DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells

Comment on: Wu R, et al. Clin Cancer Res 2011; 17:7359–72     

Mitotic catastrophe and endomitosis in tumour cells: an evolutionary key to a molecular solution

Following genotoxic insult, p53 mutated tumour cells undergo mitotic catastrophe. This is characterised by a switch from mitosis to the endocycle. The essential difference between mitosis and the endocycle is that in the latter, DNA synthesis is uncoupled from cell division, which leads to the formation of endopolyploid cells. Recent data suggests that a return from the endocycle into mitosis is also possible. Furthermore, our observations indicate that a particular type of endocycle known as endomitosis may be involved in this return. Here we review the role of endomitosis in the somatic reduction of polyploidy during development and its postulated role in the evolution of meiosis. Finally, we incorporate these evolutionary data to help interpret our most recent observations in the tumour cell system, which indicate a role for endomitosis and meiotic regulators, in particular p39mos in the segregation of genomes (somatic reduction) of these endopolyploid cells.     

Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.     

Nuclear envelope-limited chromatin sheets are part of mitotic death

Nuclear envelope-limited chromatin sheets (ELCS) are enigmatic membranous structures of uncertain function. This study describes the induction of ELCS in p53 mutated Burkitt's lymphoma cell lines after treatment with irradiation or the microtubule inhibitor, SK&F 96365. Both treatments evoked similar mitotic death, involving metaphase arrest followed by extensive endopolyploidisation and delayed apoptosis, although the kinetics were different. We found that induction of ELCS and nuclear segmentation correlated with the amount and kinetics of M-phase arrest, mitosis restitution and delayed apoptosis of endopolyploid cells. In metaphases undergoing restitution, ELCS are seen participating in the restoration of the nuclear envelope, mediating the attachment of peripheral chromatin to it. In interphase cells, ELCS join nuclear segments, ectopically linking and fusing with heterochromatin regions. In cells with segmented nuclei, continued DNA replication was observed, along with activation and redistribution of Ku70, suggestive of non-homologous DNA end-joining. Induction of ELCS also parallels the induction of cytoplasmic stacked membrane structures, such as confronting cisternae and annulate lamellae, which participate in the turnover and degeneration of ELCS. The results suggest that arrest at a spindle checkpoint and the uncoupling of mitosis from DNA replication lead to the emergence of ELCS in the resulting endopolyploid cells.     

Aberrant death in dark chondrocytes of the avian growth plate

Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion.     

Differentiation and programmed cell death in SA-45 tumour treated with bone inducer.

The relationship between differentiation and concerted cell death was studied using ultrastructural, histochemical and immunochemical methods in solid rat fibrosarcoma Sa-45 grown in the presence of demineralized bone matrix. The control tumour consisted mostly of undifferentiated cells with few poorly or moderately differentiated cells. In the presence of the inducer, cells with a more differentiated pattern appeared in the surrounding area. The proliferative activity in the presence of the inducer was 3 to 5 times lower but the apoptotic index was higher than in the controls. However, complete differentiation was induced only in stromal cells, whereas the parenchymal cells showed signs of enhanced but incomplete differentiation. The ultrastructural signs of programmed cell death progressed in them faster than the corresponding features of maturation, thus leaving differentiation incomplete.     

Emergent Self-Organized Criticality in Gene Expression Dynamics: Temporal Development of Global Phase Transition Revealed in a Cancer Cell Line

Background

The underlying mechanism of dynamic control of the genome-wide expression is a fundamental issue in bioscience. We addressed it in terms of phase transition by a systemic approach based on both density analysis and characteristics of temporal fluctuation for the time-course mRNA expression in differentiating MCF-7 breast cancer cells.

Methodology

In a recent work, we suggested criticality as an essential aspect of dynamic control of genome-wide gene expression. Criticality was evident by a unimodal-bimodal transition through flattened unimodal expression profile. The flatness on the transition suggests the existence of a critical transition at which up- and down-regulated expression is balanced. Mean field (averaging) behavior of mRNAs based on the temporal expression changes reveals a sandpile type of transition in the flattened profile. Furthermore, around the transition, a self-similar unimodal-bimodal transition of the whole expression occurs in the density profile of an ensemble of mRNA expression. These singular and scaling behaviors identify the transition as the expression phase transition driven by self-organized criticality (SOC).

Principal Findings

Emergent properties of SOC through a mean field approach are revealed: i) SOC, as a form of genomic phase transition, consolidates distinct critical states of expression, ii) Coupling of coherent stochastic oscillations between critical states on different time-scales gives rise to SOC, and iii) Specific gene clusters (barcode genes) ranging in size from kbp to Mbp reveal similar SOC to genome-wide mRNA expression and ON-OFF synchronization to critical states. This suggests that the cooperative gene regulation of topological genome sub-units is mediated by the coherent phase transitions of megadomain-scaled conformations between compact and swollen chromatin states.

Conclusion and Significance

In summary, our study provides not only a systemic method to demonstrate SOC in whole-genome expression, but also introduces novel, physically grounded concepts for a breakthrough in the study of biological regulation.