Academic literature on the topic 'Mitotic delay'

ABSTRACT Ectopic expression of Cdc6p results in mitotic delay, and this has been attributed to Cdc6p-mediated inhibition of Cdc28 protein kinase and failure to activate the anaphase-promoting complex (APC). Here we show that endogenous Cdc6p delays a specific subset of mitotic events and that Cdc28 inhibition is not sufficient to account for it. The depletion of Cdc6p in G2/M cells reveals that Cdc6p is rate limiting for the degradation of the APC/Cdc20 substrates Pds1p and Clb2p. Conversely, the premature expression of Cdc6p delays the degradation of APC/Cdc20 substrates. Abolishing Cdc6p/Cdc28p interaction does not eliminate the Cdc6-dependent delay of these anaphase events. To identify additional Cdc6-mediated, APC-inhibitory mechanisms, we looked for mutants that reversed the mitotic delay. The deletion of SWE1, RAD24, MAD2, or BUB2 had no effect. However, disrupting CDC55, a PP2A regulatory subunit, suppressed the Cdc6p-dependent delay of Pds1 and Clb2 destruction. A specific role for CDC55 was supported by demonstrating that the lethality of Cdc6 ectopic expression in a cdc16-264 mutant is suppressed by the deletion of CDC55, that endogenous Cdc6p coimmunoprecipitates with the Cdc55 and Tpd3 subunits of PP2A, that Cdc6p/Cdc55p/Tpd3 interaction occurs only during mitosis, and that Cdc6 affects PP2A-Cdc55 activity during anaphase. This demonstrates that the levels and timing of accumulation of Cdc6p in mitosis are appropriate for mediating the modulation of APC/Cdc20.

The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430–435). We show here that Chfr is a ubiquitin ligase, both in vitro and in vivo. When transfected into HEK293T cells, Myc–Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, we demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2–M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis.

ABSTRACT We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2−/− cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1−/− cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.

Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (∼7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.

Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule–chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition.

Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.

Chromatin and nuclear pore complexes (NPCs) undergo dramatic changes during mitosis, which in vertebrates and Aspergillus nidulans involves movement of Nup2 from NPCs to the chromatin region to fulfill unknown functions. This transition is shown to require the Cdk1 mitotic kinase and be promoted prematurely by ectopic expression of the NIMA kinase. Nup2 localizes with a copurifying partner termed NupA, a highly divergent yet essential NPC protein. NupA and Nup2 locate throughout the chromatin region during prophase but during anaphase move to surround segregating DNA. NupA function is shown to involve targeting Nup2 to its interphase and mitotic locations. Deletion of either Nup2 or NupA causes identical mitotic defects that initiate a spindle assembly checkpoint (SAC)–dependent mitotic delay and also cause defects in karyokinesis. These mitotic problems are not caused by overall defects in mitotic NPC disassembly–reassembly or general nuclear import. However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis. Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.

ABSTRACT HER4 expression in human breast cancers correlates with a positive prognosis. While heregulin inhibits the growth of HER4-positive breast cancer cells, it does so by undefined mechanisms. We demonstrate that heregulin-induced HER4 activity inhibits cell proliferation and delays G2/M progression of breast cancer cells. While investigating pathways of G2/M delay, we noted that heregulin increased the expression of BRCA1 in a HER4-dependent, HER2-independent manner. Induction of BRCA1 by HER4 occurred independently of the cell cycle. Moreover, BRCA1 expression was elevated in HER4-postive human breast cancer specimens. Heregulin stimulated c-Jun N-terminal kinase (JNK), and pharmacologic inhibition of JNK impaired heregulin-enhanced expression of BRCA1 and mitotic delay; inhibition of Erk1/2 did not. Knockdown of BRCA1 with small interfering RNA in a human breast cancer cell line interfered with HER4-mediated mitotic delay. Heregulin/HER4-dependent mitotic delay was examined further with an isogenic pair of mouse mammary epithelial cells (MECs) derived from mice harboring homozygous LoxP sites flanking exon 11 of BRCA1, such that one cell line expressed BRCA1 while the other cell line, after Cre-mediated excision, did not. BRCA1-positive MECs displayed heregulin-dependent mitotic delay; however, the isogenic BRCA1-negative MECs did not. These results suggest that heregulin-mediated growth inhibition in HER4-postive breast cancer cells requires BRCA1.

The spindle checkpoint delays exit from mitosis in cells with spindle defects. In this paper, we show that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mps1 protein and increased Cdk1–tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B–serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributed to prometaphase accumulation in nocodazole after partial Mps1 inhibition and was required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescued chromosome alignment or segregation in Chk2-deficient cells. We propose that Chk2 stabilizes Mps1 and phosphorylates Aurora B–serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.

Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.

Urban airborne particulate matter (PM) is known to increase morbidity and mortality due to cardiopulmonary diseases related to inflammatory processes and genotoxic effects. The study of PM-induced toxicity represents a very important field in order to understand the clinical outcomes and define the most harmful components involved. Despite the researchers’ effort, there are still unresolved questions regarding the cell mechanisms inducing the different adverse effects. Moreover, which PM components are more significant in determining the biological responses is still a debated question, although this aspect is of primary importance for the individuation of the most impacting sources, which have to be regulated. Thus, the aims of this thesis were - the analysis of the biological effects induced by PM exposure in cell lines representative of the lung apparatus, to evaluate the role of season, location and size of particles - the understanding of the mechanisms and molecular pathways activated by Milan PM fractions with particular attention to winter PM2.5, linking the outcomes to the particles physical and chemical properties. The results obtained demonstrated that the PM-induced biological effects were related to the site and season of sampling, and directly linked to the specific PM chemical composition. The PMs size was another significant factor in eliciting the toxic potential, not only for the chemical composition of particles of diverse dimension, but also for a different bioavailability of the compounds adsorbed, which is higher for the finest fractions. In particular, summer PM10 resulted to be the most cytotoxic and pro-inflammatory fraction for its high content of endotoxins and metals, known inducers of both these biological effects. Winter PMs, especially the fine ones (PM2.5, PM1 and PM0.4), produced a genotoxic effect and caused alterations of the cell cycle through the induction of DNA damage and acting as mitotic spindle poisons. Organic components, in particular PAHs, were responsible of such effects through ROS production and CYP enzymes-mediated reactive molecules formation. A peculiar aspect evidenced was the formation of abnormal mitotic spindles, and in particular the presence of tripolar spindles, which were able to satisfy the spindle assembly checkpoint and perform cell division. The data reported provide a significant contribution to the knowledge of PM toxicity, describing biological processes that can be involved in the clinical and epidemiological observations widely reported; however a complete understanding of the mechanisms of action at cell level still lacks. A further insight in the comprehension of PM health impact may derive by further investigations on different cell lines, cultures of primary pulmonary cells and by the assembly of an alveolar-capillary barrier for the study of the particles systemic effects.

Aperçu du contrôle de la dégradation des ARNm par les protéines YTHpendant la transition de la méiose à la mitose chez les levures.Le cycle cellulaire est contrôlé par des processus complexes et interconnectés. Un gène est transcrit en ARNm qui est traduit en protéines mais de nombreux processus de régulation travaillent pour contrôler chaque étape de ce processus apparemment simple. Parmi ces points de contrôle, la régulation post-transcriptionnelle est importante, et la formation d'un complexe protéine-ARN peut diriger le destin cellulaire. Parmi ces protéines de liaison à l'ARN, les protéines contenant des domaines YTH n’ont été découvertes qu’à la fin des années 90. Les protéines contenant des domaines YTH sont abondantes chez les eucaryotes et absentes chez les procaryotes. Elles constituent la majorité des protéines « readers » capables de reconnaître spécifiquement la modification m6A. L’Homme possède cinq protéines YTH, YTHDF1-3, YTHDC1,2 (Hazra, D., C. Chapat, et Graille, M. (2019). Destin de l'ARNm de m6A : enchaînés au rythme par les protéines contenant de la YTH. , 10 (1), 49.). Bien qu'il soit évident que ces protéines contrôlent le destin cellulaire, la fonction de chaque protéine et son réseau d’interaction restent à élucider. Chez les levures, une seule protéine YTH est présente: Pho92 chez Saccharomyces cerevisiae et Mmi1 chez Schizosaccharomyces pombe. Hormis le domaine YTH, il n'y a pas d'homologie de séquence entre ces deux protéines mais leur fonction cellulaire est similaire.Il est bien établi que Mmi1 est responsable de la dégradation des transcrits spécifiques de la méiose au cours de la croissance végétative des cellules chez la levure S. pombe. Mmi1 forme un complexe stable avec une petite protéine, Erh1 (complexe Erh1-Mmi1 ou EMC). Le complexe EMC peut physiquement interagir avec la sous-unité Not1 du complexe CCR4-Not et la recruter pour la dégradation des ARNm contenant des motifs DSR (déterminant de l'élimination sélective). L'action de Mmi1 est à son tour régulée par une protéine possédant un domaine RRM, Mei2. Au cours de la méiose, Mei2, avec l’aide d’un lncRNA meiRNA, séquestre Mmi1 dans un point nucléaire, le rendant inactif et assurant la continuité de la méiose. Ces trois protéines, Mmi1-Erh1-Mei2, jouent un rôle clé dans la transition de la mitose vers la méiose.Chez S. cerevisiae, Pho92 est impliquée dans la dégradation des transcrits de PHO4, contribuant à la voie du métabolisme du phosphate, pendant la privation en phosphate et participe également à la dégradation des ARNm contenant les marques épitranscriptomiques de N6-méthyladénosine (m6A). Comme pour S. pombe Mmi1, Pho92 recrute le complexe CCR4-Not via une interaction physique avec Not1.Au cours de ma thèse, j'ai tenté d'élucider le rôle de ces deux protéines du domaine YTH de deux organismes modèles, S. cerevisiae et S. pombe, dans la dégradation de l'ARNm et la régulation du cycle cellulaire par des approches biochimiques et structurales.Pho92 de S. cerevisiae interagit physiquement avec Not1 du complexe CCR4-Not, nous avons pu déterminer les limites des domaines impliqués dans cette interaction. L’interaction entre ces deux protéines a été étudiée par anisotropie de fluorescence. Le complexe protéique a été purifié avec succès et des essais de cristallisation sont en cours.Chez S. pombe, la structure de Mei2-RRM3 a été résolue avec et sans ARN. Les propriétés de liaison à l'ARN de Mei2-RRM3 ont été étudiées par ITC. La structure de Erh1 a également été résolue révélant une organisation en homodimere. Nous avons montré que la formation de cet homodimere est important pour la fonction biologique de Mmi1. Des essais de co-cristallisation ont été réalisés avec de l'ARN et les protéines Mmi1 et Mei2, mais sans succès et nous avons obtenu des cristaux de Mmi1
Insights into the control of mRNA decay by YTH proteinsduring the transition from meiosis to mitosis in yeasts.Keywords: Epitranscriptomics, mRNA decay, meiosis, multi-protein complexes, YTH domainCell cycle is controlled by multi-layered processes. A gene is transcribed in mRNA which is translated in proteins but innumerable regulation processes are working to control every step of this apparently simple process. Among these regulatory check points, post-transcriptional regulation is an important one, where formation of a protein-RNA complex may direct the cellular fate. Among these RNA binding proteins, YTH domain proteins are most novel, discovered in late 90s. YTH domain proteins are abundant in eukaryotes and absent in prokaryotes. YTH domain proteins constitute the majority of reader proteins that can specifically identify m6A modification. Human beings have five YTH domain proteins YTHDF1-3, YTHDC1-2 (Hazra, D., Chapat, C., & Graille, M. (2019). m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins. Genes, 10(1), 49.). Although it is evident that these proteins are controlling cellular fate, the function of each protein and their network is yet to be elucidated. In yeast, there is only one YTH domain protein present: Pho92 in Saccharomyces cerevisiae and Mmi1 in Schizosaccharomyces pombe. Apart from the YTH domain there is no sequence homology between these two proteins but their cellular function is similar.It is well established that Mmi1 is responsible for degradation of meiosis specific transcripts during vegetative growth of the cell. Mmi1 forms a tight complex with a small protein, Erh1 (Erh1-Mmi1 complex or EMC). EMC can physically interact with Not1 of CCR4-Not complex and recruit it for degradation of DSR (determinant of selective removal) containing RNAs. The action of Mmi1 is in turn regulated by an RRM domain protein, Mei2. During meiosis, Mei2, along with a lncRNA meiRNA sequesters Mmi1 in a nuclear dot, rendering it inactive and ensuring smooth continuance of meiosis. These three proteins, Mmi1-Erh1-Mei2 play a key role in mitosis to meiosis switch.In S. cerevisiae, Pho92 is involved in the degradation of PHO4 transcripts contributing to phosphate metabolism pathway, during phosphate starvation and also participates in the degradation of mRNAs containing the N6-methyladenosine (m6A) epitranscriptomics marks. Similarly, to S. pombe Mmi1, Pho92 recruits CCR4-Not complex by physical interaction with Not1.During my PhD, I have tried to elucidate the role of these two YTH domain proteins from two model organisms, S. cerevisiae and S. pombe, in mRNA degradation and cell cycle regulation using biochemical and structural approaches.Pho92 of S. cerevisiae physically interacts with Not1 of CCR4-Not complex, we were able to determine the boundaries of this interaction. The interaction between these two proteins was studied by Fluorescence anisotropy. The protein complex was successfully purified and crystallization trials are ongoing.From S. pombe, structure of Mei2-RRM3 was solved with and without an RNA. RNA binding properties of Mei2-RRM3 was studied by ITC. The structure of Erh1 was also solved and we tried to elucidate its importance for biological function of Mmi1. A co-crystallization trial was performed with Mmi1-Mei2-RNA but it was unsuccessful and we ended up with Mmi1 crystals

Vitamin A and its metabolites retinal and retinoic acid are important molecules for the regulation of normal cellular growth, differentiation and other important functions. Retinoids are known to exert mutagenic as well as antimutagenic activity, although conflicting reports are known. All-trans retinoic acid (ATRA) is used in the treatment of many diseases such as acne, psoriasis and ichthyosis. It is also used in differentiated therapy of acute promyelocytic leukemia; however, it is frequently observed that relapses occur when ATRA is prescribed as maintenance therapy. Therefore, understanding the mechanism of action of ATRA in cells would be helpful in the development of high potent and low toxic chemotherapeutic agents. EA-4 is a newly synthesized steroidal analogue of ATRA and is considered as a promising agent for the inhibition of human leukemic cell growth. The study of genotoxicity is an important parameter for the design and development of new chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to their possibility of inducing secondary tumours in cancer patients. Therefore, it is important to determine the genotoxic potential of a drug that will be used in chemotherapy, particularly in native human cells. Taking into consideration the above referred, it would be of interest to evaluate the genotoxic potential of EA-4 in comparison to ATRA, as to their ability to provoke micronucleus (MN) generation, due to both chromosome breakage and chromosome delay. Micronuclei originate from chromosome fragments or whole chromosomes, which lag behind at anaphase during nuclear division. According to our knowledge, there is no information on the ability of all-trans retinoic acid (ATRA) to induce micronucleus formation. To investigate the ability of ATRA and its steroidal analogue EA-4 to enhance micronucleation on human lymphocytes cultured in vitro, the Cytokinesis Block MicroNucleus (CBMN) assay was conducted. By this assay, the cytotoxic effect of the two retinoids was also estimated. To clarify the mechanism by which micronuclei are generated due to ATRA and EA-4 treatment, CBMN was combined with Fluorescence In Situ Hybridization (FISH) using an α-satellite pancentromeric probe to detect centromere inclusion and thus intact chromosome(s) in micronuclei or acentric chromosome fragments. ATRA and EA-4 were shown to be cytotoxic by decreasing CBPI (Cytokinesis Block Proliferation Index) to statistically significant levels in relation to untreated cells. A statistically significant increase in micronucleus frequency was also observed for both investigated compounds. ATRA generated micronuclei mainly via chromosome breakage while a mild effect on chromosome delay was also apparent. On the other hand, EA-4 generated micronuclei exclusively via chromosome breakage. To verify ATRA and EA-4 genotoxicity, micronucleation was investigated in a second biological system coming from a different organism, C2C12 mouse cells. Micronucleus analysis was achieved by α-tubulin/CREST immunostaining for the visualization of microtubules and the detection of kinetochore inside micronuclei and hence the inclusion of whole chromosome(s) or acentric chromosome fragments. Additionally the effect of ATRA and EA-4 on cell proliferation was investigated by the estimation of Mitotic Index (M.I.). We found that ATRA and EA-4 exerted cytotoxic activity in C2C12 mouse cells by reducing the cell proliferation rate at significant levels, as evaluated by the decrease of M.I. A statistically significant elevation in the frequency of interphase cells with micronuclei was shown. CREST analysis confirmed the clastogenic activity of the studied retinoids that was indicated in human lymphocytes. Micronucleation due to ATRA was mediated mainly by chromosome breakage and in a lesser extent by chromosome delay. EA-4 was shown to induce chromosome breakage as well as chromosome delay, as opposed to human lymphocytes at which only clastogenic effect was shown. These observations suggest that, ATRA and EA-4 are able to provoke chromosome fragmentation, but additionally and in a lesser extent to disturb chromosome segregation at anaphase due to chromosome lagging. Cell cycle analysis showed that ATRA and EA-4 accumulated cells at ana-telophase. The analysis of ana-telophases revealed micronucleation, nucleoplasmic bridges and multinucleation, phenomena that may explain the dual genetic activity of ATRA and EA-4. Multinucleated and multimicronucleated interphase cells were also apparent, the second ones generated due to both chromosome delay and breakage. To further investigate the mechanism of genotoxic activity of ATRA and EA-4 we proceeded our research on two axes based on their aneugenicity and clastogenicity. Thus we studied the effect of ATRA and EA-4: i) on the integrity of mitotic spindle, as a target of aneugens by using double immunofluorescence staining of β- and γ-tubulin in C2C12 mouse cell line, which is a convenient system to apply this experimental procedure, and ii) to investigate the ability of the studied retinoids to induce double-strand breaks on DNA by using neutral Single Cell Gel Electrophoresis (SCGE assay-Comet assay) in two different cell lines, C2C12 mouse cells and HL-60 human leukemic cells. Analysis of mitotic spindle has shown that the studied retinoids affect chromosome orientation during metaphase by inducing bipolar metaphases with non-congressed genetic material due to abnormal microtubule network. In addition defects on centrosome duplication and/or separation were observed due to the presence of monopolar metaphases. Ana-telophases as well as interphases with supernumerary centrosomes were also apparent. Additionally, interphase cells with abnormal microtubule network were observed. The above findings may explain aneugenic as well as clastogenic activity of the studied retinoids. Comet assay revealed that ATRA and its steroidal analogue EA-4 provoke DNA migration due to double strand DNA fragmentation in both C2C12 mouse cells and HL-60 human leukemic cells. EA-4 was shown to be the stronger inducer of DNA fragmentation. These results confirm the findings from FISH and CREST analysis indicating that the studied retinoids show high clastogenic activity. . Taking into account the above, we may say that our findings clarify the cytotoxic and genotoxic activity of retinoic acid and the mechanism of its action by indicating its ability to induce chromosome breakage via double-strand DNA breaks and secondary its ability to provoke chromosome delay due to defects in microtubule network and mitotic spindle integrity.
Η βιταμίνη Α και οι μεταβολίτες της, ρετινόλη και ρετινοϊκό οξύ είναι ισχυροί παράγοντες για τη ρύθμιση σημαντικών λειτουργιών, όπως της κυτταρικής ανάπτυξης, διαφοροποίησης και άλλων. Τα ρετινοειδή είναι γνωστά για την μεταλλαξιγόνο αλλά και αντιμεταλλαξιγόνο δράση τους, αν και έχουν αναφερθεί αντικρουόμενα ευρήματα. Το all-trans ρετινοϊκό οξύ (ATRA) χρησιμοποιείται στη θεραπεία πολλών ασθενειών, όπως η ακμή, ψωρίαση, ιχθύωση, αλλά και στη θεραπεία κακοηθειών όπως η μυελογενής λευχαιμία. Συχνά σε περιπτώσεις όπου το ATRA αποτελεί τη βασική θεραπεία παρατηρούνται υποτροπιάσεις Έτσι, η κατανόηση του μηχανισμού δράσης του ATRA στα κύτταρα θα αποτελέσει χρήσιμο εργαλείο για την ανάπτυξη νέων, ισχυρών και μη-τοξικών θεραπευτικών παραγόντων προερχόμενων από αυτό. Το EA-4 είναι ένα πρόσφατα συντεθέν στεροειδικό ανάλογο του ATRA, που θεωρείται υποσχόμενος παράγοντας για την αναστολή της ανάπτυξης ανθρώπινων λευχαιμικών κυττάρων. Η μελέτη της γονιδιοτοξικότητας αποτελεί σημαντική παράμετρο για το σχεδιασμό και την ανάπτυξη νέων θεραπευτικών παραγόντων. Οι γονιδιοτοξικές επιπτώσεις αντικαρκινικών φαρμάκων σε μη-καρκινικά κύτταρα είναι ιδιαίτερης σημασίας, και αποτελούν πιθανή αιτία εμφάνισης δευτερογενών όγκων σε ασθενείς. Έτσι, είναι σημαντικό να μελετηθεί η γονιδιοτοξική δράση ενός φαρμάκου που θα χρησιμοποιηθεί στη χημειοθεραπεία. Λαμβάνοντας υπόψη όλα τα παραπάνω, θεωρήθηκε ενδιαφέρον να εκτιμηθεί η γονιδιοτοξικότητα του EA-4 σε σύγκριση με το ATRA ως προς την ικανότητά τους να προκαλούν την εμφάνιση μικροπυρήνων (MN) είτε μέσω της χρωμοσωματικής θραύσης είτε μέσω της χρωμοσωματικής καθυστέρησης. Οι μικροπυρήνες προέρχονται από χρωμοσωματικά θραύσματα ή ολόκληρα χρωμοσώματα, τα οποία καθυστερούν κατά την ανάφαση της μείωσης ή της μίτωσης. Σύμφωνα με όσα μέχρι σήμερα γνωρίζουμε, δεν φαίνεται να υπάρχουν στοιχεία που αφορούν την ικανότητα του all-trans ρετινοϊκού οξέος (ATRA) να επάγει το σχηματισμό μικροπυρήνων. Για τη διερεύνηση της ικανότητας του ATRA και του στεροειδικού αναλόγου του EA-4 να επάγει την εμφάνιση μικροπυρήνων, πραγματοποιήθηκε η μέθοδος αναστολής της κυτταροκίνησης (CBMN assay) σε ανθρώπινα λεμφοκύτταρα in vitro. Με την ίδια μέθοδο εκτιμήθηκε και η κυτταροτοξικότητα των δύο ρετινοειδών. Για την διευκρίνιση του μηχανισμού δημιουργίας των μικροπυρήνων από τη δράση των ATRA και EA-4, η μέθοδος CBMN συνδυάστηκε με την in situ υβριδιποίηση με φθοροχρώματα (FISH) και χρήση α-δορυφορικού (α-satellite) πανκεντρομερικού ανιχνευτή για την επισήμανση του κεντρομέρους και την ανίχνευσή του σε μικροπυρήνες. Η παρουσία σήματος υβριδοποίησης στους μικροπυρήνες υποδηλώνει την ύπαρξη άθικτου χρωμοσώματος στο εσωτερικό τους. Το αντίθετο υποδεικνύει την παρουσία άκεντρου χρωμοσωματικού θραύσματος. Τα αποτελέσματα έδειξαν ότι και οι δύο χημικές ενώσεις προκαλούν στατιστικά σημαντική αύξηση της συχνότητας των μικροπυρήνων Το ATRA οδηγεί στην δημιουργία μικροπυρήνων κυρίως μέσω χρωμοσωματικής θραύσης, και σε ηπιότερο βαθμό μέσω χρωμοσωματικής καθυστέρησης. Αντίθετα, το EA-4 επάγει το σχηματισμό μικροπυρήνων αποκλειστικά μέσω χρωμοσωματικής θραύσης. Επίσης το ATRA και το EA-4 παρουσάζουν ισχυρή κυτταροτοξικότητα, όπως φάνηκε από τη στατιστικά σημαντική μείωση του κυτταρικού δείκτη πολλαπλασιασμού (CBPI), σε σύγκριση με τις καλλιέργειες του μάρτυρα. Προκειμένου να επιβεβαιωθεί η γονιδιοτοξικότητα του ATRA και του EA-4, διερευνήθηκε η ικανότητά τους να προκαλούν αυξημένες συχνότητες μικροπυρήνων σε ένα δεύτερο βιολογικό σύστημα, την κυτταρική σειρά ποντικού C2C12. Η ανάλυση των MN πραγματοποιήθηκε με τη μέθοδο διπλού ανοσοφθορισμού α-τουμπουλίνης/CREST, για την ανίχνευση σήματος κινητοχώρου στο εσωτερικό του μικροπυρήνα κι έτσι την παρουσία ολόκληρου χρωμοσώματος. Επίσης,η κυτταροτοξικότητα τους διερευνήθηκε με την εκτίμηση του μιτωτικού δείκτη. Με τη ίδια μέθοδο αναλύθηκε η πρόοδος του κυτταρικού κύκλου. Παρατηρήθηκε ότι το ATRA και το EA-4 παρουσιάζουν κυτταροτοξική δράση στα κύτταρα C2C12 μειώνοντας το ρυθμό κυτταρικού πολλαπλασιασμού σε στατιστικά σημαντικά επίπεδα. Επιπλέον αποκαλύφθηκε στατιστικά σημαντική αύξηση της συχνότητας κυττάρων με μικροπυρήνες. Η επισήμανση του κινητοχώρου επιβεβαίωσε τη θραυσματογόνο δράση των υπό μελέτη ρετινοειδών που παρατηρήθηκε στα ανθρώπινα λεμφοκύτταρα. Η δημιουργία μικροπυρήνων μέσω του ATRA ήταν αποτέλεσμα κυρίως χρωμοσωματικής θραύσης και σε μικρότερη έκταση χρωμοσωματικής καθυστέρησης, σε συμφωνία με τα ευρήματα από τα πειράματα στις καλλιέργειες ανθρώπινων λεμφοκυττάρων. Αντίθετα, παρατηρήθηκε ότι το EA-4, πλην της ισχυρής θραυσματογόνου δράσης, προκαλεί και χρωμοσωματική καθυστέρηση. Οι παρατηρήσεις αυτές υποδεικνύουν ότι το ATRA και το EA-4 είναι ισχυροί θραυσματογόνοι παράγοντες, αλλά σε μικρότερο βαθμό είναι ικανοί να διαταράξουν και τον χρωμοσωματικό αποχωρισμό κατά την πυρηνική διαίρεση. Η μελέτη του κυτταρικού κύκλου έδειξε ότι τόσο το ATRA και όσο και το EA-4 προκαλούν καθυστέρηση συσσωρεύοντας τα κύτταρα στα στάδια ανάφασης και τελόφασης της πυρηνικής διαίρεσης. Κύτταρα που συσσωρεύονται στα παραπάνω στάδια χαρακτηρίζονται από την εμφάνιση πυρηνοπλασματικών γεφυρών, την παρουσία περισσότερων του ενός πυρήνων, αλλά και την παρουσία μικροπυρήνων, φαινόμενα τα οποία είναι σύμφωνα με τη διττή γενετική δράση των ATRA και EA-4. Επίσης, παρατηρήθηκαν πολυπύρηνα μεσοφασικά κύτταρα και μεσοφασικά κύτταρα με πολλαπλούς μικροπυρήνες, με τον δεύτερο τύπο κυττάρων να προέρχεται τόσο από χρωμοσωματική θραύση όσο και από χρωμοσωματική καθυστέρηση. Έτσι, φαίνεται ότι τα δύο υπό μελέτη ρετινοειδή μπορούν να χαρακτηρισθούν μόρια με θραυσματογόνες αλλά και ανευπλοειδογόνες ιδιότητες. Για τη λεπτομερέστερη ανάλυση του μηχανισμού δράσης του ATRA και του EA-4 σχεδιάσθηκαν πειράματα σε δύο βασικούς άξονες που αφορούσαν την περαιτέρω μελέτη τόσο της ανευπλοειδογόνου όσο και της θραυσματογόνου δράσης τους. Έτσι, μελετήθηκε η επίδραση του ATRA και του EA-4 αντίστοιχα ως προς: α) την ακεραιότητα της μιτωτικής συσκευής, η οποία αποτελεί κυτταρικό στόχο ανευπλοειδογόνων ενώσεων. Η μελέτη πραγματοποιήθηκε στην κυτταρική σειρά C2C12, μέσω της μεθόδου διπλού ανοσοφθορισμού για τη β- και γ-τουμπουλίνη, δομικά στοιχεία των μικροσωληνίσκων και του κεντροσώματος, και β) την δημιουργία δίκλωνων ρηγμάτων στο DNA μέσω της μεθόδου ηλεκτροφόρησης μοναδιαίων κυττάρων (SCGE assay-Comet assay) σε δύο διαφορετικές κυτταρικές σειρές, στα κύτταρα ποντικού C2C12 και στα λευχαιμικά κύτταρα ανθρώπου HL-60. Τα αποτελέσματα μας έδειξαν ότι τα υπό εξέταση ρετινοειδή επηρεάζουν τον χρωμοσωματικό προσανατολισμό κατά τη μετάφαση με την εμφάνιση διπολικών μεταφάσεων με τα χρωμοσώματα μη-διατεταγμένα στο ισημερινό πεδίο, λόγω ανωμαλιών του δικτύου των μικροσωληνίσκων. Επίσης, φάνηκε ότι προκαλούν ανωμαλία στον πολλαπλασιασμό και πιθανόι στον αποχωρισμό των κεντροσωμάτων, παρατήρηση που δικαιολογείται από την παρουσία μονοπολικών μεταφάσεων, καθώς και ανάτελοφάσεων αλλά και μεσοφασικών κύττάρων με υπεράριθμο κεντροσωματικό αριθμό. Επιβεβαιώθηκε επίσης η επίδρασή τους στην πορεία του κυτταρικού κύκλου με συσσώρευση των κυττάρων στα στάδια ανάφασης-τελόφασης. Επιπρόσθετα, φάνηκε ότι το ΕΑ-4, στη μεγαλύτερη συγκέντρωση, διακόπτει τον κυτταρικό κύκλο στο στάδιο της μετάφασης. Παράλληλα, παρατηρήθηκε διαταραχή στη δομή του δικτύου των μικροσωληνίσκων. Όλα τα παραπάνω ευρήματα ερμηνεύουν τόσο την ανευπλοειδογόνο όσο και τη θραυσματογόνο δράση των δύο ρετινοειδών. Με τη μέθοδο ηλεκτροφόρησης μοναδιαίων κυττάρων δείχθηκε ότι το ATRA και το στεροειδικό του ανάλογο EA-4 προκάλεσαν τη δημιουργία «κομητών», δηλαδή πυρήνων με ανώμαλη μορφολογία μέσω του σχηματισμού δίκλωνων θραυσμάτων DNA. Το φαινόμενο αυτό παρατηρήθηκε τόσο στα κύτταρα ποντικού C2C12 όσο και στα λευχαιμικά κύτταρα ανθρώπου HL-60, με το EA-4 να παρουσιάζει ισχυρότερη επαγωγή θραύσης του DNA. Τα αποτελέσματα αυτά επιβεβαιώνουν τα ευρήματα των μεθόδων FISH και CREST, υποδεικνύοντας ότι τα υπό εξέταση ρετινοειδή παρουσιάζουν ισχυρή θραυσματογόνο δράση. Λαμβάνοντας υπόψη όλα τα παραπάνω, μπορούμε να ισχυριστούμε ότι τα ευρήματά μας διευκρινίζουν την κυτταροτοξική και γονιδιοτοξική δράση του ρετινοϊκού οξέος. Υποδεικνύουν ιδιότητες ισχυρώς θραυσματογόνων παραγόντων μέσω δημιουργίας δίκλωνων ρηγμάτων στο DNA των κυττάρων. Δευτερογενώς μπορούν να χαρακτηρισθούν ως ήπιες ανευπλοειδογόνες ενώσεις που προκαλούν ανώμαλο χρωμοσωματικό αποχωρισμό μέσω ανωμαλιών τόσο του δικτύου των μικροσωληνίσκων όσο και της ακεραιότητα της μιτωτικής συσκευής.

碩士
長榮大學
醫學研究所
104
The Ministry of Health and Welfare, National Health Department, has recently identified malignant tumors as the leading cause of death in Taiwan. Glioblastoma multiforme (GBM) is the most common malignant brain cancer, which accounts for more than half of all brain tumors. GBM has a high mortality rate and is recurrent and resistant to chemotherapy. Common cancer therapies, including surgery, chemotherapy, and targeted therapy, induce many side effects on normal cells and tissues. Extracts of herbal or natural products have proved to be complementary medicine with fewer side effects than chemotherapy drugs. Cucurbitacin E (CuE), a tetracyclic triterpene extracted from the climbing stem of melon, is shown to have anti-inflammatory and anti-cytotoxic responses and inhibitory effects on cancer cell growth. In this study, we investigated the anti-tumor effects of CuE in human brain cancer cell lines (GBM 8401, U87-MG). Cell survival was analyzed using the MTT assay. Our results showed that the GBM cell lines treated with CuE had significantly inhibited cell growth. CuE treatment was found to induced cell cycle G2/M phase arrest but not apoptosis or necrosis in the human brain cancer cell lines. The expression profile of G2 /M phase-associated proteins, such as CDC2 and cyclin B, and p38/JNK pathway regulation was analyzed by Western blotting. Our findings indicated that the expression of these proteins is downregulated in the GBM cell lines by CuE treatment. CuE may be an effective candidate for brain cancer treatment or adjuvant chemotherapy. Further, it may provide a new direction in brain cancer prevention and treatment.