The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

Alternate treatments of Allium cepa root meristems with hydroxyurea (HU) and caffeine give rise to extremely large and highly elongated cells with atypical images of mitotic divisions, including internuclear asynchrony and an unknown type of interchromosomal asynchrony observed during metaphase-to-anaphase transition. Another type of asynchrony that cannot depend solely on the increased length of cells was observed following long-term incubation of roots with HU. This kind of treatment revealed both cell nuclei entering premature mitosis and, for the first time, an uncommon form of mitotic abnormality manifested in a gradual condensation of chromatin (spanning from interphase to prometaphase). Immunocytochemical study of polykaryotic cells using anti-β tubulin antibodies revealed severe perturbations in the microtubular organization of preprophase bands. Quantitative immunofluorescence measurements of the control cells indicate that the level of cyclin B-like proteins reaches the maximum at the G2 to metaphase transition and then becomes reduced during later stages of mitosis. After long-term incubation with low doses of HU, the amount of cyclin B-like proteins considerably increases, and a significant number of elongated cells show gradients of these proteins spread along successive regions of the perinuclear cytoplasm. It is suggested that there may be a direct link between the effects of HU-mediated deceleration of S- and G2-phases and an enhanced concentration of cyclin B-like proteins. In consequence, the activation of cyclin B-CDK complexes gives rise to an abnormal pattern of premature mitotic chromosome condensation with biphasic nuclear structures having one part of chromatin decondensed, and the other part condensed.

A number of chemical agents known to influence the key cell cycle regulatory factors were used to assess the requirements of hydroxyurea-treated root meristem cells of Vicia faba for premature condensation of chromosomes (PCC). These included caffeine and 2-aminopurine (inhibitors of ATM/ATR sensor kinases activated by DNA damage or stalled replication forks), inhibitors of protein kinases (staurosporine and wortmannin), inhibitors of protein phosphatases (sodium vanadate and calyculin A), and other compounds like 1,2-dioctyl-sn-glycerol, an activator of protein kinase C, 5-azacytidine, an inhibitor of DNA methyltransferase, dithiothreitol and N-etylmaleimide, capable to up-and down-regulate the activity of Cdc25 phosphatase. Cytological parameters used to evaluate quantitative aspects of PCC allowed us to discriminate various phenotypes of cells and, consistent with the extent of chromosomal fragmentation, to classify them as S-or G2-PCC. Two significant aspects relevant to the induction of premature mitosis in plants seem to emerge: one concerns the inverse relationship between the incidence of mitotic and PCC events, the other refers to the extent with which a variety of chemical agents may activate mechanisms that override the S-M replication checkpoint. 1,2-dioctyl-sn-glycerol, an activator of protein kinase C in animal cells proved extremely effective in stimulation of PCC, in spite of evident lack of molecular targets in plants.

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