The cell cycle revisited: DNA replication past S phase preserves genome integrity.

Accurate and complete DNA duplication is critical for maintaining genome integrity. Multiple mechanisms regulate when and where DNA replication takes place, to ensure that the entire genome is duplicated once and only once per cell cycle. Although the bulk of the genome is copied during the S phase of the cell cycle, increasing evidence suggests that parts of the genome are replicated in G2 or mitosis, in a last attempt to secure that daughter cells inherit an accurate copy of parental DNA. Remaining unreplicated gaps may be passed down to progeny and replicated in the next G1 or S phase. These findings challenge the long-established view that genome duplication occurs strictly during the S phase, bridging DNA replication to DNA repair and providing novel therapeutic strategies for cancer treatment.

Alterations in the spatiotemporal expression pattern of geminin during human epidermal morphogenesis.

INTRODUCTION: Geminin, a (25 kDa) protein, was originally identified as a key regulator of DNA replication licensing in the cell cycle and of cell fate during embryonic nervous system formation. Although geminin is involved in mechanisms underlying the regulation of transcription and patterning in embryonic development, its expression and possible significance in human epidermal morphogenesis remains unknown. METHODS: Forty-one skin biopsy specimens obtained from human fetuses (10th to 23rd week of estimated gestational age) were processed for immunohistochemistry using a primary rabbit polyclonal antibody against geminin. RESULTS: Distinct and statistically significant qualitative and quantitative alterations in the spatiotemporal expression pattern of geminin were observed in the developing human epidermis. CONCLUSIONS: The highly ordered expression of geminin in different layers of fetal human epidermis reported here for the first time suggests that this protein may play a significant role in epidermal morphogenesis. However, the mechanisms underlying the alterations of the geminin expression pattern during fetal development at the molecular level remain to be elucidated. Further studies are now warranted to address whether the expression pattern of geminin in the developing human epidermis is disturbed in fetuses with genodermatoses and whether these disturbances might be important for prenatal diagnosis of genodermatoses.

Ras suppressor-1 (RSU1) exerts a tumor suppressive role with prognostic significance in lung adenocarcinoma.

Ras suppressor-1 (RSU1), originally described as a suppressor of Ras oncogenic transformation, localizes to focal adhesions interacting with the ILK-PINCH-PARVIN (IPP) complex that exerts a well-established oncogenic role in cancer. However, RSU1 implication in lung cancer is currently unknown. Our study aims to address the role of RSU1 in lung adenocarcinoma (LUADC). We here show that RSU1 protein expression by immunohistochemistry is downregulated in LUADC human tissue samples and represents a significant prognostic indicator. In silico analysis of gene chip and RNA seq data validated our findings. Depletion of RSU1 by siRNA in lung cancer cells promotes anchorage-independent cell growth, cell motility and epithelial to mesenchymal transition (EMT). Silencing of RSU1 also alters IPP complex expression in lung cancer cells. The p29 RSU1 truncated isoform is detected in lung cancer cells, and its expression is downregulated upon RSU1 silencing, whereas it is overexpressed upon ILK overexpression. These findings suggest that RSU1 exerts a tumor suppressive role with prognostic significance in LUADC.

Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

Metabolic regulation of the neural stem cell fate: Unraveling new connections, establishing new concepts.

The neural stem cell niche is a key regulator participating in the maintenance, regeneration, and repair of the brain. Within the niche neural stem cells (NSC) generate new neurons throughout life, which is important for tissue homeostasis and brain function. NSCs are regulated by intrinsic and extrinsic factors with cellular metabolism being lately recognized as one of the most important ones, with evidence suggesting that it may serve as a common signal integrator to ensure mammalian brain homeostasis. The aim of this review is to summarize recent insights into how metabolism affects NSC fate decisions in adult neural stem cell niches, with occasional referencing of embryonic neural stem cells when it is deemed necessary. Specifically, we will highlight the implication of mitochondria as crucial regulators of NSC fate decisions and the relationship between metabolism and ependymal cells. The link between primary cilia dysfunction in the region of hypothalamus and metabolic diseases will be examined as well. Lastly, the involvement of metabolic pathways in ependymal cell ciliogenesis and physiology regulation will be discussed.

Focal adhesion proteins in hepatocellular carcinoma: RSU1 a novel tumour suppressor with prognostic significance.

AIM: Hepatocellular carcinoma (HCC) is a common cause a cancer-related death. Focal adhesions (FAs) represent multiprotein complexes at integrin-mediated cell-extracellular matrix adhesion sites that orchestrate vital cellular functions. The heterotrimeric ILK-PINCH-PARVB (IPP) complex, RSU1, a PINCH binding protein and CTEN, a member of the tensin family of proteins exert a critical role in FAs, where they regulate important cancer related functions such as cell adhesion, migration, proliferation and survival. Previous studies implicate these FA proteins in liver pathophysiology but their detailed role in human HCC is not fully understood. Here in we investigated expression and function of IPP, RSU1 and CTEN in human HCC. METHODS: The expression of focal adhesion proteins was studied in human HCC by immunohistochemistry in relation to clinicopathological parameters, previous studied genomic instability markers and patient's survival. Effects on cell proliferation and FA proteins expression upon ILK inhibition and RSU1 silencing were also investigated in HCC in vitro. RESULTS: IPP complex and CTEN proteins are overexpressed while RSU1 expression is decreased in human HCC. CTEN expression correlates with reduced patients' survival while RSU1 represents an independent favorable prognostic indicator in human HCC. Nuclear ILK expression correlates with markers of genomic instability. Pharmacological targeting of ILK suppresses, while RSU1 silencing promotes cell growth of HCC cells in vitro, while in both experimental conditions expression and/or localization of focal adhesion proteins is deregulated. CONCLUSION: Our results suggest that FA signaling is implicated in hepatocellular carcinogenesis with prognostic significance. RSU1 seems to exert tumor suppressive functions in HCC and represents a novel favorable prognostic indicator.

53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling.

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double-strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.

Intrinsic neural stem cell properties define brain hypersensitivity to genotoxic stress.

Impaired replication has been previously linked to growth retardation and microcephaly; however, why the brain is critically affected compared with other organs remains elusive. Here, we report the differential response between early neural progenitors (neuroepithelial cells [NECs]) and fate-committed neural progenitors (NPs) to replication licensing defects. Our results show that, while NPs can tolerate altered expression of licensing factors, NECs undergo excessive replication stress, identified by impaired replication, increased DNA damage, and defective cell-cycle progression, leading eventually to NEC attrition and microcephaly. NECs that possess a short G1 phase license and activate more origins than NPs, by acquiring higher levels of DNA-bound MCMs. In vivo G1 shortening in NPs induces DNA damage upon impaired licensing, suggesting that G1 length correlates with replication stress hypersensitivity. Our findings propose that NECs possess distinct cell-cycle characteristics to ensure fast proliferation, although these inherent features render them susceptible to genotoxic stress.

Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.

Fanconi anemia proteins and genome fragility: unraveling replication defects for cancer therapy.

Accurate and complete genome duplication is crucial to maintain cell survival and prevent malignant transformation. The Fanconi anemia (FA) pathway has traditionally been associated with the repair of DNA interstrand crosslinks that impede the progression of the replication machinery. Recent studies demonstrate that FA proteins also regulate cell-cycle checkpoints and/or promote replication fork remodeling in response to multiple DNA impediments, and redefine the FA pathway as a fundamental mechanism to preserve genome integrity upon different insults. Alterations in FA genes fuel genomic fragility and constitute a driving force of tumorigenesis. We highlight current understanding of FA signaling in safeguarding genome stability during replication, and discuss the identification of novel determinants of cancer cell survival in FA-deficient tumors.

Ribosomal DNA and the nucleolus at the heart of aging.

The rRNA genes [ribosomal DNA (rDNA)] are organized in a prominent nuclear compartment, the nucleolus. It is now well established that the nucleolus functions beyond ribosome biosynthesis, regulating several physiological cellular responses. The nucleoli constitute dynamic genomic/nuclear hubs and demonstrate unique inherent characteristics, rendering them ideal to sense, signal, and respond to various intrinsic and environmental insults. Here, we discuss emerging findings supporting direct links between rDNA/nucleolar instability and cellular senescence/organismal aging from yeast to mammals. Moreover, we highlight evidence that nucleolar functionality and rDNA architecture impact on meiotic/transgenerational rejuvenation, thus revealing causality underlying connections between rDNA/nucleolar instability and aging.

3D Reconstitution of the Neural Stem Cell Niche: Connecting the Dots.

Neural stem cells (NSCs) are important constituents of the nervous system, and they become constrained in two specific regions during adulthood: the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. The SVZ niche is a limited-space zone where NSCs are situated and comprised of growth factors and extracellular matrix (ECM) components that shape the microenvironment of the niche. The interaction between ECM components and NSCs regulates the equilibrium between self-renewal and differentiation. To comprehend the niche physiology and how it controls NSC behavior, it is fundamental to develop in vitro models that resemble adequately the physiologic conditions present in the neural stem cell niche. These models can be developed from a variety of biomaterials, along with different biofabrication approaches that permit the organization of neural cells into tissue-like structures. This review intends to update the most recent information regarding the SVZ niche physiology and the diverse biofabrication approaches that have been used to develop suitable microenvironments ex vivo that mimic the NSC niche physiology.

Fine-tuning multiciliated cell differentiation at the post-transcriptional level: contribution of miR-34/449 family members.

Cell differentiation is a process that must be precisely regulated for the maintenance of tissue homeostasis. Differentiation towards a multiciliated cell fate is characterized by well-defined stages, where a transcriptional cascade is activated leading to the formation of multiple centrioles and cilia. Centrioles migrate and dock to the apical cell surface and, acting as basal bodies, give rise to multiple motile cilia. The concerted movement of cilia ensures directional fluid flow across epithelia and defects either in their number or structure can lead to disease phenotypes. Micro-RNAs (miRNAs; miRs) are small, non-coding RNA molecules that play an important role in post-transcriptional regulation of gene expression. miR-34b/c and miR-449a/b/c specifically function throughout the differentiation of multiciliated cells, fine-tuning the expression of many different centriole- and cilia-related genes. They strictly regulate the expression levels of genes that are required both for commitment towards the multiciliated cell fate (e.g. Notch) and for the establishment and maintenance of this fate by regulating the expression of transcription factors and structural components of the pathway. Herein we review miR-34 and miR-449 spatiotemporal regulation along with their roles during the different stages of multiciliogenesis.

Ex vivo analysis of DNA repair targeting in extreme rare cutaneous apocrine sweat gland carcinoma.

Cutaneous apocrine carcinoma is an extreme rare malignancy derived from a sweat gland. Histologically sweat gland cancers resemble metastatic mammary apocrine carcinomas, but the genetic landscape remains poorly understood. Here, we report a rare metastatic case with a PALB2 aberration identified previously as a familial susceptibility gene for breast cancer in the Finnish population. As PALB2 exhibits functions in the BRCA1/2-RAD51-dependent homologous DNA recombination repair pathway, we sought to use ex vivo functional screening to explore sensitivity of the tumor cells to therapeutic targeting of DNA repair. Drug screening suggested sensitivity of the PALB2 deficient cells to BET-bromodomain inhibition, and modest sensitivity to DNA-PKi, ATRi, WEE1i and PARPi. A phenotypic RNAi screen of 300 DNA repair genes was undertaken to assess DNA repair targeting in more detail. Core members of the HR and MMEJ pathways were identified to be essential for viability of the cells. RNAi inhibition of RAD52-dependent HR on the other hand potentiated the efficacy of a novel BETi ODM-207. Together these results describe the first ever CAC case with a BRCAness genetic background, evaluate combinatorial DNA repair targeting, and provide a data resource for further analyses of DNA repair targeting in PALB2 deficient cancers.

In silico analysis of DNA re-replication across a complete genome reveals cell-to-cell heterogeneity and genome plasticity.

DNA replication is a complex and remarkably robust process: despite its inherent uncertainty, manifested through stochastic replication timing at a single-cell level, multiple control mechanisms ensure its accurate and timely completion across a population. Disruptions in these mechanisms lead to DNA re-replication, closely connected to genomic instability and oncogenesis. Here, we present a stochastic hybrid model of DNA re-replication that accurately portrays the interplay between discrete dynamics, continuous dynamics and uncertainty. Using experimental data on the fission yeast genome, model simulations show how different regions respond to re-replication and permit insight into the key mechanisms affecting re-replication dynamics. Simulated and experimental population-level profiles exhibit a good correlation along the genome, robust to model parameters, validating our approach. At a single-cell level, copy numbers of individual loci are affected by intrinsic properties of each locus, in cis effects from adjoining loci and in trans effects from distant loci. In silico analysis and single-cell imaging reveal that cell-to-cell heterogeneity is inherent in re-replication and can lead to genome plasticity and a plethora of genotypic variations.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

BACKGROUND: Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. METHODS: Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. RESULTS: We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. CONCLUSIONS: The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.

Ιn vivo imaging of DNA-bound minichromosome maintenance complex in embryonic mouse cortex.

The recruitment of the minichromosome maintenance complex (MCM) on DNA replication origins is a critical process for faithful genome duplication termed licensing. Aberrant licensing has been associated with cancer and, recently, with neurodevelopmental diseases. Investigating MCM loading in complicated tissues, such as brain, remains challenging. Here, we describe an optimized approach for the qualitative and quantitative analysis of DNA-bound MCMs in the developing mouse cortex through direct imaging, offering an innovative insight into the research of origin licensing in vivo.

A Custom Ultra-Low-Cost 3D Bioprinter Supports Cell Growth and Differentiation.

Advances in 3D bioprinting have allowed the use of stem cells along with biomaterials and growth factors toward novel tissue engineering approaches. However, the cost of these systems along with their consumables is currently extremely high, limiting their applicability. To address this, we converted a 3D printer into an open source 3D bioprinter and produced a customized bioink based on accessible alginate/gelatin precursors, leading to a cost-effective solution. The bioprinter's resolution, including line width, spreading ratio and extrusion uniformity measurements, along with the rheological properties of the bioinks were analyzed, revealing high bioprinting accuracy within the printability window. Following the bioprinting process, cell survival and proliferation were validated on HeLa Kyoto and HEK293T cell lines. In addition, we isolated and 3D bioprinted postnatal neural stem cell progenitors derived from the mouse subventricular zone as well as mesenchymal stem cells derived from mouse bone marrow. Our results suggest that our low-cost 3D bioprinter can support cell proliferation and differentiation of two different types of primary stem cell populations, indicating that it can be used as a reliable tool for developing efficient research models for stem cell research and tissue engineering.

Chromatin and Nuclear Architecture: Shaping DNA Replication in 3D.

In eukaryotes, DNA replication progresses through a finely orchestrated temporal and spatial program. The 3D genome structure and nuclear architecture have recently emerged as fundamental determinants of the replication program. Factors with established roles in replication have been recognized as genome organization regulators. Exploiting paradigms from yeasts and mammals, we discuss how DNA replication is regulated in time and space through DNA-associated trans-acting factors, diffusible limiting replication initiation factors, higher-order chromatin folding, dynamic origin localization, and specific nuclear microenvironments. We present an integrated model for the regulation of DNA replication in 3D and highlight the importance of accurate spatio-temporal regulation of DNA replication in physiology and disease.