Nuclear DNA Synthesis Research Articles

Plasmodium falciparum OPA3-like protein (PfOPA3) is essential for maintenance of mitochondrial homeostasis and parasite proliferation.

Metabolic pathways and proteins responsible for maintaining mitochondrial dynamics and homeostasis in the Plasmodium parasite, the causative agent of malaria, remain to be elucidated. Here, we identified and functionally characterized a novel OPA3-like domain-containing protein in P. falciparum (PfOPA3). We show that PfOPA3 is expressed in the intraerythrocytic stages of the parasite and localizes to the mitochondria. Inducible knock-down of PfOPA3 using GlmS ribozyme hindered the normal intraerythrocytic cycle of the parasites; specifically, PfOPA3-iKD disrupted parasite development as well as parasite division and segregation at schizont stages, which resulted in a drastic reduction in the number of merozoites progenies. Parasites lacking PfOPA3 show severe defects in the development of functional mitochondria; the mitochondria showed reduced activity of mtETC but not ATP synthesis, as evidenced by reduced activity of complex III of the mtETC, and increased sensitivity for drugs targeting DHODH as well as complex III, but not to the drugs targeting complex V. Further, PfOPA3 downregulation leads to reduction in the level of mitochondrial proton transport uncoupling protein (PfUCP) to compensate reduced activity of complex III and maintain proton efflux across the inner membrane. The reduced activity of DHODH, which is responsible for pyrimidine biosynthesis required for nuclear DNA synthesis, resulted in a significant reduction in parasite nuclear division and generation of progeny. In conclusion, we show that PfOPA3 is essential for the functioning of mtETC and homeostasis required for the development of functional mitochondria as well as for parasite segregation, and thus PfOPA3 is crucial for parasite survival during blood stages.

Chiral Cell Nanomechanics Originated in Clockwise/Counterclockwise Biofunctional Microarrays to Govern the Nuclear Mechanotransduction of Mesenchymal Stem Cells.

Cell chirality is extremely important for the evolution of cell morphogenesis to manipulate cell performance due to left-right asymmetry. Although chiral micro- and nanoscale biomaterials have been developed to regulate cell functions, how cell chirality affects cell nanomechanics to command nuclear mechanotransduction was ambiguous. In this study, chiral engineered microcircle arrays were prepared by photosensitive cross-linking synthesis on cell culture plates to control the clockwise/counterclockwise geometric topology of stem cells. Asymmetric focal adhesion and cytoskeleton structures could induce chiral cell nanomechanics measured by atomic force microscopy (AFM) nanoindentation in left-/right-handed stem cells. Cell nanomechanics could be enhanced when the construction of mature focal adhesion and the assembly of actin and myosin cytoskeletons were well organized in chiral engineered stem cells. Curvature angles had a negative effect on cell nanomechanics, while cell chirality did not change cytoskeletal mechanics. The biased cytoskeleton tension would engender different nuclear mechanotransductions by yes-associated protein (YAP) evaluation. The chiral stimuli were delivered into the nuclei to oversee nuclear behaviors. A strong cell modulus could activate high nuclear DNA synthesis activity by mechanotransduction. The results will bring the possibility of understanding the interplay of chiral cell nanomechanics and mechanotransduction in nanomedicines and biomaterials.

Abstract 4883: Pan cancer transcriptomic response to tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy is versatile with multiple mechanisms of action, including: anti-mitotic effects, downregulation of DNA double strand break repair, generation of replication stress, upregulation of autophagy, and induction of immunogenic cell death. In this research we were interested in identifying common responses to TTFields across cancer types. We addressed this by integrating whole transcriptome analyses of cancers from various origins. Methods: Control and TTFields-treated samples examined included gastric, ovarian, pancreatic, non-small cell lung carcinoma, and glioblastoma cell lines, pleural mesothelioma patient-derived cell lines, and a hepatocellular carcinoma animal model. A list of differentially expressed genes (DEGs) was generated from transcriptomics analysis. Gene Set Enrichment Analysis (GSEA) was conducted with the MSigDB, and additional pathway databases. Significantly overlapping pathways were identified using ActivePathways package, and an enrichment map was created according to the number of datasets supporting each pathway. Results: The response to TTFields showed a positive correlation of expression across different cell lines and indications, including in an animal model. Downregulation was seen in cell cycle related pathways - E2F targets, Myc targets, G2M checkpoint, and mitotic spindle - indicative of cell cycle arrest, which was mediated by increased expression of tumor suppressors. Several DNA repair pathways were downregulated, including nucleotide excision repair, base excision repair, and the Fanconi Anemia-BRCA pathway. Downregulation was also evident in nuclear envelope, DNA and RNA synthesis, metabolism of amino acids, translation, organelle organization, and splicing. Upregulation was observed in genes associated with the cytoskeleton, adhesion, extracellular proteins and transporters. In the majority of comparisons, an upregulation in the immune response was also observed. Conclusions: Transcriptomic analysis of datasets from different tumor types revealed common expression patterns and pathways involved in the responses to TTFields. Some of the identified pathways corroborate previously described mechanisms, whereas some reveal new potential mechanisms that require further investigation. Citation Format: Kerem Wainer-Katsir, Gitit Lavy-Shahaf, Hila Fishman, Hila Ena, Roni Frechtel-Gerzi, Antonia Martinez-Conde, Eyal Dor-On, Shiri Davidi, Sara Jacobovitch, Itai Tzchori, Yaara Porat, Lianghao Ding, Michael Story, Renfei Du, Ulf Kahlert, Laura Mannarino, Federica Mirimao, Monica Lupi, Maurizio D’Incalci, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Pan cancer transcriptomic response to tumor treating fields (TTFields). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4883.

Regulation of micropatterned curvature-dependent FA heterogeneity on cytoskeleton tension and nuclear DNA synthesis of malignant breast cancer cells.

Breast cancer is considered as a worldwide disease due to its high incidence and malignant metastasis. Although numerous techniques have been developed well to conduct breast cancer therapy, the influence of micropattern-induced interfacial heterogeneity on the molecular mechanism and nuclear signalling transduction of carcinogenesis is rarely announced. In this study, PDMS stencil-assisted micropatterns were fabricated on tissue culture plates to manage cell clustering colony by adjusting initial cell seeding density and the size of microholes. The curvature of each microholes was controlled to construct the interfacial heterogeneity of MDA-MB231 cancer cells at the periphery of micropatterned colony. The distinguished focal adhesion (FA) and cytoskeleton distribution at the central and peripheral regions of the cell colony were regulated by heterogeneous properties. The interfacial heterogeneity of FA and cytoskeleton would induce the biased tension force to encourage more ezrin expression at the periphery and further promote DNA synthesis, therefore disclosing a stem-like phenotype in heterogeneous cells. This study will provide a value source of information for the development of micropattern-induced heterogeneity and the interpretation of metastatic mechanism in malignant breast cancer cells.

INVESTIGATING EARLY SIGNS OF ENDOREDUPLICATION IN ARABIDOPSIS SHOOT: AN UNKNOWN FACT

Endoreduplication is alternative form of cell cycle that involves the replication of DNA without mitosis. It commonly occurs in various tissues of plants like pavement cells of leaf, trichomes and sepals of flower and led to increase in size of the cell. Shoot apical meristem (SAM) is a region from where the aboveground organs of the plant arise. The cells in the meristem remain in meristematic state (mitotic) and get displaced from there to form the differentiated tissues. But how the nuclear DNA synthesis vary from the meristematic cells to the differentiated tissues is not very well studied. It has been observed through the present study that the cells in shoot tip of Arabidopsis are diploid in nature and starts to endoreduplicate at 1cm away from the shoot, down the stem which was justified by the flow cytometer-based DNA analysis of different Arabidopsis tissues. Petal is known to be the most variable part of the flower with different color, shape, size and fragrance but little is known about its characterization. The cell identities in petal are diverse with different cell types. One is small sized distal cells and the other is large sized proximal cells. The present study has addressed that the large size of proximal cells of petal is not merely a cell expansion but is something related to endoreduplication. This was evidenced by the DNA estimation of FACS (Florescent activated cell sorter) sorted petal cells of Arabidopsis. This is the first study in which SAM, stem, leaf and petal cells are taken into account for ploidy analysis by flow cytometry. In shoot apex we did not see endoreduplication however as the cells descend towards the stem or leaf they enter into differentiation pathways and undergo endoreduplication. On the other hand, different ploidies in petal cells shows the signs of endoreduplication which can be a way towards differentiation.

INVESTIGATING EARLY SIGNS OF ENDOREDUPLICATION IN ARABIDOPSIS SHOOT: AN UNKNOWN FACT

Endoreduplication is alternative form of cell cycle that involves the replication of DNA without mitosis. It commonly occurs in various tissues of plants like pavement cells of leaf, trichomes and sepals of flower and led to increase in size of the cell. Shoot apical meristem (SAM) is a region from where the aboveground organs of the plant arise. The cells in the meristem remain in meristematic state (mitotic) and get displaced from there to form the differentiated tissues. But how the nuclear DNA synthesis vary from the meristematic cells to the differentiated tissues is not very well studied. It has been observed through the present study that the cells in shoot tip of Arabidopsis are diploid in nature and starts to endoreduplicate at 1cm away from the shoot, down the stem which was justified by the flow cytometer-based DNA analysis of different Arabidopsis tissues. Petal is known to be the most variable part of the flower with different color, shape, size and fragrance but little is known about its characterization. The cell identities in petal are diverse with different cell types. One is small sized distal cells and the other is large sized proximal cells. The present study has addressed that the large size of proximal cells of petal is not merely a cell expansion but is something related to endoreduplication. This was evidenced by the DNA estimation of FACS (Florescent activated cell sorter) sorted petal cells of Arabidopsis. This is the first study in which SAM, stem, leaf and petal cells are taken into account for ploidy analysis by flow cytometry. In shoot apex we did not see endoreduplication however as the cells descend towards the stem or leaf they enter into differentiation pathways and undergo endoreduplication. On the other hand, different ploidies in petal cells shows the signs of endoreduplication which can be a way towards differentiation.

High-Content Image-Based Single-Cell Phenotypic Analysis for the Testicular Toxicity Prediction Induced by Bisphenol A and Its Analogs Bisphenol S, Bisphenol AF, and Tetrabromobisphenol A in a Three-Dimensional Testicular Cell Co-culture Model.

Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest.

Control of p21Cip by BRCA1-associated protein is critical for cardiomyocyte cell cycle progression and survival.

Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation. General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis. By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes.

Inhibition of Period Gene Expression Causes Repression of Cell Cycle Progression and Cell Growth in the Bombyx mori Cells.

Circadian clock system disorders can lead to uncontrolled cell proliferation, but the molecular mechanism remains unknown. We used a Bombyx mori animal model of single Period gene (BmPer) expression to investigate this mechanism. A slow growing developmental cell model (Per-KD) was isolated from a B. mori ovarian cell line (BmN) by continuous knock down of BmPer expression. The effects of BmPer expression knockdown (Per-KD) on cell proliferation and apoptosis were opposite to those of m/hPer1 and m/hPer2 in mammals. The knockdown of BmPer expression led to cell cycle deceleration with shrinking of the BmN cell nucleus, and significant inhibition of nuclear DNA synthesis and cell proliferation. It also promoted autophagy via the lysosomal pathway, and accelerated apoptosis via the caspase pathway.

Flow cytometry – a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species

Abstract Flow cytometry (FCM) has been used for plant DNA content estimation since the 1980s; however, presently, the number of laboratories equipped with flow cytometers has significantly increased and these are used extensively not only for research but also in plant breeding (especially polyploid and hybrid breeding) and seed production and technology to establish seed maturity, quality and advancement of germination. A broad spectrum of horticultural and medicinal species has been analyzed using this technique, and various FCM applications are presented in the present review. The most common application is genome size and ploidy estimation, but FCM is also very convenient for establishing cell cycle activity and endoreduplication intensity in different plant organs and tissues. It can be used to analyze plant material grown in a greenhouse/field as well as in vitro. Due to somaclonal variation, plant material grown in tissue culture is especially unstable in its DNA content and, therefore, FCM analysis is strongly recommended. Horticultural species are often used as internal standards in genome size estimation and as models for cytometrically studied cytotoxic/anticancer/allelopathic effects of different compounds. With the growing interest in genome modification, increased application of FCM is foreseen.

INDICATORS OF THE CARDIOMYOCYTES` CELLS CYCLE UNDER INFUSION OF BLOOD SUBSTITUTES AND IN THE CORRECTION OF EXPERIMENTAL BURN INJURY BY 0,9% NACL SOLUTION

According to the WHO, the thermal trauma is on the third place among other injuries. Burned injury is not only damage to the skin, but also the traumatization of all organs and systems of the body as a result of the stress response of the vascular system and the effects of toxic products coming from the area of burn injury. Firstly, such damages affect cardiomyocytes and the microcirculation vessels of the heart. The purpose of our study was to evaluate the changes in the cell cycle of myocardial cells in the left ventricle of rats under conditions of blood substitutes infusion and in the correction of experimental burn injury with a 0,9% solution of NaCl. The burn trauma was modeled using the Regas’ method and placed a catheter into the lower vena cava for intravenous infusion. The following solutions were used for infusion: 0,9% NaCl solution, lactoproteinum with sorbitol (Lactoproteinum-C) and colloidal-hyperosmolar HAES-LX-5% solution. Flow cytometry of the nuclear suspension of left ventricular cardiomyocytes was performed on the 1st, 3rd, and 7th days of the experiment. The statistical analysis of the results was carried out using the “STATISTICA 6.1” program package. The results of the performed study show a fairly stable picture of cell cycle parameters in myocardial cells of animals without burn injury with a predominance, on the one hand, of cells present in the G0G1 phase and the presence of a certain balance between the processes of creation of nuclear DNA synthesis and apoptosis. Changes in the phase of cardiac myocyte cell cycle against the background of the thermal injury of the skin throughout the observation time indicate a prolonged, uncorrected cell cycle disorder and a lack of effective normalization on the background of the physiological solution usage in the first 7 days after burning trauma of the skin. The protective effect of HAES-LX-5% prevents over-strain of cells, as evidenced by the lower synthetic activity of nuclei of cardiomyocytes at all times of the experiment.

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry.

Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2'-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIα and IIβ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected.IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIα and IIβ and PCNA, which were found associated with nascent DNA.

Regulation of chloroplast and nucleomorph replication by the cell cycle in the cryptophyte Guillardia theta

The chloroplasts of cryptophytes arose through a secondary endosymbiotic event in which a red algal endosymbiont was integrated into a previously nonphotosynthetic eukaryote. The cryptophytes retain a remnant of the endosymbiont nucleus (nucleomorph) that is replicated once in the cell cycle along with the chloroplast. To understand how the chloroplast, nucleomorph and host cell divide in a coordinated manner, we examined the expression of genes/proteins that are related to nucleomorph replication and chloroplast division as well as the timing of nuclear and nucleomorph DNA synthesis in the cryptophyte Guillardia theta. Nucleus-encoded nucleomorph HISTONE H2A mRNA specifically accumulated during the nuclear S phase. In contrast, nucleomorph-encoded genes/proteins that are related to nucleomorph replication and chloroplast division (FtsZ) are constantly expressed throughout the cell cycle. The results of this study and previous studies on chlorarachniophytes suggest that there was a common evolutionary pattern in which an endosymbiont lost its replication cycle-dependent transcription while cell-cycle-dependent transcriptional regulation of host nuclear genes came to restrict the timing of nucleomorph replication and chloroplast division.

AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome.

Trypanosoma brucei causes human African trypanosomiasis (HAT). The pyrrolopyrimidine AEE788 (a hit for anti-HAT drug discovery) associates with three trypanosome protein kinases. Herein we delineate the effects of AEE788 on T. brucei using chemical biology strategies. AEE788 treatment inhibits DNA replication in the kinetoplast (mitochondrial nucleoid) and nucleus. In addition, AEE788 blocks duplication of the basal body and the bilobe without affecting mitosis. Thus, AEE788 prevents entry into the S-phase of the cell division cycle. To study the kinetics of early events in trypanosome division, we employed an "AEE788 block and release" protocol to stage entry into the S-phase. A time-course of DNA synthesis (nuclear and kinetoplast DNA), duplication of organelles (basal body, bilobe, kinetoplast, nucleus), and cytokinesis was obtained. Unexpected findings include the following: 1) basal body and bilobe duplication are concurrent; 2) maturation of probasal bodies, marked by TbRP2 recruitment, is coupled with nascent basal body assembly, monitored by localization of TbSAS6 at newly forming basal bodies; and 3) kinetoplast division is observed in G2 after completion of nuclear DNA synthesis. Prolonged exposure of trypanosomes to AEE788 inhibited transferrin endocytosis, altered cell morphology, and decreased cell viability. To discover putative effectors for the pleiotropic effects of AEE788, proteome-wide changes in protein phosphorylation induced by the drug were determined. Putative effectors include an SR protein kinase, bilobe proteins, TbSAS4, TbRP2, and BILBO-1. Loss of function of one or more of these effectors can, from published literature, explain the polypharmacology of AEE788 on trypanosome biology.

Abstract P5-06-09: Cyclin d1 binding to chromatin and the induction of chromosomal instability requires the fuzzy domain

Abstract The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma (Rb) and the nuclear respiratory factor 1 (NRF1) proteins to regulate nuclear DNA synthesis and mitochondrial biogenesis. Cyclin D1 is required for oncogene-dependent growth and genetic ablation of the murine cyclin D1 gene resulted in resistance to Ras or ErbB2-induced mammary tumorigenesis and APC-induced gastrointestinal tumorigenesis. Cyclin D1 overexpression occurs in human breast, prostate, lung, and gastrointestinal malignancies and its abundance is induced at the level of transcription, translation and through post-translational modifications. Cyclin D1 plays a key role in transcriptional regulation inducing gene expression governing chromosomal instability (CIN) and cell-cycle progression. Cyclin D1 is also known to bind TF regulatory regions in chromatin immuno-precipitation (ChIP) assays. Genome wide analysis of cyclin D1 occupancy using ChIP-Seq identified binding sites including both the coding and non-coding genome with enrichment for genes regulating CIN and the G2/M phase (Top2A, AurkB, Cenpp, Mlf1ip, Zw10, Ckap2) consistent with enrichment of cyclin D1 at G2/M and the finding that cyclin D1 induces CIN. We sought to identify the molecular mechanisms governing the recruitment of cyclin D1 in the context of local chromatin to promote CIN. In order to define the domain of cyclin D1 involved in aneuploidy and tumorigenesis, we transduced and assessed the induction of aneuploidy in MEF cells using cyclin D1 wt (wt), cyclin D1 C-terminus domain (C4), cyclin D1 mutant lacking of the E-box motif (ΔE) or ctrl. We also searched for potential histone protein interaction motifs in cyclin D1 and determined the epigenetic motif recognized by cyclin D1 using a histone peptide array. The recognition of an epigenetic code by cyclin D1 may facilitate genome wide expression changes during cell-cycle progression and tumorigenesis. We finally identified a “fuzzy” domain of cyclin D1 which is required to local chromatin access for regulatory promoter regions governing and promoting CIN. Citation Format: Pestell RG, Di Sante G, Di Rocco A, Pupo C, Crosariol M, Tompa P, Tantos A, Wang C, Yu Z, Vadlamudi R, Mann M, Casimiro MC. Cyclin d1 binding to chromatin and the induction of chromosomal instability requires the fuzzy domain [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-09.

Intron loss in interferon genes follows a distinct set of stages, and may confer an evolutionary advantage

The promoter-intron-exon structure of genes evolve. While the structures of some IFN genes (e.g., piscine and amphibian Type I IFNs, most tetrapod IFN-λ genes) resemble those of other class II cytokines (e.g., interleukins-10, 19, 20, 22, 24, 26), the structures of other IFN genes differ significantly. Although all bony vertebrate IFN-γ genes lack the canonical third intron, and all amniote Type I IFN genes lack introns, only some IFN-λ genes lost their introns. Interestingly, these intronless IFN-λ genes are not preferentially related to one another nor are they clustered with canonical multi-intron IFN-λ genes. Hypothesizing that intronless IFN-λ genes repeatedly and independently evolved and transposed throughout the genome, we sought to understand the genetic processes involved in their intron loss and genomic migration. Utilizing the high conservation of the promoters, the UTRs and the ORFs of the IFN-λ genes, we collected data from two families of intronless IFN-λ genes, and developed a model supported by these data to explain how intronless IFN-λ genes evolved. (1) A cytoplasmic IFN-λ cDNA generated by reverse transcriptional activity enters the nucleus and attempts to recombine with its multi-exon progenitor. (2) Nuclear DNA synthesis at the 5′ and 3′ ends within recombination intermediates affixes the promoter onto the cDNA and preserves its 3′ UTR. (3) Resolution of the recombination complex releases the promoter-associated cDNA. (4) The released intronless gene co-integrates with a highly duplicated sequence undergoing transposition. We propose that this process explains not only the evolution of the gene structure of IFN genes, but also the increased transposition of intronless genes in genomes, and may confer an evolutionary advantage.

A twinfilin-like protein coordinates karyokinesis by influencing mitotic spindle elongation and DNA replication in Leishmania.

Twinfilin is an evolutionarily conserved actin-binding protein, which regulates actin-dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine.

5-Ethynyl-2′-deoxyuridine (EdU) and 5-ethynyl-2′-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.

Influence of hydroxyurea on the course of germination and growth of rape (Brasica napus L.) seedlings

The effects of continuous incubation in hydroxyurea (HU) solutions (0.2, 0.4, 0.8 mg/ml) on germination of rape seeds and growth of young seedling axes were studied during 132 hours from initial soaking. Germination turned out to be unaffected by the treatment. Root growth was first increasingly inhibited by the HU concentration tested, but after prolonged incubation a complete arrest of the root growth was noted at all HU concentrations. The elongation growth of hypocotyls was found to be stimulated by a HU 0.2 mg/ml concentration while it was markedly suppressed by 0.4 mg/ml, and completely arrested by 0.8 mg/ml Inhibition of growth of the upright hypocotyl part at higher HU concentration was found to be accompanied by the unbending of the hooked under-cotyledonary part. It is suggested that inhibition of nuclear endomitotic DNA synthesis In elongating hypocotyl cells, suppresses only partially their growth, whereas a complete inhibition of the hypocotyl growth results from arrest of the mitochondrial DNA synthesis.

Colchicine treatment reversibly blocks cytokinesis but not mitosis in Trypanosoma cruzi epimastigotes.

This work analyzes the effect of the alkaloid colchicine on the growth of Trypanosoma cruzi epimastigotes, using immunofluorescence microscopy and flow cytometry techniques. We found that colchicine reversibly inhibited cytokinesis but not synthesis or segregation of nuclear and kinetoplastid DNA, in a concentration-dependent manner. We showed that, once colchicine was removed from the growth medium, cytokinesis was restored but abnormal segregation of kinetoplasts and nuclei generated zoids and parasites with two nuclei and one kinetoplast, among other aberrant cells. After drug removal, we also observed a few anucleated cells carrying two kinetoplasts in a stage compatible with the end of cytokinesis. The anomalous subcellular localization of the kinetoplast and flagellum observed in treated parasites suggests that the effect of colchicine and its interaction with T. cruzi microtubules is cell cycle dependent. The crosstalk between nuclear and kinetoplastid mitosis and its incidence on flagellum growth and parasite cell division regulation are discussed.