Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and—recently—a promising factor in preventing cognitive decline in Alzheimer’s disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.

We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant–DSBs versus alkaline–DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

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The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even “chaotic” events are dealt with in a proper degree of order.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba’s ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

The CDC6 protein is well-known to facilitate replication origin activation in eukaryotic cells. The Cdc6 protein is part of the pre-replication complex, which initiates DNA replication by binding to specific sites on the genome called origins of replication. In doing so, it helps recruit other proteins that are required for DNA synthesis. It is also an important factor in response to replication stress, where it has been shown to physically interact with ATR in order to activate the S-phase checkpoint response. The ATR, though, can also activate the S-phase checkpoint without interacting with Cdc6. Up to this day, the Cdc6-ATR binding was thought occur where there is a low level of replication stress. Here we show that it can also occur when high levels of replication stress persist for long time. Our research shows that in root meristem cells of V. faba, subjected to long-term replication stress (24–32 hours) induced by hydroxyurea, some DNA damage sites express ATR activity through binding with Cdc6. We conclude that the Cdc6-ATR cooperation may not only depend on the dose of the stressor but also on the longitude of the treatment.

DNA replication faces many challenges, both internally and externally, generally described as factors that induce replication stress. In this article, we describe how prolonged replication stress affects the dynamics of replication and fork activity in V. faba root meristem cells. V. faba seems to have a highly effective stress response system that includes a reaction to replication stress. We used 2.5 mM hydroxyurea (HU) for prolonged stress induction (32 hours) and measured changes in replication and fork activity after initial stress induction, prolonged exposure and after regeneration time in water. We also induced premature chromosome condensation (PCC) as a reference, to compare cells that express valid ATR/Chk1 S-phase checkpoint with cells that lack ATR functions. Our results included general changes in replication activity, obtained with 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as an extended analysis of replication fork progression facilitated by double-labeling with EdU and 5-iodo-2’-deoxyuridine (IdU) which we found to be an appealing alternative to commonly used labeling with 5-chloro-2’-deoxyuridine (CldU) and IdU. A preliminary analysis of minichromo-some maintenance complex component 2 (MCM2), a subunit of minichromosome maintenance protein complex (MCM), were shown. We were able to pinpoint a mechanism that may contribute to the replication stress resistance of V. faba cells the most. We have also demonstrated that it is not only cells with ATR malfunctions where heterochromatin areas are extensively affected by replication stress.

Cell division cycle 6 (Cdc6) protein regulates the licensing of DNA replication, maintains centrosome stability, participates in DNA damage response (DDR) pathways and activates cell cycle checkpoints. The mechanisms responsible for Cdc6 relocation in the cell cycle are evolutionary conserved. Here, we analyzed changes in Cdc6 activity following hydroxyurea-induced replication stress and caffeine-induced premature chromosome condensation (PCC). We used Vicia faba root meristem cells as a model to explore the dynamics of activity changes of Cdc6, Chk1 and Chk2 proteins and their phosphorylated forms (Cdc6Ser54ph, Chk1Ser345ph and Chk2Thr68ph) under the conditions of prolonged replication stress. We observed the areas of increased Cdc6 activity that were not reported for animal cells: (i) the presence of clusters associated with low-density chromatin (euchromatin), (ii) the frequent vicinity of Cdc6 clusters to the most dense regions of constitutive heterochromatin. Cdc6 remains within the nucleus throughout the interphase which may suggest its new, yet unknown function in plants. The results of changes in Cdc6 activity during prolonged replicative stress also indicate that Cdc6 plays an important role in maintaining the cell cycle checkpoint in S phase when stress conditions last longer.