**5. Consequences of PCC induction**

196 DNA Repair

2006). Half of the γ-H2AX histones appears after a 1-minute period of irradiation, while the maximal level is reached within 3 to 10 minutes of exposure (per 1 Gy of radiation about 1% of H2AX histone molecules undergoes γ-phosphorylation, which corresponds to about 2x106 bp DNA in the area of the double-strand break of its structure) (Paull et al., 2000; Rogakou et al., 1998). It is assumed that each group of these molecules indicates a single DSB region, therefore the formation of H2AX foci phosphorylated at S139 constitutes a sensitive test revealing the presence of structural genome damage (Friesner et al., 2005). Literature reports in recent years show the occurrence of fluorescence foci of H2AX molecules within the area of S139 during PCC induction (Huang et al., 2006; Rybaczek et al., 2007; Rybaczek & Kowalewicz-Kulbat, 2011). On the other hand, Stevens and his co-workers (2010) clearly indicate that PCC is γ-H2AX negative, and that γ-H2AX phosphorylation is only a hallmark of a chromosome fragmentation phenomenon. However, simultaneously they define PCC as occurring in interphase cells exposed to active MPF vs that occurring during mitosis chromosome fragmentation. According to other investigations, however, the actual definition of drug-induced PCC involves both the described phenomena, i.e. PCC and chromosome fragmentation (Riesterer et al., 2009; Rybaczek et al., 2008;

To protect against disturbances during DNA biosynthesis, cells have developed a network of biochemical reactions known as DNA-replication-stress-response. The basic strategy of this response is retardation of processes, whose continuation would result, among other things, in the transfer of affected DNA molecules to a new cell generation. Therefore under the conditions of replication stress, the DNA biosynthesis rate is slowed down and onset of mitosis is - most frequently - completely blocked. This continues until the expression of specific genes and activation of repair factors (Deckert et al., 2009; Mosesso et al., 2010). Each structural disturbance (e.g. DSB) causes the rate of production of replication forks to slow down. Additionally, any limitation of the replication apparatus effectiveness (resulting, e.g. from the deficiency of nucleotide triphosphate or polymerase dysfunction) can facilitate DNA damage. In such a situation, the action of checkpoint sensory factors releases a cascade

The detection of double-strand breaks in DNA molecules activates the biochemical pathway in which ATM kinase is the superior element. The processes proceeding with its contribution are triggered in all the phases of cell cycle, while the factors participating here may also assist the second pathway going in parallel. ATR is also activated by damage resulting in disruption of continuity in both DNA strands, but here the induction process occurs more slowly (Scott & Pandita, 2006). The pathway subordinated to ATR kinase is specialized first of all in reacting to disturbance of the replication fork function. These disturbances can results from endogenous interactions, chemotherapy or experimental procedures leading to the inhibition or disturbance of replication processes caused by hydroxyurea (HU) or ultraviolet radiation (UV). In both biochemical pathways, Cdc25

Mec1 and Tel1 kinases in *S. cerevisiae* and Rad3 and Tel1 kinases in humans are homologues of both conservative signaling ATM and ATR kinases (Garber et al., 2005). Their substrates, kinases of the CHK family, are subordinate factors activated by ATM/ATR/Mec1/Rad3. Such groups jointly form the central pathway module of response to replication stress, which both records incoming information on the DNA condition and sends signals to replication forks. In human and *Xenopus* cells, ATR kinase is indispensable in the phosphorylation of Chk1 kinase and it occurs in stable combination with ATR-interacting

of signals supplied to various effector proteins through intermediary elements.

Terzoudi et al., 2010).

phosphatase is the target substrate.

Literature shows that proper functioning of all multicellular organisms, including *Homo sapiens,* depends not only on their ability to produce new cells but also on the ability of each cell to annihilate itself when it becomes unwanted or damaged. This takes place also when the control mechanisms of the cell cycle have been overridden during a simultaneous strong and/or long-lasting action of stress stimulus. Thus, among cells induced to enter premature, unauthorized division, there are also those that choose the apoptosis pathway (Sahu et al., 2009). Therefore premature mitoses/PCC are described as a mitotic catastrophe, abortive or suicidal. During the induction of apoptosis – following PCC induction – chromatin undergoes drastic changes: previously usually dispersed, it suddenly begins to condense into one or more aggregates in the vicinity of a nuclear membrane (Figure 2A). Changes connected with the initial phase of apoptosis also involve formation of intranuclear membranous structures (sometimes strongly developed and multi-layered) adhering to the nuclear envelope (Figure 2B-D).

Fig. 2. Ultrastructure of an apoptotic cell of *V. faba* root meristem (after PCC induction caused by 5 mM caffeine). Selected fragments of the above micro-photography (marked with arrows) at magnification. *Bar* 1μm

Among unicellular organisms, irregularities in the organization of cell cycle control systems result in a decreased reproduction potential, while among multicellular mechanisms they

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#### **6. Conclusion**

What are the practical implications and prospects of PCC induction? Could PCC induction serve as a novel anti-cancer approach? Undoubtedly, the phenomenon of premature mitosis is an essential characteristic of cell biology, therefore an important issue in respect of potential medical applications. It is so because, according to many researchers, the chemotherapy commonly used for the treatment of malignancy leads to extensive DNA damage, whereas PCC induction (resulting from the stimulation of biochemical mechanisms overriding the action of S-M checkpoint) can intensify therapeutic effects. Recently, druginduced PCC was optimized to assist analysis of the behavior of cancer cells with minimal side effects. However, PCC will also contribute to the understanding of normal cellular processes.
