Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba

Abstract

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.

Cdc6 activity analysis reveals possible unknown function in root meristem cells of Vicia faba

“…Given the fact that Cdc6 labeling density is usually lower in heterochromatin areas, our rst assumption was that the clusters of Cdc6 at euchromatin directly neighboring larger heterochromatin clusters serve as the centers of origins for heterochromatin replication. Heterochromatin areas are much more dense by default and therefore less accessible to DNA-associating proteins (so it's more di cult for the proteins of assembling replisome to actually gain access and attach to DNA within the heterochromatin) [9,44]. So it would seem logical for the cell to relocate origin sites outside of the heterochromatin.…”

“…Cdc6 is the key licensing factor for DNA as it facilitates MCM2-7 loading and further activation of replication forks [7,8]. Its expression is controlled by E2F transcription factors -the factors that regulate most of the S-phase and replication promoting genes [9][10][11][12][13]. Besides being the crucial licensing factor, Cdc6 also plays various roles in ensuring a proper replication progression.…”

Effects of Elevated Temperature on Pisum sativum Nodule Development: I—Detailed Characteristic of Unusual Apical Senescence

“…Detailed analyses of the histological and ultrastructural organization of SGE line nodules, as well as the expression of marker genes, confirmed the formation of the senescence zone in the apical part of the nodule, affecting the infection zone and the distal part of the nitrogen fixation zone (Figures 2-5). Indeed, at the early stages of degradation, the formation of multibacteroid symbiosomes was observed (Figure 3I), and coarse clumps of chromatin formed in the nuclei (Figure 3I), which can lead to inactivation of active transcription sites, considered a precursor of cell death [44,45]. Subsequently, degradation of bacteroids was observed in symbiosomes (Figure 3F), resulting in the formation of 'ghosts' of bacteroids (Figure 3G).…”

Linking replication stress with replication dynamics in Vicia faba root meristem cells

“…The checkpoint bypass can be easily forced with various non-specific ATR inhibitors such as caffeine (CF), okadaic acid or staurosporine. Caffeine has been reported to induce premature chromosome condensation (PCC) in hydroxyurea-synchronized cellsthis event was a consequence of ATR-and ATM-related checkpoint omission [47][48][49].…”