Search Results (19)

The mechanistic target of the Rapamycin complex 1 (mTORC1) signaling pathway plays a pivotal role in regulating crucial life processes, including cell growth and proliferation, by sensing and integrating various signals, such as growth factors, energy status, and amino acids. Our previous studies showed that activation of the mTORC1 signaling pathway enhances silk protein synthesis and silk gland size. Here, the potential of the molecular mechanism mTORC1 to regulate the growth and development of silk gland cells was investigated. Inhibiting mTORC1 with rapamycin decreased proliferation in the Bombyx mori embryonic (BmE) cells and endoreplication in silk gland cells, reducing CyclinB and CyclinE protein levels and DNA content, and arresting the BmE cell cycle at G2/M. Conversely, the overexpression of Ras homolog enriched in brain (Rheb) led to increased proliferation of BmE cells and endoreplication in silk gland cells, as well as a significant elevation in DNA content. This study provides a molecular explanation for the increase in silk protein synthesis and silk gland length through the activation of mTORC1, thereby refining the regulatory network of the silkworm endoreplication and providing new molecular targets for breeding high-yield varieties of Bombyx mori. Full article

Environmental concerns regarding synthetic herbicides have sparked interest in plant-derived bioactive compounds as eco-friendly alternatives. This study investigated the cellular targets of sweet flag essential oil (Acorus calamus L., SEO at IC₅₀ concentration) in root meristem cells of Fabaceae (Vicia faba, Lupinus luteus) and Brassicaceae (Brassica napus, Arabidopsis thaliana), focusing on reactive oxygen species (ROS) accumulation (DAB, NBT staining), DNA replication dynamics (EdU labeling), and genome integrity (γ-H2AX immunocytochemistry, TUNEL assay, and DNA electrophoresis). SEO induced oxidative stress (200–250% of control depending on the species) and replication stress, causing DNA double-strand breaks in 50% of proliferating cells, confirmed by γ-H2AX/TUNEL. Consequently, cells were prolonged in the G₁ phase, replication activity dropped to 70% of control in Fabaceae and 80% in Brassicaceae, and EdU incorporation intensity decreased to 80% and 70% of control, respectively. An increased proportion of cells replicating heterochromatin indicated slowed S-phase progression. Despite genotoxic effects, SEO did not trigger endoreplication, apoptotic DNA fragmentation, or extensive cell death. All species exhibited a uniform stress response, although sensitivity varied, which previously enabled the establishment of selective SEO doses between Fabaceae and Brassicaceae. These findings suggest that SEO exerts phytotoxicity by disrupting S-phase progression, supporting its potential as a selective bioherbicide. Full article

Caffeine is a nitrogenous base that naturally occurs in coffee (Cafea arabica), tea (Thea sinensis), and cocoa (Theobroma cacao). Chemically, caffeine is 1,3,5-trimethylxanthine, a purine analogue. Due to significant human consumption, caffeine effects have been widely studied. Being a natural xanthine derivative, the key degradative enzyme is xanthine oxidase, converting caffeine into 1-methyluric acid. Ecologically, caffeine is believed to act as a repellent molecule against insect feeding behavior. Caffeine’s chemical similarity to purines and plant hormones motivated this study, establishing the potential for cellular de-differentiation and re-differentiation. For this, a highly hormone-responsive plant species, Ornithogalum dubium, was used. As caffeine has been shown to induce endoreplication, the potential for new germlines in O. dubium is attractive. Using tissue culture, a range of caffeine concentrations were used (0.0125 mg/L to 2.0 mg/L) without additional hormones. A significant difference (p > 0.05) was observed for intermediate concentrations of 0.0125, 0.025, and 0.05 mg/L when compared to the control (no hormones). The highest rates of callus induction were obtained at a concentration of 0.025 mg/mL. Higher concentrations were phytotoxic (1.0 mg/L or greater). To conclude, caffeine-regenerated plants were not dissimilar to those obtained from canonical hormones. Full article

As a plant-specific endoreplication regulator, the SIAMESE-RELATED (SMR) family (a cyclin-dependent kinase inhibitor) plays an important role in plant growth and development and resistance to stress. Although the genes of the maize (Zea mays) SMR family have been studied extensively, the ZmSMR10 (Zm00001eb231280) gene has not been reported. In this study, the function of this gene was characterized by overexpression and silencing. Compared with the control, the transgenic plants exhibited the phenotypes of early maturation, dwarfing, and drought resistance. Expression of the protein in prokaryotes demonstrates that ZmSMR10 is a small protein, and the results of subcellular localization suggest that it travels functionally in the nucleus. Unlike ZmSMR4, yeast two-hybrid experiments demonstrated that ZmSMR10 does not interact strongly with with some cell cycle protein-dependent protein kinase (CDK) family members ZmCDKA;1/ZmCDKA;3/ZmCDKB1;1. Instead, it interacts strongly with ZmPCNA2 and ZmCSN5B. Based on these results, we concluded that ZmSMR10 is involved in the regulation of endoreplication through the interaction of ZmPCNA2 and ZmCSN5B. These findings provide a theoretical basis to understand the mechanism of the regulation of endoreplication and improve the yield of maize through the use of molecular techniques. Full article

Endoreplication—a process that is common in plants and also accompanies changes in the development of animal organisms—has been seen from a new perspective in recent years. In the paper, we not only shed light on this view, but we would also like to promote an understanding of the application potential of this phenomenon in plant cultivation. Endoreplication is a pathway for cell development, slightly different from the classical somatic cell cycle, which ends with mitosis. Since many rounds of DNA synthesis take place within its course, endoreplication is a kind of evolutionary compensation for the relatively small amount of genetic material that plants possess. It allows for its multiplication and active use through transcription and translation. The presence of endoreplication in plants has many positive consequences. In this case, repeatedly produced copies of genes, through the corresponding transcripts, help the plant acquire the favorable properties for which proteins are responsible directly or indirectly. These include features that are desirable in terms of cultivation and marketing: a greater saturation of fruit and flower colors, a stronger aroma, a sweeter fruit taste, an accumulation of nutrients, an increased resistance to biotic and abiotic stress, superior tolerance to adverse environmental conditions, and faster organ growth (and consequently the faster growth of the whole plant and its biomass). The two last features are related to the nuclear-cytoplasmic ratio—the greater the content of DNA in the nucleus, the higher the volume of cytoplasm, and thus the larger the cell size. Endoreplication not only allows cells to reach larger sizes but also to save the materials used to build organelles, which are then passed on to daughter cells after division, thus ending the classic cell cycle. However, the content of genetic material in the cell nucleus determines the number of corresponding organelles. The article also draws attention to the potential practical applications of the phenomenon and the factors currently limiting its use. Full article

Locus-specific gene amplification and genome-wide endoreplication generate the elevated copy number of ribosomal DNA (rDNA, 9000 C) and non-rDNA (90 C) chromosomes in the developing macronucleus of Tetrahymena thermophila. Subsequently, all macronuclear chromosomes replicate once per cell cycle during vegetative growth. Here, we describe an unanticipated, programmed switch in the regulation of replication initiation in the rDNA minichromosome. Early in development, the 21 kb rDNA minichromosome is preferentially amplified from 2 C to ~800 C from well-defined origins, concurrent with genome-wide endoreplication (2 C to 8–16 C) in starved mating Tetrahymena (endoreplication (ER) Phase 1). Upon refeeding, rDNA and non-rDNA chromosomes achieve their final copy number through resumption of just the endoreplication program (ER Phase 2). Unconventional rDNA replication intermediates are generated primarily during ER phase 2, consistent with delocalized replication initiation and possible formation of persistent RNA-DNA hybrids. Origin usage and replication fork elongation are affected in non-rDNA chromosomes as well. Despite the developmentally programmed 10-fold reduction in the ubiquitous eukaryotic initiator, the Origin Recognition Complex (ORC), active initiation sites are more closely spaced in ER phases 1 and 2 compared to vegetative growing cells. We propose that initiation site selection is relaxed in endoreplicating macronuclear chromosomes and may be less dependent on ORC. Full article

Ovarian cancer is the most lethal gynecologic malignancy in the United States. Some patients affected by ovarian cancers often present genome instability with one or more of the defects in DNA repair pathways, particularly in homologous recombination (HR), which is strictly linked to mutations in breast cancer susceptibility gene 1 (BRCA 1) or breast cancer susceptibility gene 2 (BRCA 2). The treatment of ovarian cancer remains a challenge, and the majority of patients with advanced-stage ovarian cancers experience relapse and require additional treatment despite initial therapy, including optimal cytoreductive surgery (CRS) and platinum-based chemotherapy. Targeted therapy at DNA repair genes has become a unique strategy to combat homologous recombination-deficient (HRD) cancers in recent years. Poly (ADP-ribose) polymerase (PARP), a family of proteins, plays an important role in DNA damage repair, genome stability, and apoptosis of cancer cells, especially in HRD cancers. PARP inhibitors (PARPi) have been reported to be highly effective and low-toxicity drugs that will tremendously benefit patients with HRD (i.e., BRCA 1/2 mutated) epithelial ovarian cancer (EOC) by blocking the DNA repair pathways and inducing apoptosis of cancer cells. PARP inhibitors compete with NAD⁺ at the catalytic domain (CAT) of PARP to block PARP catalytic activity and the formation of PAR polymers. These effects compromise the cellular ability to overcome DNA SSB damage. The process of HR, an essential error-free pathway to repair DNA DSBs during cell replication, will be blocked in the condition of BRCA 1/2 mutations. The PARP-associated HR pathway can also be partially interrupted by using PARP inhibitors. Grossly, PARP inhibitors have demonstrated some therapeutic benefits in many randomized phase II and III trials when combined with the standard CRS for advanced EOCs. However, similar to other chemotherapy agents, PARP inhibitors have different clinical indications and toxicity profiles and also face drug resistance, which has become a major challenge. In high-grade epithelial ovarian cancers, the cancer cells under hypoxia- or drug-induced stress have the capacity to become polyploidy giant cancer cells (PGCCs), which can survive the attack of chemotherapeutic agents and start endoreplication. These stem-like, self-renewing PGCCs generate mutations to alter the expression/function of kinases, p53, and stem cell markers, and diploid daughter cells can exhibit drug resistance and facilitate tumor growth and metastasis. In this review, we discuss the underlying molecular mechanisms of PARP inhibitors and the results from the clinical studies that investigated the effects of the FDA-approved PARP inhibitors olaparib, rucaparib, and niraparib. We also review the current research progress on PARP inhibitors, their safety, and their combined usage with antiangiogenic agents. Nevertheless, many unknown aspects of PARP inhibitors, including detailed mechanisms of actions, along with the effectiveness and safety of the treatment of EOCs, warrant further investigation. Full article

The transition from sexual reproduction to asexuality is often triggered by hybridization. The gametogenesis of many hybrid asexuals involves premeiotic genome endoreplication leading to bypass hybrid sterility and forming clonal gametes. However, it is still not clear when endoreplication occurs, how many gonial cells it affects and whether its rate differs among clonal lineages. Here, we investigated meiotic and premeiotic cells of diploid and triploid hybrids of spined loaches (Cypriniformes: Cobitis) that reproduce by gynogenesis. We found that in naturally and experimentally produced F1 hybrids asexuality is achieved by genome endoreplication, which occurs in gonocytes just before entering meiosis or, rarely, one or a few divisions before meiosis. However, genome endoreplication was observed only in a minor fraction of the hybrid’s gonocytes, while the vast majority of gonocytes were unable to duplicate their genomes and consequently could not proceed beyond pachytene due to defects in bivalent formation. We also noted that the rate of endoreplication was significantly higher among gonocytes of hybrids from natural clones than of experimentally produced F1 hybrids. Thus, asexuality and hybrid sterility are intimately related phenomena and the transition from sexual reproduction to asexuality must overcome significant problems with genome incompatibilities with a possible impact on reproductive potential. Full article

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress. Full article

The aim of the study was to demonstrate the biostimulating effect of exogenous melatonin (MEL) applied to seeds via hydroconditioning. It was indicated that only well-chosen application technique and MEL dose guarantees success concerning seed germination and young seedlings growth under stress conditions. For maize seed, 50 μM of MEL appeared to be the optimal dose. It improved seed germination and embryonic axes growth especially during chilling stress (5 °C/14 days) and during regeneration after its subsided. Unfortunately, MEL overdosing lowered IAA level in dry seeds and could disrupt the ROS-dependent signal transduction pathways. Very effective antioxidant MEL action was confirmed by low level of protein oxidative damage and smaller quantity of lipid oxidation products in embryonic axes isolated from seeds pre-treated with MEL and then exposed to cold. The stimulatory effects of MEL on antioxidant enzymes: SOD, APX and GSH-PX and on GST-a detoxifying enzyme, was also demonstrated. It was indicated for the first time, that MEL induced defence strategies against stress at the cytological level, as appearing endoreplication in embryonic axes cells even in the seeds germinating under optimal conditions (preventive action), but very intensively in those germinating under chilling stress conditions (intervention action), and after stress removal, to improve regeneration. Full article

Silkworm is an economically important insect that synthetizes silk proteins for silk production in silk gland, and silk gland cells undergo endoreplication during larval period. Transcription factor Myc is essential for cell growth and proliferation. Although silkworm Myc gene has been identified previously, its biological functions in silkworm silk gland are still largely unknown. In this study, we examined whether enhanced Myc expression in silk gland could facilitate cell growth and silk production. Based on a transgenic approach, Myc was driven by the promoter of the fibroin heavy chain (FibH) gene to be successfully overexpressed in posterior silk gland. Enhanced Myc expression in the PSG elevated FibH expression by about 20% compared to the control, and also increased the weight and shell rate of the cocoon shell. Further investigation confirmed that Myc overexpression increased nucleus size and DNA content of the PSG cells by promoting the transcription of the genes involved in DNA replication. Therefore, we conclude that enhanced Myc expression promotes DNA replication and silk protein expression in endoreplicating silk gland cells, which subsequently raises silk yield. Full article

Whole-genome doubling is the second most frequent genomic event, after TP53 alterations, in advanced solid tumors and is associated with poor prognosis. Tetraploidization step will lead to aneuploidy and chromosomic rearrangements. The mechanism leading to tetraploid cells is important since endoreplication, abortive cytokinesis and cell fusion could have distinct consequences. Unlike processes based on duplication, cell fusion involves the merging of two different genomes, epigenomes and cellular states. Since it is involved in muscle differentiation, we hypothesized that it could play a role in the oncogenesis of myogenic cancers. Spontaneous hybrids, but not their non-fused immortalized myoblast counterparts they are generated from, induced tumors in mice. Unstable upon fusion, the hybrid genome evolved from initial mitosis to tumors with a highly rearranged genome. This genome remodeling finally produced targeted DMD deletions associated with replicative stress, isoform relocalization and metastatic spreading, exactly as observed in human myogenic sarcomas. In conclusion, these results draw a model of myogenic oncogenesis in which cell fusion and oncogene activation combine to produce pleomorphic aggressive sarcomas. Full article

The quality and amount of yellow lupine yield depend on water availability. Water scarcity negatively affects germination, flowering, and pod formation, and thus introduction of an artificial irrigation system is needed. The aim of this study was to evaluate the influence of irrigation on the quality of yellow lupine seeds. Raining was applied with a semi-solid device with sprinklers during periods of greatest water demand. It was shown that watered plants produced seeds of lesser quality, having smaller size and weight. To find out why seeds of irrigated plants were of poor quality, interdisciplinary research at the cellular level was carried out. DNA cytophotometry evidenced the presence of nuclei with lower polyploidy in the apical zone of mature seeds. This may lead to formation of smaller cells and reduce depositing of storage materials. The electrophoretic and Fourier transform infrared spectroscopic analyses revealed differences in protein and cuticular wax profiles, while scanning electron microscopy and energy dispersive spectroscopy revealed, among various chemical elements, decreased calcium content in one of seed zones (near plumule). Seeds from irrigated plants showed slightly higher germination dynamics but growth rate of seedlings was slightly lower. The studies showed that irrigation of lupine affected seed features and their chemical composition, an ability to germination and seedlings growth. Full article

Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation. Full article

The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development. Full article