beta-D arabinofuranosylcytosine (ara-C) during 4 hr of incubation. This is consistent with the appearance of DNA fragmentation as determined quantitatively by diphenylamine or by agarose gel electrophoresis. The inhibition of cell growth on day 3 after drug exposure was correlated with the degree of apoptosis: Such synergistic interaction between aphidicolin and ara-C has also been observed in other human myeloid leukemia cell lines, U937 and KG-1. In addition, the induction of apoptosis by 9-beta-D arabinofuranosyladenine or 9-beta-D arabinofuranosylguanine is augmented by aphidicolin.