Hepatocyte Polyploidy: Driver or Gatekeeper of Chronic Liver Diseases

Polyploidy, also known as whole-genome amplification, is a condition in which the organism has more than two basic sets of chromosomes. Polyploidy frequently arises during tissue development and repair, and in age-associated diseases, such as cancer. Its consequences are diverse and clearly different between systems. The liver is a particularly fascinating organ in that it can adapt its ploidy to the physiological and pathological context. Polyploid hepatocytes are characterized in terms of the number of nuclei per cell (cellular ploidy; mononucleate/binucleate hepatocytes) and the number of chromosome sets in each nucleus (nuclear ploidy; diploid, tetraploid, octoploid). The advantages and disadvantages of polyploidy in mammals are not fully understood. About 30% of the hepatocytes in the human liver are polyploid. In this review, we explore the mechanisms underlying the development of polyploid cells, our current understanding of the regulation of polyploidization during development and pathophysiology and its consequences for liver function. We will also provide data shedding light on the ways in which polyploid hepatocytes cope with centrosome amplification. Finally, we discuss recent discoveries highlighting the possible roles of liver polyploidy in protecting against tumor formation, or, conversely, contributing to liver tumorigenesis.

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

Polyploid cardiomyocytes: implications for heart regeneration

ABSTRACT Terminally differentiated cells are generally thought to have arrived at their final form and function. Many terminally differentiated cell types are polyploid, i.e. they have multiple copies of the normally diploid genome. Mammalian heart muscle cells, termed cardiomyocytes, are one such example of polyploid cells. Terminally differentiated cardiomyocytes are bi- or multi-nucleated, or have polyploid nuclei. Recent mechanistic studies of polyploid cardiomyocytes indicate that they can limit cellular proliferation and, hence, heart regeneration. In this short Spotlight, we present the mechanisms generating bi- and multi-nucleated cardiomyocytes, and the mechanisms generating polyploid nuclei. Our aim is to develop hypotheses about how these mechanisms might relate to cardiomyocyte proliferation and cardiac regeneration. We also discuss how these new findings could be applied to advance cardiac regeneration research, and how they relate to studies of other polyploid cells, such as cancer cells.

INFLUENCE OF COPPER COMPLEXES [CU(PTA)4 [BF4 ] AND CU(II)2 (3,5-DIPS)4 (H2 O)3 ON THE ORGANISM OF RATS IRRADIATED WITH RADIOISOTOPE TECHNETIUM

One of the priority tasks of modern radiobiology is the search for new, effective radioprotective compounds. In this area, metal-organic complexes with high antioxidant activity are of particular interest. In order to study the possible beneficial radioprotective effect of copper complex compounds [Cu(PTA)4 [BF4 ] and Cu(II)2 (3,5-DIPS)4 (H2 O)3 on an irradiated organism, we studied cytogenetic parameters in 4 groups of experimental animals: intact animals, animals exposed to the radioisotope technetium (Tc) — "pure irradiation", animals with "irradiation + compound [Cu(PTA)4 [BF4 ]", and animals with "irradiation + compound [Cu(II)2 ( 3,5-DIPS)4 (H2 O)3 ". The survival rate and cytogenetic parameters were studied: mitotic index (MI), chromosomal aberrations (CA) and polyploid cells (PC) in the bone marrow cells of the femur. The survival rate in the first and third groups was 100%, in the second group — 40%, and in the fourth — 80%. The dynamics of survival was described by regression curves and equations, which make it possible, using extrapolation, to determine the change in the percentage of survival in the long term of the experiment and to predict the further outcome of the experiment. When analyzing the results in 4 groups of animals, we found a significant difference in cytogenetic parameters between these groups. Thus, for all 3 indicators, a significant difference is observed between intact and irradiated animals, i.e. these parameters can be considered as markers of Tc exposure. In terms of the mitotic index (proliferative activity), a significant difference was found in the irradiated compared with the groups: "irradiation + [Cu(PTA)4 [BF4 ]" and "irradiation + Cu(II)2 (3,5-DIPS)4 (H2 O)3 ", which indicates the radioprotective property of both compounds. By the number of polyploid cells, a significant difference was found between the groups: "pure irradiation" and "irradiation + Cu(II)2 (3,5-DIPS)4 (H2 O)3 ", which also proves the beneficial effect of this compound. Multiregression analysis of cytogenetic parameters along with standard statistical methods confirmed the highest efficiency of [Cu(PTA)4 [BF4] relative to Cu(II)2 (3,5-DIPS)4 (H2 O)3 .

Evaluation of mitotic activity in tapetal cells of grapevine (Vitis L.)

The knowledge with reference to the grapevine tapetum has been centered on its anatomy/morphology and hardly anything at all is known about its mitotic activity throughout the microsporogenesis. The aim of this study was to ascertain the mitotic activity in tapetal cells of some grapevines (Vitis L.) broadening knowledge about this tissue and simultaneously corroborating the viability of its use as an alternative tissue for further cytogenetic studies. Young buds of 12 grapevine varieties at different meiotic stages were squashed and tapetal cells a prometaphase/metaphase scored in each meiotic stage. Mitotic activity was observed since the beginning of microsporogenesis, where it was more intense, decreasing toward tetrad. Polyploid tapetal cells arose through endomitosis while the microsporogenesis advanced. Two types of polyploid cells were evidenced, those with two or more individualized diploid chromosome groups and those with only one polyploid group. The percentage of diploid cells and of polyploid cells with two or more individualized diploid groups was higher during the first stage of microsporogenesis, though decreasing and giving way to cells with one large polyploid group as microsporogenesis moved toward tetrad. The nucleolus number was scored at interphase at different stages. Two and four nucleoli prevailed in tapetal cells at all stages except at tetrad where one large nucleolus was seen. The results showed that despite of the squashing technique applied, grapevine tapetum has a substantial amount of cells with mitotic activity with a satisfactory chromosome spreading therefore establishing an interesting alternative and promising tissue for later cytomolecular studies.

Polyploid Giant Cancer cells and ovarian Cancer: New insights into mitotic regulators and polyploidy

Summary sentence: Cytotoxic drugs frequently used in ovarian cancer treatment impact tumor cell cycle progression and the emergence of polyploid cells where genetic and epigenetic events ultimately lead to drug resistant diploid progeny.

Chromosomal instability associated with pathogenesis in adenomyosis.

e22517 Background: Uterine adenomyosis is a benign condition in which endometrium-like epithelial and stromal tissue appear in the myometrium, but easily concurrent with uterine cancer and other diseases. To date, few investigations have explored the molecular mechanism of adenomyosis. This research is the first report that found the association between chromosomal instability and pathogenesis in adenomyosis. Methods: Whole exome (̃39Mb CDS of over 18,000 genes; WES) and RNA sequencing were conducted on a total of 57 adenomyosis patients enrolled in this study from 2018 to 2020, including 57 normal endometrium (EN), 57 adenomyosis (AM) and 11 endometrioses (EMT) samples. Genomic test results including somatic SNVs and CNVs as well as the whole genome duplication status were determined, which were all analyzed correlated with clinical factors. Results: We identified the presence of whole genome doubling event in 19 specimens from 16 adenomyosis patients (grouped as WGD, the other 41 patients grouped as noWGD), related to 7 normal endometrium (EN), 9 adenomyosis (AM) and 3 endometriosis (EMT) samples, which was significant with p value of 4.5e-12 by means of Wilcoxon rank sum test. Disease Free Survival (DFS) of WGD patients was significantly shorter than noWGD patients (Wilcoxon rank sum test: P = 2.42e-04), which indicated that WGD group has early on-set in adenomyosis. Estrogen therapy was applied to 31 patients before uterectomy (11 in WGD group and 20 in noWGD group), and the effectiveness of the treatment was significantly higher in WGD patients (Fisher’s exact test: P = 0.02). We found that one third adenomyosis performed whole genome doubling events, which leaded to the early on-set of this disease and significantly sensitive to estrogen therapy. Cellular stressors are extensive correlated with the generation of polyploid cells in a diploid organism form by cell proliferation progress. Compared with the normal and energy-intensive cell differentiation process, cell proliferation offers a “fast but cheap” solution under stress. Our results suggested that when cell differentiation in uterine could not be carried out properly, cell proliferation would have performed instead and produced more polyploid cells, shown as whole genome doubling events. While this circumstance would have been relieved when easing the metabolic stress with estrogen treatment. Conclusions: Our results explored the strong association between chromosomal instability and pathogenesis of adenomyosis and assumed that polyploidy caused by abnormal cell proliferation might be the molecular mechanism of uterine adenomyosis. More accurate verdict could be concluded through larger scale cohort study.

FC 041TUBULAR EPITHELIAL CELL POLYPLOIDY IS ESSENTIAL TO SURVIVE AKI BUT IT CONTRIBUTES TO CKD PROGRESSION

Background and Aims Acute Kidney Injury (AKI) is a syndrome characterized by an acute deterioration of renal function. Due to its high prevalence and poor short-term outcomes, AKI represents a global healthcare issue. Many epidemiologic studies have indicated that the development of Chronic Kidney Disease (CKD) features prominently among the numerous long-term complications of AKI. The pathophysiological basis for this phenomenon has remained unclear so far. Recently, we found that tubular epithelial cells (TEC) undergo endoreplication-mediated hypertrophy after AKI. Endoreplications are incomplete cell cycles that lead to the formation of polyploid cells. Physiologically, polyploidy offers several advantages such as rapid adaptation to stress, compensation for cell loss and enhanced cell function. However, as renal epithelial cells are massively lost after AKI, TEC polyploidy may constitute an effective strategy to sustain a temporary functional recovery of the kidney without restoring tissue integrity potentially leading to CKD. Therefore, we hypothesized that: 1) polyploid TEC are an adaptive stress response required to maintain kidney function after AKI; 2) polyploid TEC are involved in the AKI to CKD progression. Method To address these hypotheses, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology to monitor cell cycle phasing in combination with YAP1 overexpression or downregulation. In the in vivo models, YAP1 overexpressing mice and YAP1 knock-out mice were subjected to unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis to induce AKI. Polyploid cells have been then characterized by single cell-RNA sequencing analysis, cell sorting, super-resolution STED microscopy and transmission electron microscopy in both mouse and human. Results In vitro, human renal tubular cells undergo polyploidization. The fraction of polyploid cells significantly decreases when YAP1 nuclear translocation is blocked, indicating a possible involvement of YAP1 in regulating TEC polyploidy. After AKI in mice, YAP1 expression and nuclear translocation are significantly enhanced. The inhibition of YAP1 following AKI, reduces the number of polyploid cells impairing kidney function and causing a dramatic reduction of mouse survival. In contrast, YAP1 overexpression leads to an increase in the number of polyploid cells even in the absence of kidney damage (healthy mice). Strikingly, these healthy mice, despite having an increased percentage of polyploid cells, present an unexpected decline of renal function suggesting an association between increased polyploidy and CKD development. Indeed, they develop tubulointerstitial fibrosis acquiring a marked senescent phenotype triggering CKD. Isolation of polyploid cells proved that these cells actively transcribe and secrete pro-fibrotic factors thus confirming their role in CKD progression. Conclusion Collectively, these data suggest that: 1) polyploidization after AKI is required to maintain kidney function allowing survival; 2) polyploid cells are pro-fibrotic leading in the long run to CKD progression.

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

Regulation of DNA replication and copy number are necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rif1 is key regulator of RT and has a critical function in copy number control in polyploid cells. In a previous study (Munden et al., 2018), we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 functions both upstream and downstream of SUUR to promote UR. Our work provides new mechanistic insight into the process of UR and its links to RT.

Fifty generations of amitosis: tracing asymmetric allele segregation in polyploid cells with single-cell DNA sequencing

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis are acquired by assessing the rate of phenotypic assortment. The phenotypic diversification of heterozygous clones during successive cell divisions reveals the random segregation of alleles to daughter nuclei. Though powerful, this experimental approach relies on the availability of phenotypic markers. Here, we present an approach that overcomes the requirement for phenotypic assortment. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dimorphism and a highly polyploid somatic nucleus, we use single-cell whole-genome sequencing to track the assortment of developmentally acquired somatic DNA variants. Accounting for genome representation biases, we measure the effect of amitosis on allele segregation across the first ~50 amitotic divisions post self-fertilization and compare our empirical findings with theoretical predictions estimated via mathematical modeling. In line with our simulations, we show that amitosis in P. tetraurelia produces measurable but modest levels of somatic assortment. In forgoing the requirement for phenotypic assortment and employing developmental, environmentally induced somatic variation as molecular markers, our work provides a new powerful approach to investigate the consequences of amitosis in polyploid cells.